Plant Quality With Various Promoters

  • Published: Feb 19, 2009
  • Earliest Priority: Sep 22 1998
  • Family: 2
  • Cited Works: 0
  • Cited by: 66
  • Cites: 6
  • Sequences: 10667
  • Additional Info: Full text Sequence
  *US20090049566A1*
  US20090049566A1                                 
(19)United States 
(12)Patent Application Publication(10)Pub. No.: US 2009/0049566 A1
 Zhang et al.(43)Pub. Date:Feb.  19, 2009

(54)PLANT QUALITY WITH VARIOUS PROMOTERS 
    
(75)Inventors: James Z. Zhang,  Palo Alto, CA (US); 
  Frederick D. Hempel,  Hayward, CA (US); 
  Luc J. Adam,  Hayward, CA (US); 
  Joseph M. Palys,  Davis, CA (US) 
    
 Correspondence Address: 
 Mendel Biotechnology, Inc.  
 3935 Point Eden Way 
 Hayward, CA 94545  (US) 
    
(73)Assignee:Mendel Biotechnology, Inc.,  Hayward, CA (US), Type: US Company 
(21)Appl. No.: 12/169,527 
(22)Filed: Jul.  8, 2008 
 Related U.S. Application Data 
(60) .
Continuation-in-part of application No. 10/286,264, filed on Nov.  1, 2002 , which is a division of application No. 09/533,030, filed on Mar.  22, 2000, now abandoned , which is a continuation-in-part of application No. 10/675,852, filed on Sep.  30, 2003 , which is a continuation-in-part of application No. 11/479,226, filed on Jun.  30, 2006 , which is a continuation-in-part of application No. 09/713,994, filed on Nov.  16, 2000, now abandoned , which is a continuation-in-part of application No. 10/669,824, filed on Sep.  23, 2003 , which is a continuation-in-part of application No. 09/823,676, filed on Mar.  30, 2001, now Pat. No. 6,717,034 , which is a continuation-in-part of application No. 11/725,235, filed on Mar.  16, 2007 , which is a division of application No. 10/225,068, filed on Aug.  9, 2002, now Pat. No. 7,193,129 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned , which is a continuation-in-part of application No. 11/728,567, filed on Mar.  26, 2007 , which is a division of application No. 10/225,066, filed on Aug.  9, 2002, now Pat. No. 7,238,860 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned , which is a continuation-in-part of application No. 11/375,241, filed on Mar.  13, 2006 , which is a continuation-in-part of application No. 10/225,067, filed on Aug.  9, 2002, now Pat. No. 7,135,616 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned , which is a continuation-in-part of application No. 11/069,255, filed on Feb.  28, 2005 , which is a continuation of application No. 10/112,887, filed on Mar.  18, 2002, now abandoned , which is a continuation-in-part of application No. 10/374,780, filed on Feb.  25, 2003 , which is a continuation-in-part of application No. 09/934,455, filed on Aug.  22, 2001, now abandoned , which is a continuation-in-part of application No. 09/713,994, filed on Nov.  16, 2000, now abandoned , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned .
Said application No. 10/374,780 is a continuation-in-part of application No. 10/225,068, filed on Aug.  9, 2002, now Pat. No. 7,193,129 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned .
Said application No. 10/374,780 is a continuation-in-part of application No. 10/225,066, filed on Aug.  9, 2002, now Pat. No. 7,238,860 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned .
Said application No. 10/374,780 is a continuation-in-part of application No. 10/225,067, filed on Aug.  9, 2002, now Pat. No. 7,135,616 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned , which is a continuation-in-part of application No. 10/546,266, filed on Aug.  19, 2005 , which is a continuation-in-part of application No. 11/986,992, filed on Nov.  26, 2007 , which is a division of application No. 10/412,699, filed on Apr.  10, 2003, now Pat. No. 7,345,217 , which is a continuation-in-part of application No. 10/295,403, filed on Nov.  15, 2002, now abandoned , which is a division of application No. 09/394,519, filed on Sep.  13, 1999, now abandoned .
Said application No. 10/412,699 is a continuation-in-part of application No. 09/489,376, filed on Jan.  21, 2000, now abandoned , and which is a continuation-in-part of application No. 10/302,267, filed on Nov.  22, 2002, now Pat. No. 7,223,904 , which is a division of application No. 09/506,720, filed on Feb.  17, 2000, now abandoned .
Said application No. 10/412,699 is a continuation-in-part of application No. 10/278,173, filed on Oct.  21, 2002, now abandoned , which is a division of application No. 09/533,392, filed on Mar.  22, 2000, now abandoned .
Said application No. 10/412,699 is a continuation-in-part of application No. 09/533,029, filed on Mar.  22, 2000, now Pat. No. 6,664,446 , and which is a continuation-in-part of application No. 10/278,536, filed on Oct.  22, 2002, now abandoned , which is a division of application No. 09/532,591, filed on Mar.  22, 2000, now abandoned .
Said application No. 10/412,699 is a continuation-in-part of application No. 09/713,994, filed on Nov.  16, 2000, now abandoned , and which is a continuation-in-part of application No. 09/819,142, filed on Mar.  27, 2001, now abandoned , which is a continuation-in-part of application No. 09/934,455, filed on Aug.  22, 2001, now abandoned , which is a continuation-in-part of application No. 09/713,994, filed on Nov.  16, 2000, now abandoned , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned .
Said application No. 10/412,699 is a continuation-in-part of application No. 10/225,068, filed on Aug.  9, 2002, now Pat. No. 7,193,129 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned .
Said application No. 10/412,699 is a continuation-in-part of application No. 10/225,066, filed on Aug.  9, 2002, now Pat. No. 7,238,860 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned .
Said application No. 10/412,699 is a continuation-in-part of application No. 10/225,067, filed on Aug.  9, 2002, now Pat. No. 7,135,616 , which is a continuation-in-part of application No. 09/837,944, filed on Apr.  18, 2001, now abandoned , which is a continuation-in-part of application No. 10/171,468, filed on Jun.  14, 2002, now abandoned .
Said application No. 10/412,699 is a continuation-in-part of application No. 10/374,780, filed on Feb.  25, 2003 , which is a continuation-in-part of application No. 10/559,441, filed on Dec.  2, 2005 , which is a continuation-in-part of application No. 11/642,814, filed on Dec.  20, 2006 , which is a division of application No. 10/666,642, filed on Sep.  18, 2003, now Pat. No. 7,196,245 , which is a continuation-in-part of application No. 10/714,887, filed on Nov.  13, 2003 , which is a continuation-in-part of application No. 10/456,882, filed on Jun.  6, 2003, now abandoned , which is a continuation-in-part of application No. 10/666,642, filed on Sep.  18, 2003, now Pat. No. 7,196,245 , which is a continuation-in-part of application No. 11/435,388, filed on May  15, 2006 , which is a continuation-in-part of application No. PCT/US04/37584, filed on Nov.  12, 2004 , which is a continuation-in-part of application No. 10/714,887, filed on Nov.  13, 2003 , which is a continuation-in-part of application No. 11/632,390 , which is a continuation-in-part of application No. 12/064,961 , which is a continuation-in-part of application No. 10/903,236, filed on Jul.  30, 2004 , which is a continuation-in-part of application No. 10/456,882, filed on Jun.  6, 2003, now abandoned , which is a continuation-in-part of application No. 10/666,642, filed on Sep.  18, 2003, now Pat. No. 7,196,245 , which is a continuation-in-part of application No. 11/699,973, filed on Jan.  29, 2007 , which is a continuation-in-part of application No. PCT/US05/27151, filed on Jul.  29, 2005 , which is a continuation-in-part of application No. 10/903,236, filed on Jul.  30, 2004 , which is a continuation-in-part of application No. 10/870,198, filed on Jun.  16, 2004 , which is a continuation-in-part of application No. 10/669,824, filed on Sep.  23, 2003 , which is a continuation-in-part of application No. 09/823,676, filed on Mar.  30, 2001, now Pat. No. 6,717,034 , which is a continuation-in-part of application No. 10/838,616, filed on May  4, 2004 , which is a continuation-in-part of application No. 10/685,922, filed on Oct.  14, 2003, now abandoned , which is a continuation-in-part of application No. PCT/US07/17321, filed on Aug.  3, 2007 , which is a continuation-in-part of application No. 11/705,903, filed on Feb.  12, 2007 , which is a continuation-in-part of application No. PCT/US06/34615, filed on Aug.  31, 2006 , which is a continuation-in-part of application No. 11/821,448, filed on Jun.  22, 2007 , which is a continuation-in-part of application No. PCT/US07/09124, filed on Apr.  12, 2007 , which is a continuation-in-part of application No. 11/981,667, filed on Mar.  7, 2008 , which is a continuation-in-part of application No. 11/981,576, filed on Oct.  30, 2007 .
 
(60)Provisional application No. 60/961,403, filed on Jul.  20, 2007.
 
 Provisional application No. 60/125,814, filed on Mar.  23, 1999.
 
 Provisional application No. 60/166,228, filed on Nov.  17, 1999.
 
 Provisional application No. 60/197,899, filed on Apr.  17, 2000.
 
 Provisional application No. 60/227,439, filed on Aug.  22, 2000.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/713,952, filed on Aug.  31, 2005.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/227,439, filed on Aug.  22, 2000.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/101,349, filed on Sep.  22, 1998.
 
 Provisional application No. 60/103,312, filed on Oct.  6, 1998.
 
 Provisional application No. 60/108,734, filed on Nov.  17, 1998.
 
 Provisional application No. 60/113,409, filed on Dec.  22, 1998.
 
 Provisional application No. 60/116,841, filed on Jan.  22, 1999.
 
 Provisional application No. 60/120,880, filed on Feb.  18, 1999.
 
 Provisional application No. 60/121,037, filed on Feb.  22, 1999.
 
 Provisional application No. 60/124,278, filed on Mar.  11, 1999.
 
 Provisional application No. 60/129,450, filed on Apr.  15, 1999.
 
 Provisional application No. 60/135,134, filed on May  20, 1999.
 
 Provisional application No. 60/144,153, filed on Jul.  15, 1999.
 
 Provisional application No. 60/161,143, filed on Oct.  22, 1999.
 
 Provisional application No. 60/162,656, filed on Nov.  1, 1999.
 
 Provisional application No. 60/125,814, filed on Mar.  23, 1999.
 
 Provisional application No. 60/125,814, filed on Mar.  23, 1999.
 
 Provisional application No. 60/125,814, filed on Mar.  23, 1999.
 
 Provisional application No. 60/166,228, filed on Nov.  17, 1999.
 
 Provisional application No. 60/197,899, filed on Apr.  17, 2000.
 
 Provisional application No. 60/227,439, filed on Aug.  22, 2000.
 
 Provisional application No. 60/227,439, filed on Aug.  22, 2000.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/310,847, filed on Aug.  9, 2001.
 
 Provisional application No. 60/336,049, filed on Nov.  19, 2001.
 
 Provisional application No. 60/338,692, filed on Dec.  11, 2001.
 
 Provisional application No. 60/411,837, filed on Sep.  18, 2002.
 
 Provisional application No. 60/434,166, filed on Dec.  17, 2002.
 
 Provisional application No. 60/465,809, filed on Apr.  24, 2003.
 
 Provisional application No. 60/411,837, filed on Sep.  18, 2002.
 
 Provisional application No. 60/434,166, filed on Dec.  17, 2002.
 
 Provisional application No. 60/465,809, filed on Apr.  24, 2003.
 
 Provisional application No. 60/527,658, filed on Dec.  5, 2003.
 
 Provisional application No. 60/542,928, filed on Feb.  5, 2004.
 
 Provisional application No. 60/588,405, filed on Jul.  14, 2004.
 
 Provisional application No. 60/713,952, filed on Aug.  31, 2005.
 
 Provisional application No. 60/411,837, filed on Sep.  18, 2002.
 
 Provisional application No. 60/465,809, filed on Apr.  24, 2003.
 
 Provisional application No. 60/565,948, filed on Apr.  26, 2004.
 
 Provisional application No. 60/527,658, filed on Dec.  5, 2003.
 
 Provisional application No. 60/542,928, filed on Feb.  5, 2004.
 
 Provisional application No. 60/565,948, filed on Apr.  26, 2004.
 
 Provisional application No. 60/836,243, filed on Aug.  7, 2006.
 
 Provisional application No. 60/713,952, filed on Aug.  31, 2005.
 
 Provisional application No. 60/817,886, filed on Jun.  29, 2006.
 
 Provisional application No. 60/791,663, filed on Apr.  12, 2006.
 
 Publication Classification 
(51)Int. Cl. A01H 001/00 (20060101); C12N 015/00 (20060101); A01H 005/00 (20060101); C12N 015/82 (20060101)
(52)U.S. Cl. 800/266; 435/320.1; 800/298; 800/317.4; 800/282; 800/290

        

(57)

Abstract

The invention relates to plant transcription factor polypeptides, polynucleotides that encode them, homologs from a variety of plant species, and methods of using the polynucleotides and polypeptides to produce transgenic plants having advantageous properties, including increased soluble solids, lycopene, and improved plant volume or yield, as compared to wild-type or control plants. The invention also pertains to expression systems that may be used to regulate these transcription factor polynucleotides, providing constitutive, transient, inducible and tissue-specific regulation.
 Claim(s),  Drawing Sheet(s), and Figure(s)
 
 


RELATIONSHIP TO COPENDING APPLICATIONS

[0001] This application claims the benefit of Application No. 60/961,403, filed Jul. 20, 2007 (pending). This application is a continuation-in-part of application Ser. No. 10/286,264, filed Nov. 1, 2002 (pending), which is a divisional of application Ser. No. 09/533,030, filed Mar. 22, 2000 (abandoned), which claims the benefit of Application No. 60/125,814, filed Mar. 23, 1999. This application is a continuation-in-part of application Ser. No. 10/675,852, filed Sep. 30, 2003 (pending). This application is a continuation-in-part of application Ser. No. 11/479,226, filed Jun. 30, 2006 (pending), which is a continuation-in-part of application Ser. No. 09/713,994, filed Nov. 16, 2000 (abandoned), which claims the benefit of Application No. 60/166,228, filed Nov. 17, 1999, which also claims the benefit of Application No. 60/197,899, filed Apr. 17, 2000, which also claims the benefit of Application No. 60/227,439, filed Aug. 22, 2000. This application is a continuation-in-part of application Ser. No. 10/669,824, filed Sep. 23, 2003, which is a continuation-in-part of, 09/823,676, filed Mar. 30, 2001 (issued as U.S. Pat. No. 6,717,034). This application is a continuation-in-part of application Ser. No. 11/725,235, filed Mar. 16, 2007 (pending), which is a divisional of application Ser. No. 10/225,068, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,193,129), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, and also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, and also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001; application Ser. No. 10/225,068 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). This application is a continuation-in-part of application Ser. No. 11/728,567, filed Mar. 26, 2007, which is a divisional of application Ser. No. 10/225,066, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,238,860), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, and also claims the benefit of Application No. 60/336,049, filed Jan. 19, 2001, and also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001. Application Ser. No. 10/225,066 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). This application is a continuation-in-part of application Ser. No. 11/375,241, filed Mar. 16, 2006 (pending), which claims the benefit of Application No. 60/713,952, filed Aug. 31, 2005. Application Ser. No. 11/375,241 is also a continuation-in-part of application Ser. No. 10/225,067, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,135,616), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, which also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, and also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001. Application Ser. No. 10/225,067 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). This application is a continuation-in-part of application Ser. No. 11/069,255, filed Feb. 28, 2005 (pending), which is a continuation-in-part of application Ser. No. 10/112,887, filed Mar. 18, 2002 (abandoned). This application is a continuation-in-part of application Ser. No. 10/374,780, filed Feb. 25, 2003 (pending), which is a continuation-in-part of Application No. 09/934,455, filed Aug. 22, 2001 (abandoned), which is a continuation-in-part of application Ser. Nos. 09/713,994, 11/16/2000 (abandoned), which is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), which also claims priority to Application No. 60/227,439, filed Aug. 22, 2000. Application Ser. No. 10/374,780 is also a continuation-in-part of application Ser. No. 10/225,068, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,193,129), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, and also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, and also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001. Application Ser. No. 10/225,068 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). Application Ser. No. 10/374,780 is also a continuation-in-part of application Ser. No. 10/225,066, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,238,860), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, which also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, which also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001; application Ser. No. 10/225,066 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). Application Ser. No. 10/374,780 is also a continuation-in-part of application Ser. No. 10/225,067, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,135,616), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, and also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, and also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001; application Ser. No. 10/225,067 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). This application is a continuation-in-part of application Ser. No. 10/546,266, filed Aug. 19, 2005 (pending), which is a '371 National Stage filing of International Application No. PCT/US2004005654, filed Feb. 25, 2004 (converted), which is a continuation-in-part of application Ser. No. 10/374,780, filed Feb. 25, 2003 (pending), and is also a continuation-in-part of application Ser. No. 10/675,852, filed Sep. 30, 2003 (pending). This application is also a continuation-in-part of application Ser. No. 11/986,992, filed Nov. 26, 2007 (pending), which is a division of application Ser. No. 10/412,699, filed Apr. 10, 2003 (issued as U.S. Pat. No. 7,345,217), which is a continuation-in-part of application Ser. No. 10/295,403, filed Nov. 15, 2002 (abandoned), which is a divisional of application Ser. No. 09/394,519, filed Sep. 13, 1999 (abandoned), which claims the benefit of Application No. 60/101,349, filed Sep. 22, 1998, which also claims the benefit of Application No. 60/103,312, filed Oct. 6, 1998, which also claims the benefit of Application No. 60/108,734, filed Nov. 17, 1998, which also claims the benefit of Application No. 60/113,409, filed Dec. 22, 1998. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 09/489,376, filed Jan. 21, 2000 (abandoned), which claimed priority to Application No. 60/116,841, filed Jan. 22, 1999. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 10/302,267, filed Nov. 22, 2002 (issued as U.S. Pat. No. 7,223,904), which is a divisional of application Ser. No. 09/506,720, filed Feb. 17, 2000 (abandoned), which claims the benefit of Application No. 60/120,880, filed Feb. 18, 1999, which also claims the benefit of Application No. 60/121,037, filed Feb. 22, 1999, which also claims the benefit of Application No. 60/124,278, filed Mar. 11, 1999, which also claims the benefit of Application No. 60/129,450, filed Apr. 15, 1999, which also claims the benefit of Application No. 60/135,134, filed May 20, 1999, which also claims the benefit of Application No. 60/144,153, filed Jul. 15, 1999, which also claims the benefit of Application No. 60/161,143, filed Oct. 22, 1999, which also claims the benefit of Application No. 60/162,656, filed Nov. 1, 1999. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 10/278,173, filed Oct. 21, 2002 (abandoned), which is a divisional of application Ser. No. 09/533,392, filed Mar. 22, 2000 (abandoned), which claims the benefit of Application No. 60/125,814, filed Mar. 23, 1999. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 09/533,029, filed Mar. 22, 2000 (issued as U.S. Pat. No. 6,664,446), which claims the benefit of Application No. 60/125,814, filed Mar. 23, 1999. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 10/278,536, filed Oct. 22, 2002 (abandoned), which is a divisional of application Ser. No. 09/532,591, filed Mar. 22, 2000 (abandoned), which claims priority to Application No. 60/125,814, filed Mar. 23, 1999. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 09/713,994, filed Nov. 16, 2000 (abandoned), which claims the benefit of Application No. 60/166,228, filed Nov. 17, 1999, which also claims the benefit of Application No. 60/197,899, filed Apr. 17, 2000, which also claims the benefit of Application No. 60/227,439, filed Aug. 22, 2000. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 09/819,142, filed Mar. 27, 2001. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 09/934,455, filed Aug. 22, 2001 (abandoned), which is a continuation-in-part of application Ser. No. 09/713,994, filed Nov. 16, 2000 (abandoned), which is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), which also claim the benefit of Application No. 60/227,439, filed Aug. 22, 2000. Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 10/225,068, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,193,129), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, which also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, which also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001; and, application Ser. No. 10/225,068 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 10/225,066, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,238,860), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, which also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, which also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001; and, application Ser. No. 10/225,066 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 10/225,067, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,135,616), which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, which also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, which also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001; and, application Ser. No. 10/225,067 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001 (abandoned), and is also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002 (abandoned). Application Ser. No. 10/412,699 is also a continuation-in-part of application Ser. No. 10/374,780, filed Feb. 25, 2003 (pending). This application is a continuation-in-part of application Ser. No. 10/559,441, filed Dec. 2, 2005 (pending), which is a '371 National Stage filing of International Application No. PCT/US2004/017768, filed Jun. 4, 2004 (converted), which is a continuation-in-part of application Ser. No. 10/456,882, filed Jun. 6, 2003 (abandoned). This application is a continuation-in-part of application Ser. No. 11/642,814, filed Dec. 20, 2006 (pending), which is a divisional of application Ser. No. 10/666,642, filed Sep. 18, 2003 (issued as U.S. Pat. No. 7,196,245), which claims the benefit of Application No. 60/411,837, filed Sep. 18, 2002, and also claims the benefit of Application No. 60/434,166, filed Dec. 17, 2002, and also claims the benefit of Application No. 60/465,809, filed Apr. 24, 2003. This application is a continuation-in-part of application Ser. No. 10/714,887, filed Nov. 13, 2003 (pending), which is a continuation-in-part of application Ser. No. 10/456,882, filed Jun. 6, 2003 (abandoned); and application Ser. No. 10/714,887 is also a continuation-in-part of application Ser. No. 10/666,642, filed Sep. 18, 2003 (issued as U.S. Pat. No. 7,196,245), which claims the benefit of Application No. 60/411,837, filed Sep. 18, 2002, which also claims the benefit of Application No. 60/434,166, filed Dec. 17, 2002, which also claims the benefit of Application No. 60/465,809, filed Apr. 24, 2003. This application is a continuation-in-part of application Ser. No. 11/435,388, filed May 15, 2006 (pending), which is a continuation-in-part of International Application No. PCT/US04/37584, filed Nov. 12, 2004 (converted), which is a continuation-in-part of application Ser. No. 10/714,887, filed Nov. 13, 2003 (pending), and also claims the benefit of Application No. 60/527,658, filed Dec. 5, 2003, and also claims the benefit of Application No. 60/542,928, filed Feb. 5, 2004. This application is a continuation-in-part of application Ser. No. 11/632,390, filed Jan. 11, 2007 (pending), which is a '371 National Stage filing of International Application No. PCT/US2005/025010, filed Jul. 14, 2005 (converted), which claims the benefit of Application No. 60/588,405, filed Jul. 14, 2004. This application is a continuation-in-part of application Ser. No. 12/064,961, filed Feb. 26, 2008 (pending), which is a continuation-in-part of PCT application PCT/US06/34615, filed Aug. 31, 2006 (expired), which claims the benefit of Application No. 60/713,952, filed Aug. 31, 2005. This application is a continuation-in-part of International Application no. PCT/US2006/34615, filed Aug. 31, 2006 (pending), which claims the benefit of Application No. 60/713,952, filed Aug. 31, 2005. This application is a continuation-in-part of application Ser. No. 10/903,236, filed Jul. 30, 2004 (pending), which is a continuation-in-part of application Ser. No. 10/456,882, filed Jun. 6, 2003 (abandoned), and is also a continuation-in-part of application Ser. No. 10/666,642, filed Sep. 18, 2003 (issued as U.S. Pat. No. 7,196,245), which claims the benefit of Application No. 60/411,837, filed Sep. 18, 2002, and also claims the benefit of Application No. 60/465,809, filed Apr. 24, 2003. This application is a continuation-in-part of application Ser. No. 11/699,973, filed Jan. 29, 2007 (pending), which is a continuation-in-part of International Application No. PCT/US2005-027151, filed Jul. 29, 2005 (converted), which is a continuation-in-part of application Ser. No. 10/903,236, filed Jul. 30, 2004 (pending). This application is a continuation-in-part of application Ser. No. 10/870,198, filed Jun. 16, 2004 (pending), which claims the benefit of Application No. 60/565,948, filed Apr. 26, 2004, which also claims the benefit of Application No. 60/527,658, filed Dec. 5, 2003, which also claims the benefit of Application No. 60/542,928, filed Feb. 5, 2005; and, Application No. 10/870,198 is also a continuation-in-part of application Ser. No. 10/669,824, filed Sep. 23, 2003 (pending), which is a continuation-in-part of application Ser. No. 09/823,676, filed Mar. 30, 2001 (issued as U.S. Pat. No. 6,717,034). This application is a continuation-in-part of application Ser. No. 10/838,616, filed May 4, 2004 (pending), which claims the benefit of Application No. 60/565,948, filed Apr. 26, 2004, and is a continuation-in-part of application Ser. No. 10/685,922, filed Oct. 14, 2003 (abandoned). This application is a continuation-in-part of International Application No. PCT/US2007/17321, filed Aug. 7, 2006 (pending), which claims the benefit of Application No. 60/836,243, filed Aug. 7, 2006. This application is a continuation-in-part of application Ser. No. 11/705,903, filed Feb. 12, 2007 (pending), which is a continuation-in-part of International Application No. PCT/US2006/34615, filed Aug. 31, 2006 (converted), which claims the benefit of Application No. 60/713,952, filed Aug. 31, 2005. This application is a continuation-in-part of application Ser. No. 11/821,448, filed Jun. 22, 2007 (pending), which claims priority to Application No. 60/817,886, filed Jun. 29, 2006. This application is a continuation-in-part of application Ser. No. 11/981,667, filed Oct. 31, 2007 (pending). This application is also a continuation-in-part of application Ser. No. 11/981,576 filed Oct. 31, 2007 (pending). This application is a continuation-in-part of International Application No. PCT/US2007/09124, filed Apr. 12, 2007 (pending), which claims priority to Application No. 60/791,663, filed Apr. 12, 2006. The contents of all applications herein are incorporated by reference in their entirety.

JOINT RESEARCH AGREEMENT

[0002] The claimed invention, in the field of functional genomics and the characterization of plant genes for the improvement of plants, was made by or on behalf of Mendel Biotechnology, Inc. and Monsanto Company as a result of activities undertaken within the scope of a joint research agreement in effect on or before the date the claimed invention was made.

FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods for transforming plants for the purpose of improving plant traits, including yield and fruit quality.

