U.S. patent application number 13/664313 was filed with the patent office on 2013-05-09 for promoter, promoter control elements, and combinations, and uses thereof.
This patent application is currently assigned to CERES, INC.. The applicant listed for this patent is Ceres, Inc.. Invention is credited to Nickolai ALEXANDROV, Nestor APUYA, Zhihong COOK, David Van-Dinh DANG, Shawna DAVIS, Jonathan DONSON, Yiwen FANG, Kenneth A. FELDMANN, Danielle GRIZARD, Diane K. JOFUKU, Edward KIEGLE, Shing KWOK, Yu-Ping LU, Emilio MARGOLLES-CLARK, Leonard MEDRANO, Jack K. OKAMURO, Roger PENNELL, Michael PORTEREIKO, Dennis ROBLES, Richard SCHNEEBERGER, Tatiana TATARINOVA, Noah THEISS, Chuan-Yin WU.
Application Number | 20130117881 13/664313 |
Document ID | / |
Family ID | 48229929 |
Filed Date | 2013-05-09 |
United States Patent
Application |
20130117881 |
Kind Code |
A1 |
COOK; Zhihong ; et
al. |
May 9, 2013 |
PROMOTER, PROMOTER CONTROL ELEMENTS, AND COMBINATIONS, AND USES
THEREOF
Abstract
The present invention is directed to promoter sequences and
promoter control elements, polynucleotide constructs comprising the
promoters and control elements, and methods of identifying the
promoters, control elements, or fragments thereof. The invention
further relates to the use of the present promoters or promoter
control elements to modulate transcript levels.
Inventors: |
COOK; Zhihong; (Woodland
Hills, CA) ; FANG; Yiwen; (Los Angeles, CA) ;
FELDMANN; Kenneth A.; (Tucson, CA) ; KIEGLE;
Edward; (Chester, VT) ; KWOK; Shing; (Fairfax,
VA) ; LU; Yu-Ping; (Camarillo, CA) ; MEDRANO;
Leonard; (Azusa, CA) ; PENNELL; Roger;
(Malibu, CA) ; SCHNEEBERGER; Richard; (Carlsbad,
CA) ; WU; Chuan-Yin; (Newbury-Park, CA) ;
APUYA; Nestor; (Culver City, CA) ; OKAMURO; Jack
K.; (Oak Park, CA) ; JOFUKU; Diane K.;
(Arlington, VA) ; DONSON; Jonathan; (Oak Park,
CA) ; DANG; David Van-Dinh; (San Diego, CA) ;
MARGOLLES-CLARK; Emilio; (Miami, FL) ; ALEXANDROV;
Nickolai; (Thousand Oaks, CA) ; TATARINOVA;
Tatiana; (Los Angeles, CA) ; THEISS; Noah;
(Tucson, AZ) ; GRIZARD; Danielle; (Moorpark,
CA) ; DAVIS; Shawna; (College Station, TX) ;
ROBLES; Dennis; (Chatsworth, CA) ; PORTEREIKO;
Michael; (Thousand Oaks, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ceres, Inc.; |
Thousand Oaks |
CA |
US |
|
|
Assignee: |
CERES, INC.
Thousand Oaks
CA
|
Family ID: |
48229929 |
Appl. No.: |
13/664313 |
Filed: |
October 30, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12865719 |
Apr 19, 2011 |
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PCT/US2009/032485 |
Jan 29, 2009 |
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13664313 |
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13550341 |
Jul 16, 2012 |
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12865719 |
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12891723 |
Sep 27, 2010 |
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13550341 |
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11600953 |
Nov 14, 2006 |
7851608 |
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12891723 |
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10957569 |
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7402667 |
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11600953 |
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10950321 |
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7173121 |
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10957569 |
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12504863 |
Jul 17, 2009 |
8278434 |
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13550341 |
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11598118 |
Nov 10, 2006 |
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12504863 |
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10950321 |
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7173121 |
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11598118 |
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10965470 |
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7169915 |
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12103970 |
Apr 16, 2008 |
8389805 |
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13550341 |
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11233726 |
Sep 23, 2005 |
7378571 |
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12103970 |
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12343190 |
Dec 23, 2008 |
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11603542 |
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7868155 |
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12343190 |
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10981334 |
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7179904 |
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11603542 |
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12502117 |
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12123699 |
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12123699 |
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12575402 |
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11097589 |
Apr 1, 2005 |
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12575402 |
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12698056 |
Feb 1, 2010 |
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12294418 |
Aug 18, 2010 |
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PCT/US2007/064848 |
Mar 23, 2007 |
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12698056 |
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12891689 |
Sep 27, 2010 |
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13550341 |
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11602163 |
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7838650 |
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12891689 |
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11172703 |
Jun 30, 2005 |
7214789 |
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11602163 |
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61025697 |
Feb 1, 2008 |
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60505689 |
Sep 23, 2003 |
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60511460 |
Oct 14, 2003 |
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60518075 |
Nov 6, 2003 |
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60527611 |
Dec 4, 2003 |
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60529352 |
Dec 12, 2003 |
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60544771 |
Feb 13, 2004 |
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60583691 |
Jun 30, 2004 |
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60511460 |
Oct 14, 2003 |
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60637174 |
Dec 16, 2004 |
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60612891 |
Sep 23, 2004 |
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60613134 |
Sep 23, 2004 |
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60518075 |
Nov 6, 2003 |
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60527611 |
Dec 4, 2003 |
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60637140 |
Dec 16, 2004 |
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60558869 |
Apr 1, 2004 |
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60785794 |
Mar 24, 2006 |
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60583691 |
Jun 30, 2004 |
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60583609 |
Jun 30, 2004 |
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Current U.S.
Class: |
800/278 ;
435/254.2; 435/320.1; 435/325; 435/348; 435/419; 536/24.1;
800/298 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/823 20130101; C12N 15/8225 20130101; C12N 15/8222 20130101;
C12N 15/8227 20130101; C12N 15/8234 20130101; C12N 15/8237
20130101; C12N 15/8226 20130101 |
Class at
Publication: |
800/278 ;
435/320.1; 435/419; 800/298; 435/325; 435/348; 435/254.2;
536/24.1 |
International
Class: |
C12N 15/11 20060101
C12N015/11 |
Claims
1. An isolated nucleic acid molecule capable of modulating
transcription wherein the nucleic acid molecule shows at least 80%
sequence identity to any one of the promoter sequences in the
Sequence Listing, or a complement thereof.
2. The isolated nucleic acid molecule of claim 1, wherein said
nucleic acid is capable of functioning as a promoter.
3. The isolated nucleic acid molecule of claim 2, wherein said
nucleic acid comprises a reduced promoter nucleotide sequence
having a sequence consisting of one of the promoter sequences in
Table 1 having at least one of the corresponding optional promoter
fragments identified in Table 1.
4. The isolated nucleic acid molecule of claim 2, wherein said
nucleic acid comprises a reduced promoter nucleotide sequence
having a sequence consisting of one of the promoter sequences in
Table 1 having all of the corresponding optional promoter fragments
identified in Table 1.
5. The isolated nucleic acid molecule of claim 1, wherein said
nucleic acid molecule is capable of modulating transcription during
the developmental times, or in response to a stimulus, or in a
cell, tissue, or organ as set forth in Table 1 in the section "The
spatial expression of the promoter-marker-vector."
6. A vector construct comprising: a) a first nucleic acid capable
of modulating transcription wherein the nucleic acid molecule shows
at least 80% sequence identity to any one of the promoter sequences
in the Sequence Listing; and b) a second nucleic acid having to be
transcribed, wherein said first and second nucleic acid molecules
are heterologous to each other and are operably linked
together.
7. The vector construct according to claim 6, wherein said nucleic
acid comprises a reduced promoter nucleotide sequence having a
sequence consisting of one of the promoter sequences in Table 1
having at least one of the corresponding optional promoter
fragments identified in Table 1 deleted therefrom.
8. The vector construct according to claim 6, wherein said nucleic
acid comprises a reduced promoter nucleotide sequence having a
sequence consisting of one of the promoter sequences in Table 1
having all of the corresponding optional promoter fragments
identified in Table 1 deleted therefrom.
9. A host cell comprising an isolated nucleic acid molecule
according to claim 1, wherein said nucleic acid molecule is flanked
by exogenous sequence.
10. The host cell according to claim 9, wherein said nucleic acid
comprises a reduced promoter nucleotide sequence having a sequence
consisting of one of the promoter sequences in Table 1 having at
least one of the corresponding optional promoter fragments
identified in Table 1 deleted therefrom.
11. The host cell according to claim 9, wherein said nucleic acid
comprises a reduced promoter nucleotide sequence having a sequence
consisting of one of the promoter sequences in Table 1 having all
of the corresponding optional promoter fragments identified in
Table 1 deleted therefrom.
12. A host cell comprising a vector construct of claim 6.
13. A method of modulating transcription by combining, in an
environment suitable for transcription: a) a first nucleic acid
molecule capable of modulating transcription wherein the nucleic
acid molecule shows at least 80% sequence identity to any one of
the promoter sequences in the Sequence Listing; and b) a second
molecule to be transcribed; wherein the first and second nucleic
acid molecules are heterologous to each other and operably linked
together.
14. The method of claim 13, wherein said nucleic acid comprises a
reduced promoter nucleotide sequence having a sequence consisting
of one of the promoter sequences in Table 1 having at least one of
the corresponding optional promoter fragments identified in Table 1
deleted therefrom.
15. The method of claim 13, wherein said nucleic acid comprises a
reduced promoter nucleotide sequence having a sequence consisting
of one of the promoter sequences in Table 1 having all of the
corresponding optional promoter fragments identified in Table 1
deleted therefrom.
16. The method according to any one of claims 13-15, wherein said
first nucleic acid molecule is capable of modulating transcription
during the developmental times, or in response to a stimuli, or in
a cell tissue, or organ as set forth in Table 1 in the section
entitled "The spatial expression of the
promoter-marker-vector"wherein said first nucleic acid molecule is
inserted into a plant cell and said plant cell is regenerated into
a plant.
17. A plant comprising a vector construct according to claim 6.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of co-pending
application Ser. No. 12/865,719 filed on Apr. 19, 2011, and for
which priority is claimed under 35 U.S.C. .sctn.120. Application
Ser. No. 12/865,719 is the National Stage of PCT/US2009/032485
filed on Jan. 29, 2009 which claims priority under 35 U.S.C.
.sctn.119(e) on U.S. Provisional Application No. 61/025,697 filed
on Feb. 1, 2008; the entire contents of each of which are hereby
incorporated by reference.
[0002] This application is a Continuation-in-Part of co-pending
application Ser. No. 13/550,341 filed on Jul. 16, 2012, and for
which priority is claimed under 35 U.S.C. .sctn.120. Application
Ser. No. 13/550,341 is a Continuation-in-Part of co-pending
application Ser. No. 12/891,723 filed on Sep. 27, 2010. Application
Ser. No. 12/891,723 is a Divisional of application Ser. No.
11/600,953, now U.S. Pat. No. 7,851,608, filed on Nov. 14, 2006.
Application Ser. No. 11/600,953 is a Divisional of application Ser.
No. 10/957,569, now U.S. Pat. No. 7,402,667, filed on Sep. 30,
2004. Application Ser. No. 10/957,569 is a Continuation-in-Part of
application Ser. No. 10/950,321, now U.S. Pat. No. 7,173,121, filed
on Sep. 23, 2004 which claims priority under 35 U.S.C. .sctn.119(e)
on U.S. Provisional Application No. 60,505,689 filed on Sep. 23,
2003; Application No. 60,511,460 filed on Oct. 14, 2003;
Application No. 60/518,075 filed on Nov. 6, 2003; Application No.
60/527,611 filed on Dec. 4, 2003; Application No. 60/529,352 filed
on Dec. 12, 2003 and Application No. 60/544,771 filed on Feb. 13,
2004; and Application No. 60/583,691 filed on Jun. 30, 2004, the
entire contents of each of which are hereby incorporated by
reference, including their sequence listings.
[0003] Application Ser. No. 13/550,341 is a Continuation-in-Part of
application Ser. No. 12/504,863, now U.S. Pat. No. 8,278,434, filed
on Jul. 17, 2009, which is a Continuation of application Ser. No.
11/598,118, now abandoned, filed Nov. 10, 2006. Application Ser.
No. 11/598,118 is a Division of Ser. No. 10/950,321, now U.S. Pat.
No. 7,173,121, filed Sep. 23, 2004, which claims priority under 35
U.S.C. .sctn.119(e) on U.S. Provisional Application No. 60,505,689
filed on Sep. 23, 2003; Application No. 60,511,460 filed on Oct.
14, 2003; Application No. 60/518,075 filed on Nov. 6, 2003;
Application No. 60/527,611 filed on Dec. 4, 2003; Application No.
60/529,352 filed on Dec. 12, 2003 and Application No. 60/544,771
filed on Feb. 13, 2004; and Application No. 60/583,691 filed on
Jun. 30, 2004, the entire contents of each of which are hereby
incorporated by reference, including their sequence listings.
[0004] Application Ser. No. 13/550,341 is a Continuation-in-Part of
application Ser. No. 10/965,470, now U.S. Pat. No. 7,169,915, filed
on Oct. 13, 2004, and for which priority is claimed under 35 U.S.C.
.sctn.120; and this application claims priority under 35 U.S.C.
.sctn.119(e) of Application No. 60/511,460, filed on Oct. 13, 2003,
the entire contents of each of which are hereby incorporated by
reference, including their sequence listings.
[0005] Application Ser. No. 13/550,341 is a Continuation-in-Part of
co-pending application Ser. No. 12/103,970 filed on Apr. 16, 2008,
and for which priority is claimed under 35 U.S.C. .sctn.120.
Application Ser. No. 12/103,970 is a Divisional of application Ser.
No. 11/233,726, now U.S. Pat. No. 7,378,571, filed on Sep. 23, 2005
which claims priority under 35 U.S.C. .sctn.119(e) on U.S.
Provisional Application No. 60/612,891 filed on Sep. 23, 2004;
Application No. 60/613,134 filed on Sep. 23, 2004; and Application
No. 60/637,174 filed on Dec. 16, 2004; the entire contents of each
of which are hereby incorporated by reference, including their
sequence listings.
[0006] Application Ser. No. 13/550,341 is a Continuation-in-Part of
co-pending application Ser. No. 12/343,190 filed on Dec. 23, 2008,
and for which priority is claimed under 35 U.S.C. .sctn.120.
Application Ser. No. 12/343,190 is a Divisional of application Ser.
No. 11/603,542, now U.S. Pat. No. 7,868,155, filed on Nov. 22, 2006
which is a Divisional of application Ser. No. 10/981,334, now U.S.
Pat. No. 7,179,904, filed on Nov. 4, 2004 which claims priority
under 35 U.S.C. .sctn.119(e) on U.S. Provisional Application No.
60/518,075 filed on Nov. 6, 2003; and Application No. 60/527,611
filed on Dec. 4, 2003; the entire contents of each of which are
hereby incorporated by reference, including their sequence
listings.
[0007] Application Ser. No. 13/550,341 is a Continuation-in-Part of
co-pending application Ser. No. 12/502,117 filed on Jul. 13, 2009,
and for which priority is claimed under 35 U.S.C. .sctn.120.
Application Ser. No. 12/502,117 is a Continuation of application
Ser. No. 12/123,699, now abandoned, filed on May 20, 2008 which is
a Divisional of application Ser. No. 11/305,589, now U.S. Pat. No.
7,385,105, filed on Dec. 16, 2005 which claims priority under 35
U.S.C. .sctn.119(e) on U.S. Provisional Application No. 60/637,140
filed on Dec. 16, 2004; the entire contents of each of which are
hereby incorporated by reference, including their sequence
listings.
[0008] Application Ser. No. 13/550,341 is a Continuation-in-Part of
co-pending application Ser. No. 12/575,402 filed on Oct. 7, 2009,
and for which priority is claimed under 35 U.S.C. .sctn.120.
Application Ser. No. 12/575,402 is a Continuation of application
Ser. No. 11/097,589, now abandoned, filed on Apr. 1, 2005 which
claims priority under 35 U.S.C. .sctn.119(e) on U.S. Provisional
Application No. 60/558,869 filed on Apr. 1, 2004; the entire
contents of each of which are hereby incorporated by reference,
including their sequence listings.
[0009] Application Ser. No. 13/550,341 is a Continuation-in-Part of
co-pending application Ser. No. 12/698,056 filed on Feb. 1, 2010,
and for which priority is claimed under 35 U.S.C. .sctn.120.
Application Ser. No. 12/698,056 is a Continuation of co-pending
application Ser. No. 12/294,418 filed on Aug. 18, 2010, which is
the National Stage of PCT/US2007/64848 filed on Mar. 23, 2007 which
claims priority under 35 U.S.C. .sctn.119(e) on U.S. Provisional
Application No. 60/785,794 filed on Mar. 24, 2006; the entire
contents of each of which are hereby incorporated by reference,
including their sequence listings.
[0010] Application Ser. No. 13/550,341 is a Continuation-in-Part of
co-pending application Ser. No. 12/891,689 filed on Sep. 27, 2010,
and for which priority is claimed under 35 U.S.C. .sctn.120.
Application Ser. No. 12/981,689 is a Divisional of application Ser.
No. 11/602,163, now U.S. Pat. No. 7,838,650, filed on Nov. 20, 2006
which is a Divisional of application Ser. No. 11/172,703, now U.S.
Pat. No. 7,214,789, filed on Jun. 30, 2005 which claims priority
under 35 U.S.C. .sctn.119(e) on U.S. Provisional Application No.
60/583,691 filed on Jun. 30, 2004; and Application No. 60/583,609
filed on Jun. 30, 2004; the entire contents of each of which are
hereby incorporated by reference, including their sequence
listings.
FIELD OF THE INVENTION
[0011] The present invention relates to promoters and promoter
control elements that are useful for modulating transcription of a
desired polynucleotide. Such promoters and promoter control
elements can be included in polynucleotide constructs, expression
cassettes, vectors, or inserted into the chromosome or as an
exogenous element, to modulate in vivo and in vitro transcription
of a polynucleotide. Host cells, including plant cells, and
organisms, such as regenerated plants therefrom, with desired
traits or characteristics using polynucleotides comprising the
promoters and promoter control elements of the present invention
are also a part of the invention.
BACKGROUND OF THE INVENTION
[0012] This invention relates to the field of biotechnology and, in
particular, to specific promoter sequences and promoter control
element sequences which are useful for the transcription of
polynucleotides in a host cell or transformed host organism.
[0013] One of the primary goals of biotechnology is to obtain
organisms, such as plants, mammals, yeast, and prokaryotes having
particular desired characteristics or traits. Examples of these
characteristic or traits abound and may include, for example, in
plants, virus resistance, insect resistance, herbicide resistance,
enhanced stability or additional nutritional value. Recent advances
in genetic engineering have enabled researchers in the field to
incorporate polynucleotide sequences into host cells to obtain the
desired qualities in the organism of choice. This technology
permits one or more polynucleotides from a source different than
the organism of choice to be transcribed by the organism of choice.
If desired, the transcription and/or translation of these new
polynucleotides can be modulated in the organism to exhibit a
desired characteristic or trait. Alternatively, new patterns of
transcription and/or translation of polynucleotides endogenous to
the organism can be produced. Both approaches can be used at the
same time.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to isolated polynucleotide
sequences that comprise promoters and promoter control elements
from plants, especially Arabidopsis thaliana, Glycine max, Oryza
sativa, and Zea mays, and other promoters and promoter control
elements functional in plants.
[0015] It is an object of the present invention to provide isolated
polynucleotides that are promoter sequences. These promoter
sequences comprise, for example, [0016] (1) a polynucleotide having
a nucleotide sequence as set forth in Table 1, in the section
entitled "The predicted promoter sequence" or fragment thereof;
[0017] (2) a polynucleotide having a nucleotide sequence having at
least 80% sequence identity to a sequence as set forth in Table 1,
in the section entitled "The predicted promoter sequence" or
fragment thereof; and [0018] (3) a polynucleotide having a
nucleotide sequence which hybridizes to a sequence as set forth in
Table 1, in the section entitled "The predicted promoter sequence"
under a condition establishing a Tm-20.degree. C.
[0019] It is another object of the present invention to provide
isolated polynucleotides that are promoter control element
sequences. These promoter control element sequences comprise, for
example, [0020] (1) a polynucleotide having a nucleotide sequence
as set forth in Table 1, in the section entitled "The predicted
promoter sequence" or fragment thereof [0021] (2) a polynucleotide
having a nucleotide sequence having at least 80% sequence identity
to a sequence as set forth in Table 1, in the section entitled "The
predicted promoter sequence" or fragment thereof; and [0022] (3) a
polynucleotide having a nucleotide sequence which hybridizes to a
sequence as set forth in Table 1, in the section entitled "The
predicted promoter sequence" under a condition establishing a
Tm-20.degree. C.
[0023] Promoter or promoter control element sequences of the
present invention are capable of modulating preferential
transcription.
[0024] In another embodiment, the present promoter control elements
are capable of serving as or fulfilling the function, for example,
as a core promoter, a TATA box, a polymerase binding site, an
initiator site, a transcription binding site, an enhancer, an
inverted repeat, a locus control region, or a scaffold/matrix
attachment region.
[0025] It is yet another object of the present invention to provide
a polynucleotide that includes at least a first and a second
promoter control element. The first promoter control element is a
promoter control element sequence as discussed above, and the
second promoter control element is heterologous to the first
control element. Moreover, the first and second control elements
are operably linked. Such promoters may modulate transcript levels
preferentially in a tissue or under particular conditions.
[0026] In another embodiment, the present isolated polynucleotide
comprises a promoter or a promoter control element as described
above, wherein the promoter or promoter control element is operably
linked to a polynucleotide to be transcribed.
[0027] In another embodiment of the present vector, the promoter
and promoter control elements of the instant invention are operably
linked to a heterologous polynucleotide that is a regulatory
sequence.
[0028] It is another object of the present invention to provide a
host cell comprising an isolated polynucleotide or vector as
described above or fragment thereof. Host cells include, for
instance, bacterial, yeast, insect, mammalian, and plant. The host
cell can comprise a promoter or promoter control element exogenous
to the genome. Such a promoter can modulate transcription in cis-
and in trans-.
[0029] In yet another embodiment, the present host cell is a plant
cell capable of regenerating into a plant.
[0030] It is yet another embodiment of the present invention to
provide a plant comprising an isolated polynucleotide or vector
described above.
[0031] It is another object of the present invention to provide a
method of modulating transcription in a sample that contains either
a cell-free system of transcription or host cell. This method
comprises providing a polynucleotide or vector according to the
present invention as described above, and contacting the sample of
the polynucleotide or vector with conditions that permit
transcription.
[0032] In another embodiment of the present method, the
polynucleotide or vector preferentially modulates
[0033] (a) constitutive transcription,
[0034] (b) stress induced transcription,
[0035] (c) light induced transcription,
[0036] (d) dark induced transcription,
[0037] (e) leaf transcription,
[0038] (f) root transcription,
[0039] (g) stem or shoot transcription,
[0040] (h) silique transcription,
[0041] (i) callus transcription,
[0042] (j) flower transcription,
[0043] (k) immature bud and inflorescence specific transcription,
or
[0044] (l) senescing induced transcription
[0045] (m) germination transcription.
Other and further objects of the present invention will be made
clear or become apparent from the following description.
BRIEF DESCRIPTION OF THE TABLES AND FIGURES
Table 1
[0046] Table 1 consists of the Expression Reports for each promoter
of the invention providing the nucleotide sequence for each
promoter and details for expression driven by each of the nucleic
acid promoter sequences as observed in transgenic plants. The
results are presented as summaries of the spatial expression, which
provides information as to gross and/or specific expression in
various plant organs and tissues. The observed expression pattern
is also presented, which gives details of expression during
different generations or different developmental stages within a
generation. Additional information is provided regarding the
associated gene, the GenBank reference, the source organism of the
promoter, and the vector and marker genes used for the construct.
The following symbols are used consistently throughout the Table:
[0047] T1: First generation transformant [0048] T2: Second
generation transformant [0049] T3: Third generation transformant
[0050] (L): low expression level [0051] (M): medium expression
level [0052] (H): high expression level
[0053] Each row of the table begins with heading of the data to be
found in the section. The following provides a description of the
data to be found in each section:
TABLE-US-00001 TABLE 1 Heading Description Promoter Identifies the
particular promoter by its construct ID. Modulates the gene: This
row states the name of the gene modulated by the promoter The
GenBank description of the gene: This field gives the Locus Number
of the gene as well as the accession number. The predicted promoter
sequence: Identifies the nucleic acid promoter sequence in
question. The promoter was cloned from the organism: Identifies the
source of the DNA template used to clone the promoter. The
experimental promoter sequence: Identifies the nucleic acid
sequence in planta driving expression of the reporter gene. The
promoter was cloned in the vector: Identifies the vector used into
which a promoter was cloned. When cloned into the vector the
promoter was Identifies the type of marker linked to the promoter.
operably linked to a marker, which was the type: The marker is used
to determine patterns of gene expression in plant tissue.
Promoter-marker vector was tested in: Identifies the organism in
which the promoter- marker vector was tested. Generation screened:
.quadrature.T1 Mature .quadrature.T2 Identifies the plant
generation(s) used in the Seedling .quadrature.T2 Mature
.quadrature.T3 Seedling screening process. T1 plants are those
plants subjected to the transformation event while the T2
generation plants are from the seeds collected from the T1 plants
and T3 plants are from the seeds of T2 plants. The spatial
expression of the promoter-marker Identifies the specific parts of
the plant where vector was found observed in and would be useful
various levels of GFP expression are observed. in expression in any
or all of the following: Expression levels are noted as either low
(L), medium (M), or high (H) . Observed expression pattern of the
promoter- Identifies a general explanation of where GFP marker
vector was in: expression in different generations of plants was T1
mature: observed. T2 seedling: The promoter can be of use in the
following trait Identifies which traits and subtraits the promoter
and sub-trait areas: (search for the trait and sub- cDNA can
modulate trait table) The promoter has utility in: Identifies a
specific function or functions that can be modulated using the
promoter cDNA. Misc. promoter information: "Bidirectionality" is
determined by the number of Bidirectionality: base pairs between
the promoter and the start codon Exons: of a neighboring gene. A
promoter is considered Repeats: bidirectional if it is closer than
200 bp to a start codon of a gene 5` or 3` to the promoter. "Exons"
(or any coding sequence) identifies if the promoter has overlapped
with either the modulating gene's or other neighboring gene's
coding sequence. A "fail" for exons means that this overlap has
occurred. "Repeats" identifies the presence of normally occurring
sequence repeats that randomly exist throughout the genome. A
"pass" for repeats indicates a lack of repeats in the promoter. An
overlap in an exon with the endogenous coding Identifies the
specific nucleotides overlapping the sequence to the promoter
occurs at base pairs: UTR region or exon of a neighboring gene. The
orientation relative to the promoter is designated with a 5` or 3`.
The Ceres cDNA ID of the endogenous coding Identifies the number
associated with the Ceres sequence to the promoter: cDNA that
corresponds to the endogenous cDNA sequence of the promoter. cDNA
nucleotide sequence: The nucleic acid sequence of the Ceres cDNA
matching the endogenous cDNA region of the promoter. Coding
sequence: A translated protein sequence of the gene modulated by a
protein encoded by a cDNA Microarray Data shows that the coding
sequence Microarray data is identified along with the was expressed
in the following experiments, which corresponding experiments along
with the shows that the promoter would useful to modulate
corresponding gene expression. Gene expression is expression in
situations similar to the following: identified by a "+" or a "-"
in the "SIGN(LOG_RATIO)" column. A "+" notation indicates the cDNA
is upregulated while a "-" indicates that the cDNA is
downregulated. The "SHORT_NAME" field describes the experimental
conditions. The parameters for the microarray experiments
Parameters for microarray experiments include age, listed above by
EXPT_REP_ID and Short_Name organism, specific tissues, age,
treatments and other are as follow below: distinguishing
characteristics or features.
Table 2
[0054] Table 1 provides the results of differential expression
experiments indicating if the expression levels were increased
("+") or decreased ("-"). Such increase or decrease expression
levels indicates the utility of the corresponding promoter. The
following Table 2 correlates the various differential expression
experiments with the utility for the promoter that would be
understood from an increased or decreased expression. Table 2
includes three columns, the first column ("EXPTREP_ID") lists the
microarray experiments by their experimental prep ID number and
correspond to the same number listings in Table 1 in the
"Microarray data" section. The second column lists the Short_Name
of the experiment that corresponds to the EXPT_REP_ID. When a cDNA
is differentially expressed in an experiment, identified by its
EXPT_REP_ID, the cDNA and its endogenous promoter can be used to
modulate the traits and subtraits listed in the third column.
[0055] FIG. 1 is a schematic representation of the vector
pNewBin4-HAP1-GFP. The definitions of the abbreviations used in the
vector map are as follows: [0056] Ori--the origin of replication
used by an E. coli host [0057] RB--sequence for the right border of
the T-DNA from pMOG800 [0058] BstXI--restriction enzyme cleavage
site used for cloning [0059] HAP1VP16--coding sequence for a fusion
protein of the HAP1 and VP16 activation domains [0060]
NOS--terminator region from the nopaline synthase gene [0061]
HAP1UAS--the upstream activating sequence for HAP1 [0062]
5ERGFP--the green fluorescent protein gene that has been optimized
for localization to the endoplasmic reticulum [0063] OCS2--the
terminator sequence from the octopine synthase 2 gene [0064]
OCS--the terminator sequence from the octopine synthase gene [0065]
p28716 (a.k.a 28716 short)--promoter used to drive expression of
the PAT (BAR) gene [0066] PAT (BAR)--a marker gene conferring
herbicide resistance [0067] LB--sequence for the left border of the
T-DNA from pMOG800 [0068] Spec--a marker gene conferring
spectinomycin resistance [0069] TrfA--transcription repression
factor gene [0070] RK2-OriV--origin of replication for
Agrobacterium
[0071] FIG. 2 is a schematic representation of the vector PT0678.
The definitions of the abbreviations used in the vector map are as
described above.
[0072] FIG. 3 is a schematic of a gene.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0073] Chimeric: The term "chimeric" is used to describe
polynucleotides or genes, as defined supra, or constructs wherein
at least two of the elements of the polynucleotide or gene or
construct, such as the promoter and the polynucleotide to be
transcribed and/or other regulatory sequences and/or filler
sequences and/or complements thereof, are heterologous to each
other.
[0074] Constitutive Promoter: Promoters referred to herein as
"constitutive promoters" actively promote transcription under most,
but not necessarily all, environmental conditions and states of
development or cell differentiation. Examples of constitutive
promoters include the cauliflower mosaic virus (CaMV) .sup.35S
transcript initiation region and the 1' or 2' promoter derived from
T-DNA of Agrobacterium tumefaciens, and other transcription
initiation regions from various plant genes, such as the maize
ubiquitin-1 promoter, known to those of skill.
[0075] Core Promoter: This is the minimal stretch of contiguous DNA
sequence that is sufficient to direct accurate initiation of
transcription by the RNA polymerase II machinery (for review see:
Struhl, 1987, Cell 49: 295-297; Smale, 1994, In Transcription:
Mechanisms and Regulation (eds R. C. Conaway and J. W. Conaway), pp
63-81/Raven Press, Ltd., New York; Smale, 1997, Biochim Biophys.
Acta 1351: 73-88; Smale et al., 1998, Cold Spring Harb. Symp.
Quant. Biol. 58: 21-31; Smale, 2001, Genes & Dev. 15:
2503-2508; Weis and Reinberg, 1992, FASEB J. 6: 3300-3309; Burke et
al., 1998, Cold Spring Harb. Symp. Quant. Biol 63: 75-82). There
are several sequence motifs, including the TATA box, initiator
(Inr), TFIIB recognition element (BRE) and downstream core promoter
element (DPE), that are commonly found in core promoters, however
not all of these elements occur in all promoters and there are no
universal core promoter elements (Butler and Kadonaga, 2002, Genes
& Dev. 16: 2583-2592).
[0076] Domain: Domains are fingerprints or signatures that can be
used to characterize protein families and/or parts of proteins.
Such fingerprints or signatures can comprise conserved (1) primary
sequence, (2) secondary structure, and/or (3) three-dimensional
conformation. A similar analysis can be applied to polynucleotides.
Generally, each domain has been associated with either a conserved
primary sequence or a sequence motif. Generally these conserved
primary sequence motifs have been correlated with specific in vitro
and/or in vivo activities. A domain can be any length, including
the entirety of the polynucleotide to be transcribed. Examples of
domains include, without limitation, AP2, helicase, homeobox, zinc
finger, etc.
[0077] Endogenous: The term "endogenous," within the context of the
current invention refers to any polynucleotide, polypeptide or
protein sequence which is a natural part of a cell or organisms
regenerated from said cell. In the context of promoter, the term
"endogenous coding region" or "endogenous cDNA" refers to the
coding region that is naturally operably linked to the
promoter.
[0078] Enhancer/Suppressor: An "enhancer" is a DNA regulatory
element that can increase the steady state level of a transcript,
usually by increasing the rate of transcription initiation.
Enhancers usually exert their effect regardless of the distance,
upstream or downstream location, or orientation of the enhancer
relative to the start site of transcription. In contrast, a
"suppressor" is a corresponding DNA regulatory element that
decreases the steady state level of a transcript, again usually by
affecting the rate of transcription initiation. The essential
activity of enhancer and suppressor elements is to bind a protein
factor(s). Such binding can be assayed, for example, by methods
described below. The binding is typically in a manner that
influences the steady state level of a transcript in a cell or in
an in vitro transcription extract.
[0079] Exogenous: As referred to within, "exogenous" is any
polynucleotide, polypeptide or protein sequence, whether chimeric
or not, that is introduced into the genome of a host cell or
organism regenerated from said host cell by any means other than by
a sexual cross. Examples of means by which this can be accomplished
are described below, and include Agrobacterium-mediated
transformation (of dicots--e.g. Salomon et al. EMBO J. 3:141
(1984); Herrera-Estrella et al. EMBO J. 2:987 (1983); of monocots,
representative papers are those by Escudero et al., Plant J. 10:355
(1996), Ishida et al., Nature Biotechnology 14:745 (1996), May et
al., Bio/Technology 13:486 (1995)), biolistic methods (Armaleo et
al., Current Genetics 17:97 1990)), electroporation, in planta
techniques, and the like. Such a plant containing the exogenous
nucleic acid is referred to here as a T.sub.o for the primary
transgenic plant and T.sub.1 for the first generation. The term
"exogenous" as used herein is also intended to encompass inserting
a naturally found element into a non-naturally found location.
[0080] Gene: The term "gene," as used in the context of the current
invention, encompasses all regulatory and coding sequence
contiguously associated with a single hereditary unit with a
genetic function. Genes can include non-coding sequences that
modulate the genetic function that include, but are not limited to,
those that specify polyadenylation, transcriptional regulation, DNA
conformation, chromatin conformation, extent and position of base
methylation and binding sites of proteins that control all of
these. Genes encoding proteins are comprised of "exons" (coding
sequences), which may be interrupted by "introns" (non-coding
sequences). In some instances complexes of a plurality of protein
or nucleic acids or other molecules, or of any two of the above,
may be required for a gene's function. On the other hand a gene's
genetic function may require only RNA expression or protein
production, or may only require binding of proteins and/or nucleic
acids without associated expression. In certain cases, genes
adjacent to one another may share sequence in such a way that one
gene will overlap the other. A gene can be found within the genome
of an organism, in an artificial chromosome, in a plasmid, in any
other sort of vector, or as a separate isolated entity.
[0081] Heterologous sequences: "Heterologous sequences" are those
that are not operatively linked or are not contiguous to each other
in nature. For example, a promoter from corn is considered
heterologous to an Arabidopsis coding region sequence. Also, a
promoter from a gene encoding a growth factor from corn is
considered heterologous to a sequence encoding the corn receptor
for the growth factor. Regulatory element sequences, such as UTRS
or 3' end termination sequences that do not originate in nature
from the same gene as the coding sequence originates from, are
considered heterologous to said coding sequence. Elements
operatively linked in nature and contiguous to each other are not
heterologous to each other.
[0082] Homologous: In the current invention, a "homologous" gene or
polynucleotide or polypeptide refers to a gene or polynucleotide or
polypeptide that shares sequence similarity with the gene or
polynucleotide or polypeptide of interest. This similarity may be
in only a fragment of the sequence and often represents a
functional domain such as, examples including without limitation a
DNA binding domain or a domain with tyrosine kinase activity. The
functional activities of homologous polynucleotide are not
necessarily the same.
[0083] Inducible Promoter: An "inducible promoter" in the context
of the current invention refers to a promoter, the activity of
which is influenced by certain conditions, such as light,
temperature, chemical concentration, protein concentration,
conditions in an organism, cell, or organelle, etc. A typical
example of an inducible promoter, which can be utilized with the
polynucleotides of the present invention, is PARSK1, the promoter
from an Arabidopsis gene encoding a serine-threonine kinase enzyme,
and which promoter is induced by dehydration, abscissic acid and
sodium chloride (Wang and Goodman, Plant J. 8:37 (1995)). Examples
of environmental conditions that may affect transcription by
inducible promoters include anaerobic conditions, elevated
temperature, the presence or absence of a nutrient or other
chemical compound or the presence of light.
[0084] Modulate Transcription Level: As used herein, the phrase
"modulate transcription" describes the biological activity of a
promoter sequence or promoter control element. Such modulation
includes, without limitation, includes up- and down-regulation of
initiation of transcription, rate of transcription, and/or
transcription levels.
[0085] Mutant: In the current invention, "mutant" refers to a
heritable change in nucleotide sequence at a specific location.
Mutant genes of the current invention may or may not have an
associated identifiable phenotype.
[0086] Operable Linkage: An "operable linkage" is a linkage in
which a promoter sequence or promoter control element is connected
to a polynucleotide sequence (or sequences) in such a way as to
place transcription of the polynucleotide sequence under the
influence or control of the promoter or promoter control element.
Two DNA sequences (such as a polynucleotide to be transcribed and a
promoter sequence linked to the 5' end of the polynucleotide to be
transcribed) are said to be operably linked if induction of
promoter function results in the transcription of mRNA encoding the
polynucleotide and if the nature of the linkage between the two DNA
sequences does not (1) result in the introduction of a frame-shift
mutation, (2) interfere with the ability of the promoter sequence
to direct the expression of the protein, antisense RNA or ribozyme,
or (3) interfere with the ability of the DNA template to be
transcribed. Thus, a promoter sequence would be operably linked to
a polynucleotide sequence if the promoter was capable of effecting
transcription of that polynucleotide sequence.
[0087] Optional Promoter Fragments: The phrase "optional promoter
fragments" is used to refer to any sub-sequence of the promoter
that is not required for driving transcription of an operationally
linked coding region. These fragments comprise the 5' UTR and any
exon(s) of the endogenous coding region. The optional promoter
fragments may also comprise any exon(s) and the 3' or 5' UTR of the
gene residing upstream of the promoter (that is, 5' to the
promoter). Optional promoter fragments also include any intervening
sequences that are introns or sequence that occurs between exons or
an exon and the UTR.
[0088] Orthologous: "Orthologous" is a term used herein to describe
a relationship between two or more polynucleotides or proteins. Two
polynucleotides or proteins are "orthologous" to one another if
they serve a similar function in different organisms. In general,
orthologous polynucleotides or proteins will have similar catalytic
functions (when they encode enzymes) or will serve similar
structural functions (when they encode proteins or RNA that form
part of the ultrastructure of a cell).
[0089] Percentage of sequence identity: "Percentage of sequence
identity," as used herein, is determined by comparing two optimally
aligned sequences over a comparison window, where the fragment of
the polynucleotide or amino acid sequence in the comparison window
may comprise additions or deletions (e.g., gaps or overhangs) as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid base or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity. Optimal alignment
of sequences for comparison may be conducted by the local homology
algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by
the homology alignment algorithm of Needleman and Wunsch J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group (GCG), 575 Science Dr., Madison,
Wis.), or by inspection. Given that two sequences have been
identified for comparison, GAP and BESTFIT are preferably employed
to determine their optimal alignment. Typically, the default values
of 5.00 for gap weight and 0.30 for gap weight length are used.
[0090] Plant Promoter: A "plant promoter" is a promoter capable of
initiating transcription in plant cells and can modulate
transcription of a polynucleotide. Such promoters need not be of
plant origin. For example, promoters derived from plant viruses,
such as the CaMV35S promoter or from Agrobacterium tumefaciens such
as the T-DNA promoters, can be plant promoters. A typical example
of a plant promoter of plant origin is the maize ubiquitin-1
(ubi-1) promoter known to those of skill.
[0091] Plant Tissue: The term "plant tissue" includes
differentiated and undifferentiated tissues or plants, including
but not limited to roots, stems, shoots, cotyledons, epicotyl,
hypocotyl, leaves, pollen, seeds, tumor tissue and various forms of
cells in culture such as single cells, protoplast, embryos, and
callus tissue. The plant tissue may be in plants or in organ,
tissue or cell culture.
[0092] Preferential Transcription: "Preferential transcription" is
defined as transcription that occurs in a particular pattern of
cell types or developmental times or in response to specific
stimuli or combination thereof. Non-limitive examples of
preferential transcription include: high transcript levels of a
desired sequence in root tissues; detectable transcript levels of a
desired sequence in certain cell types during embryogenesis; and
low transcript levels of a desired sequence under drought
conditions. Such preferential transcription can be determined by
measuring initiation, rate, and/or levels of transcription.
[0093] Promoter: A "promoter" is a DNA sequence that directs the
transcription of a polynucleotide. Typically a promoter is located
in the 5' region of a polynucleotide to be transcribed, proximal to
the transcriptional start site of such polynucleotide. More
typically, promoters are defined as the region upstream of the
first exon; more typically, as a region upstream of the first of
multiple transcription start sites; more typically, as the region
downstream of the preceding gene and upstream of the first of
multiple transcription start sites; more typically, the region
downstream of the polyA signal and upstream of the first of
multiple transcription start sites; even more typically, about
3,000 nucleotides upstream of the ATG of the first exon; even more
typically, 2,000 nucleotides upstream of the first of multiple
transcription start sites. The promoters of the invention comprise
at least a core promoter as defined above. Frequently promoters are
capable of directing transcription of genes located on each of the
complementary DNA strands that are 3' to the promoter. Stated
differently, many promoters exhibit bidirectionality and can direct
transcription of a downstream gene when present in either
orientation (i.e. 5' to 3' or 3' to 5' relative to the coding
region of the gene). Additionally, the promoter may also include at
least one control element such as an upstream element. Such
elements include UARs and optionally, other DNA sequences that
affect transcription of a polynucleotide such as a synthetic
upstream element.
[0094] Promoter Control Element: The term "promoter control
element" as used herein describes elements that influence the
activity of the promoter. Promoter control elements include
transcriptional regulatory sequence determinants such as, but not
limited to, enhancers, scaffold/matrix attachment regions, TATA
boxes, transcription start locus control regions, UARs, URRs, other
transcription factor binding sites and inverted repeats.
[0095] Public sequence: The term "public sequence," as used in the
context of the instant application, refers to any sequence that has
been deposited in a publicly accessible database prior to the
filing date of the present application. This term encompasses both
amino acid to and nucleotide sequences. Such sequences are publicly
accessible, for example, on the BLAST databases on the NCBI FTP web
site (accessible at ncbi.nlm.nih.gov/ftp). The database at the NCBI
FTP site utilizes "gi" numbers assigned by NCBI as a unique
identifier for each sequence in the databases, thereby providing a
non-redundant database for sequence from various databases,
including GenBank, EMBL, DBBJ, (DNA Database of Japan) and PDB
(Brookhaven Protein Data Bank).
[0096] Regulatory Sequence: The term "regulatory sequence," as used
in the current invention, refers to any nucleotide sequence that
influences transcription or translation initiation and rate, or
stability and/or mobility of a transcript or polypeptide product.
Regulatory sequences include, but are not limited to, promoters,
promoter control elements, protein binding sequences, 5' and 3'
UTRs, transcriptional start sites, termination sequences,
polyadenylation sequences, introns, certain sequences within amino
acid coding sequences such as secretory signals, protease cleavage
sites, etc.
[0097] Related Sequences: "Related sequences" refer to either a
polypeptide or a nucleotide sequence that exhibits some degree of
sequence similarity with a reference sequence.
[0098] Specific Promoters: In the context of the current invention,
"specific promoters" refers to a subset of promoters that have a
high preference for modulating transcript levels in a specific
tissue or organ or cell and/or at a specific time during
development of an organism. By "high preference" is meant at least
3-fold, preferably 5-fold, more preferably at least 10-fold still
more preferably at least 20-fold, 50-fold or 100-fold increase in
transcript levels under the specific condition over the
transcription under any other reference condition considered.
Typical examples of temporal and/or tissue or organ specific
promoters of plant origin that can be used with the polynucleotides
of the present invention, are: PTA29, a promoter which is capable
of driving gene transcription specifically in tapetum and only
during anther development (Koltonow et al., Plant Cell 2:1201
(1990); RCc2 and RCc3, promoters that direct root-specific gene
transcription in rice (Xu et al., Plant Mol. Biol. 27:237 (1995);
TobRB27, a root-specific promoter from tobacco (Yamamoto et al.,
Plant Cell 3:371 (1991)). Examples of tissue-specific promoters
under developmental control include promoters that initiate
transcription only in certain tissues or organs, such as root,
ovule, fruit, seeds, or flowers. Other specific promoters include
those from genes encoding seed storage proteins or the lipid body
membrane protein, oleosin. A few root-specific promoters are noted
above. See also "Preferential transcription".
[0099] Stringency: "Stringency" as used herein is a function of
probe length, probe composition (G+C content), and salt
concentration, organic solvent concentration, and temperature of
hybridization or wash conditions. Stringency is typically compared
by the parameter T.sub.m, which is the temperature at which 50% of
the complementary molecules in the hybridization are hybridized, in
terms of a temperature differential from T.sub.m. High stringency
conditions are those providing a condition of T.sub.m-5.degree. C.
to T.sub.m-10.degree. C. Medium or moderate stringency conditions
are those providing T.sub.m-20.degree. C. to T.sub.m-29.degree. C.
Low stringency conditions are those providing a condition of
T.sub.m-40.degree. C. to T.sub.m-48.degree. C. The relationship of
hybridization conditions to T.sub.m (in .degree. C.) is expressed
in the mathematical equation
T.sub.m=81.5-16.6(log.sub.10[Na.sup.+])+0.41(% G+C)-(600/N) (1)
where N is the length of the probe. This equation works well for
probes 14 to 70 nucleotides in length that are identical to the
target sequence. The equation below for T.sub.m of DNA-DNA hybrids
is useful for probes in the range of 50 to greater than 500
nucleotides, and for conditions that include an organic solvent
(formamide).
T.sub.m=81.5+16.6 log {[Na.sup.+]/(1+0.7[Na.sup.+])}+0.41(%
G+C)-500/L0.63(% formamide) (2)
where L is the length of the probe in the hybrid. (P. Tijessen,
"Hybridization with Nucleic Acid Probes" in Laboratory Techniques
in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed.,
c. 1993 by Elsevier, Amsterdam.) The T.sub.m of equation (2) is
affected by the nature of the hybrid; for DNA-RNA hybrids T.sub.m
is 10-15.degree. C. higher than calculated, for RNA-RNA hybrids
T.sub.m is 20-25.degree. C. higher. Because the T.sub.m decreases
about 1.degree. C. for each 1% decrease in homology when a long
probe is used (Bonner et al., J. Mol. Biol. 81:123 (1973)),
stringency conditions can be adjusted to favor detection of
identical genes or related family members.
[0100] Equation (2) is derived assuming equilibrium and therefore,
hybridizations according to the present invention are most
preferably performed under conditions of probe excess and for
sufficient time to achieve equilibrium. The time required to reach
equilibrium can be shortened by inclusion of a hybridization
accelerator such as dextran sulfate or another high volume polymer
in the hybridization buffer.
[0101] Stringency can be controlled during the hybridization
reaction or after hybridization has occurred by altering the salt
and temperature conditions of the wash solutions used. The formulas
shown above are equally valid when used to compute the stringency
of a wash solution. Preferred wash solution stringencies lie within
the ranges stated above; high stringency is 5-8.degree. C. below
T.sub.m, medium or moderate stringency is 26-29.degree. C. below
T.sub.m and low stringency is 45-48.degree. C. below T.sub.m.
[0102] Substantially free of: A composition containing A is
"substantially free of" B when at least 85% by weight of the total
A+B in the composition is A. Preferably, A comprises at least about
90% by weight of the total of A+B in the composition, more
preferably at least about 95% or even 99% by weight. For example, a
plant gene can be substantially free of other plant genes. Other
examples include, but are not limited to, ligands substantially
free of receptors (and vice versa), a growth factor substantially
free of other growth factors and a transcription binding factor
substantially free of nucleic acids.
[0103] Suppressor: See "Enhancer/Suppressor"
[0104] TATA to start: "TATA to start" shall mean the distance, in
number of nucleotides, between the primary TATA motif and the start
of transcription.
[0105] Transgenic plant: A "transgenic plant" is a plant having one
or more plant cells that contain at least one exogenous
polynucleotide introduced by recombinant nucleic acid methods.
[0106] Translational start site: In the context of the present
invention, a "translational start site" is usually an ATG or AUG in
a transcript, often the first ATG or AUG. A single protein encoding
transcript, however, may have multiple translational start
sites.
[0107] Transcription start site: "Transcription start site" is used
in the current invention to describe the point at which
transcription is initiated. This point is typically located about
25 nucleotides downstream from a TFIID binding site, such as a TATA
box. Transcription can initiate at one or more sites within the
gene, and a single polynucleotide to be transcribed may have
multiple transcriptional start sites, some of which may be specific
for transcription in a particular cell-type or tissue or organ.
"+1" is stated relative to the transcription start site and
indicates the first nucleotide in a transcript.
[0108] Upstream Activating Region (UAR): An "Upstream Activating
Region" or "UAR" is a position or orientation dependent nucleic
acid element that primarily directs tissue, organ, cell type, or
environmental regulation of transcript level, usually by affecting
the rate of transcription initiation. Corresponding DNA elements
that have a transcription inhibitory effect are called herein
"Upstream Repressor Regions" or "URR"s. The essential activity of
these elements is to bind a protein factor. Such binding can be
assayed by methods described below. The binding is typically in a
manner that influences the steady state level of a transcript in a
cell or in vitro transcription extract.
[0109] Untranslated region (UTR): A "UTR" is any contiguous series
of nucleotide bases that is transcribed, but is not translated. A
5' UTR lies between the start site of the transcript and the
translation initiation codon and includes the +1 nucleotide. A 3'
UTR lies between the translation termination codon and the end of
the transcript. UTRs can have particular functions such as
increasing mRNA message stability or translation attenuation.
Examples of 3' UTRs include, but are not limited to polyadenylation
signals and transcription termination sequences.
[0110] Variant: The term "variant" is used herein to denote a
polypeptide or protein or polynucleotide molecule that differs from
others of its kind in some way. For example, polypeptide and
protein variants can consist of changes in amino acid sequence
and/or charge and/or post-translational modifications (such as
glycosylation, etc). Likewise, polynucleotide variants can consist
of changes that add or delete a specific UTR or exon sequence. It
will be understood that there may be sequence variations within
sequence or fragments used or disclosed in this application.
Preferably, variants will be such that the sequences have at least
80%, preferably at least 90%, 95, 97, 98, or 99% sequence identity.
Variants preferably measure the primary biological function of the
native polypeptide or protein or polynucleotide.
2. Introduction
[0111] The polynucleotides of the invention comprise promoters and
promoter control elements that are capable of modulating
transcription.
[0112] Such promoters and promoter control elements can be used in
combination with native or heterologous promoter fragments, control
elements or other regulatory sequences to modulate transcription
and/or translation.
[0113] Specifically, promoters and control elements of the
invention can be used to modulate transcription of a desired
polynucleotide, which includes without limitation: [0114] (a)
antisense; [0115] (b) ribozymes; [0116] (c) coding sequences; or
[0117] (d) fragments thereof. The promoter also can modulate
transcription in a host genome in cis- or in trans-.
[0118] In an organism, such as a plant, the promoters and promoter
control elements of the instant invention are useful to produce
preferential transcription which results in a desired pattern of
transcript levels in a particular cells, tissues, or organs, or
under particular conditions.
3. Table of Contents
[0119] The following description of the present invention is
outlined in the following table of contents.