BACKGROUND OF THE INVENTION

[0004] Biotechnological improvement of plants. Biotechnology approaches involving the expression of single transgenes in crops have resulted in the successful commercial introduction of new plant traits, including herbicide (e.g., glyphosate) resistance, insect resistance (expression of Bacillus thuringiensis toxins) and virus resistance (overexpression of viral coat proteins). Plant genomics may also be used to achieve control over polygenic traits. Some traits that may be improved, resulting in better yield and crop quality, include:
[0005] Increased lycopene levels. Lycopene is a pigment responsible for color of fruits (e.g., the red color of tomatoes). Consumers tend to prefer fruit products with good color, and often specifically buy fruit and fruit products based on lycopene levels.
[0006] Lycopene and other carotenoids are also valuable anti-oxidants. Lycopene is the subject of an increasing number of medical studies that demonstrate its efficacy in preventing certain cancers—including prostate, lung, stomach and breast cancers. Potential impacts also include ultraviolet protection and coronary heard disease prevention.
[0007] Increased soluble solids. Increased soluble solids are highly valuable to fruit processors for the production of various products. Grapes, for example, are harvested when soluble solids have reached an appropriate level, and the quality of wine produced from grapes is to a large extent dependent on soluble solid content. Increased soluble solids are also important in the production of tomato paste, sauces and ketchup. Increasing soluble solids in tomatoes increases the value of processed tomato products and decreases processing costs. Savings come from reduced processing time and less energy consumption due to shortened cooking times needed to achieve desired soluble solids levels. A one percent increase in tomato soluble solids may be worth $100 to $200 million to the tomato processing industry.
[0008] Fruit Weight. Increased fruit weight, such as the weight of tomato fruit, may directly impact yield when the fruit is the primary crop product. This is true for tomato plants, used as a model system in the present studies, and, generally, other fruit crops.
[0009] Control of cellular processes in plants with transcription factors. Strategies for manipulating traits by altering a plant cell's transcription factor content can result in plants and crops with new and/or improved commercially valuable properties. For example, manipulation of the levels of selected transcription factors may result in increased expression of economically useful proteins or biomolecules in plants or improvement in other agriculturally relevant characteristics. Conversely, blocked or reduced expression of a transcription factor may reduce biosynthesis of unwanted compounds or remove an undesirable trait. Therefore, manipulating transcription factor levels in a plant offers tremendous potential in agricultural biotechnology for modifying a plant's traits, including traits that improve a plant's survival, yield and product quality.

SUMMARY OF THE INVENTION

[0010] The present invention relates to compositions and methods for modifying the genotype of a plant for the purpose of imparting desirable characteristics, including, but not limited to, yield and/or quality-related traits, and these characteristics may pertain to the fruit of the plant. The method steps include transforming a host plant cell with a DNA construct (such as an expression vector or a plasmid). The DNA construct comprises a polynucleotide that encodes a polypeptide that may regulate transcription. The polynucleotide is homologous to a polynucleotide of the invention provided in the Sequence Listing or Tables 7 or 8.
[0011] Once the host plant cell is transformed with the DNA construct, a plant may be regenerated from the transformed host plant cell to produce a plant having the desired yield or quality characteristic. Examples of yield and quality characteristics that may be improved by these method steps include increased fungal disease resistance, increased fruit weight, increased fruit number, increased lycopene, increased soluble solids, reduced fruit softening, increased plant vigor, and increased plant size.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0012] The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.
[0013] The copy of the Sequence Listing, being submitted electronically with this patent application, provided under 37 CFR § 1.821-1.825, is a read-only memory computer-readable file in ASCII text format. The Sequence Listing is named “MMBI-0070-2CIP_ST25.txt” and is 16,074 kilobytes in size (measured in MS-WINDOWS). The Sequence Listing file was created on Jun. 20, 2008. The Sequence Listing is herein incorporated by reference in its entirety.
[0014] FIG. 1 shows a conservative estimate of phylogenetic relationships among the orders of flowering plants (modified from Angiosperm Phylogeny Group (1998)). Those plants with a single cotyledon (monocots) are a monophyletic clade nested within at least two major lineages of dicots; the eudicots are further divided into rosids and asterids. Arabidopsis is a rosid eudicot classified within the order Brassicales; rice is a member of the monocot order Poales. FIG. 1 was adapted from Daly et al. (2001).
[0015] FIG. 2 shows a phylogenic dendogram depicting phylogenetic relationships of higher plant taxa, including clades containing tomato and Arabidopsis; adapted from Ku et al. (2000) and Chase et al. (1993).
[0016] FIG. 3 is a schematic diagram of activator and target vectors used for transformation of tomato to achieve regulated expression of Arabidopsis transcription factors in tomato. The activator vector contained a promoter and a LexA-GAL4 or a-LacI-GAL4 transactivator (the transactivator comprises a LexA or LacI DNA binding domain fused to the GAL4 activation domain, and encodes a LexA-Gal4 or LacI-Gal4 transcriptional activator product), a GFP marker, and a neomycin phosphotransferase II (nptII) selectable marker. The target vector contains a transactivator binding site (opLexA) operably linked to a transgene encoding a polypeptide of interest (for example, a transcription factor of the invention), and a sulfonamide selectable marker (in this case, sulII; which encodes the dihydropteroate synthase enzyme for sulfonamide-resistance) necessary for the selection and identification of transformed plants. Binding of the transcriptional activator product encoded by the activator vector to the transactivator binding sites of the target vector initiates transcription of the transgenes of interest.

DESCRIPTION OF THE INVENTION

[0017] In an important aspect, the present invention relates to combinations of gene promoters and polynucleotides for modifying phenotypes of plants, including those associated with improved plant or fruit yield, or improved fruit quality. Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and World Wide Web browser-active and inactive page addresses, for example. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of “incorporation by reference” is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the invention.
[0018] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a plant” includes a plurality of such plants.

DEFINITIONS

[0019] “Polynucleotide” is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides, optionally at least about 30 consecutive nucleotides, or at least about 50 consecutive nucleotides. A polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof of a length listed above. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single stranded or double stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a polymerase chain reaction (PCR) product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). The polynucleotide can comprise a sequence in either sense or antisense orientations. “Oligonucleotide” is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single stranded.
[0020] A “recombinant polynucleotide” is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.
[0021] “Gene” or “gene sequence” refers to the partial or complete coding sequence of a gene, its complement, and its 5′ or 3′ untranslated regions. A gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter may be subjected to subsequent processing such as splicing and folding to obtain a functional protein or polypeptide. A gene may be isolated, partially isolated, or be found with an organism's genome. By way of example, a transcription factor gene encodes a transcription factor polypeptide, which may be functional or require processing to function as an initiator of transcription.
[0022] Operationally, genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and which may be used to determine the limits of the genetically active unit (Rieger et al. (1976)). A gene generally includes regions preceding (“leaders”; upstream) and following (“trailers”; downstream) of the coding region. A gene may also include intervening, non-coding sequences, referred to as “introns”, located between individual coding segments, referred to as “exons”. Most genes have an associated promoter region, a regulatory sequence 5′ of the transcription initiation codon (there are some genes that do not have an identifiable promoter). The function of a gene may also be regulated by enhancers, operators, and other regulatory elements.
[0023] An “isolated polynucleotide” is a polynucleotide whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not. Optionally, an isolated polynucleotide is subject to one or more enrichment or purification procedures, e.g., cell lysis, extraction, centrifugation, precipitation, or the like.
[0024] “Complementary” refers to the natural hydrogen bonding by base pairing between purines and pyrimidines. For example, the sequence A-C-G-T (5′->3′) forms hydrogen bonds with its complements A-C-G-T (5′->3′) or A-C-G-U (5′->3′). Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or “completely complementary” if all of the nucleotides bond. The degree of complementarity between nucleic acid strands affects the efficiency and strength of the hybridization and amplification reactions. “Fully complementary” refers to the case where bonding occurs between every base pair and its complement in a pair of sequences, notwithstanding gaps that may occur in an alignment due to additional bases in one of the aligned sequences.
[0025] The terms “highly stringent” or “highly stringent condition” refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs. Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are disclosed in detail by Kashima et al. (1985), Sambrook et al. (1989), and by Hames and Higgins (1985), which references are incorporated herein by reference.
[0026] In general, stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure (for a more detailed description of establishing and determining stringency, see below). The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity. Thus, similar nucleic acid sequences from a variety of sources, such as within a plant's genome (as in the case of paralogs) or from another plant (as in the case of orthologs) that may perform similar functions can be isolated on the basis of their ability to hybridize with known transcription factor sequences. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate transcription factor sequences having similarity to transcription factor sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed transcription factor sequences, such as, for example, transcription factors having 60% identity, or more preferably greater than about 70% identity, most preferably 72% or greater identity with disclosed transcription factors.
[0027] The term “transcript profile” refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state. For example, the transcript profile of a particular transcription factor in a suspension cell is the expression levels of a set of genes in a cell knocking out or overexpressing that transcription factor compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that transcription factor. The transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods.
[0028] “Ectopic expression” or “altered expression” in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type or control plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term “ectopic expression or altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.
[0029] The term “overexpression” as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more transcription factors are under the control of a strong promoter (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may also under the control of an inducible or tissue specific promoter. Thus, overexpression may occur throughout a plant, in specific tissues of the plant, or in the presence or absence of particular environmental signals, depending on the promoter used.
[0030] Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present transcription factors. Overexpression may also occur in plant cells where endogenous expression of the present transcription factors or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or “overproduction” of the transcription factor in the plant, cell or tissue.
[0031] The term “transcription regulating region” refers to a DNA regulatory sequence that regulates expression of one or more genes in a plant when a transcription factor having one or more specific binding domains binds to the DNA regulatory sequence. Transcription factors of the present invention generally possess at least one conserved domain characteristic of a particular transcription factor family. Examples of such conserved domains of the sequences of the invention may be found in Table 7. The transcription factors of the invention may also comprise an amino acid subsequence that forms a transcription activation domain that regulates expression of one or more abiotic stress tolerance genes in a plant when the transcription factor binds to the regulating region.
[0032] “Protein” refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
[0033] A “polypeptide” is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain, or 5) a DNA-binding domain, or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.
[0034] A “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide. A “synthetic polypeptide” is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art. An “isolated polypeptide,” whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.
[0035] “Homology” refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence. Additionally, the terms “homology” and “homologous sequence(s)” may refer to one or more polypeptide sequences that are modified by chemical or enzymatic means. The homologous sequence may be a sequence modified by lipids, sugars, peptides, organic or inorganic compounds, by the use of modified amino acids or the like. Protein modification techniques are illustrated in Ausubel et al. (1998).
[0036] “Identity” or “similarity” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases “percent identity” and “% identity” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. “Sequence similarity” refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.
[0037] With regard to polypeptides, the terms “substantial identity” or “substantially identical” may refer to sequences of sufficient similarity and structure to the transcription factors in the Sequence Listing to produce similar function when expressed, overexpressed, or knocked-out in a plant; in the present invention, this function is improved yield and/or fruit quality. Polypeptide sequences that are at least about 55% identical to the instant polypeptide sequences are considered to have “substantial identity” with the latter. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents. The structure required to maintain proper functionality is related to the tertiary structure of the polypeptide. There are discreet domains and motifs within a transcription factor that must be present within the polypeptide to confer function and specificity. These specific structures are required so that interactive sequences will be properly oriented to retain the desired activity. “Substantial identity” may thus also be used with regard to subsequences, for example, motifs that are of sufficient structure and similarity, being at least about 55% identical to similar motifs in other related sequences. Thus, related polypeptides within the G1421 clade have the physical characteristics of substantial identity along their full length and within their AP2-related domains. These polypeptides also share functional characteristics, as the polypeptides within this clade bind to a transcription-regulating region of DNA and improve yield and/or fruit quality in a plant when the polypeptides are overexpressed.
[0038] “Alignment” refers to a number of nucleotide or amino acid residue sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. Alignments may be used to identify conserved domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art, such as MacVector (1999) (Accelrys, Inc., San Diego, Calif.).
[0039] A “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is substantial identity between the distinct sequences. bZIPT2-related domains are examples of conserved domains. With respect to polynucleotides encoding presently disclosed transcription factors, a conserved domain is encoded by a sequence preferably at least 10 base pairs (bp) in length. A conserved domain, with respect to presently disclosed polypeptides refers to a domain within a transcription factor family that exhibits a higher degree of sequence homology or substantial identity, such as at least 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid residue sequence identity to a sequence of consecutive amino acid residues such as SEQ ID NOs 2365-4175. As one of ordinary skill in the art recognizes, conserved domains may be identified as regions or domains of identity to a specific consensus sequence. Thus, by using alignment methods well known in the art, the conserved domains of the plant transcription factors of the invention (e.g., bZIPT2, MYB-related, CCAAT-box binding, AP2, and AT-hook family transcription factors) may be determined. An alignment of any of the polypeptides of the invention with another polypeptide allows one of skill in the art to identify conserved domains for any of the polypeptides listed or referred to in this disclosure.
[0040] The terms “paralog” and “ortholog” are defined below in the section entitled “Orthologs and Paralogs”. In brief, orthologs and paralogs are evolutionarily related genes that have similar sequences and functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.
[0041] The term “equivalog” describes members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families, and otherwise into protein families with other hierarchically defined homology types. This definition is provided at the Institute for Genomic Research (TIGR) World Wide Web (www) website, “tigr.org” under the heading “Terms associated with TIGRFAMs”.
[0042] The term “variant”, as used herein, may refer to polynucleotides or polypeptides that differ from the presently disclosed polynucleotides or polypeptides, respectively, in sequence from each other, and as set forth below.
[0043] With regard to polynucleotide variants, differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences may be silent (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide. Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.
[0044] Also within the scope of the invention is a variant of a transcription factor nucleic acid listed in the Sequence Listing, that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code. Included within this definition are polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding polypeptide.
[0045] “Allelic variant” or “polynucleotide allelic variant” refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations may be “silent” or may encode polypeptides having altered amino acid sequence. “Allelic variant” and “polypeptide allelic variant” may also be used with respect to polypeptides, and in this case the terms refer to a polypeptide encoded by an allelic variant of a gene.
[0046] “Splice variant” or “polynucleotide splice variant” as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. This, splice variants may encode polypeptides having different amino acid sequences, which may or may not have similar functions in the organism. “Splice variant” or “polypeptide splice variant” may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.
[0047] As used herein, “polynucleotide variants” may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences. “Polypeptide variants” may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.
[0048] Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in a functionally equivalent transcription factor. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the transcription factors and transcription factor homolog polypeptides of the invention. A polypeptide sequence variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the functional or biological activity of the transcription factor is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine. More rarely, a variant may have “non-conservative” changes, for example, replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example, DNASTAR software (see U.S. Pat. No. 5,840,544).
[0049] “Fragment”, with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A polynucleotide fragment” refers to any subsequence of a polynucleotide, typically, of at least about 9 consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein. Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the transcription factor polynucleotides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a region that encodes an conserved domain of a transcription factor. Exemplary fragments also include fragments that comprise a conserved domain of a transcription factor. Exemplary fragments include fragments that comprise a conserved domain of a transcription factor, for example, amino acids: 84-146 of G1421, SEQ ID NO: 180, or 59-150 of G1437, SEQ ID NO: 186, or 9-111 of G663, SEQ ID NO: 1192, or 52-143 of G1957, SEQ ID NO: 446, which comprise, are comprised within, or approximate, the AP2 DNA binding domain, the QLQ/WRC protein interaction/putative DNA binding domains, the SANT/Myb DNA binding domain, or the B3 DNA binding domain of these polypeptides, respectively.
[0050] Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential. In some cases, the fragment or domain is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as three amino acid residues to the full length of the intact polypeptide, but are preferably at least about 30 amino acid residues in length and more preferably at least about 60 amino acid residues in length.
[0051] The invention also encompasses production of DNA sequences that encode transcription factors and transcription factor derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding transcription factors or any fragment thereof.
[0052] The term “plant” includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and cells for example, guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae (see for example, FIG. 1, adapted from Daly et al. (2001); FIG. 2, adapted from Ku et al. (2000); and see also Tudge (2000).
[0053] A “transgenic plant” refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
[0054] A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the controlled expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
[0055] “Wild type” or “wild-type”, as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which a transcription factor expression is altered, e.g., in that it has been knocked out, overexpressed, or ectopically expressed.
[0056] A “control plant” as used in the present invention refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transgenic or genetically modified plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.
[0057] A “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as osmotic stress tolerance or yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.
[0058] “Trait modification” refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild-type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease, or an even greater difference, in an observed trait as compared with a control or wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution and magnitude of the trait in the plants as compared to control or wild-type plants.

DETAILED DESCRIPTION

[0059] Generally, the polypeptides encoded by the present polynucleotide sequences are involved in cell differentiation, proliferation, and the regulation of growth. Accordingly, one skilled in the art would recognize that by expressing the present sequences in a plant, one may change the expression of autologous genes or induce the expression of introduced genes. By affecting the expression of similar autologous sequences in a plant that have the biological activity of the present sequences, or by introducing the present sequences into a plant, one may alter a plant's phenotype to one with improved traits related to improved yield and/or fruit quality. The sequences of the invention may also be used to transform a plant and introduce desirable traits not found in the wild-type cultivar or strain. Plants may then be selected for those that produce the most desirable degree of over- or under-expression of target genes of interest and coincident trait improvement.
[0060] The sequences of the present invention may be from any species, particularly plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant. The sequences of the invention may also include fragments of the present amino acid sequences. Where “amino acid sequence” is recited to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
[0061] In addition to methods for modifying a plant phenotype by employing one or more polynucleotides and polypeptides of the invention described herein, the polynucleotides and polypeptides of the invention have a variety of additional uses. These uses include their use in the recombinant production (i.e., expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, for example, mutation reactions, PCR reactions, or the like; as substrates for cloning for example, including digestion or ligation reactions; and for identifying exogenous or endogenous modulators of the transcription factors. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single stranded or double stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, for example, genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can comprise a sequence in either sense or antisense orientations. Expression of genes that encode transcription factors and other regulatory proteins that modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997) and Peng et al. (1999). In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response (see, for example, Fu et al. (2001); Nandi et al. (2000); Coupland (1995); and Weigel and Nilsson (1995)).
[0062] In another example, Mandel et al. (1992b) and Suzuki et al. (2001) teach that a transcription factor expressed in another plant species elicits the same or very similar phenotypic response of the endogenous sequence, as often predicted in earlier studies of Arabidopsis transcription factors in Arabidopsis (see Mandel et al. (1992b); Suzuki et al. (2001)).
[0063] Other examples include Müller et al. (2001); Kim et al. (2001); Kyozuka and Shimamoto (2002); Boss and Thomas (2002); He et al. (2000); and Robson et al. (2001).
[0064] In yet another example, Gilmour et al. (1998) teach an Arabidopsis AP2 transcription factor, CBF1, which, when overexpressed in transgenic plants, increases plant freezing tolerance. Jaglo et al. (2001) further identified sequences in Brassica napus that encode CBF-like genes and that transcripts for these genes accumulated rapidly in response to low temperature. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat, as well as in tomato. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved consecutive amino acid residues which bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family (Jaglo et al. (2001).
[0065] Transcription factors mediate cellular responses and control traits through altered expression of genes containing cis-acting nucleotide sequences that are targets of the introduced transcription factor. It is well appreciated in the art that the effect of a transcription factor on cellular responses or a cellular trait is determined by the particular genes whose expression is either directly or indirectly (for example, by a cascade of transcription factor binding events and transcriptional changes) altered by transcription factor binding. In a global analysis of transcription comparing a standard condition with one in which a transcription factor is overexpressed, the resulting transcript profile associated with transcription factor overexpression is related to the trait or cellular process controlled by that transcription factor. For example, the PAP2 gene and other genes in the MYB family have been shown to control anthocyanin biosynthesis through regulation of the expression of genes known to be involved in the anthocyanin biosynthetic pathway (Bruce et al. (2000); Borevitz et al. (2000)). Further, global transcript profiles have been used successfully as diagnostic tools for specific cellular states (for example, cancerous vs. non-cancerous; Bhattacharjee et al. (2001); Xu et al. (2001)). Consequently, it is evident to one skilled in the art that similarity of transcript profile upon overexpression of different transcription factors would indicate similarity of transcription factor function.

Polypeptides and Polynucleotides of the Invention

[0066] The present invention provides, among other things, transcription factors, and transcription factor homolog polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or polynucleotides encoding novel variants of transcription factors derived from the specific sequences provided here.
[0067] The polynucleotides of the invention can be or were ectopically expressed in overexpressor plant cells and the changes in the expression levels of a number of genes, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be employed to change expression levels of a genes, polynucleotides, and/or proteins of plants. These polypeptides and polynucleotides may be employed to modify a plant's characteristics, particularly improvement of yield and/or fruit quality. The polynucleotides of the invention can be or were ectopically expressed in overexpressor or knockout plants and the changes in the characteristic(s) or trait(s) of the plants observed. Therefore, the polynucleotides and polypeptides can be employed to improve the characteristics of plants. The polypeptide sequences of the sequence listing, including Arabidopsis sequences, such as those in Table 7, conferred improved characteristics when these polypeptides were overexpressed in tomato plants. These polynucleotides have been shown to confer increased lycopene levels and/or increased soluble solids, which impacts fruit quality, and/or increased fruit weight, which positively impacts fruit yield. Paralogs and orthologs of these sequences, listed herein, are expected to function in a similar manner by increasing these positive effects on fruit quality and/or yield.
[0068] The invention also encompasses sequences that are complementary to the polynucleotides of the invention. The polynucleotides are also useful for screening libraries of molecules or compounds for specific binding and for creating transgenic plants having improved yield and/or fruit quality. Altering the expression levels of equivalogs of these sequences, including paralogs and orthologs in the Sequence Listing, and other orthologs that are structurally and sequentially similar to the former orthologs, has been shown and is expected to confer similar phenotypes, including improved biomass, yield and/or fruit quality in plants.
[0069] In some cases, exemplary polynucleotides encoding the polypeptides of the invention were identified in the Arabidopsis thaliana GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. In addition, further exemplary polynucleotides encoding the polypeptides of the invention were identified in the plant GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. Polynucleotide sequences meeting such criteria were confirmed as transcription factors.
[0070] Additional polynucleotides of the invention were identified by screening Arabidopsis thaliana and/or other plant cDNA libraries with probes corresponding to known transcription factors under low stringency hybridization conditions. Additional sequences, including full length coding sequences were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure, using a commercially available kit according to the manufacturer's instructions. Where necessary, multiple rounds of RACE are performed to isolate 5′ and 3′ ends. The full-length cDNA was then recovered by a routine end-to-end PCR using primers specific to the isolated 5′ and 3′ ends. Exemplary sequences are provided in the Sequence Listing.
[0071] The invention also entails an agronomic composition comprising a polynucleotide of the invention in conjunction with a suitable carrier and a method for altering a plant's trait using the composition.
[0072] Examples of specific polynucleotide and polypeptides of the invention, and equivalog sequences, along with descriptions of the gene families that comprise these polynucleotides and polypeptides, are provided in Table 7, in the Sequence Listing, and in the description provided below.

Homologous Sequences

[0073] Sequences homologous, i.e., that share significant sequence identity or similarity, to those provided in the Sequence Listing, derived from Arabidopsis thaliana or from other plants of choice, are also an aspect of the invention. Homologous sequences can be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, grasses such as Miscanthus, switchgrass, and sugarcane-Miscanthus crosses, and crops such as soybean, wheat, corn (maize), potato, cotton, rice, rape, oilseed rape (including canola), sunflower, alfalfa, clover, sugarcane, and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, tomatillo, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi). Other crops, including fruits and vegetables, whose phenotype can be changed and which comprise homologous sequences include barley; rye; millet; sorghum; currant; avocado; citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries; nuts such as the walnut and peanut; endive; leek; roots such as arrowroot, beet, cassaya, turnip, radish, yam, and sweet potato; and beans. The homologous sequences may also be derived from woody species, such pine, poplar and eucalyptus, or mint or other labiates. In addition, homologous sequences may be derived from plants that are evolutionarily-related to crop plants, but which may not have yet been used as crop plants. Examples include deadly nightshade (Atropa belladona), related to tomato; jimson weed (Datura strommium), related to peyote; and teosinte (Zea species), the latter being related to corn (maize).
[0074] Homologous sequences can comprise orthologous or paralogous sequences, described below. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologs and paralogs, including phylogenetic methods, sequence similarity and hybridization methods, are described herein; an ortholog or paralog, including equivalogs, may be identified by one or more of the methods described below.