[0120] A. Identifying and Isolating Promoter Sequences of the
Invention [0121] (1) Cloning Methods [0122] (2) Chemical
Synthesis
[0123] B. Generating a "core" promoter sequence
[0124] C. Isolating Related Promoter Sequences [0125] (1) Relatives
Based on Nucleotide Sequence Identity [0126] (2) Relatives Based on
Coding Sequence Identity [0127] (3) Relatives based on Common
Function
[0128] D. Identifying Control Elements [0129] (1) Types of
Transcription Control Elements [0130] (2) Those Described by the
Examples [0131] (3) Those Identifiable by Bioinformatics [0132] (4)
Those Identifiable by In Vitro and In Vivo Assays [0133] (5)
Non-Natural Control Elements
[0134] E. Constructing Promoters and Control Elements [0135] (1)
Combining Promoters and Promoter Control Elements [0136] (2) Number
of Promoter Control Elements [0137] (3) Spacing Between Control
Elements
[0138] F. Vectors [0139] (1) Modification of Transcription by
Promoters and Promoter Control Elements [0140] (2) Polynucleotide
to be Transcribed [0141] (3) Other Regulatory Elements [0142] (4)
Other Components of Vectors
[0143] G. Insertion of Polynucleotides and Vectors Into a Host Cell
[0144] (1) Autonomous of the Host Genome [0145] (2) Integrated into
the Host Genome
[0146] H. Utility
A. Identifying and Isolating Promoter Sequences of the
Invention
[0147] The promoters and promoter control elements of the present
invention are presented in Table 1 in the section entitled "The
predicted promoter" sequence and were identified from Arabidopsis
thaliana or Oryza sativa. Additional promoter sequences encompassed
by the invention can be identified as described below.
[0148] (1) Cloning Methods
[0149] Isolation from genomic libraries of polynucleotides
comprising the sequences of the promoters and promoter control
elements of the present invention is possible using known
techniques.
[0150] For example, polymerase chain reaction (PCR) can amplify the
desired polynucleotides utilizing primers designed from sequences
in the row titled "The spatial expression of the
promoter-marker-vector". Polynucleotide libraries comprising
genomic sequences can be constructed according to Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed. (1989) Cold
Spring Harbor Press, Cold Spring Harbor, N.Y.), for example.
[0151] Other procedures for isolating polynucleotides comprising
the promoter sequences of the invention include, without
limitation, tail-PCR, and 5' rapid amplification of cDNA ends
(RACE). See, for tail-PCR, for example, Liu et al., Plant J 8(3):
457-463 (September, 1995); Liu et al., Genomics 25: 674-681 (1995);
Liu et al., Nucl. Acids Res. 21(14): 3333-3334 (1993); and Zoe et
al., BioTechniques 27(2): 240-248 (1999); for RACE, see, for
example, PCR Protocols: A Guide to Methods and Applications, (1990)
Academic Press, Inc.
[0152] (2) Chemical Synthesis
[0153] In addition, the promoters and promoter control elements
described in Table 1 in the section entitled "The predicted
promoter" sequence can be chemically synthesized according to
techniques in common use. See, for example, Beaucage et al., Tet.
Lett. (1981) 22: 1859 and U.S. Pat. No. 4,668,777.
[0154] Such chemical oligonucleotide synthesis can be carried out
using commercially available devices, such as, Biosearch 4600 or
8600 DNA synthesizer, by Applied Biosystems, a division of
Perkin-Elmer Corp., Foster City, Calif., USA; and Expedite by
Perceptive Biosystems, Framingham, Mass., USA.
[0155] Synthetic RNA, including natural and/or analog building
blocks, can be synthesized on the Biosearch 8600 machines, see
above.
[0156] Oligonucleotides can be synthesized and then ligated
together to construct the desired polynucleotide.
B. Generating Reduced and "Core" Promoter Sequences
[0157] Included in the present invention are reduced and "core"
promoter sequences. The reduced promoters can be isolated from the
promoters of the invention by deleting at least one 5' UTR, exon or
3' UTR sequence present in the promoter sequence that is associated
with a gene or coding region located 5' to the promoter sequence or
in the promoter's endogenous coding region.
[0158] Similarly, the "core" promoter sequences can be generated by
deleting all 5' UTRs, exons and 3' UTRs present in the promoter
sequence and the associated intervening sequences that are related
to the gene or coding region 5' to the promoter region and the
promoter's endogenous coding region.
[0159] This data is presented in the row titled "Optional Promoter
Fragments".
C. Isolating Related Promoter Sequences
[0160] Included in the present invention are promoter and promoter
control elements that are related to those described in Table 1 in
the section entitled "The predicted promoter sequence". Such
related sequence can be isolated utilizing
[0161] (a) nucleotide sequence identity;
[0162] (b) coding sequence identity; or
[0163] (c) common function or gene products.
Relatives can include both naturally occurring promoters and
non-natural promoter sequences. Non-natural related promoters
include nucleotide substitutions, insertions or deletions of
naturally-occurring promoter sequences that do not substantially
affect transcription modulation activity. For example, the binding
of relevant DNA binding proteins can still occur with the
non-natural promoter sequences and promoter control elements of the
present invention.
[0164] According to current knowledge, promoter sequences and
promoter control elements exist as functionally important regions,
such as protein binding sites, and spacer regions. These spacer
regions are apparently required for proper positioning of the
protein binding sites. Thus, nucleotide substitutions, insertions
and deletions can be tolerated in these spacer regions to a certain
degree without loss of function.
[0165] In contrast, less variation is permissible in the
functionally important regions, since changes in the sequence can
interfere with protein binding. Nonetheless, some variation in the
functionally important regions is permissible so long as function
is conserved.
[0166] The effects of substitutions, insertions and deletions to
the promoter sequences or promoter control elements may be to
increase or decrease the binding of relevant DNA binding proteins
to modulate transcript levels of a polynucleotide to be
transcribed. Effects may include tissue-specific or
condition-specific modulation of transcript levels of the
polypeptide to be transcribed. Polynucleotides representing changes
to the nucleotide sequence of the DNA-protein contact region by
insertion of additional nucleotides, changes to identity of
relevant nucleotides, including use of chemically-modified bases,
or deletion of one or more nucleotides are considered encompassed
by the present invention.
[0167] (1) Relatives Based on Nucleotide Sequence Identity
[0168] Included in the present invention are promoters exhibiting
nucleotide sequence identity to those described in Table 1 in the
section entitled "The predicted promoter sequence".
[0169] Definition
[0170] Typically, such related promoters exhibit at least 80%
sequence identity, preferably at least 85%, more preferably at
least 90%, and most preferably at least 95%, even more preferably,
at least 96%, 97%, 98% or 99% sequence identity compared to those
shown in Table 1 in the section entitled "The predicted promoter"
sequence. Such sequence identity can be calculated by the
algorithms and computers programs described above.
[0171] Usually, such sequence identity is exhibited in an alignment
region that is at least 75% of the length of a sequence shown in
Table 1 in the section entitled "The predicted promoter" sequence
or corresponding full-length sequence; more usually at least 80%;
more usually, at least 85%, more usually at least 90%, and most
usually at least 95%, even more usually, at least 96%, 97%, 98% or
99% of the length of a sequence shown in Table 1 in the section
entitled "The predicted promoter sequence".
[0172] The percentage of the alignment length is calculated by
counting the number of residues of the sequence in region of
strongest alignment, e.g., a continuous region of the sequence that
contains the greatest number of residues that are identical to the
residues between two sequences that are being aligned. The number
of residues in the region of strongest alignment is divided by the
total residue length of a sequence in Table 1 in the section
entitled "The predicted promoter sequence".
[0173] These related promoters may exhibit similar preferential
transcription as those promoters described in Table 1 in the
section entitled "The predicted promoter sequence".
[0174] Construction of Polynucleotides
[0175] Naturally occurring promoters that exhibit nucleotide
sequence identity to those shown in Table 1 in the section entitled
"The predicted promoter sequence" can be isolated using the
techniques as described above. More specifically, such related
promoters can be identified by varying stringencies, as defined
above, in typical hybridization procedures such as Southern blots
or probing of polynucleotide libraries, for example.
[0176] Non-natural promoter variants of those shown in Table 1 can
be constructed using cloning methods that incorporate the desired
nucleotide variation. See, for example, Ho, S. N., et al. Gene
77:51-59 1989, describing a procedure site directed mutagenesis
using PCR.
[0177] Any related promoter showing sequence identity to those
shown in Table can be chemically synthesized as described
above.
[0178] Also, the present invention includes non-natural promoters
that exhibit the above-sequence identity to those in Table 1.
[0179] The promoters and promoter control elements of the present
invention may also be synthesized with 5' or 3' extensions, to
facilitate additional manipulation, for instance.
[0180] The present invention also includes reduced promoter
sequences. These sequences have at least one of the optional
promoter fragments deleted.
[0181] Core promoter sequences are another embodiment of the
present invention. The core promoter sequences have all of the
optional promoter fragments deleted.
[0182] Testing of Polynucleotides
[0183] Polynucleotides of the invention were tested for activity by
cloning the sequence into an appropriate vector, transforming
plants with the construct and assaying for marker gene expression.
Recombinant DNA constructs were prepared which comprise the
polynucleotide sequences of the invention inserted into a vector
suitable for transformation of plant cells. The construct can be
made using standard recombinant DNA techniques (Sambrook et al.
1989) and can be introduced to the species of interest by
Agrobacterium-mediated transformation or by other means of
transformation as referenced below.
[0184] The vector backbone can be any of those typical in the art
such as plasmids, viruses, artificial chromosomes, BACs, YACs and
PACs and vectors of the sort described by [0185] (a) BAC: Shizuya
et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797 (1992); Hamilton
et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979 (1996); [0186] (b)
YAC: Burke et al., Science 236:806-812 (1987); [0187] (c) PAC:
Sternberg N. et al., Proc Natl Acad Sci USA. Jan; 87(1):103-7
(1990); [0188] (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al.,
Nucl Acids Res 23: 4850-4856 (1995); [0189] (e) Lambda Phage
Vectors: Replacement Vector, e.g., Frischauf et al., J. Mol. Biol
170: 827-842 (1983); or Insertion vector, e.g., Huynh et al., In:
Glover N M (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford:
IRL Press (1985); T-DNA gene fusion vectors: Walden et al., Mol
Cell Biol 1: 175-194 (1990); and [0190] (g) Plasmid vectors:
Sambrook et al., infra.
[0191] Typically, the construct comprises a vector containing a
sequence of the present invention operationally linked to any
marker gene. The polynucleotide was identified as a promoter by the
expression of the marker gene. Although many marker genes can be
used, Green Fluorescent Protein (GFP) is preferred. The vector may
also comprise a marker gene that confers a selectable phenotype on
plant cells. The marker may encode biocide resistance, particularly
antibiotic resistance, such as resistance to kanamycin, G418,
bleomycin, hygromycin, or herbicide resistance, such as resistance
to chlorosulfuron or phosphinotricin. Vectors can also include
origins of replication, scaffold attachment regions (SARs),
markers, homologous sequences, introns, etc.
[0192] Promoter Control Elements of the Invention
[0193] The promoter control elements of the present invention
include those that comprise a sequence shown in Table 1 in the
section entitled "The predicted promoter sequence" and fragments
thereof. The size of the fragments of the row titled "The predicted
promoter sequence" can range from 5 bases to 10 kilobases (kb).
Typically, the fragment size is no smaller than 8 bases; more
typically, no smaller than 12; more typically, no smaller than 15
bases; more typically, no smaller than 20 bases; more typically, no
smaller than 25 bases; even more typically, no more than 30, 35, 40
or 50 bases.
[0194] Usually, the fragment size in no larger than 5 kb bases;
more usually, no larger than 2 kb; more usually, no larger than 1
kb; more usually, no larger than 800 bases; more usually, no larger
than 500 bases; even more usually, no more than 250, 200, 150 or
100 bases.
[0195] Relatives Based on Nucleotide Sequence Identity
[0196] Included in the present invention are promoter control
elements exhibiting nucleotide sequence identity to those described
in Table 1 in the section entitled "The predicted promoter
sequence" of fragments thereof.
[0197] Typically, such related promoters exhibit at least 80%
sequence identity, preferably at least 85%, more preferably at
least 90%, and most preferably at least 95%, even more preferably,
at least 96%, 97%, 98% or 99% sequence identity compared to those
shown in Table 1 in the section entitled "The predicted promoter
sequence". Such sequence identity can be calculated by the
algorithms and computers programs described above.
[0198] Promoter Control Element Configuration
[0199] A common configuration of the promoter control elements in
RNA polymerase II promoters is shown below:
For more description, see, for example, "Models for prediction and
recognition of eukaryotic promoters", T. Werner, Mammalian Genome,
10, 168-175 (1999).
[0200] Promoters are generally modular in nature. Promoters can
consist of a basal promoter which functions as a site for assembly
of a transcription complex comprising an RNA polymerase, for
example RNA polymerase II. A typical transcription complex will
include additional factors such as TF.sub.IIB, TF.sub.IID, and
TF.sub.IIE. Of these, TF.sub.IID appears to be the only one to bind
DNA directly. The promoter might also contain one or more promoter
control elements such as the elements discussed above. These
additional control elements may function as binding sites for
additional transcription factors that have the function of
modulating the level of transcription with respect to tissue
specificity and of transcriptional responses to particular
environmental or nutritional factors, and the like.
[0201] One type of promoter control element is a polynucleotide
sequence representing a binding site for proteins. Typically,
within a particular functional module, protein binding sites
constitute regions of 5 to 60, preferably 10 to 30, more preferably
10 to 20 nucleotides. Within such binding sites, there are
typically 2 to 6 nucleotides which specifically contact amino acids
of the nucleic acid binding protein.
[0202] The protein binding sites are usually separated from each
other by 10 to several hundred nucleotides, typically by 15 to 150
nucleotides, often by 20 to 50 nucleotides.
[0203] Further, protein binding sites in promoter control elements
often display dyad symmetry in their sequence. Such elements can
bind several different proteins, and/or a plurality of sites can
bind the same protein. Both types of elements may be combined in a
region of 50 to 1,000 base pairs.
[0204] Binding sites for any specific factor have been known to
occur almost anywhere in a promoter. For example, functional AP-1
binding sites can be located far upstream, as in the rat bone
sialoprotein gene, where an AP-1 site located about 900 nucleotides
upstream of the transcription start site suppresses expression.
Yamauchi et al., Matrix Biol., 15, 119-130 (1996). Alternatively,
an AP-1 site located close to the transcription start site plays an
important role in the expression of Moloney murine leukemia virus.
Sap et al., Nature, 340, 242-244, (1989).
[0205] (2) Those Identifiable by Bioinformatics
[0206] Promoter control elements from the promoters of the instant
invention can be identified utilizing bioinformatic or computer
driven techniques.
[0207] One method uses a computer program AlignACE to identify
regulatory motifs in genes that exhibit common preferential
transcription across a number of time points. The programidentifies
common sequence motifs in such genes. See, Roth et al., Nature
Biotechnol. 16: 949-945 (1998); Tavazoie et al., Nat Genet. 1999
July; 22(3):281-5;
[0208] Genomatix, also makes available a GEMS Launcher program and
other programs to identify promoter control elements and
configuration of such elements. Genomatix is located in Munich,
Germany.
[0209] Other references also describe detection of promoter modules
by models independent of overall nucleotide sequence similarity.
See, for instance, Klingenhoff et al., Bioinformatics 15, 180-186
(1999).
[0210] Protein binding sites of promoters can be identified as
reported in "Computer-assisted prediction, classification, and
delimination of protein binding sites in nucleic acids", Frech, et
al., Nucleic Acids Research, Vol. 21, No. 7, 1655-1664, 1993.
[0211] Other programs used to identify protein binding sites
include, for example, Signal Scan, Prestridge et al., Comput. Appl.
Biosci. 12: 157-160 (1996); Matrix Search, Chen et al., Comput.
Appl. Biosci. 11: 563-566 (1995), available as part of Signal Scan
4.0; Matlnspector, Ghosh et al., Nucl. Acid Res. 21: 3117-3118
(1993) available http://ww.gsf.de/cgi-bin/matsearch.pl;
ConsInspector, Frech et al., Nucl. Acids Res. 21: 1655-1664 (1993),
available at ftp://ariane.gsf.de/pub/dos; TFSearch; and TESS.
[0212] Frech et al., "Software for the analysis of DNA sequence
elements of transcription", Bioinformatics & Sequence Analysis,
Vol. 13, no. 1, 89-97 (1997) is a review of different software for
analysis of promoter control elements. This paper also reports the
usefulness of matrix-based approaches to yield more specific
results.
[0213] For other procedures, see, Fickett et al., Curr. Op.
Biotechnol. 11: 19-24 (2000); and Quandt et al., Nucleic Acids
Res., 23, 4878-4884 (1995).
[0214] (3) Those Identifiable by In-Vitro and In-Vivo Assays
[0215] Promoter control elements also can be identified with
in-vitro assays, such as transcription detection methods; and with
in-vivo assays, such as enhancer trapping protocols.
[0216] In-Vitro Assays
[0217] Examples of in-vitro assays include detection of binding of
protein factors that bind promoter control elements. Fragments of
the instant promoters can be used to identify the location of
promoter control elements. Another option for obtaining a promoter
control element with desired properties is to modify known promoter
sequences. This is based on the fact that the function of a
promoter is dependent on the interplay of regulatory proteins that
bind to specific, discrete nucleotide sequences in the promoter,
termed motifs. Such interplay subsequently affects the general
transcription machinery and regulates transcription efficiency.
These proteins are positive regulators or negative regulators
(repressors), and one protein can have a dual role depending on the
context (Johnson, P. F. and McKnight, S. L. Annu. Rev. Biochem.
58:799-839 (1989)).
[0218] One type of in-vitro assay utilizes a known DNA binding
factor to isolate DNA fragments that bind. If a fragment or
promoter variant does not bind, then a promoter control element has
been removed or disrupted. For specific assays, see, for instance,
B. Luo et al., J. Mol. Biol. 266:470 (1997), S. Chusacultanachai et
al., J. Biol. Chem. 274:23591 (1999), D. Fabbro et al., Biochem.
Biophys. Res. Comm. 213:781 (1995)).
[0219] Alternatively, a fragment of DNA suspected of conferring a
particular pattern of specificity can be examined for activity in
binding transcription factors involved in that specificity by
methods such as DNA footprinting (e.g. D. J. Cousins et al.,
Immunology 99:101 (2000); V. Kolla et al., Biochem. Biophys. Res.
Comm. 266:5 (1999)) or "mobility-shift" assays (E. D. Fabiani et
al., J. Biochem. 347:147 (2000); N. Sugiura et al., J. Biochem
347:155 (2000)) or fluorescence polarization (e.g. Royer et al.,
U.S. Pat. No. 5,445,935). Both mobility shift and DNA footprinting
assays can also be used to identify portions of large DNA fragments
that are bound by proteins in unpurified transcription extracts
prepared from tissues or organs of interest.
[0220] Cell-free transcription extracts can be prepared and used to
directly assay in a reconstitutable system (Narayan et al.,
Biochemistry 39:818 (2000)).
[0221] In-Vivo Assays
[0222] Promoter control elements can be identified with reporter
genes in in-vivo assays with the use of fragments of the instant
promoters or variants of the instant promoter polynucleotides.
[0223] For example, various fragments can be inserted into a
vector, comprising a basal or "core" promoter, for example,
operably linked to a reporter sequence, which, when transcribed,
can produce a detectable label. Examples of reporter genes include
those encoding luciferase, green fluorescent protein, GUS, neo, cat
and bar. Alternatively, reporter sequence can be detected utilizing
AFLP and microarray techniques.
[0224] In promoter probe vector systems, genomic DNA fragments are
inserted upstream of the coding sequence of a reporter gene that is
expressed only when the cloned fragment contains DNA having
transcription modulation activity (Neve, R. L. et al., Nature
277:324-325 (1979)). Control elements are disrupted when fragments
or variants lacking any transcription modulation activity. Probe
vectors have been designed for assaying transcription modulation in
E. coli (An, G. et al., J. Bact. 140:400-407 (1979)) and other
bacterial hosts (Band, L. et al., Gene 26:313-315 (1983); Achen, M.
G., Gene 45:45-49 (1986)), yeast (Goodey, A. R. et al., Mol. Gen.
Genet. 204:505-511 (1986)) and mammalian cells (Pater, M. M. et
al., J. Mol. App. Gen. 2:363-371 (1984)).
[0225] A different design of a promoter/control element trap
includes packaging into retroviruses for more efficient delivery
into cells. One type of retroviral enhancer trap was described by
von Melchner et al. (Genes Dev. 1992;U.S. Pat. No. 5,364,783). The
basic design of this vector includes a reporter protein coding
sequence engineered into the U3 portion of the 3' LTR. No splice
acceptor consensus sequences are included, limiting its utility to
work as an enhancer trap only. A different approach to a gene trap
using retroviral vectors was pursued by Friedrich and Soriano
(Genes Dev. 1991), who engineered a lacZ-neo fusion protein linked
to a splicing acceptor. LacZ-neo fusion protein expression from
trapped loci allows not only for drug selection, but also for
visualization of .beta.-galatactosidase expression using the
chromogenic substrate, X-gal.
[0226] A general review of tools for identifying transcriptional
regulatory regions of genomic DNA is provided by J. W. Fickett et
al. (Curr. Opn. Biotechnol. 11:19 (2000).
[0227] (4) Non-Natural Control Elements
[0228] Non-natural control elements can be constructed by
inserting, deleting or substituting nucleotides into the promoter
control elements described above. Such control elements are capable
of transcription modulation that can be determined using any of the
assays described above.
D. Constructing Promoters with Control Elements
[0229] (1) Combining Promoters and Promoter Control Elements
[0230] The promoter polynucleotides and promoter control elements
of the present invention, both naturally occurring and synthetic,
can be combined with each other to produce the desired preferential
transcription. Also, the polynucleotides of the invention can be
combined with other known sequences to obtain other useful
promoters to modulate, for example, tissue transcription specific
or transcription specific to certain conditions. Such preferential
transcription can be determined using the techniques or assays
described above.
[0231] Fragments, variants, as well as full-length sequences those
shown in Table 1 in the section entitled "The predicted promoter
sequence" and relatives are useful alone or in combination.
[0232] The location and relation of promoter control elements
within a promoter can affect the ability of the promoter to
modulate transcription. The order and spacing of control elements
is a factor when constructing promoters.
[0233] (2) Number of Promoter Control Elements
[0234] Promoters can contain any number of control elements. For
example, a promoter can contain multiple transcription binding
sites or other control elements. One element may confer tissue or
organ specificity; another element may limit transcription to
specific time periods, etc. Typically, promoters will contain at
least a basal or core promoter as described above. Any additional
element can be included as desired. For example, a fragment
comprising a basal or "core" promoter can be fused with another
fragment with any number of additional control elements.
[0235] (3) Spacing Between Control Elements
[0236] Spacing between control elements or the configuration or
control elements can be determined or optimized to permit the
desired protein-polynucleotide or polynucleotide interactions to
occur.
[0237] For example, if two transcription factors bind to a promoter
simultaneously or relatively close in time, the binding sites are
spaced to allow each factor to bind without steric hinderance. The
spacing between two such hybridizing control elements can be as
small as a profile of a protein bound to a control element. In some
cases, two protein binding sites can be adjacent to each other when
the proteins bind at different times during the transcription
process.
[0238] Further, when two control elements hybridize the spacing
between such elements will be sufficient to allow the promoter
polynucleotide to hairpin or loop to permit the two elements to
bind. The spacing between two such hybridizing control elements can
be as small as a t-RNA loop, to as large as 10 kb.
[0239] Typically, the spacing is no smaller than 5 bases; more
typically, no smaller than 8;
[0240] more typically, no smaller than 15 bases; more typically, no
smaller than 20 bases; more typically, no smaller than 25 bases;
even more typically, no more than 30, 35, 40 or 50 bases.
[0241] Usually, the fragment size in no larger than 5 kb bases;
more usually, no larger than 2 kb; more usually, no larger than 1
kb; more usually, no larger than 800 bases; more usually, no larger
than 500 bases; even more usually, no more than 250, 200, 150 or
100 bases.
[0242] Such spacing between promoter control elements can be
determined using the techniques and assays described above.
[0243] (4) Other Promoters
[0244] The following are promoters that are induced under stress
conditions and can be combined with those of the present invention:
ldh1 (oxygen stress; tomato; see Germain and Ricard. 1997. Plant
Mol Biol 35:949-54), GPx and CAT (oxygen stress; mouse; see Franco
et al. 1999. Free Radic Biol Med 27:1122-32), ci7 (cold stress;
potato; see Kirch et al. 1997. Plant Mol. Biol. 33:897-909), Bz2
(heavy metals; maize; see Marrs and Walbot. 1997. Plant Physiol
113:93-102), HSP32 (hyperthermia; rat; see Raju and Maines. 1994.
Biochim Biophys Acta 1217:273-80); MAPKAPK-2 (heat shock;
Drosophila; see Larochelle and Suter. 1995. Gene 163:209-14).
[0245] In addition, the following examples of promoters are induced
by the presence or absence of light can be used in combination with
those of the present invention: Topoisomerase II (pea; see Reddy et
al. 1999. Plant Mol Biol 41:125-37), chalcone synthase (soybean;
see Wingender et al. 1989. Mol Gen Genet. 218:315-22) mdm2 gene
(human tumor; see Saucedo et al. 1998. Cell Growth Differ
9:119-30), Clock and BMAL1 (rat; see Namihira et al. 1999. Neurosci
Lett 271:1-4, PHYA (Arabidopsis; see Canton and Quail 1999. Plant
Physiol 121:1207-16), PRB-1b (tobacco; see Sessa et al. 1995. Plant
Mol Biol 28:537-47) and Ypr10 (common bean; see Walter et al. 1996.
Eur J Biochem 239:281-93).
[0246] The promoters and control elements of the following genes
can be used in combination with the present invention to confer
tissue specificity: MipB (iceplant; Yamada et al. 1995. Plant Cell
7:1129-42) and SUCS (root nodules; broadbean; Kuster et al. 1993.
Mol Plant Microbe Interact 6:507-14) for roots, OsSUT1 (rice;
Hirose et al. 1997. Plant Cell Physiol 38:1389-96) for leaves, Msg
(soybean; Stomvik et al. 1999. Plant Mol Biol 41:217-31) for
siliques, cell (Arabidopsis; Shani et al. 1997. Plant Mol Biol
34(6):837-42) and ACT11 (Arabidopsis; Huang et al. 1997. Plant Mol
Biol 33:125-39) for inflorescence.