Orthologs and Paralogs

[0075] Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994); Higgins et al. (1996)). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987)). For example, a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001)), and a group of very similar AP2 domain transcription factors from Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998)). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount (2001))
[0076] Transcription factor genes and other regulatory sequences are conserved across diverse eukaryotic species lines (Goodrich et al. (1993); Lin et al. (1991); Sadowski et al. (1988)). Plants are no exception to this observation; diverse plant species possess transcription factors that have similar sequences and functions. Speciation, the production of new species from a parental species, gives rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al. (1994); Higgins et al. (1996)) potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.
[0077] As described by Eisen (1998), evolutionary information may be used to predict gene function. It is common for groups of genes that are homologous in sequence to have diverse, although usually related, functions. However, in many cases, the identification of homologs is not sufficient to make specific predictions because not all homologs have the same function. Thus, an initial analysis of functional relatedness based on sequence similarity alone may not provide one with a means to determine where similarity ends and functional relatedness begins. Fortunately, it is well known in the art that protein function can be classified using phylogenetic analysis of gene trees combined with the corresponding species. Functional predictions can be greatly improved by focusing on how the genes became similar in sequence (i.e., by evolutionary processes) rather than on the sequence similarity itself (Eisen (1998)). In fact, many specific examples exist in which gene function has been shown to correlate well with gene phylogeny (Eisen (1998)). Thus, “[t]he first step in making functional predictions is the generation of a phylogenetic tree representing the evolutionary history of the gene of interest and its homologs. Such trees are distinct from clusters and other means of characterizing sequence similarity because they are inferred by techniques that help convert patterns of similarity into evolutionary relationships . . . . After the gene tree is inferred, biologically determined functions of the various homologs are overlaid onto the tree. Finally, the structure of the tree and the relative phylogenetic positions of genes of different functions are used to trace the history of functional changes, which is then used to predict functions of [as yet] uncharacterized genes” (Eisen (1998)).
[0078] By using a phylogenetic analysis, one skilled in the art would recognize that the ability to deduce similar functions conferred by closely-related polypeptides is predictable. This predictability has been confirmed by our own many studies in which we have found that a wide variety of polypeptides have orthologous or closely-related homologous sequences that function as does the first, closely-related reference sequence. For example, distinct transcription factors, including:
[0079] (i) AP2 family Arabidopsis G47 (found in U.S. Pat. No. 7,135,616), a phylogenetically-related sequence from soybean, and two phylogenetically-related homologs from rice all can confer greater tolerance to drought, hyperosmotic stress, or delayed flowering as compared to control plants;
[0080] (ii) CAAT family Arabidopsis G481 (found in PCT patent publication WO2004076638), and numerous phylogenetically-related sequences from eudicots and monocots can confer greater tolerance to drought-related stress as compared to control plants;
[0081] (iii) Myb-related Arabidopsis G682 (found in U.S. Pat. Nos. 7,223,904 and 7,193,129) and numerous phylogenetically-related sequences from eudicots and monocots can confer greater tolerance to heat, drought-related stress, cold, and salt as compared to control plants;
[0082] (iv) WRKY family Arabidopsis G 1274 (found in U.S. Pat. No. 7,196,245) and numerous closely-related sequences from eudicots and monocots have been shown to confer increased water deprivation tolerance, and
[0083] (v) AT-hook family soy sequence G3456 (found in US patent publication 20040128712A1) and numerous phylogenetically-related sequences from eudicots and monocots, increased biomass compared to control plants when these sequences are overexpressed in plants.
[0084] The polypeptides sequences belong to distinct clades of polypeptides that include members from diverse species. In each case, most or all of the clade member sequences derived from both eudicots and monocots have been shown to confer increased yield or tolerance to one or more abiotic stresses when the sequences were overexpressed. These studies each demonstrate that evolutionarily conserved genes from diverse species are likely to function similarly (i.e., by regulating similar target sequences and controlling the same traits), and that polynucleotides from one species may be transformed into closely-related or distantly-related plant species to confer or improve traits.
[0085] A method for identifying or confirming that specific homologous sequences control the same function is by comparison of the transcript profile(s) obtained upon overexpression or knockout of two or more related polypeptides. Since transcript profiles are diagnostic for specific cellular states, one skilled in the art will appreciate that genes that have a highly similar transcript profile (e.g., with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or with greater than 90% regulated transcripts in common) will have highly similar functions. Fowler and Thomashow (2002), have shown that three paralogous AP2 family genes (CBF1, CBF2 and CBF3) are induced upon cold treatment, and each of which can condition improved freezing tolerance, and all have highly similar transcript profiles. Once a polypeptide has been shown to provide a specific function, its transcript profile becomes a diagnostic tool to determine whether paralogs or orthologs have the same function.
[0086] At the polypeptide level, the sequences of the invention will typically share at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid sequence identity, and have similar functions with the polypeptides listed in Table 7 when these sequences are overexpressed in plants.
[0087] Of particular interest is the structure of a transcription factor in the region of its conserved domain(s). Structural analyses may be performed by comparing the structure of the known transcription factor around its conserved domain with those of orthologs and paralogs. Analysis of a number of polypeptides within a transcription factor group or clade, including the functionally or sequentially similar polypeptides provided in the Sequence Listing, may also provide an understanding of structural elements required to regulate transcription within a given family. Polypeptides that are phylogenetically related to the polypeptides of Table 7 may also have conserved domains that share at least 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid sequence identity, and have similar functions in that the polypeptides of the invention may, when overexpressed in plants, confer at least one regulatory activity and altered trait selected from the group consisting of greater brix, greater fruit weight, greater lycopene, greater biomass, more chlorosis, darker green fruit, darker green leaves, deeper red fruit, larger flowers, larger leaflets, larger leaflets, larger and lighter green leaves, rugulose leaves, more anthocyanin, more trichomes, paler white fruit at the green fruit stage, thicker stems, very high vigor, and waxier leaves, as compared to a control plant.
[0088] At the nucleotide level, the sequences of the invention will typically share at least about 30% or 40% nucleotide sequence identity, preferably at least about 50%, 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%, sequence identity to one or more of the listed full-length sequences, or to a listed sequence but excluding or outside of the region(s) encoding a known consensus sequence or consensus DNA-binding site, or outside of the region(s) encoding one or all conserved domains. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.

Methods for Determining Sequence Relationships

[0089] A variety of methods for determining sequence relationships can be used, including manual alignment and computer assisted sequence alignment and analysis. This later approach is a preferred approach in the present invention, due to the increased throughput afforded by computer assisted methods. As noted below, a variety of computer programs for performing sequence alignment are available, or can be produced by one of skill.
[0090] Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method (see, for example, Higgins and Sharp (1988). The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including FASTA, BLAST, or ENTREZ, FASTA and BLAST, and which may be used to calculate percent similarity. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see U.S. Pat. No. 6,262,333).
[0091] The percentage similarity between two polypeptide sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., manually, using the algorithm provided above, or by the Jotun Hein method (see, for example, Hein (1990)). Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see US Patent Application No. 20010010913).
[0092] One example algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990). Software for performing BLAST analyses is publicly available, e.g., through the National Library of Medicine's National Center for Biotechnology Information (ncbi.nlm.nih; see at world wide web (www) National Institutes of Health US government (gov) website). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1993); Altschul et al. (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992)). Unless otherwise indicated, “sequence identity” here refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter “off” (see, for example, NIH NLM NCBI website at ncbi.nlm.nih).
[0093] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. Karlin and Altschul (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence (and, therefore, in this context, homologous) if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, or less than about 0.01, and or even less than about 0.001. An additional example of a useful sequence alignment algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. The program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters.
[0094] Sequence alignment program, include, for example, the Wisconsin Package Version 10.0, such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG, Madison, Wis.). Public sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases such as PHYTOSEQ sequence database (Incyte Genomics, Wilmington, Del.) can be searched.
[0095] Alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970, by the search for similarity method of Pearson and Lipman (1988), or by computerized implementations of these algorithms. After alignment, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window can be a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions. A description of the method is provided in Ausubel (2000).
[0096] Other techniques for alignment are described by Doolittle (1996). Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments (see Shpaer (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
[0097] In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997)), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992)) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1990); Altschul et al. (1993)), BLOCKS (Henikoff and Henikoff (1991)), Hidden Markov Models (HMM; Eddy (1996); Sonnhammer et al. (1997)), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997), and in Meyers (1995).
[0098] Furthermore, methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and B-box zinc finger domains. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function and a polypeptide sequence encoded by a polynucleotide sequence that has a function not yet determined. Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.
[0099] Orthologs and paralogs of presently disclosed polypeptides may be cloned using compositions provided by the present invention according to methods well known in the art. cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present sequences. Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue. Polypeptide-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.
[0100] Examples of orthologs of the Arabidopsis polypeptide sequences and their functionally similar orthologs are listed in Table 7 and the Sequence Listing. In addition to the sequences in Table 7 and the Sequence Listing, the invention encompasses isolated nucleotide sequences that are phylogenetically and structurally similar to sequences listed in the Sequence Listing) and can function in a plant by increasing yield and/or and abiotic stress tolerance when ectopically expressed in a plant.
[0101] Since a significant number of these sequences are phylogenetically and sequentially related to each other and have been shown to increase yield from a plant and/or abiotic stress tolerance, one skilled in the art would predict that other similar, phylogenetically related sequences falling within the present clades of polypeptides would also perform similar functions when ectopically expressed.

Identifying Polynucleotides or Nucleic Acids by Hybridization

[0102] Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof), as described in more detail in the references cited below (e.g., Sambrook et al. (1989); Berger and Kimmel (1987); and Anderson and Young (1985)).
[0103] Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the transcription factor polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger (1987); and Kimmel (1987)). In addition to the nucleotide sequences in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.
[0104] With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al. (1989); Berger and Kimmel (1987) pp. 467-469; and Anderson and Young (1985).
[0105] Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson and Young (1985)). In addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinyl-pyrrolidone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.
[0106] Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency (as described by the formula above). As a general guidelines high stringency is typically performed at Tm—5° C. to Tm—20° C., moderate stringency at Tm—20° C. to Tm—35° C. and low stringency at Tm—35° C. to Tm—50° C. for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50° C. below Tm), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at Tm—25° C. for DNA-DNA duplex and Tm—15° C. for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.
[0107] High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
[0108] Hybridization and wash conditions that may be used to bind and remove polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present transcription factors include, for example:
[0109] 6×SSC and 1% SDS at 65° C.;
[0110] 50% formamide, 4×SSC at 42° C.; or
[0111] 0.5×SSC to 2.0×SSC, 0.1% SDS at 50° C. to 65° C.; or
[0112] 0.1×SSC to 2×SSC, 0.1% SDS at 50° C.-65° C.;
[0113] with a first wash step of, for example, 10 minutes at about 42° C. with about 20% (v/v) formamide in 0.1×SSC, and with, for example, a subsequent wash step with 0.2×SSC and 0.1% SDS at 65° C. for 10, 20 or 30 minutes. An example of a polynucleotide sequence of the invention would thus include a polynucleotide sequence that hybridizes to the complement of any of SEQ ID NO: 2n−1, where n=1 to 1317, or SEQ ID NO: 4176-4823, or SEQ ID NO: 4839 to 10667 under these stringent conditions. Useful variations on these conditions will be readily apparent to those skilled in the art.
[0114] A person of skill in the art would not expect substantial variation among polynucleotide species encompassed within the scope of the present invention because the highly stringent conditions set forth in the above formulae yield structurally similar polynucleotides.
[0115] For identification of less closely related homologs, wash steps may be performed at a lower temperature, e.g., 50° C. An example of a low stringency wash step employs a solution and conditions of at least 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 min. Greater stringency may be obtained at 42° C. in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 min. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, US Patent Application No. 20010010913).
[0116] If desired, one may employ wash steps of even greater stringency, including conditions of 65° C.-68° C. in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS, or about 0.2×SSC, 0.1% SDS at 65° C. and washing twice, each wash step of 10, 20 or 30 min in duration, or about 0.1×SSC, 0.1% SDS at 65° C. and washing twice for 10, 20 or 30 min. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3° C. to about 5° C., and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6° C. to about 9° C.
[0117] Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10× higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a transcription factor known as of the filing date of the application. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15× or more, is obtained. Accordingly, a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2× or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a calorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
[0118] Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the complements of the listed polynucleotide sequences, for example, to SEQ ID NO: 2n−1, where n=1 to 1317 or SEQ ID NO: 4176-4823, or SEQ ID NO: 4839-10667, and fragments thereof under various conditions of stringency (see, e.g., Wahl and Berger (1987); Kimmel (1987)). Estimates of homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins (1985). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
Identifying Polynucleotides or Nucleic Acids with Expression Libraries
[0119] In addition to hybridization methods, transcription factor homolog polypeptides can be obtained by screening an expression library using antibodies specific for one or more transcription factors. With the provision herein of the disclosed transcription factor, and transcription factor homolog nucleic acid sequences, the encoded polypeptide(s) can be expressed and purified in a heterologous expression system (e.g., E. coli) and used to raise antibodies (monoclonal or polyclonal) specific for the polypeptide(s) in question. Antibodies can also be raised against synthetic peptides derived from transcription factor, or transcription factor homolog, amino acid sequences. Methods of raising antibodies are well known in the art and are described in Harlow and Lane (1988). Such antibodies can then be used to screen an expression library produced from the plant from which it is desired to clone additional transcription factor homologs, using the methods described above. The selected cDNAs can be confirmed by sequencing and enzymatic activity.

Producing Polypeptides

[0120] The polynucleotides of the invention include sequences that encode transcription factors and transcription factor homolog polypeptides and sequences complementary thereto, as well as unique fragments of coding sequence, or sequence complementary thereto. Such polynucleotides can be, for example, DNA or RNA, the latter including mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides are either double-stranded or single-stranded, and include either, or both sense (i.e., coding) sequences and antisense (i.e., non-coding, complementary) sequences. The polynucleotides include the coding sequence of a transcription factor, or transcription factor homolog polypeptide, in isolation, in combination with additional coding sequences (e.g., a purification tag, a localization signal, as a fusion-protein, as a pre-protein, or the like), in combination with non-coding sequences (for example, introns or inteins, regulatory elements such as promoters, enhancers, terminators, and the like), and/or in a vector or host environment in which the polynucleotide encoding a transcription factor or transcription factor homolog polypeptide is an endogenous or exogenous gene.
[0121] A variety of methods exist for producing the polynucleotides of the invention. Procedures for identifying and isolating DNA clones are well known to those of skill in the art, and are described in, for example, Berger and Kimmel (1987); Sambrook et al. (1989) and Ausubel et al. (1998; supplemented through 2000).
[0122] Alternatively, polynucleotides of the invention, can be produced by a variety of in vitro amplification methods adapted to the present invention by appropriate selection of specific or degenerate primers. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase mediated techniques (for example, NASBA), e.g., for the production of the homologous nucleic acids of the invention are found in Berger and Kimmel (1987), Sambrook (1989), and Ausubel (2000), as well as Mullis et al. (1990). Improved methods for cloning in vitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) and the references cited therein, in which PCR amplicons of up to 40 kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel (2000), Sambrook (1989) and Berger and Kimmel (1987).
[0123] Alternatively, polynucleotides and oligonucleotides of the invention can be assembled from fragments produced by solid-phase synthesis methods. Typically, fragments of up to approximately 100 bases are individually synthesized and then enzymatically or chemically ligated to produce a desired sequence, e.g., a polynucleotide encoding all or part of a transcription factor. For example, chemical synthesis using the phosphoramidite method is described, e.g., by Beaucage et al. (1981) and Matthes et al. (1984). According to such methods, oligonucleotides are synthesized, purified, annealed to their complementary strand, ligated and then optionally cloned into suitable vectors. And if so desired, the polynucleotides and polypeptides of the invention can be custom ordered from any of a number of commercial suppliers.

Sequence Variations

[0124] It will readily be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the transcription factors and transcription factor homolog polypeptides of the invention. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing. Nucleic acids having a sequence that differs from the sequences shown in the Sequence Listing, or complementary sequences, that encode functionally equivalent peptides (i.e., peptides having some degree of equivalent or similar biological activity) but differ in sequence from the sequence shown in the Sequence Listing due to degeneracy in the genetic code, are also within the scope of the invention.
[0125] Altered polynucleotide sequences encoding polypeptides include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide encoding a polypeptide with at least one functional characteristic of the instant polypeptides. Included within this definition are polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding the instant polypeptides, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding the instant polypeptides.
[0126] Allelic variant refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (i.e., no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. Splice variant refers to alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
[0127] Those skilled in the art would recognize that, for example, G1421, SEQ ID NO: 180, represents a single transcription factor; allelic variation and alternative splicing may be expected to occur. Allelic variants of SEQ ID NO: 179 can be cloned by probing cDNA or genomic libraries from different individual organisms according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 179, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 180. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the transcription factor are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individual organisms or tissues according to standard procedures known in the art (see U.S. Pat. No. 6,388,064).
[0128] Thus, in addition to the sequences set forth in the Sequence Listing, the invention also encompasses related nucleic acid molecules that include allelic or splice variants of the sequences of the invention, for example, SEQ ID NO: 2n−1, where n=1 to 1317, and include sequences that are complementary to any of the above nucleotide sequences. Related nucleic acid molecules also include nucleotide sequences encoding a polypeptide comprising a substitution, modification, addition and/or deletion of one or more amino acid residues compared to the polypeptide sequences of the invention, for example, SEQ ID NO: 2n, where n=1 to 1317, or sequences comprising any of SEQ ID NOs: 2365-4175 or polypeptides encoded by any of SEQ ID NOs: 4839-10667. Such related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues.
[0129] Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed “silent” variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, e.g., site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence selected from the above table are a feature of the invention.
[0130] In addition to silent variations, other conservative variations that alter one, or a few amino acid residues in the encoded polypeptide, can be made without altering the function of the polypeptide, these conservative variants are, likewise, a feature of the invention.
[0131] For example, substitutions, deletions and insertions introduced into the sequences provided in the Sequence Listing, are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis (Wu (1993) or the other methods noted below. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotide encoding the transcription factor should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function.
[0132] Conservative substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 1 when it is desired to maintain the activity of the protein. Table 1 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.
[00001] [TABLE-US-00001]
  TABLE 1
 
  Possible conservative amino acid substitutions
    Amino Acid  
    Residue   Conservative substitutions
   
    Ala   Ser
    Arg   Lys
    Asn   Gln; His
    Asp   Glu
    Gln   Asn
    Cys   Ser
    Glu   Asp
    Gly   Pro
    His   Asn; Gln
    Ile   Leu, Val
    Leu   Ile; Val
    Lys   Arg; Gln
    Met   Leu; Ile
    Phe   Met; Leu; Tyr
    Ser   Thr; Gly
    Thr   Ser; Val
    Trp   Tyr
    Tyr   Trp; Phe
    Val   Ile; Leu
   
[0133] The sequences provided in the Sequence Listing or in Table 7 have a novel activity, being plant transcription sequences that may be used to regulate expression of proteins. Although all conservative amino acid substitutions (for example, one basic amino acid substituted for another basic amino acid) in the protein of the invention will not necessarily result in a protein that has transcriptional regulatory activity, it is expected that many of these conservative mutations would result in a protein having transcriptional regulatory activity. Most mutations, conservative or non-conservative, made to a protein of the invention, but outside of a domain critical to the activity of the protein (for example, conserved domains of SEQ ID NOs: 2365-4175) and outside of other domains essential for protein activity, will not affect the activity of the protein to any great extent.
[0134] Similar substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Substitutions that are less conservative can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

Further Modifying Sequences of the Invention—Mutation/Forced Evolution

[0135] In addition to generating silent or conservative substitutions as noted, above, the present invention optionally includes methods of modifying the sequences of the Sequence Listing. In the methods, nucleic acid or protein modification methods are used to alter the given sequences to produce new sequences and/or to chemically or enzymatically modify given sequences to change the properties of the nucleic acids or proteins.
[0136] Thus, in one embodiment, given nucleic acid sequences are modified, e.g., according to standard mutagenesis or artificial evolution methods to produce modified sequences. The modified sequences may be created using purified natural polynucleotides isolated from any organism or may be synthesized from purified compositions and chemicals using chemical means well know to those of skill in the art. For example, Ausubel (2000), provides additional details on mutagenesis methods. Artificial forced evolution methods are described, for example, by Stemmer (1994a), Stemmer (1994b), and U.S. Pat. Nos. 5,811,238, 5,837,500, and 6,242,568. Methods for engineering synthetic transcription factors and other polypeptides are described, for example, by Zhang et al. (2000), Liu et al. (2001), and Isalan et al. (2001). Many other mutation and evolution methods are also available and expected to be within the skill of the practitioner.
[0137] Similarly, chemical or enzymatic alteration of expressed nucleic acids and polypeptides can be performed by standard methods. For example, sequence can be modified by addition of lipids, sugars, peptides, organic or inorganic compounds, by the inclusion of modified nucleotides or amino acids, or the like. For example, protein modification techniques are illustrated in Ausubel (2000). Further details on chemical and enzymatic modifications can be found herein. These modification methods can be used to modify any given sequence, or to modify any sequence produced by the various mutation and artificial evolution modification methods noted herein.
[0138] Accordingly, the invention provides for modification of any given nucleic acid by mutation, evolution, chemical or enzymatic modification, or other available methods, as well as for the products produced by practicing such methods, e.g., using the sequences herein as a starting substrate for the various modification approaches.
[0139] For example, optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be used e.g., to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced using a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for Saccharomyces cerevisiae and mammals are TAA and TGA, respectively. The preferred stop codon for monocotyledonous plants is TGA, whereas insects and E. coli prefer to use TAA as the stop codon.
[0140] The polynucleotide sequences of the present invention can also be engineered in order to alter a coding sequence for a variety of reasons, including but not limited to, alterations which modify the sequence to facilitate cloning, processing and/or expression of the gene product. For example, alterations are optionally introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to introduce splice sites, etc.
[0141] Furthermore, a fragment or domain derived from any of the polypeptides of the invention can be combined with domains derived from other transcription factors or synthetic domains to modify the biological activity of a transcription factor. For instance, a DNA-binding domain derived from a transcription factor of the invention can be combined with the activation domain of another transcription factor or with a synthetic activation domain. A transcription activation domain assists in initiating transcription from a DNA-binding site. Examples include the transcription activation region of VP16 or GAL4 (Moore et al. (1998); Aoyama et al. (1995)), peptides derived from bacterial sequences (Ma and Ptashne (1987)) and synthetic peptides (Giniger and Ptashne (1987)).

Expression and Modification of Polypeptides

[0142] Typically, polynucleotide sequences of the invention are incorporated into recombinant DNA (or RNA) molecules that direct expression of polypeptides of the invention in appropriate host cells, transgenic plants, in vitro translation systems, or the like. Due to the inherent degeneracy of the genetic code, nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can be substituted for any listed sequence to provide for cloning and expressing the relevant homolog.
[0143] The transgenic plants of the present invention comprising recombinant polynucleotide sequences are generally derived from parental plants, which may themselves be non-transformed (or non-transgenic) plants. These transgenic plants may either have a transcription factor gene “knocked out” (for example, with a genomic insertion by homologous recombination, an antisense or ribozyme construct) or expressed to a normal or wild-type extent. However, overexpressing transgenic “progeny” plants will exhibit greater mRNA levels, wherein the mRNA encodes a transcription factor, that is, a DNA-binding protein that is capable of binding to a DNA regulatory sequence and inducing transcription, and preferably, expression of a plant trait gene, such as a gene that improves plant and/or fruit quality and/or yield. Preferably, the mRNA expression level will be at least three-fold greater than that of the parental plant, or more preferably at least ten-fold greater mRNA levels compared to said parental plant, and most preferably at least fifty-fold greater compared to said parental plant.

Modified Amino Acid Residues

[0144] Polypeptides of the invention may contain one or more modified amino acid residues. The presence of modified amino acids may be advantageous in, for example, increasing polypeptide half-life, reducing polypeptide antigenicity or toxicity, increasing polypeptide storage stability, or the like. Amino acid residue(s) are modified, for example, co-translationally or post-translationally during recombinant production or modified by synthetic or chemical means.
[0145] Non-limiting examples of a modified amino acid residue include incorporation or other use of acetylated amino acids, glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, PEG modified (e.g., “PEGylated”) amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References adequate to guide one of skill in the modification of amino acid residues are replete throughout the literature.
[0146] The modified amino acid residues may prevent or increase affinity of the polypeptide for another molecule, including, but not limited to, polynucleotide, proteins, carbohydrates, lipids and lipid derivatives, and other organic or synthetic compounds.

Identification of Additional Protein Factors

[0147] A transcription factor provided by the present invention can also be used to identify additional endogenous or exogenous molecules that can affect a phenotype or trait of interest. Such molecules include endogenous molecules that are acted upon either at a transcriptional level by a transcription factor of the invention to modify a phenotype as desired. For example, the transcription factors can be employed to identify one or more downstream genes that are subject to a regulatory effect of the transcription factor. In one approach, a transcription factor or transcription factor homolog of the invention is expressed in a host cell, e.g., a transgenic plant cell, tissue or explant, and expression products, either RNA or protein, of likely or random targets are monitored, e.g., by hybridization to a microarray of nucleic acid probes corresponding to genes expressed in a tissue or cell type of interest, by two-dimensional gel electrophoresis of protein products, or by any other method known in the art for assessing expression of gene products at the level of RNA or protein. Alternatively, a transcription factor of the invention can be used to identify promoter sequences (such as binding sites on DNA sequences) involved in the regulation of a downstream target. After identifying a promoter sequence, interactions between the transcription factor and the promoter sequence can be modified by changing specific nucleotides in the promoter sequence or specific amino acids in the transcription factor that interact with the promoter sequence to alter a plant trait. Typically, transcription factor DNA-binding sites are identified by gel shift assays. After identifying the promoter regions, the promoter region sequences can be employed in double-stranded DNA arrays to identify molecules that affect the interactions of the transcription factors with their promoters (Bulyk et al. (1999)).
[0148] The identified transcription factors are also useful to identify proteins that modify the activity of the transcription factor. Such modification can occur by covalent modification, such as by phosphorylation, or by protein-protein (homo or -heteropolymer) interactions. Any method suitable for detecting protein-protein interactions can be employed. Among the methods that can be employed are co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns, and the two-hybrid yeast system.
[0149] The two-hybrid system detects protein interactions in vivo and is described in Chien et al. (1991) and is commercially available from Clontech (Palo Alto, Calif.). In such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the transcription factor polypeptide and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into the plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product. Then, the library plasmids responsible for reporter gene expression are isolated and sequenced to identify the proteins encoded by the library plasmids. After identifying proteins that interact with the transcription factors, assays for compounds that interfere with the transcription factor protein-protein interactions can be preformed.

Subsequences

[0150] Also contemplated are uses of polynucleotides, also referred to herein as oligonucleotides, typically having at least 12 bases, preferably at least 50 bases, which hybridize under stringent conditions to a polynucleotide sequence described above. The polynucleotides may be used as probes, primers, sense and antisense agents, and the like, according to methods as noted above.
[0151] Subsequences of the polynucleotides of the invention, including polynucleotide fragments and oligonucleotides are useful as nucleic acid probes and primers. An oligonucleotide suitable for use as a probe or primer is at least about 15 nucleotides in length, more often at least about 18 nucleotides, often at least about 21 nucleotides, frequently at least about 30 nucleotides, or about 40 nucleotides, or more in length. A nucleic acid probe is useful in hybridization protocols, e.g., to identify additional polypeptide homologs of the invention, including protocols for microarray experiments. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods. See Sambrook (1989), and Ausubel (2000).
[0152] In addition, the invention includes an isolated or recombinant polypeptide including a subsequence of at least about 15 contiguous amino acids encoded by the recombinant or isolated polynucleotides of the invention. For example, such polypeptides, or domains or fragments thereof, can be used as immunogens, e.g., to produce antibodies specific for the polypeptide sequence, or as probes for detecting a sequence of interest. A subsequence can range in size from about 15 amino acids in length up to and including the full length of the polypeptide.
[0153] To be encompassed by the present invention, an expressed polypeptide which comprises such a polypeptide subsequence performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA binding domain that activates transcription, e.g., by binding to a specific DNA promoter region an activation domain, or a domain for protein-protein interactions.