[0247] Still other promoters are affected by hormones or
participate in specific physiological processes, which can be used
in combination with those of present invention. Some examples are
the ACC synthase gene that is induced differently by ethylene and
brassinosteroids (mung bean; Yi et al. 1999. Plant Mol
Bio141:443-54), the TAPG1 gene that is active during abscission
(tomato; Kalaitzis et al. 1995. Plant Mol Biol 28:647-56), and the
1-aminocyclopropane-1-carboxylate synthase gene (carnation; Jones
et al. 19951 Plant Mol Biol 28:505-12) and the CP-2/cathepsin L
gene (rat; Kim and Wright. 1997. Biol Reprod 57:1467-77), both
active during senescence.
E. Vectors
[0248] Vectors are a useful component of the present invention. In
particular, the present promoters and/or promoter control elements
may be delivered to a system such as a cell by way of a vector. For
the purposes of this invention, such delivery may range from simply
introducing the promoter or promoter control element by itself
randomly into a cell to integration of a cloning vector containing
the present promoter or promoter control element. Thus, a vector
need not be limited to a DNA molecule such as a plasmid, cosmid or
bacterial phage that has the capability of replicating autonomously
in a host cell. All other manner of delivery of the promoters and
promoter control elements of the invention are envisioned. The
various T-DNA vector types are a preferred vector for use with the
present invention. Many useful vectors are commercially
available.
[0249] It may also be useful to attach a marker sequence to the
present promoter and promoter control element in order to determine
activity of such sequences. Marker sequences typically include
genes that provide antibiotic resistance, such as tetracycline
resistance, hygromycin resistance or ampicillin resistance, or
provide herbicide resistance. Specific selectable marker genes may
be used to confer resistance to herbicides such as glyphosate,
glufosinate or broxynil (Comai et al., Nature 317: 741-744 (1985);
Gordon-Kamm et al., Plant Cell 2: 603-618 (1990); and Stalker et
al., Science 242: 419-423 (1988)). Other marker genes exist which
provide hormone responsiveness.
[0250] (1) Modification of Transcription by Promoters and Promoter
Control Elements
[0251] The promoter or promoter control element of the present
invention may be operably linked to a polynucleotide to be
transcribed. In this manner, the promoter or promoter control
element may modify transcription by modulate transcript levels of
that polynucleotide when inserted into a genome.
[0252] However, prior to insertion into a genome, the promoter or
promoter control element need not be linked, operably or otherwise,
to a polynucleotide to be transcribed. For example, the promoter or
promoter control element may be inserted alone into the genome in
front of a polynucleotide already present in the genome. In this
manner, the promoter or promoter control element may modulate the
transcription of a polynucleotide that was already present in the
genome. This polynucleotide may be native to the genome or inserted
at an earlier time.
[0253] Alternatively, the promoter or promoter control element may
be inserted into a genome alone to modulate transcription. See, for
example, Vaucheret, H et al. (1998) Plant J 16: 651-659. Rather,
the promoter or promoter control element may be simply inserted
into a genome or maintained extrachromosomally as a way to divert
transcription resources of the system to itself. This approach may
be used to downregulate the transcript levels of a group of
polynucleotide(s).
[0254] (2) Polynucleotide to be Transcribed
[0255] The nature of the polynucleotide to be transcribed is not
limited. Specifically, the polynucleotide may include sequences
that will have activity as RNA as well as sequences that result in
a polypeptide product. These sequences may include, but are not
limited to antisense sequences, ribozyme sequences, spliceosomes,
amino acid coding sequences, and fragments thereof.
[0256] Specific coding sequences may include, but are not limited
to endogenous proteins or fragments thereof, or heterologous
proteins including marker genes or fragments thereof.
[0257] Promoters and control elements of the present invention are
useful for modulating metabolic or catabolic processes. Such
processes include, but are not limited to, secondary product
metabolism, amino acid synthesis, seed protein storage, oil
development, pest defense and nitrogen usage. Some examples of
genes, transcripts and peptides or polypeptides participating in
these processes, which can be modulated by the present invention:
are tryptophan decarboxylase (tdc) and strictosidine synthase (str
1), dihydrodipicolinate synthase (DHDPS) and aspartate kinase (AK),
2S albumin and alpha-, beta-, and gamma-zeins, ricinoleate and
3-ketoacyl-ACP synthase (KAS), Bacillus thuringiensis (Bt)
insecticidal protein, cowpea trypsin inhibitor (CpTI), asparagine
synthetase and nitrite reductase. Alternatively, expression
constructs can be used to inhibit expression of these peptides and
polypeptides by incorporating the promoters in constructs for
antisense use, co-suppression use or for the production of dominant
negative mutations.
[0258] (3) Other Regulatory Elements
[0259] As explained above, several types of regulatory elements
exist concerning transcription regulation. Each of these regulatory
elements may be combined with the present vector if desired.
[0260] (4) Other Components of Vectors
[0261] Translation of eukaryotic mRNA is often initiated at the
codon that encodes the first methionine. Thus, when constructing a
recombinant polynucleotide according to the present invention for
expressing a protein product, it is preferable to ensure that the
linkage between the 3' portion, preferably including the TATA box,
of the promoter and the polynucleotide to be transcribed, or a
functional derivative thereof, does not contain any intervening
codons which are capable of encoding a methionine.
[0262] The vector of the present invention may contain additional
components. For example, an origin of replication allows for
replication of the vector in a host cell. Additionally, homologous
sequences flanking a specific sequence allows for specific
recombination of the specific sequence at a desired location in the
target genome. T-DNA sequences also allow for insertion of a
specific sequence randomly into a target genome.
[0263] The vector may also be provided with a plurality of
restriction sites for insertion of a polynucleotide to be
transcribed as well as the promoter and/or promoter control
elements of the present invention. The vector may additionally
contain selectable marker genes. The vector may also contain a
transcriptional and translational initiation region, and a
transcriptional and translational termination region functional in
the host cell. The termination region may be native with the
transcriptional initiation region, may be native with the
polynucleotide to be transcribed, or may be derived from another
source. Convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also, Guerineau et al.,
(1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell
64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et
al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene
91:151-158; Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903;
Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0264] Where appropriate, the polynucleotide to be transcribed may
be optimized for increased expression in a certain host cell. For
example, the polynucleotide can be synthesized using preferred
codons for improved transcription and translation. See U.S. Pat.
Nos. 5,380,831, 5,436,391; see also and Murray et al., (1989)
Nucleic Acids Res. 17:477-498.
[0265] Additional sequence modifications include elimination of
sequences encoding spurious polyadenylation signals, exon intron
splice site signals, transposon-like repeats, and other such
sequences well characterized as deleterious to expression. The G-C
content of the polynucleotide may be adjusted to levels average for
a given cellular host, as calculated by reference to known genes
expressed in the host cell. The polynucleotide sequence may be
modified to avoid hairpin secondary mRNA structures.
[0266] A general description of expression vectors and reporter
genes can be found in Gruber, et al., "Vectors for Plant
Transformation, in Methods in Plant Molecular Biology &
Biotechnology" in Glich et al., (Eds. pp. 89-119, CRC Press, 1993).
Moreover GUS expression vectors and GUS gene cassettes are
available from Clonetech Laboratories, Inc., Palo Alto, Calif.
while luciferase expression vectors and luciferase gene cassettes
are available from Promega Corp. (Madison, Wis.). GFP vectors are
available from Aurora Biosciences.
F. Polynucleotide Insertion into a Host Cell
[0267] The polynucleotides according to the present invention can
be inserted into a host cell. A host cell includes but is not
limited to a plant, mammalian, insect, yeast, and prokaryotic cell,
preferably a plant cell.
[0268] The method of insertion into the host cell genome is chosen
based on convenience. For example, the insertion into the host cell
genome may either be accomplished by vectors that integrate into
the host cell genome or by vectors which exist independent of the
host cell genome.
[0269] (1) Polynucleotides Autonomous of the Host Genome
[0270] The polynucleotides of the present invention can exist
autonomously or independent of the host cell genome. Vectors of
these types are known in the art and include, for example, certain
type of non-integrating viral vectors, autonomously replicating
plasmids, artificial chromosomes, and the like.
[0271] Additionally, in some cases transient expression of a
polynucleotide may be desired.
[0272] (2) Polynucleotides Integrated into the Host Genome The
promoter sequences, promoter control elements or vectors of the
present invention may be transformed into host cells. These
transformations may be into protoplasts or intact tissues or
isolated cells. Preferably expression vectors are introduced into
intact tissue. General methods of culturing plant tissues are
provided for example by Maki et al. "Procedures for Introducing
Foreign DNA into Plants" in Methods in Plant Molecular Biology
& Biotechnology, Glich et al. (Eds. pp. 67-88 CRC Press, 1993);
and by Phillips et al. "Cell-Tissue Culture and In-Vitro
Manipulation" in Corn & Corn Improvement, 3rd Edition 10Sprague
et al. (Eds. pp. 345-387) American Society of Agronomy Inc. et al.
1988.
[0273] Methods of introducing polynucleotides into plant tissue
include the direct infection or co-cultivation of plant cell with
Agrobacterium tumefaciens, Horsch et al., Science, 227:1229 (1985).
Descriptions of Agrobacterium vector systems and methods for
Agrobacterium-mediated gene transfer provided by Gruber et al.
supra.
[0274] Alternatively, polynucleotides are introduced into plant
cells or other plant tissues using a direct gene transfer method
such as microprojectile-mediated delivery, DNA injection,
electroporation and the like. More preferably polynucleotides are
introduced into plant tissues using the microprojectile media
delivery with the biolistic device. See, for example, Tomes et al.,
"Direct DNA transfer into intact plant cells via microprojectile
bombardment" In: Gamborg and Phillips (Eds.) Plant Cell, Tissue and
Organ Culture: Fundamental Methods, Springer Verlag, Berlin
(1995).
[0275] In another embodiment of the current invention, expression
constructs can be used for gene expression in callus culture for
the purpose of expressing marker genes encoding peptides or
polypeptides that allow identification of transformed plants. Here,
a promoter that is operatively linked to a polynucleotide to be
transcribed is transformed into plant cells and the transformed
tissue is then placed on callus-inducing media. If the
transformation is conducted with leaf discs, for example, callus
will initiate along the cut edges. Once callus growth has
initiated, callus cells can be transferred to callus shoot-inducing
or callus root-inducing media. Gene expression will occur in the
callus cells developing on the appropriate media: callus
root-inducing promoters will be activated on callus root-inducing
media, etc. Examples of such peptides or polypeptides useful as
transformation markers include, but are not limited to barstar,
glyphosate, chloramphenicol acetyltransferase (CAT), kanamycin,
spectinomycin, streptomycin or other antibiotic resistance enzymes,
green fluorescent protein (GFP), and .beta.-glucuronidase (GUS),
etc. Some of the exemplary promoters of the row titled "The
predicted promoter sequence" will also be capable of sustaining
expression in some tissues or organs after the initiation or
completion of regeneration. Examples of these tissues or organs are
somatic embryos, cotyledon, hypocotyl, epicotyl, leaf, stems,
roots, flowers and seed.
[0276] Integration into the host cell genome also can be
accomplished by methods known in the art, for example, by the
homologous sequences or T-DNA discussed above or using the cre-lox
system (A. C. Vergunst et al., Plant Mol. Biol. 38:393 (1998)).
G. Utility
[0277] Common Uses
[0278] In yet another embodiment, the promoters of the present
invention can be used to further understand developmental
mechanisms. For example, promoters that are specifically induced
during callus formation, somatic embryo formation, shoot formation
or root formation can be used to explore the effects of
overexpression, repression or ectopic expression of target genes,
or for isolation of trans-acting factors.
[0279] The vectors of the invention can be used not only for
expression of coding regions but may also be used in exon-trap
cloning, or promoter trap procedures to detect differential gene
expression in various tissues, K. Lindsey et al., 1993 "Tagging
Genomic Sequences That Direct Transgene Expression by Activation of
a Promoter Trap in Plants", Transgenic Research 2:3347. D. Auch
& Reth, et al., "Exon Trap Cloning: Using PCR to Rapidly Detect
and Clone Exons from Genomic DNA Fragments", Nucleic Acids
Research, Vol. 18, No. 22, p. 674.
[0280] Entrapment vectors, first described for use in bacteria
(Casadaban and Cohen, 1979, Proc. Nat. Aca. Sci. U.S.A., 76: 4530;
Casadaban et al., 1980, J. Bacteriol., 143: 971) permit selection
of insertional events that lie within coding sequences. Entrapment
vectors can be introduced into pluripotent ES cells in culture and
then passed into the germline via chimeras (Gossler et al., 1989,
Science, 244: 463; Skarnes, 1990, Biotechnology, 8: 827). Promoter
or gene trap vectors often contain a reporter gene, e.g., lacZ,
lacking its own promoter and/or splice acceptor sequence upstream.
That is, promoter gene traps contain a reporter gene with a splice
site but no promoter. If the vector lands in a gene and is spliced
into the gene product, then the reporter gene is expressed.
[0281] Recently, the isolation of preferentially-induced genes has
been made possible with the use of sophisticated promoter traps
(e.g. IVET) that are based on conditional auxotrophy
complementation or drug resistance. In one WET approach, various
bacterial genome fragments are placed in front of a necessary
metabolic gene coupled to a reporter gene. The DNA constructs are
inserted into a bacterial strain otherwise lacking the metabolic
gene, and the resulting bacteria are used to infect the host
organism. Only bacteria expressing the metabolic gene survive in
the host organism; consequently, inactive constructs can be
eliminated by harvesting only bacteria that survive for some
minimum period in the host. At the same time, constitutively active
constructs can be eliminated by screening only bacteria that do not
express the reporter gene under laboratory conditions. The bacteria
selected by such a method contain constructs that are selectively
induced only during infection of the host. The IVET approach can be
modified for use in plants to identify genes induced in either the
bacteria or the plant cells upon pathogen infection or root
colonization. For information on IVET see the articles by Mahan et
al. in Science 259:686-688 (1993), Mahan et al. in PNAS USA
92:669-673 (1995), Heithoff et al. in PNAS USA 94:934-939 (1997),
and Wanget al. in PNAS USA. 93:10434 (1996).
[0282] Constitutive Transcription
[0283] Use of promoters and control elements providing constitutive
transcription is desired for modulation of transcription in most
cells of an organism under most environmental conditions. In a
plant, for example, constitutive transcription is useful for
modulating genes involved in defense, pest resistance, herbicide
resistance, etc.
[0284] Constitutive up-regulation and transcription down-regulation
is useful for these applications. For instance, genes, transcripts,
and/or polypeptides that increase defense, pest and herbicide
resistance may require constitutive up-regulation of transcription.
In contrast, constitutive transcriptional down-regulation may be
desired to inhibit those genes, transcripts, and/or polypeptides
that lower defense, pest and herbicide resistance.
[0285] Typically, promoter or control elements that provide
constitutive transcription produce transcription levels that are
statistically similar in many tissues and environmental conditions
observed.
[0286] Calculation of P-value from the different observed
transcript levels is one means of determining whether a promoter or
control element is providing constitutive up-regulation. P-value is
the probability that the difference of transcript levels is not
statistically significant. The higher the P-value, the more likely
the difference of transcript levels is not significant. One formula
used to calculate P-value is as follows:
.intg. .PHI. ( x ) x , integrated from a to .infin. , where .PHI. (
x ) is a normal distribution ; ##EQU00001## where a = Sx - .mu.
.sigma. ( all Samples except Sx ) ; ##EQU00001.2## where Sx = the
intensity of the sample of interest where .mu. = is the average of
the intensities of all samples except Sx , = ( .SIGMA.S1 Sn ) - Sx
n - 1 where .sigma. ( S 1 S 11 , not including Sx ) = the standard
deviation of all sample intensities except Sx . ##EQU00001.3##
The P-value from the formula ranges from 1.0 to 0.0.
[0287] Usually, each P-value of the transcript levels observed in a
majority of cells, tissues, or organs under various environmental
conditions produced by the promoter or control to element is
greater than 10.sup.-8; more usually, greater than 10.sup.-7; even
more usually, greater than 10.sup.-6; even more usually, greater
than 10.sup.-5 or 10.sup.-4.
[0288] For up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the
assay.
[0289] Stress Induced Preferential Transcription
[0290] Promoters and control elements providing modulation of
transcription under oxidative, drought, oxygen, wound, and methyl
jasmonate stress are particularly useful for producing host cells
or organisms that are more resistant to biotic and abiotic
stresses. In a plant, for example, modulation of genes,
transcripts, and/or polypeptides in response to oxidative stress
can protect cells against damage caused by oxidative agents, such
as hydrogen peroxide and other free radicals.
[0291] Drought induction of genes, transcripts, and/or polypeptides
are useful to increase the viability of a plant, for example, when
water is a limiting factor. In contrast, genes, transcripts, and/or
polypeptides induced during oxygen stress can help the flood
tolerance of a plant.
[0292] The promoters and control elements of the present invention
can modulate stresses similar to those described in, for example,
stress conditions are VuPLD1 (drought stress; Cowpea; see Pham-Thi
et al. 1999. Plant molecular Biology. 1257-65), pyruvate
decarboxylase (oxygen stress; rice; see Rivosal et al. 1997. Plant
Physiol. 114(3): 1021-29), chromoplast specific carotenoid gene
(oxidative stress; capsicum; see Bouvier et al. 1998. Journal of
Biological Chemistry 273: 30651-59).
[0293] Promoters and control elements providing preferential
transcription during wounding or induced by methyl jasmonate can
produce a defense response in host cells or organisms. In a plant,
for example, preferential modulation of genes, transcripts, and/or
polypeptides under such conditions is useful to induce a defense
response to mechanical wounding, pest or pathogen attack or
treatment with certain chemicals.
[0294] Promoters and control elements of the present invention also
can trigger a response similar to those described for cf9 (viral
pathogen; tomato; see O'Donnell et al. 1998. The Plant journal: for
cell and molecular biology 14(1): 137-42), hepatocyte growth factor
activator inhibitor type 1 (HAI-1), which enhances tissue
regeneration (tissue injury; human; Koono et al. 1999. Journal of
Histochemistry and Cytochemistry 47: 673-82), copper amine oxidase
(CuAO), induced during ontogenesis and wound healing (wounding;
chick-pea; Rea et al. 1998. FEBS Letters 437: 177-82), proteinase
inhibitor II (wounding; potato; see Pena-Cortes et al. 1988. Planta
174: 84-89), protease inhibitor II (methyl jasmonate; tomato; see
Farmer and Ryan. 1990. Proc Natl Acad Sci USA 87: 7713-7716), two
vegetative storage protein genes VspA and VspB (wounding, jasmonic
acid, and water deficit; soybean; see Mason and Mullet. 1990. Plant
Cell 2: 569-579).
[0295] Up-regulation and transcription down-regulation are useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase oxidative, flood, or drought tolerance
may require up-regulation of transcription. In contrast,
transcriptional down-regulation may be desired to inhibit those
genes, transcripts, and/or polypeptides that lower such
tolerance.
[0296] Typically, promoter or control elements, which provide
preferential transcription in wounding or under methyl jasmonate
induction, produce transcript levels that are statistically
significant as compared to cell types, organs or tissues under
other conditions.
[0297] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0298] Light Induced Preferential Transcription
[0299] Promoters and control elements providing preferential
transcription when induced by light exposure can be utilized to
modulate growth, metabolism, and development; to increase drought
tolerance; and decrease damage from light stress for host cells or
organisms. In a plant, for example, modulation of genes,
transcripts, and/or polypeptides in response to light is useful
[0300] (1) to increase the photosynthetic rate; [0301] (2) to
increase storage of certain molecules in leaves or green parts
only, e.g., silage with high protein or starch content; [0302] (3)
to modulate production of exogenous compositions in green tissue,
e.g., certain feed enzymes; [0303] (4) to induce growth or
development, such as fruit development and maturity, during
extended exposure to light; [0304] (5) to modulate guard cells to
control the size of stomata in leaves to prevent water loss, or
[0305] (6) to induce accumulation of beta-carotene to help plants
cope with light induced stress. The promoters and control elements
of the present invention also can trigger responses similar to
those described in: abscisic acid insensitive3 (ABI3) (dark-grown
Arabidopsis seedlings, see Rohde et al. 2000. The Plant Cell 12:
35-52), asparagine synthetase (pea root nodules, see Tsai, F. Y.;
Coruzzi, G. M. 1990. EMBO J. 9: 323-32), mdm2 gene (human tumor;
see Saucedo et al. 1998. Cell Growth Differ 9: 119-30).
[0306] Up-regulation and transcription down-regulation are useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase drought or light tolerance may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to inhibit those genes, transcripts,
and/or polypeptides that lower such tolerance.
[0307] Typically, promoter or control elements, which provide
preferential transcription in cells, tissues or organs exposed to
light, produce transcript levels that are statistically significant
as compared to cells, tissues, or organs under decreased light
exposure (intensity or length of time).
[0308] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0309] Dark Induced Preferential Transcription
[0310] Promoters and control elements providing preferential
transcription when induced by dark or decreased light intensity or
decreased light exposure time can be utilized to time growth,
metabolism, and development, to modulate photosynthesis
capabilities for host cells or organisms. In a plant, for example,
modulation of genes, transcripts, and/or polypeptides in response
to dark is useful, for example, [0311] (1) to induce growth or
development, such as fruit development and maturity, despite lack
of light; [0312] (2) to modulate genes, transcripts, and/or
polypeptide active at night or on cloudy days; or [0313] (3) to
preserve the plastid ultra structure present at the onset of
darkness. The present promoters and control elements can also
trigger response similar to those described in the section
above.
[0314] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase growth and development may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to inhibit those genes, transcripts,
and/or polypeptides that modulate photosynthesis capabilities.
[0315] Typically, promoter or control elements, which provide
preferential transcription under exposure to dark or decrease light
intensity or decrease exposure time, produce transcript levels that
are statistically significant.
[0316] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0317] Leaf Preferential Transcription
[0318] Promoters and control elements providing preferential
transcription in a leaf can modulate growth, metabolism, and
development or modulate energy and nutrient utilization in host
cells or organisms. In a plant, for example, preferential
modulation of genes, transcripts, and/or polypeptide in a leaf, is
useful, for example,
[0319] (1) to modulate leaf size, shape, and development;
[0320] (2) to modulate the number of leaves; or
[0321] (3) to modulate energy or nutrient usage in relation to
other organs and tissues
[0322] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase growth, for example, may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to inhibit energy usage in a leaf to
be directed to the fruit instead, for instance.
[0323] Typically, promoter or control elements, which provide
preferential transcription in the cells, tissues, or organs of a
leaf, produce transcript levels that are statistically significant
as compared to other cells, organs or tissues.
[0324] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0325] Root Preferential Transcription
[0326] Promoters and control elements providing preferential
transcription in a root can modulate growth, metabolism,
development, nutrient uptake, nitrogen fixation, or modulate energy
and nutrient utilization in host cells or organisms. In a plant,
for example, preferential modulation of genes, transcripts, and/or
in a leaf, is useful
[0327] (1) to modulate root size, shape, and development;
[0328] (2) to modulate the number of roots, or root hairs;
[0329] (3) to modulate mineral, fertilizer, or water uptake;
[0330] (4) to modulate transport of nutrients; or
[0331] (4) to modulate energy or nutrient usage in relation to
other organs and tissues.
[0332] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase growth, for example, may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to inhibit nutrient usage in a root
to be directed to the leaf instead, for instance.
[0333] Typically, promoter or control elements, which provide
preferential transcription in cells, tissues, or organs of a root,
produce transcript levels that are statistically significant as
compared to other cells, organs or tissues.
[0334] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0335] Stem/Shoot Preferential Transcription
[0336] Promoters and control elements providing preferential
transcription in a stem or shoot can modulate growth, metabolism,
and development or modulate energy and nutrient utilization in host
cells or organisms. In a plant, for example, preferential
modulation of genes, transcripts, and/or polypeptide in a stem or
shoot, is useful, for example,
[0337] (1) to modulate stem/shoot size, shape, and development;
or
[0338] (2) to modulate energy or nutrient usage in relation to
other organs and tissues
[0339] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase growth, for example, may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to inhibit energy usage in a
stem/shoot to be directed to the fruit instead, for instance.
[0340] Typically, promoter or control elements, which provide
preferential transcription in the cells, tissues, or organs of a
stem or shoot, produce transcript levels that are statistically
significant as compared to other cells, organs or tissues.
[0341] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0342] Fruit and Seed Preferential Transcription
[0343] Promoters and control elements providing preferential
transcription in a silique or fruit can time growth, development,
or maturity; or modulate fertility; or modulate energy and nutrient
utilization in host cells or organisms. In a plant, for example,
preferential modulation of genes, transcripts, and/or polypeptides
in a fruit, is useful [0344] (1) to modulate fruit size, shape,
development, and maturity; [0345] (2) to modulate the number of
fruit or seeds; [0346] (3) to modulate seed shattering; [0347] (4)
to modulate components of seeds, such as, storage molecules,
starch, protein, oil, vitamins, anti-nutritional components, such
as phytic acid; [0348] (5) to modulate seed and/or seedling vigor
or viability; [0349] (6) to incorporate exogenous compositions into
a seed, such as lysine rich proteins; [0350] (7) to permit similar
fruit maturity timing for early and late blooming flowers; or
[0351] (8) to modulate energy or nutrient usage in relation to
other organs and tissues.
[0352] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase growth, for example, may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to inhibit late fruit maturity, for
instance.
[0353] Typically, promoter or control elements, which provide
preferential transcription in the cells, tissues, or organs of
siliques or fruits, produce transcript levels that are
statistically significant as compared to other cells, organs or
tissues.
[0354] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0355] Callus Preferential Transcription
[0356] Promoters and control elements providing preferential
transcription in a callus can be useful to modulating transcription
in dedifferentiated host cells. In a plant transformation, for
example, preferential modulation of genes, transcripts, in callus
is useful to modulate transcription of a marker gene, which can
facilitate selection of cells that are transformed with exogenous
polynucleotides.
[0357] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase marker gene detectability, for example,
may require up-regulation of transcription. In contrast,
transcriptional down-regulation may be desired to increase the
ability of the calluses to later differentiate, for instance.