Vectors Promoters and Expression Systems

[0154] This section describes vectors, promoters, and expression systems that may be used with the present invention. Expression constructs that have been used to transform plants for testing in field trials are also described in Example III. The present invention includes recombinant constructs comprising one or more of the nucleic acid sequences herein. The constructs typically comprise a vector, such as a plasmid, a cosmid, a phage, a virus (e.g., a plant virus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
[0155] General texts that describe molecular biological techniques useful herein, including the use and production of vectors, promoters and many other relevant topics, include Berger and Kimmel (1987), Sambrook (1989) and Ausubel (2000). Any of the identified sequences can be incorporated into a cassette or vector, e.g., for expression in plants. A number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach (1989) and Gelvin et al. (1990). Specific examples include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed by Herrera-Estrella et al. (1983), Bevan (1984), and Klee (1985) for dicotyledonous plants.
[0156] Alternatively, non-Ti vectors can be used to transfer the DNA into monocotyledonous plants and cells by using free DNA delivery techniques. Such methods can involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. By using these methods transgenic plants such as wheat, rice (Christou (1991) and corn (Gordon-Kamm (1990) can be produced. An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks et al. (1993); Vasil (1993a); Wan and Lemeaux (1994), and for Agrobacterium-mediated DNA transfer (Ishida et al. (1996)).
[0157] Typically, plant transformation vectors include one or more cloned plant coding sequence (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally—or developmentally—regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.
[0158] A potential utility for the transcription factor polynucleotides disclosed herein is the isolation of promoter elements from these genes that can be used to program expression in plants of any genes. Each transcription factor gene disclosed herein is expressed in a unique fashion, as determined by promoter elements located upstream of the start of translation, and additionally within an intron of the transcription factor gene or downstream of the termination codon of the gene. As is well known in the art, for a significant portion of genes, the promoter sequences are located entirely in the region directly upstream of the start of translation. In such cases, typically the promoter sequences are located within 2.0 KB of the start of translation, or within 1.5 KB of the start of translation, frequently within 1.0 KB of the start of translation, and sometimes within 0.5 KB of the start of translation.
[0159] The promoter sequences can be isolated according to methods known to one skilled in the art.
[0160] Examples of constitutive plant promoters which can be useful for expressing the transcription factor sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odell et al. (1985)); the nopaline synthase promoter (An et al. (1988)); and the octopine synthase promoter (Fromm et al. (1989)).
[0161] The transcription factors of the invention may be operably linked with a specific promoter that causes the transcription factor to be expressed in response to environmental, tissue-specific or temporal signals. A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for regulating the expression of a polypeptide sequence of the invention in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like. Numerous known promoters have been characterized and can favorably be employed to promote expression of a polynucleotide of the invention in a transgenic plant or cell of interest. For example, tissue specific promoters include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (Bird et al. (1988)), root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such as PTA29, PTA26 and PTA13 (U.S. Pat. No. 5,792,929), promoters active in vascular tissue (Ringli and Keller (1998)), flower-specific (Kaiser et al. (1995)), pollen (Baerson et al. (1994)), carpels (Ohl et al. (1990)), pollen and ovules (Baerson et al. (1993)), auxin-inducible promoters (such as that described in van der Kop et al. (1999) or Baumann et al. (1999)), cytokinin-inducible promoter (Guevara-Garcia (1998)), promoters responsive to gibberellin (Shi et al. (1998), Willmott et al. (1998)) and the like. Additional promoters are those that elicit expression in response to heat (Ainley et al. (1993)), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al. (1989)), and the maize rbcS promoter, Schaffner and Sheen (1991)); wounding (e.g., wuni, Siebertz et al. (1989)); pathogens (such as the PR-1 promoter described in Buchel et al. (1999) and the PDF1.2 promoter described in Manners et al. (1998), and chemicals such as methyl jasmonate or salicylic acid (Gatz (1997)). In addition, the timing of the expression can be controlled by using promoters such as those acting at senescence (Gan and Amasino (1995)); or late seed development (Odell et al. (1994)).
[0162] Plant expression vectors can also include RNA processing signals that can be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors can include additional regulatory sequences from the 3′-untranslated region of plant genes, e.g., a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.

Expression Hosts

[0163] The present invention also relates to host cells which are transduced with vectors of the invention, and the production of polypeptides of the invention (including fragments thereof) by recombinant techniques. Host cells are genetically engineered (i.e., nucleic acids are introduced, e.g., transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector comprising the relevant nucleic acids herein. The vector is optionally a plasmid, a viral particle, a phage, a naked nucleic acid, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the relevant gene. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, Sambrook (1989) and Ausubel (2000).
[0164] The host cell can be a eukaryotic cell, such as a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Plant protoplasts are also suitable for some applications. For example, the DNA fragments are introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods including electroporation (Fromm et al. (1985)), infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al. (1982); U.S. Pat. No. 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al. (1987)), use of pollen as vector (WO 85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al. (1984); Fraley et al. (1983)).
[0165] The cell can include a nucleic acid of the invention that encodes a polypeptide, wherein the cell expresses a polypeptide of the invention. The cell can also include vector sequences, or the like. Furthermore, cells and transgenic plants that include any polypeptide or nucleic acid above or throughout this specification, e.g., produced by transduction of a vector of the invention, are an additional feature of the invention.
[0166] For long-term, high-yield production of recombinant proteins, stable expression can be used. Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides encoding mature proteins of the invention can be designed with signal sequences which direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane.
Potential Applications of the Presently Disclosed Sequences that Improve Plant Yield and/or Fruit Yield or Quality
[0167] The genes identified by the experiment presently disclosed represent potential regulators of plant yield and/or fruit yield or quality. As such, these sequences, or their functional equivalogs, orthologs or paralogs, can be introduced into plant species, including commercial plant species, in order to produce higher yield and/or quality, including higher fruit yield and/or quality.

Production of Transgenic Plants and Modification of Traits

[0168] The polynucleotides of the invention are favorably employed to produce transgenic plants with various traits, or characteristics, that have been modified in a desirable manner, e.g., to improve the fruit quality characteristics of a plant. For example, alteration of expression levels or patterns (e.g., spatial or temporal expression patterns) of one or more of the transcription factors (or transcription factor homologs) of the invention, as compared with the levels of the same protein found in a wild-type plant, can be used to modify a plant's traits. An illustrative example of trait modification, improved characteristics, by altering expression levels of a particular transcription factor is described further in the Examples and the Sequence Listing.
Homologous Genes Introduced into Transgenic Plants.
[0169] Homologous genes that may be derived from any plant, or from any source whether natural, synthetic, semi-synthetic or recombinant, and that share significant sequence identity or similarity to those provided by the present invention, may be introduced into plants, for example, crop plants, to confer desirable or improved traits. Consequently, transgenic plants may be produced that comprise a recombinant expression vector or cassette with a promoter operably linked to one or more sequences homologous to presently disclosed sequences. The promoter may be, for example, a plant or viral promoter.
[0170] The invention thus provides for methods for preparing transgenic plants, and for modifying plant traits. These methods include introducing into a plant a recombinant expression vector or cassette comprising a functional promoter operably linked to one or more sequences homologous to presently disclosed sequences. Plants and kits for producing these plants that result from the application of these methods are also encompassed by the present invention.
Genes, Traits and Utilities that Affect Plant Characteristics
[0171] Plant transcription factors can modulate gene expression, and, in turn, be modulated by the environmental experience of a plant. Significant alterations in a plant's environment invariably result in a change in the plant's transcription factor gene expression pattern. Altered transcription factor expression patterns generally result in phenotypic changes in the plant. Transcription factor gene product(s) in transgenic plants then differ(s) in amounts or proportions from that found in wild-type or non-transformed plants, and those transcription factors likely represent polypeptides that are used to alter the response to the environmental change. By way of example, it is well accepted in the art that analytical methods based on altered expression patterns may be used to screen for phenotypic changes in a plant far more effectively than can be achieved using traditional methods.

Antisense and Co-Suppression

[0172] In addition to expression of the nucleic acids of the invention as gene replacement or plant phenotype modification nucleic acids, the nucleic acids are also useful for sense and anti-sense suppression of expression, e.g. to down-regulate expression of a nucleic acid of the invention, e.g. as a further mechanism for modulating plant phenotype. That is, the nucleic acids of the invention, or subsequences or anti-sense sequences thereof, can be used to block expression of naturally occurring homologous nucleic acids. A variety of sense and anti-sense technologies are known in the art, e.g. as set forth in Lichtenstein and Nellen (1997). Antisense regulation is also described in Crowley et al. (1985); Rosenberg et al. (1985); Preiss et al. (1985); Melton (1985); Izant and Weintraub (1985); and Kim and Wold (1985). Additional methods for antisense regulation are known in the art. Antisense regulation has been used to reduce or inhibit expression of plant genes in, for example in European Patent Publication No. 271988. Antisense RNA may be used to reduce gene expression to produce a visible or biochemical phenotypic change in a plant (Smith et al. (1988); Smith et al. (1990)). In general, sense or anti-sense sequences are introduced into a cell, where they are optionally amplified, e.g. by transcription. Such sequences include both simple oligonucleotide sequences and catalytic sequences such as ribozymes.
[0173] For example, a reduction or elimination of expression (i.e., a “knock-out”) of a transcription factor or transcription factor homolog polypeptide in a transgenic plant, e.g., to modify a plant trait, can be obtained by introducing an antisense construct corresponding to the polypeptide of interest as a cDNA. For antisense suppression, the transcription factor or homolog cDNA is arranged in reverse orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector. The introduced sequence need not be the full-length cDNA or gene, and need not be identical to the cDNA or gene found in the plant type to be transformed. Typically, the antisense sequence need only be capable of hybridizing to the target gene or RNA of interest. Thus, where the introduced sequence is of shorter length, a higher degree of homology to the endogenous transcription factor sequence will be needed for effective antisense suppression. While antisense sequences of various lengths can be utilized, preferably, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. Preferably, the length of the antisense sequence in the vector will be greater than 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous transcription factor gene in the plant cell.
[0174] Suppression of endogenous transcription factor gene expression can also be achieved using RNA interference, or RNAi. RNAi is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to incite degradation of messenger RNA (mRNA) containing the same sequence as the dsRNA (Constans (2002)). Small interfering RNAs, or siRNAs are produced in at least two steps: an endogenous ribonuclease cleaves longer dsRNA into shorter, 21-23 nucleotide-long RNAs. The siRNA segments then mediate the degradation of the target mRNA (Zamore (2001). RNAi has been used for gene function determination in a manner similar to antisense oligonucleotides (Constans (2002)). Expression vectors that continually express siRNAs in transiently and stably transfected have been engineered to express small hairpin RNAs (shRNAs), which get processed in vivo into siRNAs-like molecules capable of carrying out gene-specific silencing (Brummelkamp et al. (2002), and Paddison, et al. (2002)). Post-transcriptional gene silencing by double-stranded RNA is discussed in further detail by Hammond et al. (2001), Fire et al. (1998) and Timmons and Fire (1998). Vectors in which RNA encoded by a transcription factor or transcription factor homolog cDNA is over-expressed can also be used to obtain co-suppression of a corresponding endogenous gene, e.g., in the manner described in U.S. Pat. No. 5,231,020 to Jorgensen. Such co-suppression (also termed sense suppression) does not require that the entire transcription factor cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly identical to the endogenous transcription factor gene of interest. However, as with antisense suppression, the suppressive efficiency will be enhanced as specificity of hybridization is increased, e.g., as the introduced sequence is lengthened, and/or as the sequence similarity between the introduced sequence and the endogenous transcription factor gene is increased.
[0175] Vectors expressing an untranslatable form of the transcription factor mRNA, e.g., sequences comprising one or more stop codon, or nonsense mutation) can also be used to suppress expression of an endogenous transcription factor, thereby reducing or eliminating its activity and modifying one or more traits. Methods for producing such constructs are described in U.S. Pat. No. 5,583,021. Preferably, such constructs are made by introducing a premature stop codon into the transcription factor gene. Alternatively, a plant trait can be modified by gene silencing using double-strand RNA (Sharp (1999)). Another method for abolishing the expression of a gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens. After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a transcription factor or transcription factor homolog gene. Plants containing a single transgene insertion event at the desired gene can be crossed to generate homozygous plants for the mutation. Such methods are well known to those of skill in the art (see for example Koncz et al. (1992a, 1992b)).
[0176] Alternatively, a plant phenotype can be altered by eliminating an endogenous gene, such as a transcription factor or transcription factor homolog, e.g., by homologous recombination (Kempin et al. (1997)).
[0177] A plant trait can also be modified by using the Cre-lox system (for example, as described in U.S. Pat. No. 5,658,772). A plant genome can be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.
[0178] The polynucleotides and polypeptides of this invention can also be expressed in a plant in the absence of an expression cassette by manipulating the activity or expression level of the endogenous gene by other means, such as, for example, by ectopically expressing a gene by T-DNA activation tagging (Ichikawa et al. (1997); Kakimoto et al. (1996)). This method entails transforming a plant with a gene tag containing multiple transcriptional enhancers and once the tag has inserted into the genome, expression of a flanking gene coding sequence becomes deregulated. In another example, the transcriptional machinery in a plant can be modified so as to increase transcription levels of a polynucleotide of the invention (see, e.g., PCT Publications WO 96/06166 and WO 98/53057 which describe the modification of the DNA-binding specificity of zinc finger proteins by changing particular amino acids in the DNA-binding motif).
[0179] The transgenic plant can also include the machinery necessary for expressing or altering the activity of a polypeptide encoded by an endogenous gene, for example, by altering the phosphorylation state of the polypeptide to maintain it in an activated state.
[0180] Transgenic plants (or plant cells, or plant explants, or plant tissues) incorporating the polynucleotides of the invention and/or expressing the polypeptides of the invention can be produced by a variety of well established techniques as described above. Following construction of a vector, most typically an expression cassette, including a polynucleotide, e.g., encoding a transcription factor or transcription factor homolog, of the invention, standard techniques can be used to introduce the polynucleotide into a plant, a plant cell, a plant explant or a plant tissue of interest. Optionally, the plant cell, explant or tissue can be regenerated to produce a transgenic plant.
[0181] The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledonous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al. (1984); Shimamoto et al. (1989); Fromm et al. (1990); and Vasil et al. (1990).
[0182] Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens mediated transformation. Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence.
[0183] Successful examples of the modification of plant characteristics by transformation with cloned sequences which serve to illustrate the current knowledge in this field of technology, and which are herein incorporated by reference, include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.
[0184] Following transformation, plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.
[0185] After transformed plants are selected and grown to maturity, those plants showing a modified trait are identified using methods well known in the art that are specifically directed to improved fruit or yield characteristics. Methods that may be used are provided in Examples II through VI. The modified trait can be any of those traits described above. Additionally, to confirm that the modified trait is due to changes in expression levels or activity of the polypeptide or polynucleotide of the invention can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.

Methods for Increasing Plant Yield or Quality by Modifying Transcription Factor Expression

[0186] The present invention includes compositions and methods for increasing the yield and quality of a plant or its products, including those derived from a crop plant. These methods incorporate steps described in the Examples listed below, and may be achieved by inserting a nucleic acid sequence of the invention into the genome of a plant cell: (i) a promoter that functions in the cell; and (ii) a nucleic acid sequence that is substantially identical to any of SEQ ID NO: 2n−1, where n=1 to 1317, or SEQ ID NO: 4839-10667, where the promoter is operably linked to the nucleic acid sequence. A transformed plant may then be generated from the cell. One may either obtain transformed seeds from that plant or its progeny, or propagate the transformed plant asexually. Alternatively, the transformed plant may be grown and harvested for plant products directly.
[0187] The methods encompassed by the invention may be extended to propagation techniques used to generate plants. For example, a target plant is transformed with a polynucleotide encoding provided in the Sequence Listing, or a polynucleotide encoding a polypeptide that is an equivalog of one of the polypeptides provided in the Sequence Listing, and that has an improved trait relative to a control plant such as one or more traits provided in Table 7, and the transformed target plant may be “selfed” (i.e., self-pollinated) or crossed with another plant to produce transgenic seed. A progeny plant may be grown from this seed, thus generating a transformed progeny plant with the improved trait as compared to the control plant.

EXAMPLES

[0188] It is to be understood that this invention is not limited to the particular devices, machines, materials and methods described. Although particular embodiments are described, equivalent embodiments may be used to practice the invention.
[0189] The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. It will be recognized by one of skill in the art that a transcription factor that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the first trait.

Example I

Isolation and Cloning of Full-Length Plant Transcription Factor cDNAs

[0190] Putative transcription factor sequences (genomic or ESTs) related to known transcription factors were identified in the Arabidopsis thaliana GenBank database using the tblastn sequence analysis program using default parameters and a P-value cutoff threshold of B4 or B5 or lower, depending on the length of the query sequence. Putative transcription factor sequence hits were then screened to identify those containing particular sequence strings. If the sequence hits contained such sequence strings, the sequences were confirmed as transcription factors.
[0191] Alternatively, Arabidopsis thaliana cDNA libraries derived from different tissues or treatments, or genomic libraries were screened to identify novel members of a transcription family using a low stringency hybridization approach. Probes were synthesized using gene specific primers in a standard PCR reaction (annealing temperature 600 C) and labeled with 32P dCTP using the High Prime DNA Labeling Kit (Roche Diagnostics Corp., Indianapolis, Ind.). Purified radiolabelled probes were added to filters immersed in Church hybridization medium (0.5 M NaPO4 pH 7.0, 7% SDS, 1% w/v bovine serum albumin) and hybridized overnight at 60° C. with shaking. Filters were washed two times for 45 to 60 minutes with 1×SCC, 1% SDS at 60° C.
[0192] To identify additional sequence 5′ or 3′ of a partial cDNA sequence in a cDNA library, 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed using the MARATHON cDNA amplification kit (Clontech, Palo Alto, Calif.). Generally, the method entailed first isolating poly(A) mRNA, performing first and second strand cDNA synthesis to generate double stranded cDNA, blunting cDNA ends, followed by ligation of the MARATHON Adaptor to the cDNA to form a library of adaptor-ligated ds cDNA.
[0193] Gene-specific primers were designed to be used along with adaptor specific primers for both 5′ and 3′ RACE reactions. Nested primers, rather than single primers, were used to increase PCR specificity. Using 5′ and 3′ RACE reactions, 5′ and 3′ RACE fragments were obtained, sequenced and cloned. The process can be repeated until 5′ and 3′ ends of the full-length gene were identified. Then the full-length cDNA was generated by PCR using primers specific to 5′ and 3′ ends of the gene by end-to-end PCR.

Example II

Strategy to Produce a Tomato Population Expressing all Transcription Factors Driven by Various Promoters

[0194] Ten promoters were chosen to control the expression of transcription factors in tomato for the purpose of evaluating complex traits in fruit development. All ten are expressed in fruit tissues, although the temporal and spatial expression patterns in the fruit vary (Table 2). All of the promoters have been characterized in tomato using a LexA-GAL4 two-component activation system. The PID (Plasmid IDentifier) and PID SEQ ID NOs used in the field study to drive expression of the sequences of the invention are listed in the second column.
[00002] [TABLE-US-00002]
  TABLE 2
 
  Promoters, promoter constructs and expression patterns used in the field study
    PID and SEQ    
    ID NO: of
    promoter
  Promoter   construct   General expression patterns   References
 
  CaMV35S (“35S”)   P6506   Constitutive, high levels of expression in   Odell et al (1985)
    SEQ ID NO:   all throughout the plant and fruit
    4837
  SHOOT MERISTEMLESS   P5318   Expressed in meristematic tissues,   Long and Barton
  (STM)   SEQ ID NO:   including apical meristems, cambium.   (1998)
    4832   Low levels of expression also in some   Long and Barton
      differentiating tissues. In fruit, most   (2000)
      strongly expressed in vascular tissues
      and endosperm.
  ASYMMETRIC LEAVES 1   P5319   Expressed predominately in   Byrne et al
  (AS1)   SEQ ID NO:   differentiating tissues. In fruit, most   (2000)
    4833   strongly expressed in vascular tissues   Ori et al. (2000)
      and in endosperm.
  LIPID TRANSFER   P5287   In vegetative tissues, expression   Thoma et al.
  PROTEIN 1 (LTP1)   SEQ ID NO:   predominately in epidermis. Low levels   (1994)
    4825   of expression also evident in vascular
      tissue. In fruit, expression is strongest in
      the pith-like columella/placental tissue.
  RIBULOSE-1,5-   P5284   Expression predominately in highly   Wanner and
  BISPHOSPHATE   SEQ ID NO:   photosynthetic vegetative tissues. Fruit   Gruissem (1991)
  CARBOXYLASE, SMALL   4824   expression predominately in the pericarp.
  SUBUNIT 3 (RbcS3)
  ROOT SYSTEM   P5310   Expression generally limited to roots.   Taylor and
  INDUCIBLE 1 (RSI-1)   SEQ ID NO:   Also expressed in the vascular tissues of   Scheuring (1994)
    4830   the fruit.
  APETALA 1   P5326   Light expression in leaves increases with   Mandel et al.
  (AP1)   SEQ ID NO:   maturation. Highest expression in flower   (1992a)
    4835   primordia and flower organs. In fruits,   Hempel et al.
      predominately in pith-like   (1997)
      columella/placental tissue.
  POLYGALACTURONASE   P5297   High expression throughout the fruit,   Nicholass et al.
  (PG)   SEQ ID NO:   comparable to 35S. Strongest late in fruit   (1995)
    4828   development.   Montgomery et
        al. (1993)
  PHYTOENE   P5303   Moderate expression in fruit tissues.   Corona et al.
  DESATURASE   SEQ ID NO:     (1996)
  (PD)   4829
  CRUCIFERIN 1   P5324   Expressed at low levels in fruit vascular   Breen and
  (Cru)   SEQ ID NO:   tissue and columella, endosperm   Crouch (1992)
    4834   expression.   Sjodahl et al.
        (1995)
 
[0195] Transgenic tomato lines expressing all Arabidopsis transcription factors driven by ten tissue and/or developmentally regulated promoters relied on the use of a two-component system similar to that developed by Guyer et al. (1998) that uses the DNA binding domain of the yeast GAL4 transcriptional activator fused to the activation domains of the maize C1 or the herpes simplex virus VP16 transcriptional activators, respectively. Modifications used either the E. coli lactose repressor DNA binding domain (LacI) or the E. coli LexA DNA binding domain fused to the GAL4 activation domain. The LexA-based system was the most reliable in activating tissue-specific GFP expression in tomato and was used to generate the tomato population. A diagram of the test transformation vectors is shown in FIG. 3. Arabidopsis transcription factor genes replaced the GFP gene in the target vector. As shown in Table 7, various promoters were used in the activator plasmid. Both families of vectors were used to transform tomato to yield one set of transgenic lines harboring different target vector constructs of transcription factor genes and a second population harboring the activator vector constructs of promoter-LexA/GAL4 fusions. Transgenic plants harboring the activator vector construct of promoter-LexA/GAL4 fusions were screened to identify plants with appropriate and high level expression of GUS. In addition, five of each of the transgenic plants harboring the target vector constructs of transcription factor genes were grown and crossed with a 35S activator line. F1 progeny were assayed to ensure that the transgene was capable of being activated by the LexA/GAL4 activator protein. The best plants constitutively expressing transcription factors were selected for subsequent crossing to the ten transgenic activator lines. Several of these initial lines have been evaluated and preliminary results of seedling traits indicate that similar phenotypes observed in Arabidopsis were also observed in tomato when the same transcription factor was constitutively overexpressed. Thus, each parental line harboring either a promoter-LexA/GAL4 activator or an activatible Arabidopsis transcription factors gene were pre-selected based on a functional assessment. These parental lines were used in sexual crosses to generate F1 (hemizygous for the activator and target genes) lines representing the complete set of Arabidopsis transcription factors under the regulation of the promoter. The transgenic tomato population was grown field conditions for evaluation.

Example III

Test Constructs

[0196] The Two-Component Multiplication System vectors have an activator vector and a target vector. The LexA version of these is shown in FIG. 3. The LacI versions are identical except that LacI replaces LexA portions. Both LacI and LexA DNA binding regions were tested in otherwise identical vectors. These regions were made from portions of the test vectors described above, using standard cloning methods. They were cloned into a binary vector that had been previously tested in tomato transformations. These vectors were then introduced into Arabidopsis and tomato plants to verify their functionality. The LexA-based system was determined to be the most reliable in activating tissue-specific GFP expression in tomato and was used to generate the tomato population.
[0197] A useful feature of the PTF Tool Kit vectors described in FIG. 3 is the use of two different resistance markers, one in the activator vector and another in the target vector. This greatly facilitates identifying the activator and target plant transcription factor genes in plants following crosses. The presence of both the activator and target in the same plant can be confirmed by resistance to both markers. Additionally, plants homozygous for one or both genes can be identified by scoring the segregation ratios of resistant progeny. These resistance markers are useful for making the technology easier to use for the breeder.
[0198] Another useful feature of the PTF Tool Kit activator vector described in FIG. 3 is the use of a target GFP construct to characterize the expression pattern of each of the 10 activator promoters. The Activator vector contains a construct consisting of multiple copies of the LexA (or LacI) binding sites and a TATA box upstream of the gene encoding the green fluorescence protein (GFP). This GFP reporter construct verifies that the activator gene is functional and that the promoter has the desired expression pattern before extensive plant crossing and characterizations proceed. The GFP reporter gene is also useful in plants derived from crossing the activator and target parents because it provides an easy method to detect the pattern of expression of expressed plant transcription factor genes.

Example IV

Tomato Transformation and Sulfonamide Selection

[0199] After the activator and target vectors were constructed, the vectors were used to transform Agrobacterium tumefaciens cells. Since the target vector comprised a polypeptide or interest (in the example given in FIG. 3, the polypeptide of interest was green fluorescent protein; other polypeptides of interest may include transcription factor polypeptides of the invention), it was expected that plants containing both vectors would be conferred with improved and useful traits. Methods for generating transformed plants with expression vectors are well known in the art; this Example also describes a novel method for transforming tomato plants with a sulfonamide selection marker. In this Example, tomato cotyledon explants were transformed with Agrobacterium cultures comprising target vectors having a sulfonamide selection marker.
[0200] Seed sterilization. T63 seeds were surface sterilized in a sterilization solution of 20% bleach (containing 6% sodium hypochlorite) for 20 minutes with constant stirring. Two drops of Tween 20 were added to the sterilization solution as a wetting agent. Seeds were rinsed five times with sterile distilled water, blotted dry with sterile filter paper and transferred to Sigma P4928 phytacons (25 seeds per phytacon) containing 84 ml of MSO medium (the formula for MS medium may be found in Murashige and Skoog (1962); MSO is supplemented as indicated in Table 3).
[0201] Seed germination and explanting. Phytacons were placed in a growth room at 24° C. with a 16 hour photoperiod. Seedlings were grown for seven days.
[0202] Explanting plates were prepared by placing a 9 cm Whatman No. 2 filter paper onto a plate of 100 mm×25 mm Petri dish containing 25 ml of R1F medium. Tomato seedlings were cut and placed into a 100 mm×25 mm Petri dish containing a 9 cm Whatman No. 2 filter paper and 3 ml of distilled water. Explants were prepared by cutting cotyledons into three pieces. The two proximal pieces were transferred onto the explanting plate, and the distal section was discarded. One hundred twenty explants were placed on each plate. A control plate was also prepared that was not subjected to the Agrobacterium transformation procedure. Explants were kept in the dark at 24° C. for 24 hours.
[0203] Agrobacterium culture preparation and cocultivation. The stock of Agrobacterium tumefaciens cells for transformation were made as described by Nagel et al. (1990). Agrobacterium strain ABI was grown in 250 ml LB medium (Sigma) overnight at 28° C. with shaking until an absorbance over 1 cm at 600 nm (A600) of 0.5-1.0 was reached. Cells were harvested by centrifugation at 4,000×g for 15 minutes at 4° C. Cells were then resuspended in 2501 chilled buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells were centrifuged again as described above and resuspended in 125 μL chilled buffer. Cells were then centrifuged and resuspended two more times in the same HEPES buffer as described above at a volume of 100 μL and 750 μL, respectively. Resuspended cells were then distributed into 40 μL aliquots, quickly frozen in liquid nitrogen, and stored at −80° C.
[0204] Agrobacterium cells were transformed with vectors prepared as described above following the protocol described by Nagel et al. (1990). For each DNA construct to be transformed, 50 to 100 ng DNA (generally resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) were mixed with 40 μL of Agrobacterium cells. The DNA/cell mixture was then transferred to a chilled cuvette with a 2 mm electrode gap and subject to a 2.5 kV charge dissipated at 25 RF and 200 RF using a Gene Pulser TI apparatus (Bio-Rad, Hercules, Calif.). After electroporation, cells were immediately resuspended in 1.0 ml LB and allowed to recover without antibiotic selection for 2 B 4 hours at 28° C. in a shaking incubator. After recovery, cells were plated onto selective medium of LB broth containing 100 μg/ml spectinomycin (Sigma) and incubated for 24-48 hours at 28° C. Single colonies were then picked and inoculated in fresh medium. The presence of the vector construct was verified by PCR amplification and sequence analysis.
[0205] Agrobacteria were cultured in two sequential overnight cultures. On day 1, the agrobacteria containing the target vectors having the sulfonamide selection vector (FIG. 3) were grown in 25 ml of liquid 523 medium (Moore et al. (1988)) plus 100 mg spectinomycin, 50 mg kanamycin, and 25 mg chloramphenicol per liter. On day 2, five ml of the first overnight suspension were added to 25 ml of AB medium to which is added 100 mg spectinomycin, 50 mg kanamycin, and 25 mg chloramphenicol per liter. Cultures were grown at 28° C. with constant shaking on a gyratory shaker. The second overnight suspension was centrifuged in a 38 ml sterile Oakridge tubes for 5 minutes at 8000 rpm in a Beckman JA20 rotor. The pellet was resuspended in 10 ml of MSO liquid medium containing 600 μm acetosyringone (for each 20 ml of MSO medium, 40 μl of 0.3 M stock acetosyringone were added). The Agrobacterium concentration was adjusted to an A600 of 0.25.
[0206] Seven milliliters of this Agrobacterium suspension were added to each of explanting plates. After 20 minutes, the Agrobacterium suspension was aspirated and the explants were blotted dry three times with sterile filter paper. The plates were sealed with Parafilm® and incubated in the dark at 21° C. for 48 hours.
[0207] Regeneration. Cocultivated explants were transferred after 48 hours in the dark to 100 mm×25 mm Petri plates (20 explants per plate) containing 25 ml of R1SB10 medium (this medium and subsequently used media contained sulfadiazine, the sulfonamide antibiotic used to select transformants). Plates were kept in the dark for 72 hours and then placed in low light. After 14 days, the explants were transferred to fresh RZ1/2SB25 medium. After an additional 14 days, the regenerating tissues at the edge of the explants were excised away from the primary explants and were transferred onto fresh RZ1/2SB25 medium. After another 14 day interval, regenerating tissues were again transferred to fresh ROSB25 medium. After this period, the regenerating tissues were subsequently rotated between ROSB25 and RZ1/2SB25 media at two week intervals. The well defined shoots that appeared were excised and transferred to ROSB100 medium for rooting.
[0208] Shoot Analysis. Once shoots were rooted on ROSB100 medium, small leaf pieces from the rooted shoots were sampled and analyzed with a polymerase chain reaction procedure (PCR) for the presence of the SulA gene. The PCR-positive shoots (TO) were then grown to maturity in the greenhouses. Some TO plants were crossed to plants containing the CaMV 35S activator vector. The TO self pollinated seeds were saved for later crosses to different activator promoters.
[00003] [TABLE-US-00003]
  TABLE 3
 
  Media Compositions (amounts per liter)
    MSO   R1F   R1SB10   RZ1/2SB25   ROSB25   ROSB100
   
  Gibco ® MS Salts   4.3   g   4.3   g   4.3   g   4.3   g   4.3   g   4.3   g
  RO Vitamins (100X)   10   ml           5   ml   10   ml   10   ml
  R1 Vitamins (100X)       10   ml   10   ml
  RZ Vitamins (100X)               5   ml
  Glucose   16.0   g   16.0   g   16.0   g   16.0   g   16.0   g   16.0   g
  Timentin ®           100   mg
  Carbenicillin               350   mg   350   mg   350   mg
  Noble Agar   8   11.5   10.3   10.45   10.45   10.45
  MES           0.6   g   0.6   g   0.6   g   0.6   g
  Sulfadiazine free acid           1   ml   2.5   ml   2.5   ml   10   ml
  (10 mg/ml stock)
  pH   5.7   5.7   5.7   5.7   5.7   5.7
 
[00004] [TABLE-US-00004]
  TABLE 4
 
  100x Vitamins (amounts per liter)
    RO   R1   RZ
   
    Nicotinic acid   500 mg    500 mg    500 mg 
    Thiamine HCl   50 mg   50 mg   50 mg
    Pyridoxine HCl   50 mg   50 mg   50 mg
    Myo-inositol20 g  20 g  20 g  
    Glycine   200 mg    200 mg    200 mg 
    Zeatin     0.65 mg     0.65 mg  
    IAA     1.0 mg 
    pH   5.7   5.7   5.7
   
[00005] [TABLE-US-00005]
  TABLE 5
 
  523 Medium (amounts per liter)
 
 
    Sucrose   10 g 
    Casein Enzymatic Hydrolysate   8 g
    Yeast Extract   4 g
  K2HPO4   2 g
  MgSO4•7H2O   0.3 g  
    pH   7.00
   
[00006] [TABLE-US-00006]
  TABLE 6
 
  AB Medium
    Part A     Part B (10X stock)
   
  K2HPO4   3   gMgSO4•7H2O   3   g
  NaH2PO4   1   gCaCl2   0.1   g
  NH4Cl   1   gFeSO4•7H2O   0.025   g
    KCl   0.15   g   Glucose   50   g
    pH   7.00     7.00
    Volume   900   ml     1000   ml
   
    Prepared by autoclaving and mixing 900 ml Part A with 100 ml Part B.

Example V

Population Characterization and Measurements

[0209] After the crosses were made (to generate plants having both activator and target vectors), general characterization of the F1 population was performed in the field. General evaluation included photographs of seedling and adult plant morphology, photographs of leaf shape, open flower morphology and of mature green and ripe fruit. Vegetative plant size, a measure of plant biomass, was measured by ruler at approximately two months after transplant. Plant volume was obtained by the multiplication of the three dimensions. In addition, observations were made to determine fruit number per plant. Three red-ripe fruit were harvested from each individual plant when possible and were used for the lycopene and Brix assays. Two weeks later, six fruits per promoter::gene grouping were harvested, with two fruits per plant harvested when possible. The fruits were pooled, weighed, and seeds collected.
[0210] Measurement of soluble solids. “Brix” measurement was used to determine the amount of sugar in solution. For example, 18 degree Brix sugar solution contains 18% sugar (w/w basis). Brix was measured using a refractometer (which measures refractive index). Brix measurements were performed by the follow protocol:
[0211] 1. Three red ripe fruit were harvest from each plant sampled.
[0212] 2. Each sample of three fruit was weighed together
[0213] 3. The three fruit were then quartered and blended together at high speed in a blender for approximately four minutes, until a fine puree was produced.
[0214] 4. Two 40 ml aliquots were decanted from the pureed sampled into 50 ml polypropylene tubes.
[0215] 5. Samples were then kept frozen at −20° C. until analysis
[0216] 6. For analysis samples were thawed in warm water.
[0217] 7. Approximately 15 ml of thawed tomato puree was filtered and placed onto the reading surface of a digital refractometer, and the reading recorded.
[0218] Fruit lycopene measurements. Lycopene is a pigment responsible for color of fruits (e.g., the red color of tomatoes). It was measured by the following procedure:
[0219] 1. Three red ripe fruit were harvest from each plant sampled.
[0220] 2. Each sample of three fruit was weighed together
[0221] 3. The three fruit were then quartered and blended together at high speed in a blender for approximately four minutes, until a fine puree was produced.
[0222] 4. Two 40 ml aliquots were decanted from the pureed sampled into 50 ml polypropylene tubes.
[0223] 5. Samples were frozen at −20° C. until further analysis was performed
[0224] 6. Samples were thawed in warm water.
[0225] 7. Lycopene level was measured using high performance liquid chromatography (HPLC).
[0226] Source/sink activities. Source/sink activities were determined by screening for lines in which Arabidopsis transcription factors were driven by the RbcS-3 (leaf mesophyll expression), LTP1 (epidermis and vascular expression) and the PD (early fruit development) promoters. These promoters target source processes localized in photosynthetically active cells (RbcS-3), sink processes localized in developing fruit (PD) or transport processes active in vascular tissues (LTP1) that link source and sink activities. Leaf punches were collected within one hour of sunrise, in the seventh week after transplant, and stored in ethanol. The leaves were then stained with iodine, and plants with notably high or low levels of starch were noted.
[0227] Fruit ripening regulation. Screening for traits associated with fruit ripening focused on transgenic tomato lines in which Arabidopsis transcription factors are driven by the PD (early fruit development) and PG (fruit ripening) promoters. These promoters target fruit regulatory processes that lead to fruit maturation or which trigger ripening or components of the ripening process. In order to identify lines expressing transcription factors that impact ripening, fruits at 1 cm stage, a developmental time 7-10 days post anthesis and shortly after fruit set were tagged. Tagging occurred over a single two-day period per field trial at a time when plants are in the early fruiting stage to ensure tagging of one to two fruits per plant, and four to six fruits per line. Tagged fruit at the “breaker” stage on any given inspection were marked with a second colored and dated tag. Later inspections included monitoring of breaker-tagged fruit to identify any that have reached the full red ripe stage. To assess the regulation of components of the ripening process, fruit at the mature green and red ripe stage have been harvested and fruit texture analyzed by force necessary to compress equator of the fruit by 2 mm.

Example VI

Screening CaMV 35S Activator Line Progeny with the Transcription Factor Target Lines to Identify Lines Expressing Plant Transcription Factors

[0228] The plant transcription factor target plants that were initially prepared lacked an activator gene to facilitate later crosses to various activator promoter lines. In order to find transformants that were adequately expressed in the presence of an activator, the plant transcription factor plants were crossed to the CaMV 35S promoter activator line and screened for transcription factor expression by RT-PCR. The mRNA was reverse transcribed into cDNA and the amount of product was measured by quantitative PCR.
[0229] Because the parental lines were each heterozygous for the transgenes, T1 hybrid progeny were sprayed with chlorsulfuron and cyanamide to find the 25% of the progeny containing both the activator (chlorsulfuron resistant) and target (cyanamide resistant) transgenes. Segregation ratios were measured and lines with abnormal ratios were discarded. Too high a ratio indicated multiple inserts, while too low a ratio indicated a variety of possible problems. The ideal inserts produced 50% resistant progeny. Progeny containing both inserts appeared at 25% because they also required the other herbicidal markers from the Activator parental line (50%×50%).
[0230] These T1 hybrid progeny were then screened in a 96 well format for plant transcription factor gene expression by RT-PCR to ensure expression of the target plant transcription factor gene, as certain chromosomal positions can be silent or very poorly expressed or the gene can be disrupted during the integration process. The 96 well format was also used for cDNA synthesis and PCR. This procedure involves the use of one primer in the transcribed portion of the vector and a second gene-specific primer.
[0231] Because both the activator and target genes are dominant in their effects, phenotypes were observable in hybrid progeny containing both genes. These T1F1 plants were examined for visual phenotypes. However, more detailed analysis for increased color, high solids and disease resistance were also conducted once the best lines were identified and reproduced on a larger scale.

Example VII

Results of Overexpressing Specific Promoter::Transcription Factor Combinations in Tomato Plants

[0232] Using the methods described in the above Examples, a number of Arabidopsis sequences were identified that resulted, when expressed in tomato plants, in greater brix, greater fruit weight, greater lycopene, greater biomass, more chlorosis, darker green fruit, darker green leaves, deeper red fruit, larger flowers, larger leaflets, larger leaflets, larger and lighter green leaves, rugulose leaves, more anthocyanin, more trichomes, paler white fruit at the green fruit stage, thicker stems, very high vigor, and/or waxier leaves, as compared to a control plant. Table 7 shows a number of polypeptides of the invention shown to improve fruit or yield characteristics. SEQ ID NOs and GID (Gene IDentifiers) are listed in Columns 1 and 2. The conserved domains in amino acid coordinates (beginning from the n-terminus of each polypeptide) of each polypeptide associates with particular Transcription Factor Family, and the Transcription Factor Families to which the polypeptide belongs, are listed in Columns 3 and 4, respectively. The PID (Plasmid IDentifier) and PID SEQ ID NOs are listed in Columns 5 and 6, respectively. The promoters used to drive expression of the polynucleotides encoding the polypeptides and the traits that were observed in tomato plants when each polypeptide sequence was overexpressed in tomato plants, relative to traits observed in control tomato plants, are listed in Column 7. Sequences listed below for the traits of increased lycopene, brix and fruit weight were in the top 5% of all tomato lycopene, brix and fruit weight measurements, respectively.
[00007] [TABLE-US-00007]
  TABLE 7
 
  Polypeptides of the invention, their conserved domains, families and the traits conferred by
  overexpressing the polypeptides in tomato plants
          Col. 5   Col. 6   Col. 7
      Col. 3     Second   SEQ ID   Experimental
    Col. 2   TF family (conserved   Col. 4   construct   NO: of   observation
  Col. 1   SEQ ID   domain amino acid   First construct (expression   containing   second   (trait relative
  GID   NO:   coordinates)   system)   TF   construct   to controls)
 