[0358] Typically, promoter or control elements, which provide
preferential transcription in callus, produce transcript levels
that are statistically significant as compared to other cell types,
tissues, or organs. Calculation of P-value from the different
observed transcript levels is one means of determining whether a
promoter or control element is providing such preferential
transcription.
[0359] Usually, each P-value of the transcript levels observed in
callus as compared to, at least one other cell type, tissue or
organ, is less than 10.sup.-4; more usually, less than 10.sup.-5;
even more usually, less than 10.sup.-6; even more usually, less
than 10.sup.-7 or 10.sup.-8.
[0360] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0361] Flower Specific Transcription
[0362] Promoters and control elements providing preferential
transcription in flowers can modulate pigmentation; or modulate
fertility in host cells or organisms. In a plant, for example,
preferential modulation of genes, transcripts, and/or polypeptides
in a flower, is useful,
[0363] (1) to modulate petal color; or
[0364] (2) to modulate the fertility of pistil and/or stamen.
[0365] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase pigmentation, for example, may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to inhibit fertility, for
instance.
[0366] Typically, promoter or control elements, which provide
preferential transcription in flowers, produce transcript levels
that are statistically significant as compared to other cells,
organs or tissues.
[0367] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0368] Immature Bud and Inflorescence Preferential
Transcription
[0369] Promoters and control elements providing preferential
transcription in a immature bud or inflorescence can time growth,
development, or maturity; or modulate fertility or viability in
host cells or organisms. In a plant, for example, preferential
modulation of genes, transcripts, and/or polypeptide in a fruit, is
useful,
[0370] (1) to modulate embryo development, size, and maturity;
[0371] (2) to modulate endosperm development, size, and
composition;
[0372] (3) to modulate the number of seeds and fruits; or
[0373] (4) to modulate seed development and viability.
[0374] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase growth, for example, may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to decrease endosperm size, for
instance.
[0375] Typically, promoter or control elements, which provide
preferential transcription in immature buds and inflorescences,
produce transcript levels that are statistically significant as
compared to other cell types, organs or tissues.
[0376] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0377] Senescence Preferential Transcription
[0378] Promoters and control elements providing preferential
transcription during senescence can be used to modulate cell
degeneration, nutrient mobilization, and scavenging of free
radicals in host cells or organisms. Other types of responses that
can be modulated include, for example, senescence associated genes
(SAG) that encode enzymes thought to be involved in cell
degeneration and nutrient mobilization (Arabidopsis; see Hensel et
al. 1993. Plant Cell 5: 553-64), and the CP-2/cathepsin L gene
(rat; Kim and Wright. 1997. Biol Reprod 57: 1467-77), both induced
during senescence.
[0379] In a plant, for example, preferential modulation of genes,
transcripts, and/or polypeptides during senescencing is useful to
modulate fruit ripening.
[0380] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase scavenging of free radicals, for
example, may require up-regulation of transcription. In contrast,
transcriptional down-regulation may be desired to inhibit cell
degeneration, for instance.
[0381] Typically, promoter or control elements, which provide
preferential transcription in cells, tissues, or organs during
senescence, produce transcript levels that are statistically
significant as compared to other conditions.
[0382] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
[0383] Germination Preferential Transcription
[0384] Promoters and control elements providing preferential
transcription in a germinating seed can time growth, development,
or maturity; or modulate viability in host cells or organisms. In a
plant, for example, preferential modulation of genes, transcripts,
and/or polypeptide in a germinating seed, is useful,
[0385] (1) to modulate the emergence of they hypocotyls, cotyledons
and radical; or
[0386] (2) to modulate shoot and primary root growth and
development;
[0387] Up-regulation and transcription down-regulation is useful
for these applications. For instance, genes, transcripts, and/or
polypeptides that increase growth, for example, may require
up-regulation of transcription. In contrast, transcriptional
down-regulation may be desired to decrease endosperm size, for
instance.
[0388] Typically, promoter or control elements, which provide
preferential transcription in a germinating seed, produce
transcript levels that are statistically significant as compared to
other cell types, organs or tissues.
[0389] For preferential up-regulation of transcription, promoter
and control elements produce transcript levels that are above
background of the assay.
Microarray Analysis
[0390] A major way that a cell controls its response to internal or
external stimuli is by regulating the rate of transcription of
specific genes. For example, the differentiation of cells during
organogenensis into forms characteristic of the organ is associated
with the selective activation and repression of large numbers of
genes. Thus, specific organs, tissues and cells are functionally
distinct due to the different populations of mRNAs and protein
products they possess. Internal signals program the selective
activation and repression programs. For example, internally
synthesized hormones produce such signals. The level of hormone can
be raised by increasing the level of transcription of genes
encoding proteins concerned with hormone synthesis.
[0391] To measure how a cell reacts to internal and/or external
stimuli, individual mRNA levels can be measured and used as an
indicator for the extent of transcription of the gene. Cells can be
exposed to a stimulus, and mRNA can be isolated and assayed at
different time points after stimulation. The mRNA from the
stimulated cells can be compared to control cells that were not
stimulated. The mRNA levels that are higher in the stimulated cell
versus the control indicate a stimulus-specific response of the
cell. The same is true of mRNA levels that are lower in stimulated
cells versus the control condition.
[0392] Similar studies can be performed with cells taken from an
organism with a defined mutation in their genome as compared with
cells without the mutation. Altered mRNA levels in the mutated
cells indicate how the mutation causes transcriptional changes.
These transcriptional changes are associated with the phenotype
that the mutated cells exhibit that is different from the phenotype
exhibited by the control cells.
[0393] Applicants have utilized microarray techniques to measure
the levels of mRNAs in cells from plants transformed with a
construct containing the promoter or control elements of the
present invention together with their endogenous cDNA sequences. In
general, transformants with the constructs were grown to an
appropriate stage, and tissue samples were prepared for the
microarray differential expression analysis. In this manner it is
possible to determine the differential expression for the cDNAs
under the control of the endogenous promoter under various
conditions.
Microarray Experimental Procedures and Results
Procedures
1. Sample Tissue Preparation
[0394] Tissue samples for each of the expression analysis
experiments were prepared as follows:
[0395] (a) Roots
[0396] Seeds of Arabidopsis thaliana (Ws) were sterilized in full
strength bleach for less than 5 min., washed more than 3 times in
sterile distilled deionized water and plated on MS agar plates. The
plates were placed at 4.degree. C. for 3 nights and then placed
vertically into a growth chamber having 16 hr light/8 hr dark
cycles, 23.degree. C., 70% relative humidity and .about.11,000 LUX.
After 2 weeks, the roots were cut from the agar, flash frozen in
liquid nitrogen and stored at -80.degree. C.
[0397] (b) Rosette Leaves, Stems, and Siliques
[0398] Arabidopsis thaliana (Ws) seed was vernalized at4.degree. C.
for 3 days before sowing in Metro-mix soil type 350. Flats were
placed in a growth chamber having 16 hr light/8 hr dark, 80%
relative humidity, 23.degree. C. and 13,000 LUX for germination and
growth. After 3 weeks, rosette leaves, stems, and siliques were
harvested, flash frozen in liquid nitrogen and stored at
-80.degree. C. until use. After 4 weeks, siliques (<5 mm, 5-10
mm and >10 mm) were harvested, flash frozen in liquid nitrogen
and stored at -80.degree. C. until use. 5 week old whole plants
(used as controls) were harvested, flash frozen in liquid nitrogen
and kept at -80.degree. C. until RNA was isolated.
[0399] (c) Germination
[0400] Arabidopsis thaliana seeds (ecotype Ws) were sterilized in
bleach and rinsed with sterile water. The seeds were placed in 100
mm petri plates containing soaked autoclaved filter paper. Plates
were foil-wrapped and left at 4.degree. C. for 3 nights to
vernalize. After cold treatment, the foil was removed and plates
were placed into a growth chamber having 16 hr light/8 hr dark
cycles, 23.degree. C., 70% relative humidity and .about.11,000 lux.
Seeds were collected 1 d, 2 d, 3 d and 4 d later, flash frozen in
liquid nitrogen and stored at -80.degree. C. until RNA was
isolated.
[0401] (d) Abscissic Acid (ABA)
[0402] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in trays and left at 4.degree. C. for 4 days to vernalize.
They were then transferred to a growth chamber having grown 16 hr
light/8 hr dark, 13,000 LUX, 70% humidity, and 20.degree. C. and
watered twice a week with 1 L of 1.times. Hoagland's solution.
Approximately 1,000 14 day old plants were spayed with 200-250 mls
of 100 .mu.M ABA in a 0.02% solution of the detergent Silwet L-77.
Whole seedlings, including roots, were harvested within a 15 to 20
minute time period at 1 hr and 6 hr after treatment, flash-frozen
in liquid nitrogen and stored at -80.degree. C.
[0403] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 100 .mu.M ABA for
treatment. Control plants were treated with water. After 6 hr and
24 hr, aerial and root tissues were separated and flash frozen in
liquid nitrogen prior to storage at -80.degree. C.
[0404] (e) Brassinosteroid Responsive
[0405] Two separate experiments were performed, one with
epi-brassinolide and one with the brassinosteroid biosynthetic
inhibitor brassinazole. In the epi-brassinolide experiments, seeds
of wild-type Arabidopsis thaliana (ecotype Wassilewskija) and the
brassinosteroid biosynthetic mutant dwf4-1 were sown in trays and
left at 4.degree. C. for 4 days to vernalize. They were then
transferred to a growth chamber having 16 hr light/8 hr dark,
11,000 LUX, 70% humidity and 22.degree. C. temperature. Four week
old plants were spayed with a 1 .mu.M solution of epi-brassinolide
and shoot parts (unopened floral primordia and shoot apical
meristems) harvested three hours later. Tissue was flash-frozen in
liquid nitrogen and stored at -80.degree. C. In the brassinazole
experiments, seeds of wild-type Arabidopsis thaliana (ecotype
Wassilewskija) were grown as described above. Four week old plants
were spayed with a 1 .mu.M solution of brassinazole and shoot parts
(unopened floral primordia and shoot apical meristems) harvested
three hours later. Tissue was flash-frozen in liquid nitrogen and
stored at -80.degree. C.
[0406] In addition to the spray experiments, tissue was prepared
from two different mutants; (1) a dwf4-1 knock out mutant and (2) a
mutant overexpressing the dwf4-1 gene.
[0407] Seeds of wild-type Arabidopsis thaliana (ecotype
Wassilewskija) and of the dwf4-1 knock out and overexpressor
mutants were sown in trays and left at 4.degree. C. for 4 days to
vernalize. They were then transferred to a growth chamber having 16
hr light/8 hr dark, 11,000 LUX, 70% humidity and 22.degree. C.
temperature. Tissue from shoot parts (unopened floral primordia and
shoot apical meristems) was flash-frozen in liquid nitrogen and
stored at -80.degree. C.
[0408] Another experiment was completed with seeds of Arabidopsis
thaliana (ecotype Wassilewskija) were sown in trays and left at
4.degree. C. for 4 days to vernalize. They were then transferred to
a growth chamber. Plants were grown under long-day (16 hr light: 8
hr. dark) conditions, 13,000 LUX light intensity, 70% humidity,
20.degree. C. temperature and watered twice a week with 1 L
1.times. Hoagland's solution (recipe recited in Feldmann et al.,
(1987) Mol. Gen. Genet. 208: 1-9 and described as complete nutrient
solution). Approximately 1,000 14 day old plants were spayed with
200-250 mls of 0.1 .mu.M Epi-Brassinolite in 0.02% solution of the
detergent Silwet L-77. At 1 hr. and 6 hrs. after treatment aerial
tissues were harvested within a 15 to 20 minute time period and
flash-frozen in liquid nitrogen.
[0409] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 0.1 .mu.M
epi-brassinolide for treatment. Control plants were treated with
distilled deionized water. After 24 hr, aerial and root tissues
were separated and flash frozen in liquid nitrogen prior to storage
at -80.degree. C.
[0410] (f) Nitrogen: High to Low
[0411] Wild type Arabidopsis thaliana seeds (ecotpye Ws) were
surface sterilized with 30% Clorox, 0.1% Triton X-100 for 5
minutes. Seeds were then rinsed with 4-5 exchanges of sterile
double distilled deionized water. Seeds were vernalized at
4.degree. C. for 2-4 days in darkness. After cold treatment, seeds
were plated on modified 1.times.MS media (without NH.sub.4NO.sub.3
or KNO.sub.3), 0.5% sucrose, 0.5 g/L MES pH5.7, 1% phytagar and
supplemented with KNO.sub.3 to a final concentration of 60 mM (high
nitrate modified 1.times.MS media). Plates were then grown for 7
days in a Percival growth chamber at 22.degree. C. with 16 hr.
light/8 hr dark. Germinated seedlings were then transferred to a
sterile flask containing 50 mL of high nitrate modified 1.times.MS
liquid media. Seedlings were grown with mild shaking for 3
additional days at 22.degree. C. in 16 hr. light/8 hr dark (in a
Percival growth chamber) on the high nitrate modified 1.times.MS
liquid media.
[0412] After three days of growth on high nitrate modified
1.times.MS liquid media, seedlings were transferred either to a new
sterile flask containing 50 mL of high nitrate modified 1.times. MS
liquid media or to low nitrate modified 1.times.MS liquid media
(containing 20.quadrature. M KNQ. Seedlings were grown in these
media conditions with mild shaking at 22.degree. C. in 16 hr
light/8 hr dark for the appropriate time points and whole seedlings
harvested for total RNA isolation via the Trizol method
(LifeTech.). The time points used for the microarray experiments
were 10 min and 1 hour time points for both the high and low
nitrate modified 1.times.MS media.
[0413] Alternatively, seeds that were surface sterilized in 30%
bleach containing 0.1% Triton X-100 and further rinsed in sterile
water, were planted on MS agar, (0.5% sucrose) plates containing 50
mM KNO.sub.3 (potassium nitrate). The seedlings were grown under
constant light (3500 LUX) at 22.degree. C. After 12 days, seedlings
were transferred to MS agar plates containing either 1 mM KNO.sub.3
or 50 mM KNO.sub.3. Seedlings transferred to agar plates containing
50 mM KNO.sub.3 were treated as controls in the experiment.
Seedlings transferred to plates with 1 mM KNO.sub.3 were rinsed
thoroughly with sterile MS solution containing 1 mM KNO.sub.3.
There were ten plates per transfer. Root tissue was collected and
frozen in 15 mL Falcon tubes at various time points which included
1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 9 hours, 12 hours, 16
hours, and 24 hours.
[0414] Maize 35A19 Pioneer hybrid seeds were sown on flats
containing sand and grown in a Conviron growth chamber at
25.degree. C., 16 hr light/8 hr dark, .about.13,000 LUX and 80%
relative humidity. Plants were watered every three days with double
distilled deionized water. Germinated seedlings are allowed to grow
for 10 days and were watered with high nitrate modified 1.times.MS
liquid media (see above). On day 11, young corn seedlings were
removed from the sand (with their roots intact) and rinsed briefly
in high nitrate modified 1.times.MS liquid media. The equivalent of
half a flat of seedlings were then submerged (up to their roots) in
a beaker containing either 500 mL of high or low nitrate modified
1.times.MS liquid media (see above for details).
[0415] At appropriate time points, seedlings were removed from
their respective liquid media, the roots separated from the shoots
and each tissue type flash frozen in liquid nitrogen and stored at
-80.degree. C. This was repeated for each time point. Total RNA was
isolated using the Trizol method (see above) with root tissues
only.
[0416] Corn root tissues isolated at the 4 hr and 16 hr time points
were used for the microarray experiments. Both the high and low
nitrate modified 1.times.MS media were used.
[0417] (g) Nitrogen: Low to High
[0418] Arabidopsis thaliana ecotype Ws seeds were sown on flats
containing 4 L of a 1:2 mixture of Grace Zonolite vermiculite and
soil. Flats were watered with 3 L of water and vernalized at
4.degree. C. for five days. Flats were placed in a Conviron growth
chamber having 16 hr light/8 hr dark at 20.degree. C., 80% humidity
and 17,450 LUX. Flats were watered with approximately 1.5 L of
water every four days. Mature, bolting plants (24 days after
germination) were bottom treated with 2 L of either a control (100
mM mannitol pH 5.5) or an experimental (50 mM ammonium nitrate, pH
5.5) solution. Roots, leaves and siliques were harvested separately
30, 120 and 240 minutes after treatment, flash frozen in liquid
nitrogen and stored at -80.degree. C.
[0419] Hybrid maize seed (Pioneer hybrid 35A19) were aerated
overnight in deionized water. Thirty seeds were plated in each
flat, which contained 4 liters of Grace zonolite vermiculite. Two
liters of water were bottom fed and flats were kept in a Conviron
growth chamber with 16 hr light/8 hr dark at 20.degree. C. and 80%
humidity. Flats were watered with 1 L of tap water every three
days. Five day old seedlings were treated as described above with 2
L of either a control (100 mM mannitol pH 6.5) solution or 1 L of
an experimental (50 mM ammonium nitrate, pH 6.8) solution. Fifteen
shoots per time point per treatment were harvested 10, 90 and 180
minutes after treatment, flash frozen in liquid nitrogen and stored
at -80.degree. C.
[0420] Alternatively, seeds of Arabidopsis thaliana (ecotype
Wassilewskija) were left at 4.degree. C. for 3 days to vernalize.
They were then sown on vermiculite in a growth chamber having 16
hours light/8 hours dark, 12,000-14,000 LUX, 70% humidity, and
20.degree. C. They were bottom-watered with tap water, twice
weekly. Twenty-four days old plants were sprayed with either water
(control) or 0.6% ammonium nitrate at 4 .mu.L/cm.sup.2 of tray
surface. Total shoots and some primary roots were cleaned of
vermiculite, flash-frozen in liquid nitrogen and stored at
-80.degree. C.
[0421] (h) Methyl Jasmonate
[0422] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in trays and left at 4.degree. C. for 4 days to vernalize
before being transferred to a growth chamber having 16 hr light/8
hr. dark, 13,000 LUX, 70% humidity, 20.degree. C. temperature and
watered twice a week with 1 L of a 1.times. Hoagland's solution.
Approximately 1,000 14 day old plants were spayed with 200-250 mls
of 0.001% methyl jasmonate in a 0.02% solution of the detergent
Silwet L-77. At 1 hr and 6 hrs after treatment, whole seedlings,
including roots, were harvested within a 15 to 20 minute time
period, flash-frozen in liquid nitrogen and stored at -80.degree.
C.
[0423] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 0.001% methyl jasmonate
for treatment. Control plants were treated with water. After 24 hr,
aerial and root tissues were separated and flash frozen in liquid
nitrogen prior to storage at -80.degree. C.
[0424] (i) Salicylic Acid
[0425] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in trays and left at 4.degree. C. for 4 days to vernalize
before being transferred to a growth chamber having 16 hr light/8
hr. dark, 13,000 LUX, 70% humidity, 20.degree. C. temperature and
watered twice a week with 1 L of a 1.times. Hoagland's solution.
Approximately 1,000 14 day old plants were spayed with 200-250 mls
of 5 mM salicylic acid (solubilized in 70% ethanol) in a 0.02%
solution of the detergent Silwet L-77. At 1 hr and 6 hrs after
treatment, whole seedlings, including roots, were harvested within
a 15 to 20 minute time period flash-frozen in liquid nitrogen and
stored at -80.degree. C.
[0426] Alternatively, seeds of wild-type Arabidopsis thaliana
(ecotype Columbia) and mutant CS3726 were sown in soil type 200
mixed with osmocote fertilizer and Marathon insecticide and left at
4.degree. C. for 3 days to vernalize. Flats were incubated at room
temperature with continuous light. Sixteen days post germination
plants were sprayed with 2 mM SA, 0.02% SilwettL-77 or control
solution (0.02% SilwettL-77. Aerial parts or flowers were harvested
1 hr, 4 hr, 6 hr, 24 hr and 3 weeks post-treatment flash frozen and
stored at -80.degree. C.
[0427] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 2 mM SA for
treatment.
[0428] Control plants were treated with water. After 12 hr and 24
hr, aerial and root tissues were separated and flash frozen in
liquid nitrogen prior to storage at -80.degree. C.
[0429] (j) Drought Stress
[0430] Seeds of Arabidopsis thaliana (Wassilewskija) were sown in
pots and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber having 16 hr light/8 hr dark,
150,000-160,000 LUX, 20.degree. C. and 70% humidity. After 14 days,
aerial tissues were cut and left to dry on 3 mM Whatman paper in a
Petri-plate for 1 hour and 6 hours. Aerial tissues exposed for 1
hour and 6 hours to 3 mM Whatman paper wetted with 1.times.
Hoagland's solution served as controls. Tissues were harvested,
flash-frozen in liquid nitrogen and stored at -80.degree. C.
[0431] Alternatively, Arabidopsis thaliana (Ws) seed was vernalized
at 4.degree. C. for 3 days before sowing in Metromix soil type 350.
Flats were placed in a growth chamber with 23.degree. C., 16 hr
light/8 hr. dark, 80% relative humidity, 13,000 LUX for germination
and growth. Plants were watered with 1-1.5 L of water every four
days. Watering was stopped 16 days after germination for the
treated samples, but continued for the control samples. Rosette
leaves and stems, flowers and siliques were harvested 2 d, 3 d, 4
d, 5 d, 6 d and 7 d after watering was stopped. Tissue was flash
frozen in liquid nitrogen and kept at -80.degree. C. until RNA was
isolated. Flowers and siliques were also harvested on day 8 from
plants that had undergone a 7 d drought treatment followed by 1 day
of watering. Control plants (whole plants) were harvested after 5
weeks, flash frozen in liquid nitrogen and stored as above.
[0432] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in empty 1-liter beakers at room temperature
for treatment. Control plants were placed in water. After 1 hr, 6
hr, 12 hr and 24 hr aerial and root tissues were separated and
flash frozen in liquid nitrogen prior to storage at -80.degree.
C.
[0433] (k) Osmotic Stress
[0434] Seeds of Arabidopsis thaliana (Wassilewskija) were sown in
trays and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber having 16 hr light/8 hr dark,
12,000-14,000 LUX, 20.degree. C., and 70% humidity. After 14 days,
the aerial tissues were cut and placed on 3 MM Whatman paper in a
petri-plate wetted with 20% PEG (polyethylene glycol-M.sub.r 8,000)
in 1.times. Hoagland's solution. Aerial tissues on 3 MM Whatman
paper containing 1.times. Hoagland's solution alone served as the
control. Aerial tissues were harvested at 1 hour and 6 hours after
treatment, flash-frozen in liquid nitrogen and stored at
-80.degree. C.
[0435] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 10% PEG (polyethylene
glycol-M.sub.r 8,000) for treatment. Control plants were treated
with water. After 1 hr and 6 hr aerial and root tissues were
separated and flash frozen in liquid nitrogen prior to storage at
-80.degree. C.
[0436] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 150 mM NaCl for
treatment. Control plants were treated with water. After 1 hr, 6
hr, and 24 hr aerial and root tissues were separated and flash
frozen in liquid nitrogen prior to storage at -80.degree. C.
[0437] (l) Heat Shock Treatment
[0438] Seeds of Arabidopsis Thaliana (Wassilewskija) were sown in
trays and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber with 16 hr light/8 hr dark,
12,000-14,000 Lux, 70% humidity and 20.degree. C., fourteen day old
plants were transferred to a 42.degree. C. growth chamber and
aerial tissues were harvested 1 hr and 6 hr after transfer. Control
plants were left at 20.degree. C. and aerial tissues were
harvested. Tissues were flash-frozen in liquid nitrogen and stored
at -80.degree. C.
[0439] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers containing 42.degree. C.
water for treatment. Control plants were treated with water at
25.degree. C. After 1 hr and 6 hr aerial and root tissues were
separated and flash frozen in liquid nitrogen prior to storage at
-80.degree. C.
[0440] (m) Cold Shock Treatment
[0441] Seeds of Arabidopsis thaliana (Wassilewskija) were sown in
trays and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber having 16 hr light/8 hr dark,
12,000-14,000 LUX, 20.degree. C. and 70% humidity. Fourteen day old
plants were transferred to a 4.degree. C. dark growth chamber and
aerial tissues were harvested 1 hour and 6 hours later.
[0442] Control plants were maintained at 20.degree. C. and covered
with foil to avoid exposure to light. Tissues were flash-frozen in
liquid nitrogen and stored at -80.degree. C.
[0443] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers containing 4.degree. C.
water for treatment. Control plants were treated with water at
25.degree. C. After 1 hr and 6 hr aerial and root tissues were
separated and flash frozen in liquid nitrogen prior to storage at
-80.degree. C.
[0444] (n) Arabidopsis Seeds
[0445] Fruits (Pod+Seed) 0-5 mm
[0446] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Siliques 0-5 mm in
length containing post fertilization through pre-heart stage [0-72
hours after fertilization (HAF)] embryos were harvested and flash
frozen in liquid nitrogen.
[0447] Fruits (Pod+Seed) 5-10 mm
[0448] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Siliques 5-10 mm in
length containing heart- through early upturned-U-stage [72-120
hours after fertilization (HAF)] embryos were harvested and flash
frozen in liquid nitrogen.
[0449] Fruits (Pod+Seed)>10 mm
[0450] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Siliques >10 mm in
length containing green, late upturned-U-stage [>120 hours after
fertilization (HAF)-9 days after flowering (DAF)] embryos were
harvested and flash frozen in liquid nitrogen.
[0451] Green Pods 5-10 mm (Control Tissue for Samples 72-74)
[0452] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Green siliques 5-10 mm
in length containing developing seeds 72-120 hours after
fertilization (HAF)] were opened and the seeds removed. The
remaining tissues (green pods minus seed) were harvested and flash
frozen in liquid nitrogen.
[0453] Green Seeds from Fruits >10 mm
[0454] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what to developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Green siliques >10
mm in length containing developing seeds up to 9 days after
flowering (DAF)] were opened and the seeds removed and harvested
and flash frozen in liquid nitrogen.
[0455] Brown Seeds from Fruits >10 mm
[0456] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Yellowing siliques
>10 mm in length containing brown, dessicating seeds >11 days
after flowering (DAF)] were opened and the seeds removed and
harvested and flash frozen in liquid nitrogen.
[0457] Green/Brown Seeds from Fruits >10 mm
[0458] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Green siliques >10
mm in length containing both green and brown seeds >9 days after
flowering (DAF)] were opened and the seeds removed and harvested
and flash frozen in liquid nitrogen.