  G2   464   AP2 (129-195, 221-288)   P5310 (root-expressed   P8197   4657   Inc. brix
        RSI1 prom.)
  G2   464   AP2 (129-195, 221-288)   P5284 (leaf-expressed   P8197   4657   Inc. lycopene
        RbcS3 prom.)
  G2   464   AP2 (129-195, 221-288)   P5310 (root-expressed   P8197   4657   Inc. lycopene
        RSI1 prom.)
  G3   944   AP2 (28-95)   P5284 (leaf-expressed   P3375   4181   Inc. lycopene
        RbcS3 prom.)
  G3   944   AP2 (28-95)   P5326 (floral meristem-   P3375   4181   Inc. lycopene
        expressed AP1 prom.)
  G4   1054   AP2 (121-183)   P5326 (floral meristem-   P7794   4619   Inc. lycopene
        expressed AP1 prom.)
  G7   1210   AP2 (58-125)   P5319 (emergent leaf   P6439   4454   Inc. fruit
        primordia-expressed AS1       weight
        prom.)
  G8   1256   AP2 (151-217, 243-293)   P5297 (fruit tissue-   P6038   4416   Inc. brix
        expressed PG prom.)
  G8   1256   AP2 (151-217, 243-293)   P5303 (fruit tissue-   P6038   4416   Inc. brix
        expressed PD prom.)
  G8   1256   AP2 (151-217, 243-293)   P5318 (shoot apical   P6038   4416   Inc. brix
        meristem-expressed STM
        prom.)
  G15   230   AP2 (281-357, 383-451)   P5284 (leaf-expressed   P9218   4812   Inc. lycopene
        RbcS3 prom.)
  G19   416   AP2 (76-143)   P5297 (fruit tissue-   P5056   4370   Inc. brix
        expressed PG prom.)
  G19   416   AP2 (76-143)   P5297 (fruit tissue-   P5056   4370   Inc. lycopene
        expressed PG prom.)
  G20   466   AP2 (68-144)   P5284 (leaf-expressed   P9251   4816   Inc. brix
        RbcS3 prom.)
  G20   466   AP2 (68-144)   P5297 (fruit tissue-   P9251   4816   Inc. lycopene
        expressed PG prom.)
  G20   466   AP2 (68-144)   P5284 (leaf-expressed   P9251   4816   Inc. lycopene
        RbcS3 prom.)
  G21   516   AP2 (97-164)   P5324 (fruit vascular   P4364   4290   Inc. lycopene
        tissue-expressed Cru
        prom.)
  G22   570   AP2 (88-152)   P5326 (floral meristem-   P3376   4182   Inc. brix
        expressed AP1 prom.)
  G22   570   AP2 (88-152)   P5284 (leaf-expressed   P3376   4182   Inc. lycopene
        RbcS3 prom.)
  G22   570   AP2 (88-152)   P5303 (fruit tissue-   P3376   4182   Inc. lycopene
        expressed PD prom.)
  G22   570   AP2 (88-152)   P5326 (floral meristem-   P3376   4182   Inc. lycopene
        expressed AP1 prom.)
  G26   728   AP2 (67-134)   P5297 (fruit tissue-   P9131   4790   Inc. lycopene
        expressed PG prom.)
  G28   828   AP2 (145-208)   P5326 (floral meristem-   P7826   4620   Inc. lycopene
        expressed AP1 prom.)
  G28   828   AP2 (145-208)   P5297 (fruit tissue-   P7826   4620   Inc. lycopene
        expressed PG prom.)
  G28   828   AP2 (145-208)   P5287 (epidermal tissue-   P7826   4620   Inc. lycopene
        expressed LTP1 prom.)
  G29   884   AP2 (134-197)   P5284 (leaf-expressed   P6441   4455   Inc. brix
        RbcS3 prom.)
  G30   946   AP2 (16-80)   P5297 (fruit tissue-   P3852   4223   Inc. brix
        expressed PG prom.)
  G32   1002   AP2 (17-84)   P6506 (constitutive CaMv   P6849   4480   Inc. brix
        35S prom.)
  G32   1002   AP2 (17-84)   P5324 (fruit vascular   P6849   4480   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G35   1022   AP2 (NA)   P5326 (floral meristem-   P5130   4383   Inc. fruit
        expressed AP1 prom.)       weight
  G35   1022   AP2 (NA)   P5318 (shoot apical   P5130   4383   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G36   1034   AP2 (NA)   P5297 (fruit tissue-   P6149   4426   Inc. brix
        expressed PG prom.)
  G43   1072   AP2 (104-172)   P5287 (epidermal tissue-   P9092   4778   Inc. fruit
        expressed LTP1 prom.)       weight
  G43   1072   AP2 (104-172)   P5326 (floral meristem-   P9092   4778   Inc. fruit
        expressed AP1 prom.)       weight
  G47   1096   AP2 (10-75)   P5318 (shoot apical   P3853   4224   Inc. lycopene
        meristem-expressed STM
        prom.)
  G137   154   MADS (1-57)   P5297 (fruit tissue-   P7474   4555   Inc. brix
        expressed PG prom.)
  G137   154   MADS (1-57)   P6506 (constitutive CaMv   P7474   4555   Inc. brix
        35S prom.)
  G137   154   MADS (1-57)   P5324 (fruit vascular   P7474   4555   Inc. brix
        tissue-expressed Cru
        prom.)
  G138   158   MADS (1-57)   P5284 (leaf-expressed   P7475   4556   Inc. brix
        RbcS3 prom.)
  G139   166   MADS (1-57)   P5319 (emergent leaf   P7476   4557   Inc. fruit
        primordia-expressed AS1       weight
        prom.)
  G140   172   MADS (16-72)   P5318 (shoot apical   P7771   4611   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G145   196   MADS (1-57)   P5297 (fruit tissue-   P8652   4730   Inc. fruit
        expressed PG prom.)       weight
  G146   202   MADS (1-57)   P5318 (shoot apical   P7477   4558   Inc. lycopene
        meristem-expressed STM
        prom.)
  G148   216   MADS (1-57)   P5326 (floral meristem-   P8537   4695   Inc. fruit
        expressed AP1 prom.)       weight
  G154   254   MADS (2-57)   P5326 (floral meristem-   P9152   4799   Inc. fruit
        expressed AP1 prom.)       weight
  G156   270   MADS (2-57)   P5326 (floral meristem-   P3354   4176   Inc. lycopene
        expressed AP1 prom.)
  G157   272   MADS (2-57)   P6506 (constitutive CaMv   P6885   4492   Inc. brix
        35S prom.)
  G159   274   MADS (7-61)   P5287 (epidermal tissue-   P4955   4369   Inc. lycopene
        expressed LTP1 prom.)
  G165   292   MADS (7-62)   P5284 (leaf-expressed   P7065   4510   Inc. brix
        RbcS3 prom.)
  G166   300   MADS (2-56)   P5297 (fruit tissue-   P7056   4507   Inc. lycopene
        expressed PG prom.)
  G167   310   MADS (2-57)   P5318 (shoot apical   P6872   4487   Inc. brix
        meristem-expressed STM
        prom.)
  G168   314   MADS (1-57)   P5326 (floral meristem-   P7059   4508   Inc. fruit
        expressed AP1 prom.)       weight
  G168   314   MADS (1-57)   P5287 (epidermal tissue-   P7059   4508   Inc. fruit
        expressed LTP1 prom.)       weight
  G179   356   WRKY (65-121)   P6506 (constitutive CaMv   P6892   4493   Inc. lycopene
        35S prom.)
  G179   356   WRKY (65-121)   P5287 (epidermal tissue-   P6892   4493   Inc. lycopene
        expressed LTP1 prom.)
  G179   356   WRKY (65-121)   P5297 (fruit tissue-   P6892   4493   Inc. lycopene
        expressed PG prom.)
  G189   410   WRKY (240-297)   P5318 (shoot apical   P3357   4177   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G190   418   WRKY (110-169)   P5318 (shoot apical   P5142   4384   Inc. lycopene
        meristem-expressed STM
        prom.)
  G190   418   WRKY (110-169)   P5326 (floral meristem-   P5142   4384   Inc. lycopene
        expressed AP1 prom.)
  G194   438   WRKY (174-230)   P5318 (shoot apical   P3582   4194   Inc. lycopene
        meristem-expressed STM
        prom.)
  G194   438   WRKY (174-230)   P5284 (leaf-expressed   P3582   4194   Inc. lycopene
        RbcS3 prom.)
  G194   438   WRKY (174-230)   P5284 (leaf-expressed   P3582   4194   Inc. brix
        RbcS3 prom.)
  G204   482   MYB-related (6-54)   P5318 (shoot apical   P9076   4774   Inc. brix
        meristem-expressed STM
        prom.)
  G204   482   MYB-related (6-54)   P5287 (epidermal tissue-   P9076   4774   Inc. brix
        expressed LTP1 prom.)
  G206   490   MYB-(R1)R2R3 (13-   P5287 (epidermal tissue-   P9137   4793   Inc. brix
      116)   expressed LTP1 prom.)
  G206   490   MYB-(R1)R2R3 (13-   P5326 (floral meristem-   P9137   4793   Inc. lycopene
      116)   expressed AP1 prom.)
  G206   490   MYB-(R1)R2R3 (13-   P5310 (root-expressed   P9137   4793   Inc. lycopene
      116)   RSI1 prom.)
  G209   510   MYB-related (36-82)   P5318 (shoot apical   P3765   4213   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G210   518   MYB-(R1)R2R3 (14-   P5297 (fruit tissue-   P9123   4788   Inc. fruit
      114)   expressed PG prom.)       weight
  G210   518   MYB-(R1)R2R3 (14-   P5318 (shoot apical   P9123   4788   Inc. fruit
      114)   meristem-expressed STM       weight
        prom.)
  G210   518   MYB-(R1)R2R3 (14-   P5287 (epidermal tissue-   P9123   4788   Inc. fruit
      114)   expressed LTP1 prom.)       weight
  G210   518   MYB-(R1)R2R3 (14-   P5284 (leaf-expressed   P9123   4788   Inc. fruit
      114)   RbcS3 prom.)       weight
  G210   518   MYB-(R1)R2R3 (14-   P5326 (floral meristem-   P9123   4788   Inc. fruit
      114)   expressed AP1 prom.)       weight
  G211   528   MYB-(R1)R2R3 (24-   P5287 (epidermal tissue-   P4359   4288   Inc. lycopene
      137)   expressed LTP1 prom.)
  G211   528   MYB-(R1)R2R3 (24-   P5318 (shoot apical   P4359   4288   Inc. lycopene
      137)   meristem-expressed STM
        prom.)
  G216   564   MYB-(R1)R2R3 (49-   P5310 (root-expressed   P9134   4791   Inc. brix
      151)   RSI1 prom.)
  G217   566   MYB-related (8-55)   P5287 (epidermal tissue-   P5842   4406   Inc. lycopene
        expressed LTP1 prom.)
  G219   568   MYB-related (98-146)   P5303 (fruit tissue-   P7082   4514   Inc. brix
        expressed PD prom.)
  G219   568   MYB-related (98-146)   P5318 (shoot apical   P7082   4514   Inc. brix
        meristem-expressed STM
        prom.)
  G219   568   MYB-related (98-146)   P5284 (leaf-expressed   P7082   4514   Inc. brix
        RbcS3 prom.)
  G219   568   MYB-related (98-146)   P5326 (floral meristem-   P7082   4514   Inc. brix
        expressed AP1 prom.)
  G222   576   MYB-(R1)R2R3 (13-   P5310 (root-expressed   P5839   4405   Inc. lycopene
      119)   RSI1 prom.)
  G225   588   MYB-related (36-80)   P5310 (root-expressed   P9125   4789   Inc. brix
        RSI1 prom.)
  G225   588   MYB-related (36-80)   P5326 (floral meristem-   P9125   4789   Inc. fruit
        expressed AP1 prom.)       weight
  G227   596   MYB-(R1)R2R3 (13-   P5297 (fruit tissue-   P4021   4247   Inc. brix
      113)   expressed PG prom.)
  G229   598   MYB-(R1)R2R3 (14-   P5284 (leaf-expressed   P7860   4627   Inc. brix
      120)   RbcS3 prom.)
  G232   622   MYB-(R1)R2R3 (14-   P5284 (leaf-expressed   P9119   4786   Inc. brix
      115)   RbcS3 prom.)
  G236   632   MYB-(R1)R2R3 (NA)   P5287 (epidermal tissue-   P5843   4407   Inc. brix
        expressed LTP1 prom.)
  G236   632   MYB-(R1)R2R3 (NA)   P6506 (constitutive CaMv   P5843   4407   Inc. biomass
        35S prom.)
  G236   632   MYB-(R1)R2R3 (NA)   P5284 (leaf-expressed   P5843   4407   Inc. biomass
        RbcS3 prom.)
  G237   634   MYB-(R1)R2R3 (11-   P5303 (fruit tissue-   P4877   4361   Inc. lycopene
      113)   expressed PD prom.)
  G237   634   MYB-(R1)R2R3 (11-   P5297 (fruit tissue-   P4877   4361   Inc. lycopene
      113)   expressed PG prom.)
  G237   634   MYB-(R1)R2R3 (11-   P5284 (leaf-expressed   P4877   4361   Dark green
      113)   RbcS3 prom.)       leaves
  G239   644   MYB-(R1)R2R3 (21-   P5287 (epidermal tissue-   P5843   4407   Inc. brix
      125)   expressed LTP1 prom.)
  G239   644   MYB-(R1)R2R3 (21-   P5287 (epidermal tissue-   P5843   4407   Inc. lycopene
      125)   expressed LTP1 prom.)
  G239   644   MYB-(R1)R2R3 (21-   P5318 (shoot apical   P5843   4407   Inc. lycopene
      125)   meristem-expressed STM
        prom.)
  G241   650   MYB-(R1)R2R3 (14-   P5297 (fruit tissue-   P9136   4792   Inc. lycopene
      114)   expressed PG prom.)
  G242   654   MYB-(R1)R2R3 (6-   P5310 (root-expressed   P9121   4787   Inc. lycopene
      106)   RSI1 prom.)
  G242   654   MYB-(R1)R2R3 (6-   P5326 (floral meristem-   P9121   4787   Inc. fruit
      106)   expressed AP1 prom.)       weight
  G242   654   MYB-(R1)R2R3 (6-   P5297 (fruit tissue-   P9121   4787   Inc. fruit
      106)   expressed PG prom.)       weight
  G242   654   MYB-(R1)R2R3 (6-   P5284 (leaf-expressed   P9121   4787   Inc. fruit
      106)   RbcS3 prom.)       weight
  G242   654   MYB-(R1)R2R3 (6-   P5287 (epidermal tissue-   P9121   4787   Inc. fruit
      106)   expressed LTP1 prom.)       weight
  G243   666   MYB-(R1)R2R3 (12-   P5326 (floral meristem-   P7459   4551   Inc. brix
      128)   expressed AP1 prom.)
  G245   670   MYB-(R1)R2R3 (14-   P5284 (leaf-expressed   P7007   4500   Inc. fruit
      114)   RbcS3 prom.)       weight
  G246   678   MYB-(R1)R2R3 (57-   P5297 (fruit tissue-   P9046   4770   Inc. lycopene
      159)   expressed PG prom.)
  G255   696   MYB-(R1)R2R3 (14-   P5318 (shoot apical   P6841   4479   Inc. fruit
      116)   meristem-expressed STM       weight
        prom.)
  G258   720   MYB-(R1)R2R3 (24-   P5297 (fruit tissue-   P4194   4267   Inc. brix
      124)   expressed PG prom.)
  G258   720   MYB-(R1)R2R3 (24-   P5326 (floral meristem-   P4194   4267   Inc. fruit
      124)   expressed AP1 prom.)       weight
  G259   726   HS (40-131)   P5287 (epidermal tissue-   P4172   4261   Inc. lycopene
        expressed LTP1 prom.)
  G261   734   HS (15-106)   P5297 (fruit tissue-   P5145   4385   Inc. fruit
        expressed PG prom.)       weight
  G262   738   HS (52-143)   P5284 (leaf-expressed   P4759   4347   Inc. lycopene
        RbcS3 prom.)
  G264   746   HS (23-114)   P5284 (leaf-expressed   P8948   4764   Inc. fruit
        RbcS3 prom.)       weight
  G264   746   HS (23-114)   P5318 (shoot apical   P8948   4764   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G264   746   HS (23-114)   P5287 (epidermal tissue-   P8948   4764   Inc. fruit
        expressed LTP1 prom.)       weight
  G265   750   HS (13-104)   P5297 (fruit tissue-   P7086   4517   Inc. brix
        expressed PG prom.)
  G268   762   AKR (186-689)   P5287 (epidermal tissue-   P8695   4748   Inc. fruit
        expressed LTP1 prom.)       weight
  G273   794   AKR (93-610)   P5318 (shoot apical   P6817   4474   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G274   798   AKR (94-600)   P5287 (epidermal tissue-   P7087   4518   Inc. fruit
        expressed LTP1 prom.)       weight
  G277   812   AKR (28-173)   P5319 (emergent leaf   P7162   4541   Inc. fruit
        primordia-expressed AS1       weight
        prom.)
  G279   820   HMG (31-103)   P5303 (fruit tissue-   P7021   4502   Inc. brix
        expressed PD prom.)
  G280   830   AT-hook (97-104, 130-   P5324 (fruit vascular   P6901   4497   Inc. brix
      137-155-162, 185-192)   tissue-expressed Cru
        prom.)
  G285   860   MISC (NA)   P5297 (fruit tissue-   P5248   4386   Inc. brix
        expressed PG prom.)
  G286   866   ENBP (206-252, 332-   P5284 (leaf-expressed   P5249   4387   Inc. brix
      409, 588-786)   RbcS3 prom.)
  G286   866   ENBP (206-252, 332-   P5297 (fruit tissue-   P5249   4387   Inc. brix
      409, 588-786)   expressed PG prom.)
  G286   866   ENBP (206-252, 332-   P6506 (constitutive CaMv   P5249   4387   Inc. brix
      409, 588-786)   35S prom.)
  G286   866   ENBP (206-252, 332-   P5319 (emergent leaf   P5249   4387   Inc. brix
      409, 588-786)   primordia-expressed AS1
        prom.)
  G286   866   ENBP (206-252, 332-   P5326 (floral meristem-   P5249   4387   Inc. brix
      409, 588-786)   expressed AP1 prom.)
  G288   870   MISC (309-361)   P5287 (epidermal tissue-   P6452   4458   Inc. fruit
        expressed LTP1 prom.)       weight
  G288   870   MISC (309-361)   P5284 (leaf-expressed   P6452   4458   Inc. fruit
        RbcS3 prom.)       weight
  G290   886   SWI/SNF (538-   P5284 (leaf-expressed   P4785   4360   Inc. fruit
      784, 919-958, 1086-   RbcS3 prom.)       weight
      1169)
  G290   886   SWI/SNF (538-   P5287 (epidermal tissue-   P4785   4360   Inc. fruit
      784, 919-958, 1086-   expressed LTP1 prom.)       weight
      1169)
  G294   908   MISC (NA)   P5297 (fruit tissue-   P4560   4313   Inc. brix
        expressed PG prom.)
  G295   910   bZIP (287-354)   P5318 (shoot apical   P4783   4359   Inc. brix
        meristem-expressed STM
        prom.)
  G306   974   SCR (370-435, 486-   P5324 (fruit vascular       Inc. lycopene
      573, 576-649)   tissue-expressed Cru
        prom.)
  G309   992   SCR (223-288, 342-   P5287 (epidermal tissue-   P9195   4811   Inc. fruit
      427, 431-505)   expressed LTP1 prom.)       weight
  G321   1004   RING/C3HC4 (NA)   P5297 (fruit tissue-       Inc. fruit
        expressed PG prom.)       weight
  G322   1006   RING/C3HC4 (NA)   P5287 (epidermal tissue-       Inc. lycopene
        expressed LTP1 prom.)
  G323   1008   RING/C3HC4 (48-96)   P5297 (fruit tissue-   P4201   4269   Inc. brix
        expressed PG prom.)
  G323   1008   RING/C3HC4 (48-96)   P5318 (shoot apical   P4201   4269   Inc. lycopene
        meristem-expressed STM
        prom.)
  G326   1010   Z-CO-like (11-94, 354-   P5303 (fruit tissue-   P5256   4388   Inc. brix
      400)   expressed PD prom.)
  G326   1010   Z-CO-like (11-94, 354-   P6506 (constitutive CaMv   P5256   4388   Inc. brix
      400)   35S prom.)
  G326   1010   Z-CO-like (11-94, 354-   P5284 (leaf-expressed   P5256   4388   Inc. brix
      400)   RbcS3 prom.)
  G328   1012   Z-CO-like (12-78)   P5297 (fruit tissue-   P3955   4227   Inc. lycopene
        expressed PG prom.)
  G328   1012   Z-CO-like (12-78)   P5326 (floral meristem-   P3955   4227   Inc. lycopene
        expressed AP1 prom.)
  G335   1014   Z-Tall-1 (205-218)   P5287 (epidermal tissue-   P8167   4653   Inc. brix
        expressed LTP1 prom.)
  G335   1014   Z-Tall-1 (205-218)   P5287 (epidermal tissue-   P8167   4653   Inc. lycopene
        expressed LTP1 prom.)
  G341   1016   Z-C3H (254-374)   P5297 (fruit tissue-   P8566   4703   Inc. brix
        expressed PG prom.)
  G344   1018   GATA/Zn (166-192)   P5287 (epidermal tissue-   P6181   4431   Inc. brix
        expressed LTP1 prom.)
  G344   1018   GATA/Zn (166-192)   P5326 (floral meristem-   P6181   4431   Inc. brix
        expressed AP1 prom.)
  G345   1020   GATA/Zn (NA)   P5287 (epidermal tissue-   P6819   4475   Inc. brix
        expressed LTP1 prom.)
  G350   1024   Z-C2H2 (91-113, 150-   P5326 (floral meristem-   P6197   4434   Inc. brix
      170)   expressed AP1 prom.)
  G351   1026   Z-C2H2 (77-97, 118-   P5284 (leaf-expressed   P7773   4612   Inc. lycopene
      140)   RbcS3 prom.)
  G351   1026   Z-C2H2 (77-97, 118-   P5297 (fruit tissue-   P7773   4612   Inc. lycopene
      140)   expressed PG prom.)
  G352   1028   Z-C2H2 (99-119, 166-   P5284 (leaf-expressed   P4225   4276   Inc. brix
      186)   RbcS3 prom.)
  G354   1030   Z-C2H2 (42-62, 88-109)   P5297 (fruit tissue-   P4535   4310   Inc. brix
        expressed PG prom.)
  G354   1030   Z-C2H2 (42-62, 88-109)   P5284 (leaf-expressed   P4535   4310   Inc. brix
        RbcS3 prom.)
  G354   1030   Z-C2H2 (42-62, 88-109)   P5287 (epidermal tissue-   P4535   4310   Inc. fruit
        expressed LTP1 prom.)       weight
  G357   1032   Z-C2H2 (7-29)   P5326 (floral meristem-       Inc. lycopene
        expressed AP1 prom.)
  G362   1036   Z-C2H2 (62-82)   P5324 (fruit vascular   P6468   4464   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G363   1038   Z-C2H2 (87-108)   P5287 (epidermal tissue-       Inc. lycopene
        expressed LTP1 prom.)
  G365   1040   Z-C2H2 (70-90)   P5303 (fruit tissue-   P6820   4476   Inc. brix
        expressed PD prom.)
  G368   1042   Z-C2H2 (NA)   P5324 (fruit vascular   P5262   4389   Inc. lycopene
        tissue-expressed Cru
        prom.)
  G379   1044   RING/C3HC4 (16-56)   P5318 (shoot apical   P8225   4665   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G383   1046   GATA/Zn (77-102)   P5318 (shoot apical   P4352   4285   Inc. lycopene
        meristem-expressed STM
        prom.)
  G383   1046   GATA/Zn (77-102)   P5303 (fruit tissue-   P4352   4285   Inc. fruit
        expressed PD prom.)       weight
  G385   1048   HB (60-123)   P5287 (epidermal tissue-   P8937   4762   Inc. fruit
        expressed LTP1 prom.)       weight
  G385   1048   HB (60-123)   P5284 (leaf-expressed   P8937   4762   Inc. fruit
        RbcS3 prom.)       weight
  G385   1048   HB (60-123)   P5318 (shoot apical   P8937   4762   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G392   1050   HB (15-75)   P5318 (shoot apical   P9145   4796   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G399   1052   HB (126-186)   P5287 (epidermal tissue-   P5060   4371   Inc. lycopene
        expressed LTP1 prom.)
  G399   1052   HB (126-186)   P5326 (floral meristem-   P5060   4371   Inc. lycopene
        expressed AP1 prom.)
  G406   1056   HB (58-118)   P5326 (floral meristem-   P4533   4309   Inc. lycopene
        expressed AP1 prom.)
  G409   1058   HB (64-124)   P5318 (shoot apical   P9141   4794   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G411   1060   HB (NA)   P5319 (emergent leaf   P9142   4795   Inc. brix
        primordia-expressed AS1
        prom.)
  G411   1060   HB (NA)   P5310 (root-expressed   P9142   4795   Inc. brix
        RSI1 prom.)
  G411   1060   HB (NA)   P5310 (root-expressed   P9142   4795   Inc. lycopene
        RSI1 prom.)
  G413   1062   HB (37-97)   P5318 (shoot apical   P8730   4754   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G413   1062   HB (37-97)   P5326 (floral meristem-   P8730   4754   Inc. fruit
        expressed AP1 prom.)       weight
  G413   1062   HB (37-97)   P5297 (fruit tissue-   P8730   4754   Inc. fruit
        expressed PG prom.)       weight
  G415   1064   HB (550-610)   P5297 (fruit tissue-   P6044   4418   Inc. brix
        expressed PG prom.)
  G415   1064   HB (550-610)   P5284 (leaf-expressed   P6044   4418   Inc. brix
        RbcS3 prom.)
  G421   1066   HB (371-434)   P5297 (fruit tissue-   P8206   4658   Inc. lycopene
        expressed PG prom.)
  G426   1068   HB (346-406)   P5326 (floral meristem-   P6445   4456   Inc. fruit
        expressed AP1 prom.)       weight
  G428   1070   HB (229-292)   P5297 (fruit tissue-   P5061   4372   Inc. brix
        expressed PG prom.)
  G432   1074   HB (NA)   P5287 (epidermal tissue-   P3967   4230   Inc. lycopene
        expressed LTP1 prom.)
  G432   1074   HB (NA)   P5318 (shoot apical   P3967   4230   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G435   1076   HB (4-67)   P5284 (leaf-expressed   P3771   4214   Inc. lycopene
        RbcS3 prom.)
  G440   1078   AP2 (122-184)   P5303 (fruit tissue-   P5265   4390   Inc. brix
        expressed PD prom.)
  G443   1080   IAA (NA)   P5297 (fruit tissue-   P9079   4775   Inc. brix
        expressed PG prom.)
  G443   1080   IAA (NA)   P5297 (fruit tissue-   P9079   4775   Inc. lycopene
        expressed PG prom.)
  G443   1080   IAA (NA)   P5310 (root-expressed   P9079   4775   Inc. lycopene
        RSI1 prom.)
  G449   1082   IAA (NA)   P5326 (floral meristem-   P5266   4391   Inc. brix
        expressed AP1 prom.)
  G450   1084   IAA (6-14, 78-89, 112-   P5318 (shoot apical   P4012   4244   Inc. lycopene
      128, 180-217)   meristem-expressed STM
        prom.)
  G450   1084   IAA (6-14, 78-89, 112-   P5326 (floral meristem-   P4012   4244   Inc. lycopene
      128, 180-217)   expressed AP1 prom.)
  G451   1086   IAA (12-20, 57-68, 76-   P5297 (fruit tissue-   P9081   4776   Inc. brix
      92, 131-164)   expressed PG prom.)
  G452   1088   IAA (NA)   P5287 (epidermal tissue-   P5065   4373   Inc. brix
        expressed LTP1 prom.)
  G463   1090   IAA (14-23, 77-88, 130-   P5318 (shoot apical   P5067   4374   Inc. lycopene
      146, 194-227)   meristem-expressed STM
        prom.)
  G463   1090   IAA (14-23, 77-88, 130-   P5284 (leaf-expressed   P5067   4374   Inc. lycopene
      146, 194-227)   RbcS3 prom.)
  G467   1092   IAA (NA)   P5287 (epidermal tissue-   P7027   4503   Inc. fruit
        expressed LTP1 prom.)       weight
  G468   1094   IAA (86-102, 141-171)   P5297 (fruit tissue-   P7537   4576   Inc. brix
        expressed PG prom.)
  G468   1094   IAA (86-102, 141-171)   P5297 (fruit tissue-   P7537   4576   Inc. lycopene
        expressed PG prom.)
  G481   1098   CAAT (20-109)   P5324 (fruit vascular   P6812   4473   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G481   1098   CAAT (20-109)   P5326 (floral meristem-   P6812   4473   Inc. fruit
        expressed AP1 prom.)       weight
  G482   1100   CAAT (26-115)   P5287 (epidermal tissue-   P5072   4375   Inc. brix
        expressed LTP1 prom.)
  G485   1102   CAAT (20-109)   P5318 (shoot apical   P4190   4266   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G489   1104   CAAT (68-164)   P5297 (fruit tissue-   P3404   4183   Inc. brix
        expressed PG prom.)
  G501   1106   NAC (10-131)   P5318 (shoot apical   P5272   4392   Inc. brix
        meristem-expressed STM
        prom.)
  G501   1106   NAC (10-131)   P5303 (fruit tissue-   P5272   4392   Inc. brix
        expressed PD prom.)
  G508   1108   NAC (NA)   P5319 (emergent leaf   P5274   4393   Inc. brix
        primordia-expressed AS1
        prom.)
  G508   1108   NAC (NA)   P5303 (fruit tissue-   P5274   4393   Inc. brix
        expressed PD prom.)
  G513   1110   NAC (16-161)   P5287 (epidermal tissue-   P5507   4394   Inc. fruit
        expressed LTP1 prom.)       weight
  G519   1112   NAC (10-131)   P5297 (fruit tissue-   P9098   4779   Inc. brix
        expressed PG prom.)
  G522   1114   NAC (10-165)   P6506 (constitutive CaMv   P4942   4366   Inc. brix
        35S prom.)
  G525   1116   NAC (23-167)   P5287 (epidermal tissue-   P5076   4376   Inc. fruit
        expressed LTP1 prom.)       weight
  G525   1116   NAC (23-167)   P5318 (shoot apical   P5076   4376   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G525   1116   NAC (23-167)   P5326 (floral meristem-   P5076   4376   Inc. fruit
        expressed AP1 prom.)       weight
  G527   1118   NAC (NA)   P5326 (floral meristem-   P6470   4465   Inc. brix
        expressed AP1 prom.)
  G527   1118   NAC (NA)   P5297 (fruit tissue-   P6470   4465   Inc. brix
        expressed PG prom.)
  G527   1118   NAC (NA)   P5303 (fruit tissue-   P6470   4465   Inc. brix
        expressed PD prom.)
  G527   1118   NAC (NA)   P6506 (constitutive CaMv   P6470   4465   Inc. brix
        35S prom.)
  G529   1120   GF14 (229-236)   P5303 (fruit tissue-   P5513   4395   Inc. brix
        expressed PD prom.)
  G530   1122   GF14 (227-235)   P5284 (leaf-expressed   P4523   4307   Inc. brix
        RbcS3 prom.)
  G530   1122   GF14 (227-235)   P5287 (epidermal tissue-   P4523   4307   Inc. fruit
        expressed LTP1 prom.)       weight
  G531   1124   GF14 (227-234)   P5326 (floral meristem-   P7130   4530   Inc. brix
        expressed AP1 prom.)
  G531   1124   GF14 (227-234)   P5287 (epidermal tissue-   P7130   4530   Inc. brix
        expressed LTP1 prom.)
  G532   1126   GF14 (NA)   P5297 (fruit tissue-   P7131   4531   Inc. brix
        expressed PG prom.)
  G547   1128   Z-C2H2 (79-99, 164-   P5326 (floral meristem-   P8614   4716   Inc. fruit
      184, 222-244)   expressed AP1 prom.)       weight
  G553   1130   bZIP (94-160)   P6506 (constitutive CaMv   P3778   4215   Inc. lycopene
        35S prom.)
  G553   1130   bZIP (94-160)   P5303 (fruit tissue-   P3778   4215   Inc. fruit
        expressed PD prom.)       weight
  G556   1132   bZIP (83-143)   P5287 (epidermal tissue-   P4162   4259   Inc. fruit
        expressed LTP1 prom.)       weight
  G558   1134   bZIP (45-105)   P5319 (emergent leaf   P3573   4191   Inc. lycopene
        primordia-expressed AS1
        prom.)
  G558   1134   bZIP (45-105)   P5318 (shoot apical   P3573   4191   Inc. lycopene
        meristem-expressed STM
        prom.)
  G559   1136   bZIP (203-264)   P5326 (floral meristem-   P3585   4195   Inc. lycopene
        expressed AP1 prom.)
  G562   1138   bZIP (253-315)   P5318 (shoot apical   P6000   4412   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G563   1140   bZIP (186-248)   P5318 (shoot apical   P4896   4363   Large leaflets
        meristem-expressed STM
        prom.)
  G563   1140   bZIP (186-248)   P5324 (fruit vascular   P4896   4363   Large leaflets
        tissue-expressed Cru
        prom.)
  G567   1142   bZIP (210-270)   P5297 (fruit tissue-   P4762   4348   Inc. brix
        expressed PG prom.)
  G567   1142   bZIP (210-270)   P5297 (fruit tissue-   P4762   4348   Inc. lycopene
        expressed PG prom.)
  G567   1142   bZIP (210-270)   P5319 (emergent leaf   P4762   4348   Inc. lycopene
        primordia-expressed AS1
        prom.)
  G568   1144   bZIP (215-265)   P5297 (fruit tissue-   P7120   4527   Inc. brix
        expressed PG prom.)
  G577   1146   BZIPT2 (1-53, 356-466)   P5326 (floral meristem-   P4026   4250   Inc. brix
        expressed AP1 prom.)
  G577   1146   BZIPT2 (1-53, 356-466)   P5326 (floral meristem-   P4026   4250   Inc. lycopene
        expressed AP1 prom.)
  G579   1148   bZIP (167-227)   P5287 (epidermal tissue-   P3981   4237   Inc. lycopene
        expressed LTP1 prom.)
  G579   1148   bZIP (167-227)   P5287 (epidermal tissue-   P4025   4249   Inc. lycopene
        expressed LTP1 prom.)
  G580   1150   bZIP (162-218)   P5318 (shoot apical   P3657   4212   Inc. brix
        meristem-expressed STM
        prom.)
  G580   1150   bZIP (162-218)   P5318 (shoot apical   P3657   4212   Inc. lycopene
        meristem-expressed STM
        prom.)
  G580   1150   bZIP (162-218)   P6506 (constitutive CaMv   P3657   4212   Inc. lycopene
        35S prom.)
  G580   1150   bZIP (162-218)   P5287 (epidermal tissue-   P6493   4469   Inc. fruit
        expressed LTP1 prom.)       weight
  G588   1152   HLH/MYC (313-370)   P5284 (leaf-expressed   P7856   4625   Inc. fruit
        RbcS3 prom.)       weight
  G591   1154   HLH/MYC (149-206)   P5318 (shoot apical   P5085   4377   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G595   1156   HLH/MYC (NA)   P5287 (epidermal tissue-   P9103   4780   Inc. brix
        expressed LTP1 prom.)
  G596   1158   AT-hook (89-97, 98-   P5318 (shoot apical   P7033   4504   Inc. fruit
      244)   meristem-expressed STM       weight
        prom.)
  G598   1160   DBP (205-263)   P5319 (emergent leaf   P6001   4413   Inc. brix
        primordia-expressed AS1
        prom.)
  G598   1160   DBP (205-263)   P5326 (floral meristem-   P6001   4413   Inc. lycopene
        expressed AP1 prom.)
  G599   1162   DBP (187-219, 264-   P5303 (fruit tissue-   P6417   4445   Inc. brix
      300)   expressed PD prom.)
  G599   1162   DBP (187-219, 264-   P5326 (floral meristem-   P6417   4445   Inc. brix
      300)   expressed AP1 prom.)
  G599   1162   DBP (187-219, 264-   P5324 (fruit vascular   P6417   4445   Inc. brix
      300)   tissue-expressed Cru
        prom.)
  G615   1164   TEO (88-147)   P5284 (leaf-expressed   P7159   4540   Inc. brix
        RbcS3 prom.)
  G619   1166   ARF (64-406)   P6506 (constitutive CaMv   P5706   4403   Inc. brix
        35S prom.)
  G619   1166   ARF (64-406)   P5326 (floral meristem-   P5706   4403   Inc. brix
        expressed AP1 prom.)
  G625   1168   AP2 (52-119)   P5297 (fruit tissue-   P6837   4478   Inc. brix
        expressed PG prom.)
  G631   1170   bZIP (212-282)   P5326 (floral meristem-   P6460   4461   Inc. lycopene
        expressed AP1 prom.)
  G634   1172   TH (62-147, 189-245)   P5297 (fruit tissue-   P7198   4545   Inc. lycopene
        expressed PG prom.)
  G635   1174   TH (239-323)   P5303 (fruit tissue-   P3619   4203   Inc. brix
        expressed PD prom.)
  G635   1174   TH (239-323)   P5303 (fruit tissue-   P3619   4203   Inc. lycopene
        expressed PD prom.)
  G639   1176   TH (304-389)   P5284 (leaf-expressed   P3641   4206   Inc. lycopene
        RbcS3 prom.)
  G641   1178   TH (23-102)   P5326 (floral meristem-   P7134   4532   Inc. fruit
        expressed AP1 prom.)       weight
  G645   1180   SCR (276-341, 392-   P5297 (fruit tissue-   P6053   4420   Inc. brix
      479, 480-553)   expressed PG prom.)
  G645   1180   SCR (276-341, 392-   P5284 (leaf-expressed   P6053   4420   Inc. lycopene
      479, 480-553)   RbcS3 prom.)
  G649   1182   RING/C3H2C3 (NA)   P5326 (floral meristem-   P5091   4378   Inc. brix
        expressed AP1 prom.)
  G653   1184   Z-LIM (10-61, 109-160)   P5284 (leaf-expressed   P9248   4815   Inc. lycopene
        RbcS3 prom.)
  G655   1186   MYB-(R1)R2R3 (44-   P5284 (leaf-expressed   P6430   4448   Inc. lycopene
      206)   RbcS3 prom.)
  G655   1186   MYB-(R1)R2R3 (44-   P5324 (fruit vascular   P6430   4448   Inc. fruit
      206)   tissue-expressed Cru       weight
        prom.)
  G658   1188   MYB-(R1)R2R3 (2-   P5324 (fruit vascular   P6002   4414   Inc. brix
      105)   tissue-expressed Cru
        prom.)
  G658   1188   MYB-(R1)R2R3 (2-   P5284 (leaf-expressed   P6002   4414   Inc. lycopene
      105)   RbcS3 prom.)
  G659   1190   MYB-(R1)R2R3 (16-   P5326 (floral meristem-   P6429   4447   Inc. fruit
      116)   expressed AP1 prom.)       weight
  G663   1192   MYB-(R1)R2R3 (9-   P6506 (constitutive CaMv   P5094   4379   Inc. fruit
      111)   35S prom.)       weight
  G663   1192   MYB-(R1)R2R3 (9-   P5303 (fruit tissue-   P5094   4379   Inc. fruit
      111)   expressed PD prom.)       weight
  G664   1194   MYB-(R1)R2R3 (14-   P5287 (epidermal tissue-   P7015   4501   Inc. lycopene
      116)   expressed LTP1 prom.)
  G665   1196   MYB-related (91-139)   P5284 (leaf-expressed   P6430   4448   Inc. lycopene
        RbcS3 prom.)
  G666   1198   MYB-(R1)R2R3 (14-   P5297 (fruit tissue-   P6431   4449   Inc. biomass
      116)   expressed PG prom.)
  G674   1200   MYB-(R1)R2R3 (20-   P5284 (leaf-expressed   P7123   4528   Inc. lycopene
      120)   RbcS3 prom.)
  G675   1202   MYB-(R1)R2R3 (13-   P5284 (leaf-expressed   P4019   4245   Inc. lycopene
      116)   RbcS3 prom.)
  G675   1202   MYB-(R1)R2R3 (13-   P5318 (shoot apical   P4019   4245   Inc. lycopene
      116)   meristem-expressed STM
        prom.)
  G675   1202   MYB-(R1)R2R3 (13-   P5319 (emergent leaf   P4019   4245   Inc. lycopene
      116)   primordia-expressed AS1
        prom.)
  G675   1202   MYB-(R1)R2R3 (13-   P5326 (floral meristem-   P4019   4245   Inc. lycopene
      116)   expressed AP1 prom.)
  G679   1204   MYB-related (102-150)   P6506 (constitutive CaMv   P6433   4450   Inc. brix
        35S prom.)
  G682   1206   MYB-related (33-77)   P5287 (epidermal tissue-   P5099   4380   Inc. lycopene
        expressed LTP1 prom.)
  G699   1208   HB (52-115)   P5287 (epidermal tissue-   P6494   4470   Inc. fruit
        expressed LTP1 prom.)       weight
  G699   1208   HB (52-115)   P5284 (leaf-expressed   P6494   4470   Inc. fruit
        RbcS3 prom.)       weight
  G707   1212   HB (109-169)   P5284 (leaf-expressed   P8734   4757   Inc. brix
        RbcS3 prom.)
  G707   1212   HB (109-169)   P5287 (epidermal tissue-   P8734   4757   Inc. brix
        expressed LTP1 prom.)
  G714   1214   CAAT (58-150)   P5284 (leaf-expressed   P7861   4628   Inc. lycopene
        RbcS3 prom.)
  G714   1214   CAAT (58-150)   P5297 (fruit tissue-   P7861   4628   Inc. lycopene
        expressed PG prom.)
  G715   1216   CAAT (53-149)   P5287 (epidermal tissue-   P8668   4736   Inc. fruit
        expressed LTP1 prom.)       weight
  G720   1218   GARP (301-349)   P5284 (leaf-expressed   P6076   4425   Inc. lycopene
        RbcS3 prom.)
  G720   1218   GARP (301-349)   P5287 (epidermal tissue-   P6076   4425   Inc. lycopene
        expressed LTP1 prom.)
  G721   1220   GARP (NA)   P5326 (floral meristem-   P4888   4362   Inc. brix
        expressed AP1 prom.)
  G722   1222   GARP (188-236)   P5324 (fruit vascular   P6042   4417   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G724   1224   GARP (86-134)   P5326 (floral meristem-   P8493   4688   Inc. brix
        expressed AP1 prom.)
  G724   1224   GARP (86-134)   P5310 (root-expressed   P8493   4688   Inc. lycopene
        RSI1 prom.)
  G724   1224   GARP (86-134)   P5287 (epidermal tissue-   P8493   4688   Inc. lycopene
        expressed LTP1 prom.)
  G724   1224   GARP (86-134)   P5284 (leaf-expressed   P8493   4688   Inc. fruit
        RbcS3 prom.)       weight
  G727   1226   GARP (226-269)   P5287 (epidermal tissue-   P6406   4441   Inc. fruit
        expressed LTP1 prom.)       weight
  G730   1228   GARP (169-217)   P5287 (epidermal tissue-   P8528   4693   Inc. lycopene
        expressed LTP1 prom.)
  G732   1230   bZIP (31-91)   P5318 (shoot apical   P7084   4515   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G748   1232   Z-Dof (112-140)   P5324 (fruit vascular   P3363   4178   Inc. lycopene
        tissue-expressed Cru
        prom.)
  G748   1232   Z-Dof (112-140)   P6506 (constitutive CaMv   P3363   4178   Inc. lycopene
        35S prom.)
  G748   1232   Z-Dof (112-140)   P5318 (shoot apical   P3363   4178   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G749   1234   Z-C3H (125-177)   P5287 (epidermal tissue-   P5105   4381   Inc. fruit
        expressed LTP1 prom.)       weight
  G751   1236   Z-Dof (37-82)   P5284 (leaf-expressed   P6495   4471   Inc. brix
        RbcS3 prom.)
  G751   1236   Z-Dof (37-82)   P5287 (epidermal tissue-   P6495   4471   Inc. brix
        expressed LTP1 prom.)
  G756   1238   Z-C3H (199-319)   P5287 (epidermal tissue-   P7748   4603   Inc. brix
        expressed LTP1 prom.)
  G763   1240   NAC (17-157)   P5318 (shoot apical   P5541   4396   Inc. brix
        meristem-expressed STM
        prom.)
  G763   1240   NAC (17-157)   P5297 (fruit tissue-   P5541   4396   Inc. brix
        expressed PG prom.)
  G764   1242   NAC (27-171)   P5297 (fruit tissue-   P6496   4472   Inc. brix
        expressed PG prom.)
  G764   1242   NAC (27-171)   P5297 (fruit tissue-   P6496   4472   Inc. lycopene
        expressed PG prom.)
  G783   1244   HLH/MYC (24-82)   P5319 (emergent leaf       Inc. lycopene
        primordia-expressed AS1
        prom.)
  G783   1244   HLH/MYC (24-82)   P5318 (shoot apical       Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G783   1244   HLH/MYC (24-82)   P5287 (epidermal tissue-       Inc. fruit
        expressed LTP1 prom.)       weight
  G783   1244   HLH/MYC (24-82)   P5297 (fruit tissue-       Inc. fruit
        expressed PG prom.)       weight
  G787   1246   HLH/MYC (49-105)   P5297 (fruit tissue-   P9254   4817   Inc. brix
        expressed PG prom.)
  G787   1246   HLH/MYC (49-105)   P5287 (epidermal tissue-   P9254   4817   Inc. brix
        expressed LTP1 prom.)
  G787   1246   HLH/MYC (49-105)   P5326 (floral meristem-   P9254   4817   Inc. brix
        expressed AP1 prom.)
  G789   1248   HLH/MYC (253-310)   P5324 (fruit vascular   P7068   4511   Inc. brix
        tissue-expressed Cru
        prom.)
  G790   1250   HLH/MYC (NA)   P5284 (leaf-expressed   P4044   4254   Inc. brix
        RbcS3 prom.)
  G792   1252   HLH/MYC (63-122)   P5318 (shoot apical   P7110   4524   Inc. brix
        meristem-expressed STM
        prom.)
  G792   1252   HLH/MYC (63-122)   P5319 (emergent leaf   P7110   4524   Inc. brix
        primordia-expressed AS1
        prom.)
  G793   1254   HLH/MYC (147-204)   P5287 (epidermal tissue-   P5111   4382   Inc. brix
        expressed LTP1 prom.)
  G793   1254   HLH/MYC (147-204)   P5287 (epidermal tissue-   P5111   4382   Inc. lycopene
        expressed LTP1 prom.)
  G810   1258   HS (23-114)   P5284 (leaf-expressed   P4763   4349   Inc. brix
        RbcS3 prom.)
  G811   1260   HS (17-108)   P5326 (floral meristem-   P8573   4704   Inc. fruit
        expressed AP1 prom.)       weight
  G811   1260   HS (17-108)   P5287 (epidermal tissue-   P8573   4704   Inc. fruit
        expressed LTP1 prom.)       weight
  G811   1260   HS (17-108)   P5318 (shoot apical   P8573   4704   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G812   1262   HS (29-120)   P5284 (leaf-expressed   P3650   4209   Inc. lycopene
        RbcS3 prom.)
  G819   1264   AKR (88-598)   P5284 (leaf-expressed   P8953   4765   Inc. brix
        RbcS3 prom.)
  G830   1266   AKR (89-601)   P5324 (fruit vascular   P6165   4428   Inc. brix
        tissue-expressed Cru
        prom.)
  G843   1268   MISC (60-119, 270-   P6506 (constitutive CaMv   P4559   4312   Inc. lycopene
      350)   35S prom.)
  G843   1268   MISC (60-119, 270-   P5326 (floral meristem-   P4559   4312   Inc. lycopene
      350)   expressed AP1 prom.)
  G865   1270   AP2 (36-103)   P5297 (fruit tissue-   P7088   4519   Inc. brix
        expressed PG prom.)
  G865   1270   AP2 (36-103)   P5318 (shoot apical   P7088   4519   Inc. brix
        meristem-expressed STM
        prom.)
  G865   1270   AP2 (36-103)   P5319 (emergent leaf   P7088   4519   Inc. brix
        primordia-expressed AS1
        prom.)
  G869   1272   AP2 (110-165)   P5297 (fruit tissue-   P9105   4781   Inc. lycopene
        expressed PG prom.)
  G869   1272   AP2 (110-165)   P5310 (root-expressed   P9105   4781   Inc. lycopene
        RSI1 prom.)
  G881   1274   WRKY (176-233)   P5318 (shoot apical   P5557   4397   Inc. lycopene
        meristem-expressed STM
        prom.)
  G886   1276   BZIPT2 (1-53, 542-652)   P5287 (epidermal tissue-   P6828   4477   Inc. brix
        expressed LTP1 prom.)
  G896   1278   Z-LSDlike (18-39)   P5303 (fruit tissue-   P7452   4548   Inc. fruit
        expressed PD prom.)       weight
  G896   1278   Z-LSDlike (18-39)   P5284 (leaf-expressed   P7452   4548   Inc. fruit
        RbcS3 prom.)       weight
  G896   1278   Z-LSDlike (18-39)   P5297 (fruit tissue-   P7452   4548   Inc. fruit
        expressed PG prom.)       weight
  G896   1278   Z-LSDlike (18-39)   P5324 (fruit vascular   P7452   4548   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G896   1278   Z-LSDlike (18-39)   P5287 (epidermal tissue-   P7452   4548   Inc. fruit
        expressed LTP1 prom.)       weight
  G896   1278   Z-LSDlike (18-39)   P5318 (shoot apical   P7452   4548   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G897   1280   Z-CO-like (8-39, 51-82)   P5318 (shoot apical   P7085   4516   Inc. lycopene
        meristem-expressed STM
        prom.)
  G902   1282   Z-CO-like (NA)   P5319 (emergent leaf   P9089   4777   Inc. fruit
        primordia-expressed AS1       weight
        prom.)
  G902   1282   Z-CO-like (NA)   P5324 (fruit vascular   P9089   4777   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G902   1282   Z-CO-like (NA)   P5318 (shoot apical   P9089   4777   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G904   1284   RING/C3H2C3 (117-   P5318 (shoot apical   P4748   4342   Inc. lycopene
      158)   meristem-expressed STM
        prom.)
  G905   1286   RING/C3H2C3 (118-   P5318 (shoot apical   P8548   4697   Inc. brix
      159)   meristem-expressed STM
        prom.)
  G910   1288   Z-CO-like (14-37, 77-   P5297 (fruit tissue-   P6867   4486   Inc. brix
      103)   expressed PG prom.)
  G920   1290   WRKY (152-211)   P5326 (floral meristem-   P8941   4763   Inc. lycopene
        expressed AP1 prom.)
  G921   1292   WRKY (146-203)   P5287 (epidermal tissue-   P5826   4404   Inc. lycopene
        expressed LTP1 prom.)
  G927   1294   CAAT (136-199)   P5284 (leaf-expressed   P6434   4451   Inc. brix
        RbcS3 prom.)
  G927   1294   CAAT (136-199)   P5297 (fruit tissue-   P6434   4451   Inc. brix
        expressed PG prom.)
  G927   1294   CAAT (136-199)   P5284 (leaf-expressed   P6434   4451   Inc. lycopene
        RbcS3 prom.)
  G927   1294   CAAT (136-199)   P5297 (fruit tissue-   P6434   4451   Inc. lycopene
        expressed PG prom.)
  G929   1296   CAAT (98-157)   P5324 (fruit vascular   P9107   4782   Very high
        tissue-expressed Cru       vigor
        prom.)
  G934   1298   ARF (NA)   P5326 (floral meristem-   P8669   4737   Inc. brix
        expressed AP1 prom.)
  G934   1298   ARF (NA)   P5284 (leaf-expressed   P8669   4737   Inc. lycopene
        RbcS3 prom.)
  G936   1300   GARP (59-107)   P5318 (shoot apical   P7536   4575   Inc. brix
        meristem-expressed STM
        prom.)
  G937   1302   GARP (197-246)   P5324 (fruit vascular   P4527   4308   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G938   1304   EIL (96-104)   P5310 (root-expressed       Inc. lycopene
        RSI1 prom.)
  G939   1306   EIL (97-106)   P5318 (shoot apical   P3590   4196   Inc. brix
        meristem-expressed STM
        prom.)
  G939   1306   EIL (97-106)   P5318 (shoot apical   P3590   4196   Inc. lycopene
        meristem-expressed STM
        prom.)
  G941   1308   EIL (95-103)   P5297 (fruit tissue-   P5565   4398   Inc. lycopene
        expressed PG prom.)
  G961   1310   NAC (12-180)   P5326 (floral meristem-   P8222   4663   Inc. fruit
        expressed AP1 prom.)       weight
  G961   1310   NAC (12-180)   P5310 (root-expressed   P8222   4663   Inc. fruit
        RSI1 prom.)       weight
  G962   1312   NAC (53-175)   P5297 (fruit tissue-   P6873   4488   Inc. brix
        expressed PG prom.)
  G963   1314   NAC (NA)   P5324 (fruit vascular   P4330   4279   Inc. lycopene
        tissue-expressed Cru
        prom.)
  G975   1316   AP2 (4-71)   P5324 (fruit vascular   P3367   4179   Inc. lycopene
        tissue-expressed Cru
        prom.)
  G976   1318   AP2 (87-153)   P5318 (shoot apical   P3823   4216   Inc. brix
        meristem-expressed STM
        prom.)
  G976   1318   AP2 (87-153)   P5326 (floral meristem-   P3823   4216   Inc. brix
        expressed AP1 prom.)
  G976   1318   AP2 (87-153)   P5287 (epidermal tissue-   P3823   4216   Inc. brix
        expressed LTP1 prom.)
  G979   1320   AP2 (63-139, 165-233)   P5287 (epidermal tissue-   P3635   4205   Inc. fruit
        expressed LTP1 prom.)       weight
  G988   1322   SCR (150-217, 277-   P5326 (floral meristem-   P4204   4270   Inc. brix
      366, 371-444)   expressed AP1 prom.)
  G991   1324   IAA (7-14, 48-59, 82-   P5297 (fruit tissue-   P5703   4402   Inc. lycopene
      115, 128-164)   expressed PG prom.)
  G993   1326   AP2 (69-134, 191-290)   P5324 (fruit vascular       Deep red fruit
        tissue-expressed Cru
        prom.)
  G997   1328   MYB-related (9-59)   P5297 (fruit tissue-   P7039   4505   Inc. brix
        expressed PG prom.)
  G999   1330   MYB-(R1)R2R3 (28-   P5297 (fruit tissue-   P8150   4648   Inc. lycopene
      363)   expressed PG prom.)
  G1004   2   AP2 (153-221)   P5297 (fruit tissue-   P4764   4350   Inc. fruit
        expressed PG prom.)       weight
  G1006   4   AP2 (113-177)   P5303 (fruit tissue-   P7145   4533   Inc. brix
        expressed PD prom.)
  G1006   4   AP2 (113-177)   P5324 (fruit vascular   P7145   4533   Inc. biomass
        tissue-expressed Cru
        prom.)
  G1007   6   AP2 (23-90)   P5326 (floral meristem-   P4002   4243   Inc. lycopene
        expressed AP1 prom.)
  G1008   8   AP2 (96-163)   P5297 (fruit tissue-   P7040   4506   Inc. lycopene
        expressed PG prom.)
  G1014   10   ABI3/VP-1 (90-172)   P5318 (shoot apical   P9109   4783   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1018   12   ABI3/VP-1 (222-311)   P5318 (shoot apical   P8584   4706   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1021   14   PMR (NA)   P5297 (fruit tissue-   P4777   4356   Inc. brix
        expressed PG prom.)
  G1021   14   PMR (NA)   P5284 (leaf-expressed   P4777   4356   Inc. lycopene
        RbcS3 prom.)
  G1022   16   WRKY (281-340)   P5318 (shoot apical   P7865   4631   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1033   18   HMG (49-121)   P5326 (floral meristem-   P7786   4615   Inc. lycopene
        expressed AP1 prom.)
  G1048   20   bZIP (138-190)   P5324 (fruit vascular   P6435   4452   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G1048   20   bZIP (138-190)   P5326 (floral meristem-   P6435   4452   Inc. fruit
        expressed AP1 prom.)       weight
  G1053   22   bZIP (74-120)   P5318 (shoot apical   P3599   4197   Inc. lycopene
        meristem-expressed STM
        prom.)
  G1057   24   bZIP (305-365)   P5284 (leaf-expressed   P6436   4453   Inc. lycopene
        RbcS3 prom.)
  G1062   26   HLH/MYC (300-357)   P5303 (fruit tissue-   P6854   4481   Inc. brix
        expressed PD prom.)
  G1065   28   DBP (101-210)   P5326 (floral meristem-   P3579   4192   Inc. fruit
        expressed AP1 prom.)       weight
  G1067   30   AT-hook (86-94, 95-   P5326 (floral meristem-   P7832   4621   Inc. brix
      246)   expressed AP1 prom.)
  G1067   30   AT-hook (86-94, 95-   P5326 (floral meristem-   P7832   4621   Inc. lycopene
      246)   expressed AP1 prom.)
  G1067   30   AT-hook (86-94, 95-   P5297 (fruit tissue-   P7832   4621   Inc. lycopene
      246)   expressed PG prom.)
  G1072   32   AT-hook (56-64, 123-   P5318 (shoot apical   P6160   4427   Inc. brix
      238)   meristem-expressed STM
        prom.)
  G1076   34   AT-hook (82-90, 91-   P5284 (leaf-expressed   P7092   4520   Inc. lycopene
      233)   RbcS3 prom.)
  G1078   36   BZIPT2 (1-53, 440-550)   P5284 (leaf-expressed   P3580   4193   Inc. lycopene
        RbcS3 prom.)
  G1080   38   BZIPT2 (1-50)   P5297 (fruit tissue-   P6453   4459   Inc. brix
        expressed PG prom.)
  G1080   38   BZIPT2 (1-50)   P5318 (shoot apical   P6453   4459   Inc. brix
        meristem-expressed STM
        prom.)
  G1080   38   BZIPT2 (1-50)   P5287 (epidermal tissue-   P6453   4459   Inc. lycopene
        expressed LTP1 prom.)
  G1084   40   BZIPT2 (1-53, 490-619)   P5318 (shoot apical   P4779   4357   Inc. lycopene
        meristem-expressed STM
        prom.)
  G1089   42   BZIPT2 (425-500)   P5284 (leaf-expressed   P6421   4446   Inc. brix
        RbcS3 prom.)
  G1090   44   AP2 (17-84)   P5318 (shoot apical   P7093   4521   Inc. brix
        meristem-expressed STM
        prom.)
  G1091   46   WRKY (262-319)   P5318 (shoot apical       Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1091   46   WRKY (262-319)   P5284 (leaf-expressed       Inc. fruit
        RbcS3 prom.)       weight
  G1098   48   RING/C3H2C3 (10-   P5326 (floral meristem-   P8230   4667   Inc. fruit
      29, 190-230, 255-   expressed AP1 prom.)       weight
      262, 286-290)
  G1098   48   RING/C3H2C3 (10-   P5297 (fruit tissue-   P8230   4667   Inc. fruit
      29, 190-230, 255-   expressed PG prom.)       weight