[0459] Mature Seeds (24 hours after imbibition)
[0460] Mature dry seeds of Arabidopsis thaliana (ecotype
Wassilewskija) were sown onto moistened filter paper and left at
4.degree. C. for two to three days to vernalize. Imbibed seeds were
then transferred to a growth chamber [16 hr light: 8 hr dark
conditions, 7000-8000 LUX light intensity, 70% humidity, and
22.degree. C. temperature], the emerging seedlings harvested after
48 hours and flash frozen in liquid nitrogen.
[0461] Mature Seeds (Dry)
[0462] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature and taken to maturity. Mature dry seeds are collected,
dried for one week at 28.degree. C., and vernalized for one week at
4.degree. C. before used as a source of RNA.
[0463] (o) Herbicide Treatment
[0464] Arabidopsis thaliana (Ws) seeds were sterilized for 5 min.
with 30% bleach, 50 .mu.l Triton in a total volume of 50 ml. Seeds
were vernalized at 4.degree. C. for 3 days before being plated onto
GM agar plates at a density of about 144 seeds per plate. Plates
were incubated in a Percival growth chamber having 16 hr light/8 hr
dark, 80% relative humidity, 22.degree. C. and 11,000 LUX for 14
days.
[0465] Plates were sprayed (.about.0.5 mls/plate) with water,
Finale (1.128 g/L), Glean (1.88 g/L), RoundUp (0.01 g/L) or Trimec
(0.08 g/L). Tissue was collected and flash frozen in liquid
nitrogen at the following time points: 0, 1, 2, 4, 8, 12 and 24
hours. Frozen tissue was stored at -80.degree. C. prior to RNA
isolation.
[0466] (p) Root Tips
[0467] Seeds of Arabidopsis thaliana (ecotype Ws) were placed on MS
plates and vernalized at 4.degree. C. for 3 days before being
placed in a 25.degree. C. growth chamber having 16 hr light/8 hr
dark, 70% relative humidity and about 3 W/m.sup.2. After 6 days,
young seedlings were transferred to flasks containing B5 liquid
medium, 1% sucrose and 0.05 mg/l indole-3-butyric acid. Flasks were
incubated at room temperature with 100 rpm agitation. Media was
replaced weekly. After three weeks, roots were harvested and
incubated for 1 hr with 2% pectinase, 0.2% cellulase, pH 7 before
straining through a #80 (Sigma) sieve. The root body material
remaining on the sieve (used as the control) was flash frozen and
stored at -80.degree. C. until use. The material that passed
through the #80 sieve was strained through a #200 (Sigma) sieve and
the material remaining on the sieve (root tips) was flash frozen
and stored at -80.degree. C. until use. Approximately 10 mg of root
tips were collected from one flask of root culture.
[0468] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 8 days. Seedlings were carefully removed from
the sand and the root tips (.about.2 mm long) were removed and
flash frozen in liquid nitrogen prior to storage at -80.degree. C.
The tissues above the root tips (.about.1 cm long) were cut,
treated as above and used as control tissue.
[0469] (q) Imbibed Seed
[0470] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in covered flats (10 rows, 5-6 seed/row) and
covered with clear, plastic lids before being placed in a growth
chamber having 16 hr light (25.degree. C.)/8 hr dark (20.degree.
C.), 75% relative humidity and 13,000-14,000 LUX. One day after
sowing, whole seeds were flash frozen in liquid nitrogen prior to
storage at -80.degree. C. Two days after sowing, embryos and
endosperm were isolated and flash frozen in liquid nitrogen prior
to storage at -80.degree. C. On days 3-6, aerial tissues, roots and
endosperm were isolated and flash frozen in liquid nitrogen prior
to storage at -80.degree. C.
[0471] (r) Flowers (Green, White or Buds)
[0472] Approximately 10 .mu.l of Arabidopsis thaliana seeds
(ecotype Ws) were sown on 350 soil (containing 0.03% marathon) and
vernalized at 4 C for 3 days. Plants were then grown at room
temperature under fluorescent lighting until flowering. Flowers
were harvested after 28 days in three different categories. Buds
that had not opened at all and were completely green were
categorized as "flower buds" (also referred to as green buds by the
investigator). Buds that had started to open, with white petals
emerging slightly were categorized as "green flowers" (also
referred to as white buds by the investigator). Flowers that had
opened mostly (with no silique elongation) with white petals
completely visible were categorized as "white flowers" (also
referred to as open flowers by the investigator). Buds and flowers
were harvested with forceps, flash frozen in liquid nitrogen and
stored at -80 C until RNA was isolated.
[0473] s) Ovules
[0474] Seeds of Arabidopsis thaliana heterozygous for pistillata
(pi) [ecotype Landsberg erecta (Ler)] were sown in pots and left at
4.degree. C. for two to three days to vernalize. They were then
transferred to a growth chamber. Plants were grown under long-day
(16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity,
76% humidity, and 24.degree. C. temperature. Inflorescences were
harvested from seedlings about 40 days old. The inflorescences were
cut into small pieces and incubated in the following enzyme
solution (pH 5) at room temperature for 0.5-1 hr.: 0.2% pectolyase
Y-23, 0.04% pectinase, 5 mM MES, 3% Sucrose and MS salts (1900 mg/l
KNO.sub.3, 1650 mg/l NH.sub.4NO.sub.3, 370 mg/l MgSO.sub.4.7
H.sub.2O, 170 mg/l KH.sub.2PO.sub.4, 440 mg/l CaCl.sub.2.2
H.sub.2O, 6.2 mg/l H.sub.2BO.sub.3, 15.6 mg/l MnSO.sub.4.4
H.sub.2O, 8.6 mg/l ZnSO.sub.4.7 H.sub.2O, 0.25 mg/l NaMoO.sub.4.2
H.sub.2O, 0.025 mg/l CuCO.sub.4.5 H.sub.2O, 0.025 mg/l CoCl.sub.2.6
H.sub.2O, 0.83 mg/l KI, 27.8 mg/l FeSO.sub.4.7 H.sub.2O, 37.3 mg/l
Disodium EDTA, pH 5.8). At the end of the incubation the mixture of
inflorescence material and enzyme solution was passed through a
size 60 sieve and then through a sieve with a pore size of 125 p.m.
Ovules greater than 125 .mu.m in diameter were collected, rinsed
twice in B5 liquid medium (2500 mg/l KNO.sub.3, 250 mg/l
MgSO.sub.4.7 H.sub.2O, 150 mg/l NaH2PO4.H.sub.2O, 150 mg/l
CaCl.sub.2.2 H.sub.2O, 134 mg/l (NH4)2 CaCl.sub.2.SO.sub.4, 3 mg/l
H.sub.2BO.sub.3, 10 mg/l MnSO.sub.4.4 H.sub.2O, 2 ZnSO.sub.4.7
H.sub.2O, 0.25 mg/l NaMoO.sub.4. 2 H.sub.2O, 0.025 mg/l
CuCO.sub.4.5 H.sub.2O, 0.025 mg/l CoCl.sub.2.6 H.sub.2O, 0.75 mg/l
KI, 40 mg/l EDTA sodium ferric salt, 20 g/l sucrose, 10 mg/l
Thiamine hydrochloride, 1 mg/l Pyridoxine hydrochloride, 1 mg/l
Nicotinic acid, 100 mg/l myo-inositol, pH 5.5)), rinsed once in
deionized water and flash frozen in liquid nitrogen. The
supernatant from the 125 .mu.m sieving was passed through
subsequent sieves of 50 .mu.m and 32 .mu.m. The tissue retained in
the 32 .mu.m sieve was collected and mRNA prepared for use as a
control.
[0475] t) Wounding
[0476] Seeds of Arabidopsis thaliana (Wassilewskija) were sown in
trays and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber having 16 hr light/8 hr dark,
12,000-14,000 LUX, 70% humidity and 20.degree. C. After 14 days,
the leaves were wounded with forceps. Aerial tissues were harvested
1 hour and 6 hours after wounding. Aerial tissues from unwounded
plants served as controls. Tissues were flash-frozen in liquid
nitrogen and stored at -80.degree. C.
[0477] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were wounded (one leaf
nicked by scissors) and placed in 1-liter beakers of water for
treatment. Control plants were treated not wounded. After 1 hr and
6 hr aerial and root tissues were separated and flash frozen in
liquid nitrogen prior to storage at -80.degree. C.
[0478] u) Nitric Oxide Treatment
[0479] Seeds of Arabidopsis thaliana (Wassilewskija) were sown in
trays and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber having 16 hr light/8 hr dark,
12,000-14,000 LUX, 20.degree. C. and 70% humidity. Fourteen day old
plants were sprayed with 5 mM sodium nitroprusside in a 0.02%
Silwett L-77 solution. Control plants were sprayed with a 0.02%
Silwett L-77solution. Aerial tissues were harvested 1 hour and 6
hours after spraying, flash-frozen in liquid nitrogen and stored at
-80.degree. C.
[0480] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 5 mM nitroprusside for
treatment. Control plants were treated with water. After 1 hr, 6 hr
and 12 hr, aerial and root tissues were separated and flash frozen
in liquid nitrogen prior to storage at -80.degree. C.
[0481] V) Root Hairless Mutants
[0482] Plants mutant at the rhl gene locus lack root hairs. This
mutation is maintained as a heterozygote.
[0483] Seeds of Arabidopsis thaliana (Landsberg erecta) mutated at
the rhl gene locus were sterilized using 30% bleach with 1 ul/ml
20% Triton-X 100 and then vernalized at 4.degree. C. for 3 days
before being plated onto GM agar plates. Plates were placed in
growth chamber with 16 hr light/8 hr. dark, 23.degree. C.,
14,500-15,900 LUX, and 70% relative humidity for germination and
growth.
[0484] After 7 days, seedlings were inspected for root hairs using
a dissecting microscope. Mutants were harvested and the cotyledons
removed so that only root tissue remained. Tissue was then flash
frozen in liquid nitrogen and stored at -80 C.
[0485] Arabidopsis thaliana (Landsberg erecta) seedlings grown and
prepared as above were used as controls.
[0486] Alternatively, seeds of Arabidopsis thaliana (Landsberg
erecta), heterozygous for the rhll (root hairless) mutation, were
surface-sterilized in 30% bleach containing 0.1% Triton X-100 and
further rinsed in sterile water. They were then vernalized at
4.degree. C. for 4 days before being plated onto MS agar plates.
The plates were maintained in a growth chamber at 24.degree. C.
with 16 hr light/8 hr dark for germination and growth. After 10
days, seedling roots that expressed the phenotype (i.e. lacking
root hairs) were cut below the hypocotyl junction, frozen in liquid
nitrogen and stored at -80.degree. C. Those seedlings with the
normal root phenotype (heterozygous or wt) were collected as
described for the mutant and used as controls.
[0487] w) Ap2
[0488] Seeds of Arabidopsis thaliana (ecotype Landesberg erecta)
and floral mutant apetala2 (Jofuku et al., 1994, Plant Cell
6:1211-1225) were sown in pots and left at 4.degree. C. for two to
three days to vernalize. They were then transferred to a growth
chamber. Plants were grown under long-day (16 hr light, 8 hr dark)
conditions 7000-8000 LUX light intensity, 70% humidity and
22.degree. C. temperature. Inflorescences containing immature
floral buds (stages 1-7; Bowman, 1994) as well as the inflorescence
meristem were harvested and flashfrozen. Polysomal polyA+ RNA was
isolated from tissue according to Cox and Goldberg, 1988).
[0489] x) Salt
[0490] Arabidopsis thaliana ecotype Ws seeds were vernalized at
4.degree. C. for 3 days before sowing in flats containing
vermiculite soil. Flats were placed at 20.degree. C. in a Conviron
growth chamber having 16 hr light/8 hr dark. Whole plants (used as
controls) received water. Other plants were treated with 100 mM
NaCl. After 6 hr and 72 hr, aerial and root tissues were harvested
and flash frozen in liquid nitrogen prior to storage at -80.degree.
C.
[0491] y) Petals
[0492] Arabidopsis thaliana ecotype Ws seeds were vernalized at
4.degree. C. for 3 days before sowing in flats containing
vermiculite soil. Flats were watered placed at 20.degree. C. in a
Conviron growth chamber having 16 hr light/8 hr dark. Whole plants
(used as the control) and petals from inflorescences 23-25 days
after germination were harvested, flash frozen in liquid nitrogen
and stored at -80.degree. C.
[0493] z) Pollen
[0494] Arabidopsis thaliana ecotype Ws seeds were vernalized at
4.degree. C. for 3 days before sowing in flats containing
vermiculite soil. Flats were watered and placed at 20.degree. C. in
a Conviron growth chamber having 16 hr light/8 hr dark. Whole
plants (used as controls) and pollen from plants 38 dap was
harvested, flash frozen in liquid nitrogen and stored at
-80.degree. C.
[0495] aa) Interploidy Crosses
[0496] Interploidy crosses involving a 6.times. parent are lethal.
Crosses involving a 4.times. parent are compelte and analyzed. The
imbalance in the maternal/paternal ratio produced from the cross
can lead to big seeds. Arabidopsis thaliana ecotype Ws seeds were
vernalized at 4.degree. C. for 3 days before sowing. Small siliques
were harvested at 5 days after pollination, flash frozen in liquid
nitrogen and stored at -80.degree. C.
[0497] bb) Line comparisons
[0498] Alkaloid 35S over-expressing lines were used to monitor the
expression levels of terpenoid/alkaloid biosynthetic and P450 genes
to identify the transcriptional regulatory points I the
biosynthesis pathway and the related P450 genes. Arabidopsis
thaliana ecotype Ws seeds were vernalized at 4.degree. C. for 3
days before sowing in vermiculite soil (Zonolite) supplemented by
Hoagland solution. Flats were placed in Conviron growth chambers
under long day conditions (16 hr light, 23.degree. C./8 hr dark,
20.degree. C.) Basta spray and selection of the overexpressing
lines was conducted about 2 weeks after germination. Approximately
2-3 weeks after bolting (approximately 5-6 weeks after
germination), stem and siliques from the over-expressing lines and
from wild-type plants were harvested, flash frozen in liquid
nitrogen and stored at -80.degree. C.
[0499] cc) DMT-II
[0500] Demeter (dirt) is a mutant of a methyl transferase gene and
is similar to fie. Arabidopsis thaliana ecotype Ws seeds were
vernalized at 4.degree. C. for 3 days before sowing. Cauline leaves
and closed flowers were isolated from 35S::DMT and dmt -/- plant
lines, flash frozen in liquid nitrogen and stored at -80.degree.
C.
[0501] dd) CS6630 Roots and shoots
[0502] Arabidopsis thaliana ecotype Ws seeds were vernalized at
4.degree. C. for 3 days before sowing on MS media (1%) sucrose on
bactor-agar. Roots and shoots were separated 14 days after
germination, flash frozen in liquid nitrogen and stored at
-80.degree. C.
[0503] ee) CS237 CS237 is an ethylene triple response mutant that
is insensitive to ethylene and which has an etr1-1 phenotype.
Arabidopsis thaliana CS237 seeds were vernalized at 4.degree. C.
for 3 days before sowing. Aerial tissue was collected from mutants
and wild-type Columbia ecotype plants, flash frozen in liquid
nitrogen and stored at -80.degree. C.
[0504] ff) Guard Cells
[0505] Arabidopsis thaliana ecotype Ws seeds were vernalized at
4.degree. C. for 3 days before sowing. Leaves were harvested,
homogenized and centrifuged to isolate the guard cell containing
fraction. Homogenate from leaves served as the control. Samples
were flash frozen in liquid nitrogen and stored at -80.degree. C.
Identical experiments using leaf tissue from canola were
performed.
[0506] gg) 3642-1
[0507] 3642-1 is a T-DNA mutant that affects leaf development. This
mutant segregates 3:1, wild-type:mutant. Arabidopsis thaliana
3642-1 mutant seeds were vernalized at 4.degree. C. for 3 days
before sowing in flats of MetroMix 200. Flats were placed in the
greenhouse, watered and grown to the 8 leaf, pre-flower stage.
Stems and rosette leaves were harvested from the mutants and the
wild-type segregants, flash frozen and stored at -80.degree. C.
[0508] hh) Caf
[0509] Carple factory (Caf) is a double-stranded RNAse protein that
is hypothesized to process small RNAs in Arabidopsis. The protein
is closely related to a Drosophila protein named DICER that
functions in the RNA degradation steps of RNA interference.
Arabidopsis thaliana Caf mutant seeds were vernalized at 4.degree.
C. for 3 days before sowing in flats of MetroMix 200. Flats were
placed in the greenhouse, watered and grown to the 8 leaf,
pre-flower stage. Stems and rosette leaves were harvested from the
mutants and the wild-type segregants, flash frozen and stored at
-80.degree. C.
2. Microarray Hybridization Procedures
[0510] Microarray technology provides the ability to monitor mRNA
transcript levels of thousands of genes in a single experiment.
These experiments simultaneously hybridize two differentially
labeled fluorescent cDNA pools to glass slides that have been
previously spotted with cDNA clones of the same species. Each
arrayed cDNA spot will have a corresponding ratio of fluorescence
that represents the level of disparity between the respective mRNA
species in the two sample pools. Thousands of polynucleotides can
be spotted on one slide, and each experiment generates a global
expression pattern.
Coating Slides
[0511] The microarray consists of a chemically coated microscope
slide, referred herein as a "chip" with numerous polynucleotide
samples arrayed at a high density. The poly-L-lysine coating allows
for this spotting at high density by providing a hydrophobic
surface, reducing the spreading of spots of DNA solution arrayed on
the slides. Glass microscope slides (Gold Seal #3010 manufactured
by Gold Seal Products, Portsmouth, N.H., USA) were coated with a
0.1% WN solution of Poly-L-lysine (Sigma, St. Louis, Mo.) using the
following protocol: [0512] 1. Slides were placed in slide racks
(Shandon Lipshaw #121). The racks were then put in chambers
(Shandon Lipshaw #121). [0513] 2. Cleaning solution was prepared:
[0514] 70 g NaOH was dissolved in 280 mL ddH2O. [0515] 420 mL 95%
ethanol was added. The total volume was 700 mL (=2.times.350 mL);
it was stirred until completely mixed. If the solution remained
cloudy, ddH.sub.2O was added until clear. [0516] 3. The solution
was poured into chambers with slides; the chambers were covered
with glass lids. The solution was mixed on an orbital shaker for 2
hr. [0517] 4. The racks were quickly transferred to fresh chambers
filled with ddH.sub.2O. They were rinsed vigorously by plunging
racks up and down. Rinses were repeated 4.times. with fresh
ddH.sub.2O each time, to remove all traces of NaOH-ethanol. [0518]
5. Polylysine solution was prepared: [0519] 0 mL poly-L-lysine+70
mL tissue culture PBS in 560 mL water, using plastic graduated
cylinder and beaker. [0520] 6. Slides were transferred to
polylysine solution and shaken for 1 hr. [0521] 7. The rack was
transferred to a fresh chambers filled with ddH.sub.2O. It was
plunged up and down 5.times. to rinse. [0522] 8. The slides were
centrifuged on microtiter plate carriers (paper towels were placed
below the rack to absorb liquid) for 5 min. @ 500 rpm. The slide
racks were transferred to empty chambers with covers. [0523] 9.
Slide racks were dried in a 45 C oven for 10 min. [0524] 10. The
slides were stored in a closed plastic slide box. [0525] 11.
Normally, the surface of lysine coated slides was not very
hydrophobic immediately after this process, but became increasingly
hydrophobic with storage. A hydrophobic surface helped ensure that
spots didn't run together while printing at high densities. After
they aged for 10 days to a month the slides were ready to use.
However, coated slides that have been sitting around for long
periods of time were usually too old to be used. This was because
they developed opaque patches, visible when held to the light, and
these resulted in high background hybridization from the
fluorescent probe. Alternatively, pre-coated glass slides were
purchased from TeleChem International, Inc. (Sunnyvale, Calif.,
94089; catalog number SMM-25, Superamine substrates). PCR
Amplification of cDNA Clone Inserts
[0526] Polynucleotides were amplified from Arabidopsis cDNA clones
using insert specific probes. The resulting 100 uL PCR reactions
were purified with Qiaquick 96 PCR purification columns (Qiagen,
Valencia, Calif., USA) and eluted in 30 uL of 5 mM Tris. 8.5 uL of
the elution were mixed with 1.5 uL of 20.times.SSC to give a final
spotting solution of DNA in 3.times.SSC. The concentrations of DNA
generated from each clone varied between 10-100 ng/ul, but were
usually about 50 ng/ul.
Arraying of PCR Products on Glass Slides
[0527] PCR products from cDNA clones were spotted onto the
poly-L-Lysine coated glass slides using an arrangement of quill-tip
pins (ChipMaker 3 spotting pins; Telechem, International, Inc.,
Sunnyvale, Calif., USA) and a robotic arrayer (PixSys 3500,
Cartesian Technologies, Irvine, Calif., USA). Around 0.5 n1 of a
prepared PCR product was spotted at each location to produce spots
with approximately 100 um diameters. Spot center-to-center spacing
was from 180 um to 210 um depending on the array. Printing was
conducted in a chamber with relative humidity set at 50%.
[0528] Slides containing maize sequences were purchased from
Agilent Technology (Palo Alto, Calif. 94304).
Post-Processing of Slides
[0529] After arraying, slides were processed through a series of
steps--rehydration, UV cross-linking, blocking and
denaturation--required prior to hybridization. Slides were
rehydrated by placing them over a beaker of warm water (DNA face
down), for 2-3 sec, to distribute the DNA more evenly within the
spots, and then snap dried on a hot plate (DNA side, face up). The
DNA was then cross-linked to the slides by UV irradiation (60-65
mJ; 2400 Stratalinker, Stratagene, La Jolla, Calif., USA).
[0530] Following this a blocking step was performed to modify
remaining free lysine groups, and hence minimize their ability to
bind labeled probe DNA. To achieve this the arrays were placed in a
slide rack. An empty slide chamber was left ready on an orbital
shaker. The rack was bent slightly inwards in the middle, to ensure
the slides would not run into each other while shaking. The
blocking solution was prepared as follows:
3.times.350-ml glass chambers (with metal tops) were set to one
side, and a large round Pyrex dish with dH.sub.2O was placed ready
in the microwave. At this time, 15 ml sodium borate was prepared in
a 50 ml conical tube.
[0531] 6-g succinic anhydride was dissolved in approx. 325-350 mL
1-methyl-2-pyrrolidinone. Rapid addition of reagent was
crucial.
[0532] a Immediately after the last flake of the succinic anhydride
dissolved, the 15-mL sodium borate was added.
[0533] b Immediately after the sodium borate solution mixed in, the
solution was poured into an empty slide chamber.
[0534] c. The slide rack was plunged rapidly and evenly in the
solution. It was vigorously shaken up and down for a few seconds,
making sure slides never left the solution.
[0535] d. It was mixed on an orbital shaker for 15-20 min.
Meanwhile, the water in the Pyrex dish (enough to cover slide rack)
was heated to boiling.
[0536] Following this, the slide rack was gently plunge in the 95 C
water (just stopped boiling) for 2 min. Then the slide rack was
plunged 5.times. in 95% ethanol. The slides and rack were
centrifuged for 5 min. @ 500 rpm. The slides were loaded quickly
and evenly onto the carriers to avoid streaking. The arrays were
used immediately or store in slide box.
[0537] The Hybridization process began with the isolation of mRNA
from the two tissues (see "Isolation of total RNA" and "Isolation
of mRNA", below) in question followed by their conversion to single
stranded cDNA (see "Generation of probes for hybridization",
below). The cDNA from each tissue was independently labeled with a
different fluorescent dye and then both samples were pooled
together. This final differentially labeled cDNA pool was then
placed on a processed microarray and allowed to hybridize (see
"Hybridization and wash conditions", below).
Isolation of Total RNA
[0538] Approximately 1 g of plant tissue was ground in liquid
nitrogen to a fine powder and transferred into a 50-ml centrifuge
tube containing 10 ml of Trizol reagent. The tube was vigorously
vortexed for 1 min and then incubated at room temperature for 10-20
min. on an orbital shaker at 220 rpm. Two ml of chloroform was
added to the tube and the solution vortexed vigorously for at least
30-sec before again incubating at room temperature with shaking.
The sample was then centrifuged at 12,000.times.g (10,000 rpm) for
15-20 min at 4.degree. C. The aqueous layer was removed and mixed
by inversion with 2.5 ml of 1.2 M NaCl/0.8 M Sodium Citrate and 2.5
ml of isopropyl alcohol added. After a 10 min. incubation at room
temperature, the sample was centrifuged at 12,000.times.g (10,000
rpm) for 15 min at 4.degree. C. The pellet was washed with 70%
ethanol, re-centrifuged at 8,000 rpm for 5 min and then air dried
at room temperature for 10 min. The resulting total RNA was
dissolved in either TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or DEPC
(diethylpyrocarbonate) treated deionized water (RNAse-free water).
For subsequent isolation of mRNA using the Qiagen kit, the total
RNA pellet was dissolved in RNAse-free water.
Isolation of mRNA
[0539] mRNA was isolated using the Qiagen Oligotex mRNA Spin-Column
protocol (Qiagen, Valencia, Calif.). Briefly, 500 .mu.l OBB buffer
(20 mM Tris-C1, pH 7.5, 1 M NaCl, 2 mM EDTA, 0.2% SDS) was added to
500 .mu.l of total RNA (0.5-0.75 mg) and mixed thoroughly. The
sample was first incubated at 70.degree. C. for 3 min, then at room
temperature for 10 minutes and finally centrifuged for 2 min at
14,000-18,000.times.g. The pellet was resuspended in 400 .mu.l OW2
buffer (10 mM Tris-C1, pH 7.5, 150 mM NaCl, 1 mM EDTA) by
vortexing, the resulting solution placed on a small spin column in
a 1.5 ml RNase-free microcentrifuge tube and centrifuged for 1 min
at 14,000-18,000.times.g. The spin column was transferred to a new
1.5 ml RNase-free microcentrifuge tube and washed with 400 .mu.l of
OW2 buffer. To release the isolated mRNA from the resin, the spin
column was again transferred to a new RNase-free 1.5 ml
microcentrifuge tube, 20-100 .mu.l 70.degree. C. OEB buffer (5 mM
Tris-Cl, pH 7.5) added and the resin resuspended in the resulting
solution via pipeting. The mRNA solution was collected after
centrifuging for 1 min at 14,000-18,000.times.g.
[0540] Alternatively, mRNA was isolated using the Stratagene
Poly(A) Quik mRNA Isolation Kit (Startagene, La Jolla, Calif.).
Here, up to 0.5 mg of total RNA (maximum volume of 1 ml) was
incubated at 65.degree. C. for 5 minutes, snap cooled on ice and
0.1.times. volumes of 10.times. sample buffer (10 mM Tris-HCl (pH
7.5), 1 mM EDTA (pH 8.0) 5 M NaCl) added. The RNA sample was
applied to a prepared push column and passed through the column at
a rate of .about.1 drop every 2 sec. The solution collected was
reapplied to the column and collected as above. 200 .mu.l of high
salt buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 NaCl) was
applied to the column and passed through the column at a rate of
.about.1 drop every 2 sec. This step was repeated and followed by
three low salt buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M
NaCl) washes preformed in a similar manner. mRNA was eluted by
applying to the column four separate 200 .mu.l aliquots of elution
buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) preheated to 65.degree.
C. Here, the elution buffer was passed through the column at a rate
of 1 drop/sec. The resulting mRNA solution was precipitated by
adding 0.1.times. volumes of 10.times. sample buffer, 2.5 volumes
of ice-cold 100% ethanol, incubating overnight at -20.degree. C.
and centrifuging at 14,000-18,000.times.g for 20-30 min at
4.degree. C. The pellet was washed with 70% ethanol and air dried
for 10 min at room temperature before resuspension in RNase-free
deionized water.
Preparation of Yeast Controls
[0541] Plasmid DNA was isolated from the following yeast clones
using Qiagen filtered maxiprep kits (Qiagen, Valencia, Calif.):
YAL022c(Fun26), YAL031c(Fun21), YBR032w, YDL131w, YDL182w, YDL194w,
YDL196w, YDR050c and YDR116c. Plasmid DNA was linearized with
either BsrBI (YAL022c(Fun26), YAL031c(Fun21), YDL131w, YDL182w,
YDL194w, YDL196w, YDR050c) or AflIII (YBR032w, YDR116c) and
isolated.
In Vitro Transcription of Yeast Clones
[0542] The following solution was incubated at 37.degree. C. for 2
hours: 17 .mu.l of isolated yeast insert DNA (1 .mu.g), 20 .mu.l
5.times. buffer, 10 .mu.l 100 mM DTT, 2.5 .mu.l (100 U) RNasin, 20
.mu.l 2.5 mM (ea.).sub.rNTPs, 2.7 .mu.l (40 U) SP6 polymerase and
27.8 .mu.l RNase-free deionized water. 2 .mu.l (2 U) Ampli DNase I
was added and the incubation continued for another 15 min. 10 .mu.l
5M NH.sub.4OAC and 100 .mu.l phenol:chloroform:isoamyl alcohol
(25:24:1) were added, the solution vortexed and then centrifuged to
separate the phases. To precipitate the RNA, 250 .mu.l ethanol was
added and the solution incubated at -20.degree. C. for at least one
hour. The sample was then centrifuged for 20 min at 4.degree. C. at
14,000-18,000.times.g, the pellet washed with 500 .mu.l of 70%
ethanol, air dried at room temperature for 10 min and resuspended
in 100 .mu.l of RNase-free deionized water. The precipitation
procedure was then repeated.
[0543] Alternatively, after the two-hour incubation, the solution
was extracted with phenol/chloroform once before adding 0.1 volume
3M sodium acetate and 2.5 volumes of 100% ethanol. The solution was
centrifuged at 15,000 rpm, 4.degree. C. for 20 minutes and the
pellet resuspended in RNase-free deionized water. The DNase I
treatment was carried out at 37.degree. C. for 30 minutes using 2 U
of Ampli DNase I in the following reaction condition: 50 mM
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2. The DNase I reaction was then
stopped with the addition of NH.sub.4OAC and
phenol:chloroform:isoamyl alcohol (25:24:1), and RNA isolated as
described above.
[0544] 0.15-2.5 ng of the in vitro transcript RNA from each yeast
clone were added to each plant mRNA sample prior to labeling to
serve as positive (internal) probe controls.
Generation of Probes for Hybridization
[0545] Generation of Labeled Probes for Hybridization from
First-Strand cDNA
[0546] Hybridization probes were generated from isolated mRNA using
an Atlas.TM. Glass Fluorescent Labeling Kit (Clontech Laboratories,
Inc., Palo Alto, Calif., USA). This entails a two step labeling
procedure that first incorporates primary aliphatic amino groups
during cDNA synthesis and then couples fluorescent dye to the cDNA
by reaction with the amino functional groups. Briefly, 5 .mu.g of
oligo(dT).sub.18 primer d(TTTTTTTTTTTTTTTTTTV) was mixed with Poly
A+ mRNA (1.5-2 .mu.g mRNA isolated using the Qiagen Oligotex mRNA
Spin-Column protocol or-the Stratagene Poly(A) Quik mRNA Isolation
protocol (Stratagene, La Jolla, Calif., USA)) in a total volume of
25 .mu.l. The sample was incubated in a thermocycler at 70.degree.
C. for 5 min, cooled to 48.degree. C. and 10 .mu.l of 5.times.cDNA
Synthesis Buffer (kit supplied), 5 .mu.l 10.times. dNTP mix (dATP,
dCTP, dGTP, dTTP and aminoallyl-dUTP; kit supplied), 7.5 .mu.l
deionized water and 2.5 .mu.l MMLV Reverse Transcriptase (500 U)
added. The reaction was then incubated at 48.degree. C. for 30
minutes, followed by 1 hr incubation at 42.degree. C.
[0547] At the end of the incubation the reaction was heated to
70.degree. C. for 10 min, cooled to 37.degree. C. and 0.5 .mu.l (5
U) RNase H added, before incubating for 15 min at 37.degree. C. The
solution was vortexed for 1 min after the addition of 0.5 .mu.l 0.5
M EDTA and 5 .mu.l of QuickClean Resin (kit supplied) then
centrifuged at 14,000-18,000.times.g for 1 min. After removing the
supernatant to a 0.45 .mu.m spin filter (kit supplied), the sample
was again centrifuged at 14,000-18,000.times.g for 1 min, and 5.5
.mu.l 3 M sodium acetate and 137.5 .mu.l of 100% ethanol added to
the sample before incubating at -20.degree. C. for at least 1 hr.
The sample was then centrifuged at 14,000-18,000.times.g at
4.degree. C. for 20 min, the resulting pellet washed with 500 .mu.l
70% ethanol, air-dried at room temperature for 10 min and
resuspended in 10 .mu.l of 2.times. fluorescent labeling buffer
(kit provided). 10 .mu.l each of the fluorescent dyes Cy3 and Cy5
(Amersham Pharmacia (Piscataway, N.J., USA); prepared according to
Atlas.TM. kit directions of Clontech) were added and the sample
incubated in the dark at room temperature for 30 min.
[0548] The fluorescently labeled first strand cDNA was precipitated
by adding 2 .mu.l 3M sodium acetate and 50 .mu.l 100% ethanol,
incubated at -20.degree. C. for at least 2 hrs, centrifuged at
14,000-18,000.times.g for 20 min, washed with 70% ethanol,
air-dried for 10 min and dissolved in 100 .mu.l of water.
[0549] Alternatively, 3-4 .mu.g mRNA, 2.5 (.about.8.9 ng of in
vitro translated mRNA) .mu.l yeast control and 3 .mu.g oligo dTV
(TTTTTTTTTTTTTTTTTT(A/C/G) were mixed in a total volume of 24.7
.mu.l. The sample was incubated in a thermocycler at 70.degree. C.
for 10 min. before chilling on ice. To this, 8 .mu.l of 5.times.
first strand buffer (SuperScript II RNase H-Reverse Transcriptase
kit from Invitrogen (Carlsbad, Calif. 92008); cat no. 18064022),
0.8.degree. C. of aa-dUTP/dNTP mix (50.times.; 25 mM dATP, 25 mM
dGTP, 25 mM dCTP, 15 mM dTTP, 10 mM aminoallyl-dUTP), 4 .mu.l of
0.1 M DTT and 2.5 .mu.l (500 units) of Superscript R.T.II enzyme
(Stratagene) were added. The sample was incubated at 42.degree. C.
for 2 hours before a mixture of 10.degree. C. of 1M NaOH and
10.degree. C. of 0.5 M EDTA were added. After a 15 minute
incubation at 65.degree. C., 25 .mu.l of 1 M Tris pH 7.4 was added.
This was mixed with 450 .mu.l of water in a Microcon 30 column
before centrifugation at 11,000.times.g for 12 min. The column was
washed twice with 450 .mu.l (centrifugation at 11,000 g, 12 min.)
before eluting the sample by inverting the Microcon column and
centrifuging at 11,000.times.g for 20 seconds. Sample was
dehydrated by centrifugation under vacuum and stored at -20.degree.
C.
[0550] Each reaction pellet was dissolved in 9 .mu.l of 0.1 M
carbonate buffer (0.1M sodium carbonate and sodium bicarbonate,
pH=8.5-9) and 4.5 .mu.l of this placed in two microfuge tubes. 4.5
.mu.l of each dye (in DMSO) were added and the mixture incubated in
the dark for 1 hour. 4.5 .mu.l of 4 M hydroxylamine was added and
again incubated in the dark for 15 minutes.
[0551] Regardless of the method used for probe generation, the
probe was purified using a Qiagen PCR cleanup kit (Qiagen,
Valencia, Calif., USA), and eluted with 100 ul EB (kit provided).
The sample was loaded on a Microcon YM-30 (Millipore, Bedford,
Mass., USA) spin column and concentrated to 4-5 ul in volume.
Probes for the maize microarrays were generated using the
Fluorescent Linear Amplification Kit (cat. No. G2556A) from Agilent
Technologies (Palo Alto, Calif.).
Hybridization and Wash Conditions
[0552] The following Hybridization and Washing Condition were
developed:
Hybridization Conditions.
[0553] Labeled probe was heated at 95.degree. C. for 3 min and
chilled on ice. Then 25.quadrature. L of the hybridization buffer
which was warmed at 42 C was added to the probe, mixing by
pipeting, to give a final concentration of:
50% formamide
[0554] 4.times.SSC
[0555] 0.03% SDS
5.times.Denhardt's solution 0.1 .mu.g/ml single-stranded salmon
sperm DNA
[0556] The probe was kept at 42 C. Prior to the hybridization, the
probe was heated for 1 more min., added to the array, and then
covered with a glass cover slip. Slides were placed in
hybridization chambers (Telechem, Sunnyvale, Calif.) and incubated
at 42.degree. C. overnight.
Washing Conditions:
[0557] A. Slides were washed in 1.times.SSC+0.03% SDS solution at
room temperature for 5 minutes, [0558] B. Slides were washed in
0.2.times.SSC at room temperature for 5 minutes, [0559] C. Slides
were washed in 0.05.times.SSC at room temperature for 5
minutes.
[0560] After A, B, and C, slides were spun at 800.times.g for 2 min
to dry. They were then scanned.
[0561] Maize microarrays were hybridized according to the
instructions included Fluorescent Linear Amplification Kit (cat.
No. G2556A) from Agilent Technologies (Palo Alto, Calif.).
Scanning of Slides
[0562] The chips were scanned using a ScanArray 3000 or 5000
(General Scanning, Watertown, Mass., USA). The chips were scanned
at 543 and 633 nm, at 10 um resolution to measure the intensity of
the two fluorescent dyes incorporated into the samples hybridized
to the chips.
Data Extraction and Analysis
[0563] The images generated by scanning slides consisted of two
16-bit TIFF images representing the fluorescent emissions of the
two samples at each arrayed spot. These images were then quantified
and processed for expression analysis using the data extraction
software Imagene.TM. (Biodiscovery, Los Angeles, Calif., USA).
Imagene output was subsequently analyzed using the analysis program
Genespring.TM. (Silicon Genetics, San Carlos, Calif., USA). In
Genespring, the data was imported using median pixel intensity
measurements derived from Imagene output. Background subtraction,
ratio calculation and normalization were all conducted in
Genespring. Normalization was achieved by breaking the data in to
32 groups, each of which represented one of the 32 pin printing
regions on the microarray. Groups consist of 360 to 550 spots. Each
group was independently normalized by setting the median of ratios
to one and multiplying ratios by the appropriate factor.
[0564] Results
[0565] The results of the microaray experiments are set forth in
Table 1 in the section entitled "Microarray Data" which shows the
results of the differential expression experiments for the mRNAs,
as reported by their corresponding cDNA ID number, that were
differentially transcribed under a particular set of conditions as
compared to a control sample. The cDNA ID numbers correspond to
those utilized. Increases in mRNA abundance levels in experimental
plants versus the controls are denoted with the plus sign (+).
Likewise, reductions in mRNA abundance levels in the experimental
plants are denoted with the minus (-) sign.
[0566] The Table 1 section entitled "Microarray Data" is organized
according to the clone number with each set of experimental
conditions being denoted by the term "Expt Rep ID:" followed by a
"short name". The row titled "Microarray Experiment Parameters"
links each "short name" with a short description of the experiment
and the parameters. The sequences showing differential expression
in a particular experiment (denoted by either a "+" or "-" in the
column in Table 1 entitled "SIGNCLOG_RATIO") thereby show utility
for a function in a plant, and these functions/utilities are
described in detail below, where the title of each section (i.e. a
"utlity section") is correlated with the particular differential
expression experiment in the section of Table 1 entitled"Microarray
Experiment Parameters".
Organ-Affecting Genes, Gene Components, Products (Including
Differentiation and Function)
Root Genes
[0567] The economic values of roots arise not only from harvested
adventitious roots or tubers, but also from the ability of roots to
funnel nutrients to support growth of all plants and increase their
vegetative material, seeds, fruits, etc. Roots have four main
functions. First, they anchor the plant in the soil. Second, they
facilitate and regulate the molecular signals and molecular traffic
between the plant, soil, and soil fauna. Third, the root provides a
plant with nutrients gained from the soil or growth medium. Fourth,
they condition local soil chemical and physical properties.
[0568] Root genes are active or potentially active to a greater
extent in roots than in most other organs of the plant. These genes
and gene products can regulate many plant traits from yield to
stress tolerance. Root genes can be used to modulate root growth
and development.
[0569] Differential Expression of the Sequences in Roots
[0570] The relative levels of mRNA product in the root versus the
aerial portion of the plant was measured. Specifically, mRNA was
isolated from roots and root tips of Arabidopsis plants and
compared to mRNA isolated from the aerial portion of the plants
utilizing microanay procedures.
Root Hair Genes, Gene Components and Products
[0571] Root hairs are specialized outgrowths of single epidermal
cells termed trichoblasts. In many and perhaps all species of
plants, the trichoblasts are regularly arranged around the
perimeter of the root. In Arabidopsis, for example, trichoblasts
tend to alternate with non-hair cells or atrichoblasts. This
spatial patterning of the root epidermis is under genetic control,
and a variety of mutants have been isolated in which this spacing
is altered or in which root hairs are completely absent.
[0572] The root hair development genes of the instant invention are
useful to modulate one or more processes of root hair structure
and/or function including (1) development; (2) interaction with the
soil and soil contents; (3) uptake and transport in the plant; and
(4) interaction with microorganisms.
[0573] 1.) Development
[0574] The surface cells of roots can develop into single epidermal
cells termed trichoblasts or root hairs. Some of the root hairs
will persist for the life of the plant; others will gradually die
back; some may cease to function due to external influences. These
genes and gene products can be used to modulate root hair density
or root hair growth; including rate, timing, direction, and size,
for example. These genes and gene products can also be used to
modulate cell properties such as cell size, cell division, rate and
direction and number, cell elongation, cell differentiation,
lignified cell walls, epidermal cells (including trichoblasts) and
root apical meristem cells (growth and initiation); and root hair
architecture such as leaf cells under the trichome, cells forming
the base of the trichome, trichome cells, and root hair
responses.
[0575] In addition these genes and gene products can be used to
modulate one or more of the growth and development processes in
response to internal plant programs or environmental stimuli in,
for example, the seminal system, nodal system, hormone responses,
Auxin, root cap abscission, root senescence, gravitropism,
coordination of root growth and development with that of other
organs (including leaves, flowers, seeds, fruits, and stems), and
changes in soil environment (including water, minerals, Ph, and
microfauna and flora).
[0576] 2.) Interaction with Soil and Soil Contents
[0577] Root hairs are sites of intense chemical and biological
activity and as a result can strongly modify the soil they contact.
Roots hairs can be coated with surfactants and mucilage to
facilitate these activities. Specifically, roots hairs are
responsible for nutrient uptake by mobilizing and assimilating
water, reluctant ions, organic and inorganic compounds and
chemicals. In addition, they attract and interact with beneficial
microfauna and flora. Root hairs also help to mitigate the effects
of toxic ions, pathogens and stress. Thus, root hair genes and gene
products can be used to modulate traits such as root hair
surfactant and mucilage (including composition and secretion rate
and time); nutrient uptake (including water, nitrate and other
sources of nitrogen, phosphate, potassium, and micronutrients (e.g.
iron, copper, etc.); microbe and nematode associations (such as
bacteria including nitrogen-fixing bacteria, mycorrhizae,
nodule-forming and other nematodes, and nitrogen fixation); oxygen
transpiration; detoxification effects of iron, aluminum, cadium,
mercury, salt, and other soil constituents; pathogens (including
chemical repellents) glucosinolates (GSL1), which release
pathogen-controlling isothiocyanates; and changes in soil (such as
Ph, mineral excess and depletion), and rhizosheath.
[0578] 3.) Transport of Materials in Plants
[0579] Uptake of the nutrients by the root and root hairs
contributes a source-sink effect in a plant. The greater source of
nutrients, the more sinks, such as stems, leaves, flowers, seeds,
fruits, etc. can draw sustenance to grow. Thus, root hair
development genes and gene products can be used to modulate the
vigor and yield of the overall plant as well as distinct cells,
organs, or tissues of a plant. The genes and gene products,
therefore, can modulate plant nutrition, growth rate (such as whole
plant, including height, flowering time, etc., seedling, coleoptile
elongation, young leaves, stems, flowers, seeds and fruit) and
yield, including biomass (fresh and dry weight during any time in
plant life, including maturation and senescence), number of
flowers, number of seeds, seed yield, number, size, weight and
harvest index (content and composition, e.g. amino acid, jasmonate,
oil, protein and starch) and fruit yield (number, size, weight,
harvest index, and post harvest quality).
Reproduction Genes, Gene Components and Products
[0580] Reproduction genes are defined as genes or components of
genes capable of modulating any aspect of sexual reproduction from
flowering time and inflorescence development to fertilization and
finally seed and fruit development. These genes are of great
economic interest as well as biological importance. The fruit and
vegeTable industry grosses over $1 billion USD a year. The seed
market, valued at approximately $15 billion USD annually, is even
more lucrative.
Inflorescence and Floral Development Genes, Gene Components and
Products
[0581] During reproductive growth the plant enters a program of
floral development that culminates in fertilization, followed by
the production of seeds. Senescence may or may not follow. The
flower formation is a precondition for the sexual propagation of
plants and is therefore essential for the propagation of plants
that cannot be propagated vegetatively as well as for the formation
of seeds and fruits. The point of time at which the merely
vegetative growth of plants changes into flower formation is of
vital importance for example in agriculture, horticulture and plant
breeding. Also the number of flowers is often of economic
importance, for example in the case of various useful plants
(tomato, cucumber, zucchini, cotton etc.) with which an increased
number of flowers may lead to an increased yield, or in the case of
growing ornamental plants and cut flowers.
[0582] Flowering plants exhibit one of two types of inflorescence
architecture: indeterminate, in which the inflorescence grows
indefinitely, or determinate, in which a terminal flower is
produced. Adult organs of flowering plants develop from groups of
stem cells called meristems. The identity of a meristem is inferred
from structures it produces: vegetative meristems give rise to
roots and leaves, inflorescence meristems give rise to flower
meristems, and flower meristems give rise to floral organs such as
sepals and petals. Not only are meristems capable of generating new
meristems of different identity, but their own identity can change
during development. For example, a vegetative shoot meristem can be
transformed into an inflorescence meristem upon floral induction,
and in some species, the inflorescence meristem itself will
eventually become a flower meristem. Despite the importance of
meristem transitions in plant development, little is known about
the underlying mechanisms.
[0583] Following germination, the shoot meristem produces a series
of leaf meristems on its flanks. However, once floral induction has
occurred, the shoot meristem switches to the production of flower
meristems. Flower meristems produce floral organ primordia, which
develop individually into sepals, petals, stamens or carpels. Thus,
flower formation can be thought of as a series of distinct
developmental steps, i.e. floral induction, the formation of flower
primordia and the production of flower organs. Mutations disrupting
each of the steps have been isolated in a variety of species,
suggesting that a genetic hierarchy directs the flowering process
(see for review, Weigel and Meyerowitz, In Molecular Basis of
Morphogenesis (ed. M. Bernfield). 51st Annual Symposium of the
Society for Developmental Biology, pp. 93-107, New York, 1993).
[0584] Expression of many reproduction genes and gene products is
orchestrated by internal programs or the surrounding environment of
a plant. These genes can be used to modulate traits such as fruit
and seed yield
Seed and Fruit Development Genes, Gene Components and Products
[0585] The ovule is the primary female sexual reproductive organ of
flowering plants. At maturity it contains the egg cell and one
large central cell containing two polar nuclei encased by two
integuments that, after fertilization, develops into the embryo,
endosperm, and seed coat of the mature seed, respectively. As the
ovule develops into the seed, the ovary matures into the fruit or
silique. As such, seed and fruit development requires the
orchestrated transcription of numerous polynucleotides, some of
which are ubiquitous, others that are embryo-specific and still
others that are expressed only in the endosperm, seed coat, or
fruit. Such genes are termed fruit development responsive genes and
can be used to modulate seed and fruit growth and development such
as seed size, seed yield, seed composition and seed dormancy.
[0586] Differential Expression of the Sequences in Siliques,
Inflorescences and Flowers
[0587] The relative levels of mRNA product in the siliques relative
to the plant as a whole was measured.
[0588] Differential Expression of the Sequences in Hybrid Seed
Development
[0589] The levels of mRNA product in the seeds relative to those in
a leaf and floral stems was measured.
Development Genes, Gene Components and Products
[0590] Imbibition and Germination Responsive Genes, Gene Components
and Products
[0591] Seeds are a vital component of the world's diet. Cereal
grains alone, which comprise .about.90% of all cultivated seeds,
contribute up to half of the global per capita energy intake. The
primary organ system for seed production in flowering plants is the
ovule. At maturity, the ovule consists of a haploid female
gametophyte or embryo sac surrounded by several layers of maternal
tissue including the nucleus and the integuments. The embryo sac
typically contains seven cells including the egg cell, two
synergids, a large central cell containing two polar nuclei, and
three antipodal cells. That pollination results in the
fertilization of both egg and central cell. The fertilized egg
develops into the embryo. The fertilized central cell develops into
the endosperm. And the integuments mature into the seed coat. As
the ovule develops into the seed, the ovary matures into the fruit
or silique. Late in development, the developing seed ends a period
of extensive biosynthetic and cellular activity and begins to
desiccate to complete its development and enter a dormant,
metabolically quiescent state. Seed dormancy is generally an
undesirable characteristic in agricultural crops, where rapid
germination and growth are required. However, some degree of
dormancy is advantageous, at least during seed development. This is
particularly true for cereal crops because it prevents germination
of grains while still on the ear of the parent plant (preharvest
sprouting), a phenomenon that results in major losses to the
agricultural industry. Extensive domestication and breeding of crop
species have ostensibly reduced the level of dormancy mechanisms
present in the seeds of their wild ancestors, although under some
adverse environmental conditions, dormancy may reappear. By
contrast, weed seeds frequently mature with inherent dormancy
mechanisms that allow some seeds to persist in the soil for many
years before completing germination.
[0592] Germination commences with imbibition, the uptake of water
by the dry seed, and the activation of the quiescent embryo and
endosperm. The result is a burst of intense metabolic activity. At
the cellular level, the genome is transformed from an inactive
state to one of intense transcriptional activity. Stored lipids,
carbohydrates and proteins are catabolized fueling seedling growth
and development. DNA and organelles are repaired, replicated and
begin functioning. Cell expansion and cell division are triggered.
The shoot and root apical meristem are activated and begin growth
and organogenesis. Schematic 4 summarizes some of the metabolic and
cellular processes that occur during imbibition. Germination is
complete when a part of the embryo, the radicle, extends to
penetrate the structures that surround it. In Arabidopsis, seed
germination takes place within twenty-four (24) hours after
imbibition. As such, germination requires the rapid and
orchestrated transcription of numerous polynucleotides. Germination
is followed by expansion of the hypocotyl and opening of the
cotyledons. Meristem development continues to promote root growth
and shoot growth, which is followed by early leaf formation.
Imbibition and Germination Genes
[0593] Imbibition and germination includes those events that
commence with the uptake of water by the quiescent dry seed and
terminate with the expansion and elongation of the shoots and
roots. The germination period exists from imbibition to when part
of the embryo, usually the radicle, extends to penetrate the seed
coat that surrounds it. Imbibition and germination genes are
defined as genes, gene components and products capable of
modulating one or more processes of imbibition and germination
described above. They are useful to modulate many plant traits from
early vigor to yield to stress tolerance.
[0594] Differential Expression of the Sequences in Germinating
Seeds and Imbibed Embryos
[0595] The levels of mRNA product in the seeds versus the plant as
a whole was measured.
Hormone Responsive Genes, Gene Components and Products
Abscissic Acid Responsive Genes, Gene Components and Products
[0596] Plant hormones are naturally occurring substances, effective
in very small amounts, which act as signals to stimulate or inhibit
growth or regulate developmental processes in plants. Abscisic acid
(ABA) is a ubiquitous hormone in vascular plants that has been
detected in every major organ or living tissue from the root to the
apical bud. The major physiological responses affected by ABA are
dormancy, stress stomatal closure, water uptake, abscission and
senescence. In contrast to Auxins, cytokinins and gibberellins,
which are principally growth promoters, ABA primarily acts as an
inhibitor of growth and metabolic processes.
[0597] Changes in ABA concentration internally or in the
surrounding environment in contact with a plant results in
modulation of many genes and gene products. These genes and/or
products are responsible for effects on traits such as plant vigor
and seed yield.