      262, 286-290)
  G1113   50   RING/C3H2C3 (85-   P5310 (root-expressed   P8232   4668   Inc. lycopene
      128)   RSI1 prom.)
  G1113   50   RING/C3H2C3 (85-   P5297 (fruit tissue-   P8232   4668   Inc. fruit
      128)   expressed PG prom.)       weight
  G1127   52   AT-hook (102-110, 155-   P5297 (fruit tissue-   P4185   4264   Inc. fruit
      162, 180-295)   expressed PG prom.)       weight
  G1131   54   HLH/MYC (160-217)   P5297 (fruit tissue-   P3950   4225   Inc. lycopene
        expressed PG prom.)
  G1131   54   HLH/MYC (160-217)   P5326 (floral meristem-   P3950   4225   Inc. fruit
        expressed AP1 prom.)       weight
  G1133   56   HLH/MYC (260-317)   P5319 (emergent leaf   P7148   4534   Inc. brix
        primordia-expressed AS1
        prom.)
  G1134   58   HLH/MYC (187-245)   P6506 (constitutive CaMv   P7112   4525   Inc. brix
        35S prom.)
  G1136   60   HLH/MYC (408-465)   P5287 (epidermal tissue-   P7769   4609   Inc. fruit
        expressed LTP1 prom.)       weight
  G1137   62   HLH/MYC (257-314)   P5297 (fruit tissue-   P3410   4184   Inc. brix
        expressed PG prom.)
  G1140   64   MADS (2-57)   P5326 (floral meristem-   P4765   4351   Inc. brix
        expressed AP1 prom.)
  G1141   66   AP2 (75-142)   P5284 (leaf-expressed   P7149   4535   Inc. brix
        RbcS3 prom.)
  G1141   66   AP2 (75-142)   P5326 (floral meristem-   P7149   4535   Inc. brix
        expressed AP1 prom.)
  G1141   66   AP2 (75-142)   P5319 (emergent leaf   P7149   4535   Inc. brix
        primordia-expressed AS1
        prom.)
  G1141   66   AP2 (75-142)   P5297 (fruit tissue-   P7149   4535   Inc. lycopene
        expressed PG prom.)
  G1141   66   AP2 (75-142)   P5319 (emergent leaf   P7149   4535   Inc. lycopene
        primordia-expressed AS1
        prom.)
  G1146   68   PAZ (886-896)   P6506 (constitutive CaMv   P7061   4509   Inc. brix
        35S prom.)
  G1146   68   PAZ (886-896)   P5297 (fruit tissue-   P7061   4509   Inc. brix
        expressed PG prom.)
  G1148   70   PAZ (770-807)   P5318 (shoot apical   P8627   4723   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1148   70   PAZ (770-807)   P5326 (floral meristem-   P8627   4723   Inc. fruit
        expressed AP1 prom.)       weight
  G1150   72   PAZ (887-907)   P5287 (epidermal tissue-       Inc. lycopene
        expressed LTP1 prom.)
  G1198   74   bZIP (173-223)   P5287 (epidermal tissue-   P4766   4352   Inc. fruit
        expressed LTP1 prom.)       weight
  G1211   76   MISC (123-179)   P5297 (fruit tissue-   P6173   4430   Inc. brix
        expressed PG prom.)
  G1211   76   MISC (123-179)   P5319 (emergent leaf   P6173   4430   Inc. brix
        primordia-expressed AS1
        prom.)
  G1211   76   MISC (123-179)   P5324 (fruit vascular   P6173   4430   Inc. brix
        tissue-expressed Cru
        prom.)
  G1216   78   BPF-1 (293-373, 464-   P5297 (fruit tissue-   P7850   4624   Inc. fruit
      550)   expressed PG prom.)       weight
  G1226   80   HLH/MYC (109-168)   P5284 (leaf-expressed   P3647   4208   Inc. lycopene
        RbcS3 prom.)
  G1228   82   HLH/MYC (172-231)   P5326 (floral meristem-   P3411   4185   Inc. lycopene
        expressed AP1 prom.)
  G1228   82   HLH/MYC (172-231)   P5297 (fruit tissue-   P3411   4185   Inc. lycopene
        expressed PG prom.)
  G1231   84   Z-C4HC3 (201-249)   P5318 (shoot apical   P7870   4634   Inc. lycopene
        meristem-expressed STM
        prom.)
  G1233   86   Z-C4HC3 (192-240)   P5318 (shoot apical   P7152   4536   Inc. lycopene
        meristem-expressed STM
        prom.)
  G1240   88   MISC (NA)   P5318 (shoot apical   P7153   4537   Inc. brix
        meristem-expressed STM
        prom.)
  G1240   88   MISC (NA)   P5297 (fruit tissue-   P7153   4537   Inc. brix
        expressed PG prom.)
  G1243   90   SWI/SNF (185-   P5326 (floral meristem-   P3998   4242   Inc. fruit
      377, 520-604, 842-   expressed AP1 prom.)       weight
      891, 944-1005)
  G1246   92   MYB-(R1)R2R3 (27-   P5303 (fruit tissue-   P6062   4423   Inc. fruit
      139)   expressed PD prom.)       weight
  G1247   94   MYB-(R1)R2R3 (18-   P5318 (shoot apical   P7737   4599   Inc. lycopene
      141)   meristem-expressed STM
        prom.)
  G1249   96   CAAT (13-89)   P5318 (shoot apical   P3993   4240   Inc. brix
        meristem-expressed STM
        prom.)
  G1256   98   Z-C2H2 (67-87, 143-   P5287 (epidermal tissue-   P8611   4715   Inc. fruit
      163)   expressed LTP1 prom.)       weight
  G1266   100   AP2 (79-147)   P5303 (fruit tissue-   P7154   4538   Inc. brix
        expressed PD prom.)
  G1272   102   PAZ (800-837)   P5297 (fruit tissue-   P4181   4263   Inc. lycopene
        expressed PG prom.)
  G1273   104   WRKY (163-218, 347-   P5326 (floral meristem-   P3994   4241   Inc. lycopene
      403)   expressed AP1 prom.)
  G1274   106   WRKY (110-166)   P5297 (fruit tissue-   P8239   4675   Inc. brix
        expressed PG prom.)
  G1275   108   WRKY (113-169)   P5287 (epidermal tissue-   P3412   4186   Inc. lycopene
        expressed LTP1 prom.)
  G1278   110   bZIP (230-328)   P5284 (leaf-expressed   P4780   4358   Inc. lycopene
        RbcS3 prom.)
  G1293   112   AKR (NA)   P5318 (shoot apical   P9114   4784   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1293   112   AKR (NA)   P5297 (fruit tissue-   P9114   4784   Inc. fruit
        expressed PG prom.)       weight
  G1293   112   AKR (NA)   P6506 (constitutive CaMv   P9114   4784   More
        35S prom.)       trichomes
  G1297   114   ENBP (77-124, 480-   P5297 (fruit tissue-   P8552   4698   Inc. fruit
      795)   expressed PG prom.)       weight
  G1303   116   Z-C4HC3 (187-235)   P5284 (leaf-expressed   P7871   4635   Inc. brix
        RbcS3 prom.)
  G1303   116   Z-C4HC3 (187-235)   P5318 (shoot apical   P7871   4635   Inc. brix
        meristem-expressed STM
        prom.)
  G1303   116   Z-C4HC3 (187-235)   P5284 (leaf-expressed   P7871   4635   Inc. lycopene
        RbcS3 prom.)
  G1305   118   MYB-(R1)R2R3 (15-   P5326 (floral meristem-   P6207   4437   Inc. brix
      118)   expressed AP1 prom.)
  G1308   120   MYB-(R1)R2R3 (1-   P5287 (epidermal tissue-   P3830   4217   Inc. fruit
      128)   expressed LTP1 prom.)       weight
  G1309   122   MYB-(R1)R2R3 (13-   P5297 (fruit tissue-   P3413   4187   Inc. lycopene
      115)   expressed PG prom.)
  G1309   122   MYB-(R1)R2R3 (13-   P5287 (epidermal tissue-   P3413   4187   Inc. fruit
      115)   expressed LTP1 prom.)       weight
  G1313   124   MYB-(R1)R2R3 (32-   P5297 (fruit tissue-   P7462   4552   Inc. lycopene
      135)   expressed PG prom.)
  G1313   124   MYB-(R1)R2R3 (32-   P5326 (floral meristem-   P7462   4552   Inc. fruit
      135)   expressed AP1 prom.)       weight
  G1315   126   MYB-(R1)R2R3 (14-   P5326 (floral meristem-   P3832   4218   Inc. fruit
      115)   expressed AP1 prom.)       weight
  G1319   128   MYB-(R1)R2R3 (14-   P5318 (shoot apical   P4020   4246   Inc. fruit
      114)   meristem-expressed STM       weight
        prom.)
  G1320   130   MYB-(R1)R2R3 (5-   P5287 (epidermal tissue-   P3834   4219   Inc. brix
      108)   expressed LTP1 prom.)
  G1324   132   MYB-(R1)R2R3 (20-   P5297 (fruit tissue-   P4914   4364   Inc. lycopene
      118)   expressed PG prom.)
  G1326   134   MYB-(R1)R2R3 (18-   P5287 (epidermal tissue-   P3417   4188   Inc. fruit
      121)   expressed LTP1 prom.)       weight
  G1335   136   Z-CLDSH (24-43, 131-   P6506 (constitutive CaMv   P5689   4399   Inc. brix
      144, 185-203)   35S prom.)
  G1335   136   Z-CLDSH (24-43, 131-   P5326 (floral meristem-   P5689   4399   Inc. brix
      144, 185-203)   expressed AP1 prom.)
  G1341   138   BZIPT2 (1-34, 288-398)   P5287 (epidermal tissue-   P8628   4724   Inc. lycopene
        expressed LTP1 prom.)
  G1349   140   Z-LSDlike (6-39)   P5287 (epidermal tissue-   P5692   4400   Inc. brix
        expressed LTP1 prom.)
  G1349   140   Z-LSDlike (6-39)   P5319 (emergent leaf   P5692   4400   Inc. brix
        primordia-expressed AS1
        prom.)
  G1349   140   Z-LSDlike (6-39)   P5324 (fruit vascular   P5692   4400   Inc. brix
        tissue-expressed Cru
        prom.)
  G1349   140   Z-LSDlike (6-39)   P5324 (fruit vascular   P5692   4400   Inc. brix
        tissue-expressed Cru
        prom.)
  G1352   142   Z-C2H2 (108-129, 167-   P5310 (root-expressed   P9257   4818   Inc. lycopene
      188)   RSI1 prom.)
  G1352   142   Z-C2H2 (108-129, 167-   P5287 (epidermal tissue-   P9257   4818   Inc. fruit
      188)   expressed LTP1 prom.)       weight
  G1354   144   NAC (7-157)   P5297 (fruit tissue-   P3976   4235   Inc. brix
        expressed PG prom.)
  G1354   144   NAC (7-157)   P5326 (floral meristem-   P3976   4235   Inc. brix
        expressed AP1 prom.)
  G1354   144   NAC (7-157)   P5318 (shoot apical   P3976   4235   Inc. brix
        meristem-expressed STM
        prom.)
  G1354   144   NAC (7-157)   P5310 (root-expressed   P3976   4235   Inc. brix
        RSI1 prom.)
  G1355   146   NAC (9-159)   P5287 (epidermal tissue-   P7864   4630   Inc. fruit
        expressed LTP1 prom.)       weight
  G1361   148   NAC (59-200)   P5297 (fruit tissue-   P7770   4610   Inc. brix
        expressed PG prom.)
  G1361   148   NAC (59-200)   P5326 (floral meristem-   P7770   4610   Inc. brix
        expressed AP1 prom.)
  G1364   150   CAAT (29-118)   P5287 (epidermal tissue-   P4357   4286   Inc. fruit
        expressed LTP1 prom.)       weight
  G1367   152   AT-hook (179-201, 262-   P5297 (fruit tissue-   P7184   4544   Inc. lycopene
      285, 298-319, 335-357)   expressed PG prom.)
  G1379   156   AP2 (18-85)   P5319 (emergent leaf   P6858   4483   Inc. brix
        primordia-expressed AS1
        prom.)
  G1379   156   AP2 (18-85)   P5318 (shoot apical   P6858   4483   Inc. brix
        meristem-expressed STM
        prom.)
  G1379   156   AP2 (18-85)   P5326 (floral meristem-   P6858   4483   Inc. brix
        expressed AP1 prom.)
  G1379   156   AP2 (18-85)   P5297 (fruit tissue-   P6858   4483   Inc. brix
        expressed PG prom.)
  G1382   160   WRKY (210-266, 385-   P5326 (floral meristem-   P9268   4821   Inc. fruit
      437)   expressed AP1 prom.)       weight
  G1382   160   WRKY (210-266, 385-   P5287 (epidermal tissue-   P9268   4821   Inc. fruit
      437)   expressed LTP1 prom.)       weight
  G1382   160   WRKY (210-266, 385-   P5318 (shoot apical   P9268   4821   Inc. fruit
      437)   meristem-expressed STM       weight
        prom.)
  G1384   162   AP2 (127-194)   P5297 (fruit tissue-   P7479   4559   Inc. brix
        expressed PG prom.)
  G1384   162   AP2 (127-194)   P5284 (leaf-expressed   P7479   4559   Inc. brix
        RbcS3 prom.)
  G1389   164   TEO (30-87)   P5318 (shoot apical   P6866   4485   Inc. brix
        meristem-expressed STM
        prom.)
  G1394   168   S1FA (13-63)   P5287 (epidermal tissue-   P6857   4482   Inc. brix
        expressed LTP1 prom.)
  G1394   168   S1FA (13-63)   P6506 (constitutive CaMv   P6857   4482   Inc. brix
        35S prom.)
  G1395   170   S1FA (1-72)   P5284 (leaf-expressed   P7157   4539   Inc. fruit
        RbcS3 prom.)       weight
  G1403   174   GF14 (16-61)   P5326 (floral meristem-       Inc. lycopene
        expressed AP1 prom.)
  G1410   176   HS (206-298)   P5287 (epidermal tissue-   P4771   4353   Inc. fruit
        expressed LTP1 prom.)       weight
  G1419   178   AP2 (69-137)   P6506 (constitutive CaMv   P3616   4201   Inc. lycopene
        35S prom.)
  G1421   180   AP2 (84-146)   P5326 (floral meristem-   P3631   4204   Inc. brix
        expressed AP1 prom.)
  G1421   180   AP2 (84-146)   P5319 (emergent leaf   P3631   4204   Inc. brix
        primordia-expressed AS1
        prom.)
  G1421   180   AP2 (84-146)   P5287 (epidermal tissue-   P3631   4204   Inc. brix
        expressed LTP1 prom.)
  G1421   180   AP2 (84-146)   P5297 (fruit tissue-   P3631   4204   Inc. brix
        expressed PG prom.)
  G1421   180   AP2 (84-146)   P5318 (shoot apical   P3631   4204   Inc. brix
        meristem-expressed STM
        prom.)
  G1421   180   AP2 (84-146)   P5287 (epidermal tissue-   P3631   4204   Inc. lycopene
        expressed LTP1 prom.)
  G1421   180   AP2 (84-146)   P5324 (fruit vascular   P3631   4204   Inc. lycopene
        tissue-expressed Cru
        prom.)
  G1421   180   AP2 (84-146)   P5303 (fruit tissue-   P3631   4204   Inc. lycopene
        expressed PD prom.)
  G1421   180   AP2 (84-146)   P5284 (leaf-expressed   P3631   4204   Inc. lycopene
        RbcS3 prom.)
  G1421   180   AP2 (84-146)   P5297 (fruit tissue-   P3631   4204   Inc. lycopene
        expressed PG prom.)
  G1421   180   AP2 (84-146)   P5319 (emergent leaf   P3631   4204   Inc. lycopene
        primordia-expressed AS1
        prom.)
  G1421   180   AP2 (84-146)   P5318 (shoot apical   P3631   4204   Inc. lycopene
        meristem-expressed STM
        prom.)
  G1423   182   MADS (6-62)   P6506 (constitutive CaMv   P7511   4567   Inc. brix
        35S prom.)
  G1435   184   GARP (146-194)   P5326 (floral meristem-   P9116   4785   Inc. fruit
        expressed AP1 prom.)       weight
  G1437   186   GRF-like (59-150)   P5303 (fruit tissue-   P4918   4365   Inc. fruit
        expressed PD prom.)       weight
  G1437   186   GRF-like (59-150)   P5287 (epidermal tissue-   P4918   4365   Inc. fruit
        expressed LTP1 prom.)       weight
  G1437   186   GRF-like (59-150)   P5297 (fruit tissue-   P4918   4365   Inc. fruit
        expressed PG prom.)       weight
  G1438   188   GRF-like (16-124)   P6506 (constitutive CaMv   P3840   4220   Larger
        35S prom.)       leaflets
  G1439   190   GRF-like (133-239)   P5284 (leaf-expressed   P6019   4415   Inc. lycopene
        RbcS3 prom.)
  G1442   192   GRF-like (111-223)   P6506 (constitutive CaMv   P6050   4419   Large light
        35S prom.)       green leaves
  G1442   192   GRF-like (111-223)   P6506 (constitutive CaMv   P6050   4419   Rugulose
        35S prom.)       leaves (small
              wrinkles)
  G1443   194   GRF-like (24-132)   P5284 (leaf-expressed   P3841   4221   Inc. fruit
        RbcS3 prom.)       weight
  G1451   198   ARF (22-357)   P5319 (emergent leaf   P8610   4714   Inc. fruit
        primordia-expressed AS1       weight
        prom.)
  G1451   198   ARF (22-357)   P5318 (shoot apical   P8610   4714   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1451   198   ARF (22-357)   P5287 (epidermal tissue-   P8610   4714   Inc. fruit
        expressed LTP1 prom.)       weight
  G1456   200   NAC (NA)   P5318 (shoot apical   P3978   4236   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1460   204   NAC (NA)   P5284 (leaf-expressed   P4946   4367   Inc. brix
        RbcS3 prom.)
  G1462   206   NAC (14-273)   P5326 (floral meristem-   P4336   4280   Inc. lycopene
        expressed AP1 prom.)
  G1469   208   Z-C2H2 (11-31, 230-   P5297 (fruit tissue-   P8615   4717   Inc. fruit
      250, 276-296)   expressed PG prom.)       weight
  G1469   208   Z-C2H2 (11-31, 230-   P5318 (shoot apical   P8615   4717   Inc. fruit
      250, 276-296)   meristem-expressed STM       weight
        prom.)
  G1469   208   Z-C2H2 (11-31, 230-   P5326 (floral meristem-   P8615   4717   Inc. fruit
      250, 276-296)   expressed AP1 prom.)       weight
  G1469   208   Z-C2H2 (11-31, 230-   P5287 (epidermal tissue-   P8615   4717   Inc. fruit
      250, 276-296)   expressed LTP1 prom.)       weight
  G1471   210   Z-C2H2 (49-70)   P5297 (fruit tissue-   P4375   4292   Inc. fruit
        expressed PG prom.)       weight
  G1471   210   Z-C2H2 (49-70)   P5287 (epidermal tissue-   P4375   4292   Inc. fruit
        expressed LTP1 prom.)       weight
  G1474   212   Z-C2H2 (41-68)   P5319 (emergent leaf   P7456   4549   Inc. fruit
        primordia-expressed AS1       weight
        prom.)
  G1474   212   Z-C2H2 (41-68)   P5324 (fruit vascular   P7456   4549   Thick stems
        tissue-expressed Cru
        prom.)
  G1478   214   Z-CO-like (32-76)   P5318 (shoot apical   P4378   4293   Inc. brix
        meristem-expressed STM
        prom.)
  G1480   218   Z-CO-like (50-73, 92-   P5326 (floral meristem-   P5883   4408   Inc. fruit
      116)   expressed AP1 prom.)       weight
  G1487   220   GATA/Zn (251-276)   P5287 (epidermal tissue-   P9266   4819   Inc. fruit
        expressed LTP1 prom.)       weight
  G1487   220   GATA/Zn (251-276)   P5284 (leaf-expressed   P9266   4819   Inc. fruit
        RbcS3 prom.)       weight
  G1487   220   GATA/Zn (251-276)   P5318 (shoot apical   P9266   4819   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1488   222   GATA/Zn (221-246)   P5287 (epidermal tissue-   P9267   4820   Inc. fruit
        expressed LTP1 prom.)       weight
  G1488   222   GATA/Zn (221-246)   P5297 (fruit tissue-   P9267   4820   Inc. fruit
        expressed PG prom.)       weight
  G1488   222   GATA/Zn (221-246)   P5284 (leaf-expressed   P9267   4820   Inc. fruit
        RbcS3 prom.)       weight
  G1491   224   GARP (50-100)   P5287 (epidermal tissue-   P4583   4317   Inc. lycopene
        expressed LTP1 prom.)
  G1494   226   HLH/MYC (254-311)   P5284 (leaf-expressed   P4772   4354   Inc. lycopene
        RbcS3 prom.)
  G1498   228   HLH/MYC (281-338)   P5284 (leaf-expressed   P3952   4226   Inc. brix
        RbcS3 prom.)
  G1498   228   HLH/MYC (281-338)   P5326 (floral meristem-   P3952   4226   Inc. brix
        expressed AP1 prom.)
  G1505   232   GATA/Zn (NA)   P5318 (shoot apical   P4414   4303   Inc. lycopene
        meristem-expressed STM
        prom.)
  G1506   234   GATA/Zn (7-33)   P5287 (epidermal tissue-   P4024   4248   Inc. fruit
        expressed LTP1 prom.)       weight
  G1506   234   GATA/Zn (7-33)   P5297 (fruit tissue-   P4024   4248   Inc. fruit
        expressed PG prom.)       weight
  G1510   236   GATA/Zn (230-263)   P5297 (fruit tissue-   P8244   4677   Inc. lycopene
        expressed PG prom.)
  G1517   238   RING/C3HC4 (312-   P5310 (root-expressed   P9147   4798   Inc. lycopene
      349)   RSI1 prom.)
  G1518   240   RING/C3HC4 (51-   P5287 (epidermal tissue-   P6449   4457   Inc. brix
      93, 126-209, 374-670)   expressed LTP1 prom.)
  G1518   240   RING/C3HC4 (51-   P5324 (fruit vascular   P6449   4457   Inc. fruit
      93, 126-209, 374-670)   tissue-expressed Cru       weight
        prom.)
  G1521   242   RING/C3HC4 (39-80)   P5310 (root-expressed   P3652   4211   Inc. brix
        RSI1 prom.)
  G1521   242   RING/C3HC4 (39-80)   P5310 (root-expressed   P3652   4211   Inc. lycopene
        RSI1 prom.)
  G1527   244   RING/C3HC4 (129-   P5326 (floral meristem-   P9146   4797   Inc. fruit
      166)   expressed AP1 prom.)       weight
  G1530   246   RING/C3H2C3 (102-   P5287 (epidermal tissue-   P8233   4669   Inc. fruit
      144)   expressed LTP1 prom.)       weight
  G1530   246   RING/C3H2C3 (102-   P5319 (emergent leaf   P8233   4669   Inc. fruit
      144)   primordia-expressed AS1       weight
        prom.)
  G1530   246   RING/C3H2C3 (102-   P5318 (shoot apical   P8233   4669   Inc. fruit
      144)   meristem-expressed STM       weight
        prom.)
  G1530   246   RING/C3H2C3 (102-   P5297 (fruit tissue-   P8233   4669   Inc. fruit
      144)   expressed PG prom.)       weight
  G1531   248   RING/C3HC4 (41-77)   P5284 (leaf-expressed   P6473   4466   Inc. brix
        RbcS3 prom.)
  G1531   248   RING/C3HC4 (41-77)   P5324 (fruit vascular   P6473   4466   Inc. brix
        tissue-expressed Cru
        prom.)
  G1531   248   RING/C3HC4 (41-77)   P5297 (fruit tissue-   P6473   4466   Inc. brix
        expressed PG prom.)
  G1535   250   HB (109-169)   P5287 (epidermal tissue-   P8616   4718   Inc. brix
        expressed LTP1 prom.)
  G1538   252   HB (66-126)   P5287 (epidermal tissue-   P3968   4231   Inc. lycopene
        expressed LTP1 prom.)
  G1540   256   HB (35-98)   P5326 (floral meristem-   P3423   4189   Inc. fruit
        expressed AP1 prom.)       weight
  G1540   256   HB (35-98)   P5318 (shoot apical   P3423   4189   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1543   258   HB (135-195)   P5284 (leaf-expressed   P3424   4190   Inc. brix
        RbcS3 prom.)
  G1543   258   HB (135-195)   P5284 (leaf-expressed   P3424   4190   Inc. lycopene
        RbcS3 prom.)
  G1549   260   HB (75-135)   P5287 (epidermal tissue-   P7720   4595   Inc. fruit
        expressed LTP1 prom.)       weight
  G1550   262   HB (NA)   P5319 (emergent leaf   P3970   4232   Inc. brix
        primordia-expressed AS1
        prom.)
  G1551   264   HB (99-160)   P5319 (emergent leaf   P4575   4316   Inc. lycopene
        primordia-expressed AS1
        prom.)
  G1551   264   HB (99-160)   P5284 (leaf-expressed   P4575   4316   Inc. fruit
        RbcS3 prom.)       weight
  G1553   266   ARF (20-351)   P5284 (leaf-expressed   P9057   4771   Inc. fruit
        RbcS3 prom.)       weight
  G1553   266   ARF (20-351)   P5326 (floral meristem-   P9057   4771   Inc. fruit
        expressed AP1 prom.)       weight
  G1553   266   ARF (20-351)   P5318 (shoot apical   P9057   4771   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1553   266   ARF (20-351)   P5287 (epidermal tissue-   P9057   4771   Inc. fruit
        expressed LTP1 prom.)       weight
  G1559   268   TH (55-154)   P5287 (epidermal tissue-   P7788   4617   Inc. brix
        expressed LTP1 prom.)
  G1559   268   TH (55-154)   P5297 (fruit tissue-   P7788   4617   Inc. lycopene
        expressed PG prom.)
  G1559   268   TH (55-154)   P5287 (epidermal tissue-   P7788   4617   Inc. lycopene
        expressed LTP1 prom.)
  G1592   276   HB (NA)   P5326 (floral meristem-   P4153   4257   Inc. fruit
        expressed AP1 prom.)       weight
  G1592   276   HB (NA)   P5318 (shoot apical   P4153   4257   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1634   278   MYB-related (29-   P5287 (epidermal tissue-   P7863   4629   Inc. brix
      79, 131-179)   expressed LTP1 prom.)
  G1635   280   MYB-related (56-102)   P5318 (shoot apical   P3606   4200   Inc. lycopene
        meristem-expressed STM
        prom.)
  G1635   280   MYB-related (56-102)   P5287 (epidermal tissue-   P3606   4200   Inc. lycopene
        expressed LTP1 prom.)
  G1637   282   MYB-related (108-156)   P5318 (shoot apical   P6168   4429   Inc. brix
        meristem-expressed STM
        prom.)
  G1638   284   MYB-related (27-   P5297 (fruit tissue-   P3843   4222   Inc. lycopene
      77, 141-189)   expressed PG prom.)
  G1640   286   MYB-(R1)R2R3 (14-   P5324 (fruit vascular   P3604   4199   Inc. lycopene
      115)   tissue-expressed Cru
        prom.)
  G1642   288   MYB-(R1)R2R3 (NA)   P5324 (fruit vascular   P6893   4494   Inc. fruit
        tissue-expressed Cru       weight
        prom.)
  G1646   290   CAAT (66-162)   P5297 (fruit tissue-   P7118   4526   Inc. fruit
        expressed PG prom.)       weight
  G1656   294   HLH/MYC (112-169)   P5326 (floral meristem-   P4156   4258   Inc. lycopene
        expressed AP1 prom.)
  G1657   296   DBP (56-97, 149-268)   P5319 (emergent leaf   P6184   4432   Inc. fruit
        primordia-expressed AS1       weight
        prom.)
  G1659   298   DBP (17-116)   P5319 (emergent leaf   P4594   4320   Inc. lycopene
        primordia-expressed AS1
        prom.)
  G1659   298   DBP (17-116)   P5318 (shoot apical   P4594   4320   Inc. lycopene
        meristem-expressed STM
        prom.)
  G1659   298   DBP (17-116)   P5324 (fruit vascular   P4594   4320   Inc. lycopene
        tissue-expressed Cru
        prom.)
  G1660   302   DBP (362-476)   P5297 (fruit tissue-       Inc. fruit
        expressed PG prom.)       weight
  G1660   302   DBP (362-476)   P5284 (leaf-expressed       Inc. fruit
        RbcS3 prom.)       weight
  G1660   302   DBP (362-476)   P5326 (floral meristem-       Inc. fruit
        expressed AP1 prom.)       weight
  G1665   304   HLH/MYC (NA)   P5297 (fruit tissue-   P4186   4265   Inc. lycopene
        expressed PG prom.)
  G1666   306   HLH/MYC (356-413)   P5318 (shoot apical   P4595   4321   Inc. fruit
        meristem-expressed STM       weight
        prom.)
  G1666   306   HLH/MYC (356-413)   P5284 (leaf-expressed   P4595   4321   More
        RbcS3 prom.)       anthocyanin
  G1669   308   Z-CO-like (NA)   P5287 (epidermal tissue-   P5702   4401   Inc. brix
        expressed LTP1 prom.)
  G1670   312   NAC (15-198)   P5326 (floral meristem-   P4340   4281   Inc. fruit
        expressed AP1 prom.)       weight
  G1730   316   RING/C3H2C3 (103-   P5287 (epidermal tissue-   P8234   4670   Inc. brix
      144)   expressed LTP1 prom.)
  G1730   316   RING/C3H2C3 (103-   P5297 (fruit tissue-   P8234   4670   Inc. brix
      144)   expressed PG prom.)
  G1730   316   RING/C3H2C3 (103-   P5284 (leaf-expressed   P8234   4670   Inc. lycopene
      144)   RbcS3 prom.)
  G1730   316   RING/C3H2C3 (103-   P5287 (epidermal tissue-   P8234   4670   Inc. lycopene