[0598] While ABA responsive polynucleotides and gene products can
act alone, combinations of these polynucleotides also affect growth
and development. Useful combinations include different ABA
responsive polynucleotides and/or gene products that have similar
transcription profiles or similar biological activities, and
members of the same or similar biochemical pathways. Whole pathways
or segments of pathways are controlled by transcription factor
proteins and proteins controlling the activity of signal
transduction pathways. Therefore, manipulation of such protein
levels is especially useful for altering phenotypes and biochemical
activities of plants. In addition, the combination of an ABA
responsive polynucleotide and/or gene product with another
environmentally responsive polynucleotide is also useful because of
the interactions that exist between hormone-regulated pathways,
stress and defense induced pathways, nutritional pathways and
development.
[0599] Differential Expression of the Sequences in ABA Treated
Plants
[0600] The relative levels of mRNA product in plants treated with
ABA versus controls treated with water were measured.
Brassinosteroid Responsive Genes, Gene Components and Products
[0601] Plant hormones are naturally occurring substances, effective
in very small amounts, which act as signals to stimulate or inhibit
growth or regulate developmental processes in plants.
Brassinosteroids (BRs) are the most recently discovered, and least
studied, class of plant hormones. The major physiological response
affected by BRs is the longitudinal growth of young tissue via cell
elongation and possibly cell division. Consequently, disruptions in
BR metabolism, perception and activity frequently result in a dwarf
phenotype. In addition, because BRs are derived from the sterol
metabolic pathway, any perturbations to the sterol pathway can
affect the BR pathway. In the same way, perturbations in the BR
pathway can have effects on the later part of the sterol pathway
and thus the sterol composition of membranes.
[0602] Changes in BR concentration in the surrounding environment
or in contact with a plant result in modulation of many genes and
gene products. These genes and/or products are responsible for
effects on traits such as plant biomass and seed yield. These genes
were discovered and characterized from a much larger set of genes
by experiments designed to find genes whose mRNA abundance changed
in response to application of BRs to plants.
[0603] While BR responsive polynucleotides and gene products can
act alone, combinations of these polynucleotides also affect growth
and development. Useful combinations include different BR
responsive polynucleotides and/or gene products that have similar
transcription profiles or similar biological activities, and
members of the same or functionally related biochemical pathways.
Whole pathways or segments of pathways are controlled by
transcription factors and proteins controlling the activity of
signal transduction pathways. Therefore, manipulation of such
protein levels is especially useful for altering phenotypes and
biochemical activities of plants. In addition, the combination of a
BR responsive polynucleotide and/or gene product with another
environmentally responsive polynucleotide is useful because of the
interactions that exist between hormone-regulated pathways, stress
pathways, nutritional pathways and development. Here, in addition
to polynucleotides having similar transcription profiles and/or
biological activities, useful combinations include polynucleotides
that may have different transcription profiles but which
participate in common or overlapping pathways.
[0604] Differential Expression of the Sequences in Epi-Brassinolide
or Brassinozole Plants
[0605] The relative levels of mRNA product in plants treated with
either epi-brassinolide or brassinozole were measured.
Metabolism Affecting Genes, Gene Components and Products
Nitrogen Responsive Genes, Gene Components and Products
[0606] Nitrogen is often the rate-limiting element in plant growth,
and all field crops have a fundamental dependence on exogenous
nitrogen sources. Nitrogenous fertilizer, which is usually supplied
as ammonium nitrate, potassium nitrate, or urea, typically accounts
for 40% of the costs associated with crops, such as corn and wheat
in intensive agriculture. Increased efficiency of nitrogen use by
plants should enable the production of higher yields with existing
fertilizer inputs and/or enable existing yields of crops to be
obtained with lower fertilizer input, or better yields on soils of
poorer quality. Also, higher amounts of proteins in the crops could
also be produced more cost-effectively. "Nitrogen responsive" genes
and gene products can be used to alter or modulate plant growth and
development.
[0607] Differential Expression of the Sequences in Whole Seedlings,
Shoots and Roots
[0608] The relative levels of mRNA product in whole seedlings,
shoots and roots treated with either high or low nitrogen media
were compared to controls.
Viability Genes, Gene Components and Products
[0609] Plants contain many proteins and pathways that when blocked
or induced lead to cell, organ or whole plant death. Gene variants
that influence these pathways can have profound effects on plant
survival, vigor and performance. The critical pathways include
those concerned with metabolism and development or protection
against stresses, diseases and pests. They also include those
involved in apoptosis and necrosis. Viability genes can be
modulated to affect cell or plant death.
[0610] Herbicides are, by definition, chemicals that cause death of
tissues, organs and whole plants. The genes and pathways that are
activated or inactivated by herbicides include those that cause
cell death as well as those that function to provide
protection.
[0611] Differential Expression of the Sequences in Herbicide
Treated Plants and Herbicide Resistant Mutants
[0612] The relative levels of mRNA product in plants treated with
herbicide and mutants resistant to herbicides were compared to
control plants.
Stress Responsive Genes, Gene Components and Products
Wounding Responsive Genes, Gene Components and Products
[0613] Plants are continuously subjected to various forms of
wounding from physical attacks including the damage created by
pathogens and pests, wind, and contact with other objects.
Therefore, survival and agricultural yields depend on constraining
the damage created by the wounding process and inducing defense
mechanisms against future damage.
[0614] Plants have evolved complex systems to minimize and/or
repair local damage and to minimize subsequent attacks by pathogens
or pests or their effects. These involve stimulation of cell
division and cell elongation to repair tissues, induction of
programmed cell death to isolate the damage caused mechanically and
by invading pests and pathogens, and induction of long-range
signaling systems to induce protecting molecules, in case of future
attack. The genetic and biochemical systems associated with
responses to wounding are connected with those associated with
other stresses such as pathogen attack and drought.
[0615] Wounding responsive genes and gene products can be used to
alter or modulate traits such as growth rate; whole plant height,
width, or flowering time; organ development (such as coleoptile
elongation, young leaves, roots, lateral roots, tuber formation,
flowers, fruit, and seeds); biomass; fresh and dry weight during
any time in plant life, such as at maturation; number of flowers;
number of seeds; seed yield, number, size, weight, harvest index
(such as content and composition, e.g., amino acid, nitrogen, oil,
protein, and carbohydrate); fruit yield, number, size, weight,
harvest index, post harvest quality, content and composition (e.g.,
amino acid, carotenoid, jasmonate, protein, and starch); seed and
fruit development; germination of dormant and non-dormant seeds;
seed viability, seed reserve mobilization, fruit ripening,
initiation of the reproductive cycle from a vegetative state,
flower development time, insect attraction for fertilization, time
to fruit maturity, senescence; fruits, fruit drop; leaves; stress
and disease responses; drought; heat and cold; wounding by any
source, including wind, objects, pests and pathogens; uv and high
light damage (insect, fungus, virus, worm, nematode damage).
Cold Responsive Genes, Gene Components and Products
[0616] The ability to endure low temperatures and freezing is a
major determinant of the geographical distribution and productivity
of agricultural crops. Even in areas considered suiTable for the
cultivation of a given species or cultivar, can give rise to yield
decreases and crop failures as a result of aberrant, freezing
temperatures. Even modest increases (1-2.degree. C.) in the
freezing tolerance of certain crop species would have a dramatic
impact on agricultural productivity in some areas. The development
of genotypes with increased freezing tolerance would provide a more
reliable means to minimize crop losses and diminish the use of
energy-costly practices to modify the microclimate.
[0617] Sudden cold temperatures result in modulation of many genes
and gene products, including promoters. These genes and/or products
are responsible for effects on traits such as plant vigor and seed
yield.
[0618] Manipulation of one or more cold responsive gene activities
is useful to modulate growth and development.
[0619] Differential Expression of the Sequences in Cold Treated
Plants
[0620] The relative levels of mRNA product in cold treated plants
were compared to control plants.
Heat Responsive Genes, Gene Components and Products
[0621] The ability to endure high temperatures is a major
determinant of the geographical distribution and productivity of
agricultural crops. Decreases in yield and crop failure frequently
occur as a result of aberrant, hot conditions even in areas
considered suiTable for the cultivation of a given species or
cultivar. Only modest increases in the heat tolerance of crop
species would have a dramatic impact on agricultural productivity.
The development of genotypes with increased heat tolerance would
provide a more reliable means to minimize crop losses and diminish
the use of energy-costly practices to modify the microclimate.
[0622] Changes in temperature in the surrounding environment or in
a plant microclimate results in modulation of many genes and gene
products.
[0623] Differential Expression of the Sequences in Heat Treated
Plants
[0624] The relative levels of mRNA product in heat treated plants
were compared to control plants.
Drought Responsive Genes, Gene Components and Products
[0625] The ability to endure drought conditions is a major
determinant of the geographical distribution and productivity of
agricultural crops. Decreases in yield and crop failure frequently
occur as a result of aberrant, drought conditions even in areas
considered suiTable for the cultivation of a given species or
cultivar. Only modest increases in the drought tolerance of crop
species would have a dramatic impact on agricultural productivity.
The development of genotypes with increased drought tolerance would
provide a more reliable means to minimize crop losses and diminish
the use of energy-costly practices to modify the microclimate.
[0626] Drought conditions in the surrounding environment or within
a plant, results in modulation of many genes and gene products.
[0627] Differential Expression of the Sequences in Drought Treated
Plants and Drought Mutants
[0628] The relative levels of mRNA product in drought treated
plants and drought mutants were compared to control plants.
Methyl Jasmonate (Jasmonate) Responsive Genes, Gene Components and
Products
[0629] Jasmonic acid and its derivatives, collectively referred to
as jasmonates, are naturally occurring derivatives of plant lipids.
These substances are synthesized from linolenic acid in a
lipoxygenase-dependent biosynthetic pathway. Jasmonates are
signalling molecules which have been shown to be growth regulators
as well as regulators of defense and stress responses. As such,
jasmonates represent a separate class of plant hormones. Jasmonate
responsive genes can be used to modulate plant growth and
development.
[0630] Differential Expression of the Sequences in Methyl Jasmonate
Treated Plants
[0631] The relative levels of mRNA product in methyl jasmonate
treated plants were compared to control plants.
Salicylic Acid Responsive Genes, Gene Components and Products
[0632] Plant defense responses can be divided into two groups:
constitutive and induced. Salicylic acid (SA) is a signaling
molecule necessary for activation of the plant induced defense
system known as systemic acquired resistance or SAR. This response,
which is triggered by prior exposure to avirulent pathogens, is
long lasting and provides protection against a broad spectrum of
pathogens. Another induced defense system is the hypersensitive
response (HR). HR is far more rapid, occurs at the sites of
pathogen (avirulent pathogens) entry and precedes SAR. SA is also
the key signaling molecule for this defense pathway.
[0633] Differential Expression of the Sequences in Salicylic Acid
Treated Plants
[0634] The relative levels of mRNA product in salicylic acid
treated plants were compared to control plants.
Osmotic Stress Responsive Genes, Gene Components and Products
[0635] The ability to endure and recover from osmotic and salt
related stress is a major determinant of the geographical
distribution and productivity of agricultural crops. Osmotic stress
is a major component of stress imposed by saline soil and water
deficit. Decreases in yield and crop failure frequently occur as a
result of aberrant or transient environmental stress conditions
even in areas considered suitable for the cultivation of a given
species or cultivar. Only modest increases in the osmotic and salt
tolerance of a crop species would have a dramatic impact on
agricultural productivity. The development of genotypes with
increased osmotic tolerance would provide a more reliable means to
minimize crop losses and diminish the use of energy-costly
practices to modify the soil environment. Thus, osmotic stress
responsive genes can be used to modulate plant growth and
development.
[0636] Differential Expression of the Sequences in PEG Treated
Plants
[0637] The relative levels of mRNA product in PEG treated plants
were compared to control plants.
Shade Responsive Genes, Gene Components and Products
[0638] Plants sense the ratio of Red (R): Far Red (FR) light in
their environment and respond differently to particular ratios. A
low R:FR ratio, for example, enhances cell elongation and favors
flowering over leaf production. The changes in R:FR ratios mimic
and cause the shading response effects in plants. The response of a
plant to shade in the canopy structures of agricultural crop fields
influences crop yields significantly. Therefore manipulation of
genes regulating the shade avoidance responses can improve crop
yields. While phytochromes mediate the shade avoidance response,
the down-stream factors participating in this pathway are largely
unknown. One potential downstream participant, ATHB-2, is a member
of the HD-Zip class of transcription factors and shows a strong and
rapid response to changes in the R:FR ratio. ATHB-2 overexpressors
have a thinner root mass, smaller and fewer leaves and longer
hypocotyls and petioles. This elongation arises from longer
epidermal and cortical cells, and a decrease in secondary vascular
tissues, paralleling the changes observed in wild-type seedlings
grown under conditions simulating canopy shade. On the other hand,
plants with reduced ATHB-2 expression have a thick root mass and
many larger leaves and shorter hypocotyls and petioles. Here, the
changes in the hypocotyl result from shorter epidermal and cortical
cells and increased proliferation of vascular tissue.
Interestingly, application of Auxin is able to reverse the root
phenotypic consequences of high ATHB-2 levels, restoring the
wild-type phenotype. Consequently, given that ATHB-2 is tightly
regulated by phytochrome, these data suggest that ATHB-2 may link
the Auxin and phytochrome pathways in the shade avoidance response
pathway.
[0639] Shade responsive genes can be used to modulate plant growth
and development.
[0640] Differential Expression of the Sequences in Far-Red Light
Treated Plants
[0641] The relative levels of mRNA product in far-red light treated
plants were compared to control plants.
Viability Genes, Gene Components and Products
[0642] Plants contain many proteins and pathways that when blocked
or induced lead to cell, organ or whole plant death. Gene variants
that influence these pathways can have profound effects on plant
survival, vigor and performance. The critical pathways include
those concerned with metabolism and development or protection
against stresses, diseases and pests. They also include those
involved in apoptosis and necrosis. The applicants have elucidated
many such genes and pathways by discovering genes that when
inactivated lead to cell or plant death.
[0643] Herbicides are, by definition, chemicals that cause death of
tissues, organs and whole plants. The genes and pathways that are
activated or inactivated by herbicides include those that cause
cell death as well as those that function to provide protection.
The applicants have elucidated these genes.
[0644] The genes defined in this section have many uses including
manipulating which cells, tissues and organs are selectively
killed, which are protected, making plants resistant to herbicides,
discovering new herbicides and making plants resistant to various
stresses.
[0645] Viability genes were also identified from a much larger set
of genes by experiments designed to find genes whose mRNA products
changed in concentration in response to applications of different
herbicides to plants. Viability genes are characteristically
differentially transcribed in response to fluctuating herbicide
levels or concentrations, whether internal or external to an
organism or cell. The MA_diff Table reports the changes in
transcript levels of various viability genes.
Early Seedling-Phase Specific Responsive Genes, Gene Components and
Products
[0646] One of the more active stages of the plant life cycle is a
few days after germination is complete, also referred to as the
early seedling phase. During this period the plant begins
development and growth of the first leaves, roots, and other organs
not found in the embryo. Generally this stage begins when
germination ends. The first sign that germination has been
completed is usually that there is an increase in length and fresh
weight of the radicle. Such genes and gene products can regulate a
number of plant traits to modulate yield. For example, these genes
are active or potentially active to a greater extent in developing
and rapidly growing cells, tissues and organs, as exemplified by
development and growth of a seedling 3 or 4 days after planting a
seed.
[0647] Rapid, efficient establishment of a seedling is very
important in commercial agriculture and horticulture. It is also
vital that resources are approximately partitioned between shoot
and root to facilitate adaptive growth. Phototropism and geotropism
need to be established. All these require post-germination process
to be sustained to ensure that vigorous seedlings are produced.
Early seedling phase genes, gene components and products are useful
to manipulate these and other processes.
Guard Cell Genes, Gene Components and Products
[0648] Scattered throughout the epidermis of the shoot are minute
pores called stomata. Each stomal pore is surrounded by two guard
cells. The guard cells control the size of the stomal pore, which
is critical since the stomata control the exchange of carbon
dioxide, oxygen, and water vapor between the interior of the plant
and the outside atmosphere. Stomata open and close through turgor
changes driven by ion fluxes, which occur mainly through the guard
cell plasma membrane and tonoplast. Guard cells are known to
respond to a number of external stimuli such as changes in light
intensity, carbon dioxide and water vapor, for example. Guard cells
can also sense and rapidly respond to internal stimuli including
changes in ABA, auxin and calcium ion flux.
[0649] Thus, genes, gene products, and fragments thereof
differentially transcribed and/or translated in guard cells can be
useful to modulate ABA responses, drought tolerance, respiration,
water potential, and water management as examples. All of which can
in turn affect plant yield including seed yield, harvest index,
fruit yield, etc.
[0650] To identify such guard cell genes, gene products, and
fragments thereof, Applicants have performed a microarray
experiment comparing the transcript levels of genes in guard cells
versus leaves. Experimental data is shown below.
Nitric Oxide Responsive Genes, Gene Components and Products
[0651] The rate-limiting element in plant growth and yield is often
its ability to tolerate suboptimal or stress conditions, including
pathogen attack conditions, wounding and the presence of various
other factors. To combat such conditions, plant cells deploy a
battery of inducible defense responses, including synergistic
interactions between nitric oxide (NO), reactive oxygen
intermediates (ROS), and salicylic acid (SA). NO has been shown to
play a critical role in the activation of innate immune and
inflammatory responses in animals. At least part of this mammalian
signaling pathway is present in plants, where NO is known to
potentiate the hypersensitive response (HR). In addition, NO is a
stimulator molecule in plant photomorphogenesis.
[0652] Changes in nitric oxide concentration in the internal or
surrounding environment, or in contact with a plant, results in
modulation of many genes and gene products.
[0653] In addition, the combination of a nitric oxide responsive
polynucleotide and/or gene product with other environmentally
responsive polynucleotides is also useful because of the
interactions that exist between hormone regulated pathways, stress
pathways, pathogen stimulated pathways, nutritional pathways and
development.
[0654] Nitric oxide responsive genes and gene products can function
either to increase or dampen the above phenotypes or activities
either in response to changes in nitric oxide concentration or in
the absence of nitric oxide fluctuations. More specifically, these
genes and gene products can modulate stress responses in an
organism. In plants, these genes and gene products are useful for
modulating yield under stress conditions. Measurements of yield
include seed yield, seed size, fruit yield, fruit size, etc.
Shoot-Apical Meristem Genes, Gene Components and Products
[0655] New organs, stems, leaves, branches and inflorescences
develop from the stem apical meristem (SAM). The growth structure
and architecture of the plant therefore depends on the behavior of
SAMs. Shoot apical meristems (SAMs) are comprised of a number of
morphologically undifferentiated, dividing cells located at the
tips of shoots. SAM genes elucidated here are capable of modifying
the activity of SAMs and thereby many traits of economic interest
from ornamental leaf shape to organ number to responses to plant
density.
[0656] In addition, a key attribute of the SAM is its capacity for
self-renewal. Thus, SAM genes of the instant invention are useful
for modulating one or more processes of SAM structure and/or
function including (I) cell size and division; (II) cell
differentiation and organ primordia. The genes and gene components
of this invention are useful for modulating any one or all of these
cell division processes generally, as in timing and rate, for
example. In addition, the polynucleotides and polypeptides of the
invention can control the response of these processes to the
internal plant programs associated with embryogenesis, and hormone
responses, for example.
[0657] Because SAMs determine the architecture of the plant,
modified plants will be useful in many agricultural, horticultural,
forestry and other industrial sectors. Plants with a different
shape, numbers of flowers and seed and fruits will have altered
yields of plant parts. For example, plants with more branches can
produce more flowers, seed or fruits. Trees without lateral
branches will produce long lengths of clean timber. Plants with
greater yields of specific plant parts will be useful sources of
constituent chemicals.
GFP Experimental Procedures and Results
Procedures
[0658] The polynucleotide sequences of the present invention were
tested for promoter activity using Green Fluorescent Protein (GFP)
assays in the following manner.
[0659] Approximately 1-2 kb of genomic sequence occurring
immediately upstream of the ATG translational start site of the
gene of interest was isolated using appropriate primers tailed with
BstXI restriction sites. Standard PCR reactions using these primers
and genomic DNA were conducted. The resulting product was isolated,
cleaved with BstXI and cloned into the BstXI site of an appropriate
vector, such as pNewBin4-HAP1-GFP (see FIG. 1).
[0660] Transformation
[0661] The following procedure was used for transformation of
plants
1. Stratification of WS-2 Seed.
[0662] Add 0.5 ml WS-2 (CS2360) seed to 50 ml of 0.2% Phytagar in a
50 ml Corning tube and vortex until seeds and Phytagar form a
homogenous mixture. [0663] Cover tube with foil and stratify at
4.degree. C. for 3 days.
2. Preparation of Seed Mixture.
[0663] [0664] Obtain stratified seed from cooler. [0665] Add seed
mixture to a 1000 ml beaker. [0666] Add an additional 950 ml of
0.2% Phytagar and mix to homogenize.
3. Preparation of Soil Mixture.
[0666] [0667] Mix 24 L SunshineMix #5 soil with 16 L Therm-O-Rock
vermiculite in cement mixer to make a 60:40 soil mixture. [0668]
Amend soil mixture by adding 2 Tbsp Marathon and 3 Tbsp Osmocote
and mix contents thoroughly. [0669] Add 1 Tbsp Peters fertilizer to
3 gallons of water and add to soil mixture and mix thoroughly.
[0670] Fill 4-inch pots with soil mixture and round the surface to
create a slight dome. [0671] Cover pots with 8-inch squares of
nylon netting and fasten using rubber bands. [0672] Place 14 4-inch
pots into each no-hole utility flat.
4. Planting.
[0672] [0673] Using a 60 ml syringe, aspirate 35 ml of the seed
mixture. [0674] Exude 25 drops of the seed mixture onto each pot.
[0675] Repeat until all pots have been seeded. [0676] Place flats
on greenhouse bench, cover flat with clear propagation domes, place
55% shade cloth on top of flats and subirrigate by adding 1 inch of
water to bottom of each flat.
5. Plant Maintenance.
[0676] [0677] 3 to 4 days after planting, remove clear lids and
shade cloth. [0678] Subirrigate flats with water as needed. [0679]
After 7-10 days, thin pots to 20 plants per pot using forceps.
[0680] After 2 weeks, subirrigate all plants with Peters fertilizer
at a rate of 1 Tsp per gallon water. [0681] When bolts are about
5-10 cm long, clip them between the first node and the base of stem
to induce secondary bolts. [0682] 6 to 7 days after clipping,
perform dipping infiltration.
6. Preparation of Agrobacterium.
[0682] [0683] Add 150 ml fresh YEB to 250 ml centrifuge bottles and
cap each with a foam plug
[0684] (Identi-Plug). [0685] Autoclave for 40 min at 121.degree. C.
[0686] After cooling to room temperature, uncap and add 0.1 ml each
of carbenicillin, spectinomycin and rifampicin stock solutions to
each culture vessel. [0687] Obtain Agrobacterium starter block
(96-well block with Agrobacterium cultures grown to an OD.sub.600
of approximately 1.0) and inoculate one culture vessel per
construct by transferring 1 ml from appropriate well in the starter
block. [0688] Cap culture vessels and place on Lab-Line incubator
shaker set at 27.degree. C. and 250 RPM. [0689] Remove after
Agrobacterium cultures reach an OD.sub.600 of approximately 1.0
(about 24 hours), cap culture vessels with plastic caps, place in
Sorvall SLA 1500 rotor and centrifuge at 8000 RPM for 8 min at
4.degree. C. [0690] Pour out supernatant and put bottles on ice
until ready to use. [0691] Add 200 ml Infiltration Media (IM) to
each bottle, resuspend Agrobacterium pellets and store on ice.
7. Dipping Infiltration.
[0691] [0692] Pour resuspended Agrobacterium into 16 oz
polypropylene containers. [0693] Invert 4-inch pots and submerge
the aerial portion of the plants into the Agrobacterium suspension
and let stand for 5 min [0694] Pour out Agrobacterium suspension
into waste bucket while keeping polypropylene container in place
and return the plants to the upright position. [0695] Place 10
covered pots per flat. [0696] Fill each flat with 1-inch of water
and cover with shade cloth. [0697] Keep covered for 24 hr and then
remove shade cloth and polypropylene containers. [0698] Resume
normal plant maintenance. [0699] When plants have finished
flowering cover each pot with a ciber plant sleeve. [0700] After
plants are completely dry, collect seed and place into 2.0 ml micro
tubes and store in 100-place cryogenic boxes.
RECIPES:
0.2% Phytagar
[0701] 2 g Phytagar
[0702] 1 L nanopure water [0703] Shake until Phytagar suspended
[0704] Autoclave 20 min
YEB (for 1 L)
[0705] 5 g extract of meat
[0706] 5 g Bacto peptone
[0707] 1 g yeast extract
[0708] 5 g sucrose
[0709] 0.24 g magnesium sulfate [0710] While stirring, add
ingredients, in order, to 900 ml nanopure water [0711] When
dissolved, adjust pH to 7.2 [0712] Fill to 1 L with nanopure water
[0713] Autoclave 35 min
Infiltration Medium (IM) (for 1 L)
[0714] 2.2 g MS salts
[0715] 50 g sucrose
[0716] 5 ul BAP solution (stock is 2 mg/ml) [0717] While stirring,
add ingredients in order listed to 900 ml nanopure water [0718]
When dissolved, adjust pH to 5.8. [0719] Volume up to 1 L with
nanopure water. [0720] Add 0.02% Silwet L-77 just prior to
resuspending Agrobacterium
[0721] High Throughput Screening--T1 Generation
1. Soil Preparation. Wear gloves at all times. [0722] In a large
container, mix 60% autoclaved SunshineMix #5 with 40% vermiculite.
[0723] Add 2.5 Tbsp of Osmocote, and 2.5 Tbsp of 1% granular
Marathon per 25 L of soil. [0724] Mix thoroughly.
2. Fill Com-Packs With Soil.
[0724] [0725] Loosely fill D601 Com-Packs level to the rim with the
prepared soil. [0726] Place filled pot into utility flat with
holes, within a no-hole utility flat. [0727] Repeat as necessary
for planting. One flat set should contain 6 pots.
3. Saturate Soil.
[0727] [0728] Evenly water all pots until the soil is saturated and
water is collecting in the bottom of the flats. [0729] After the
soil is completely saturated, dump out the excess water.
4. Plant the Seed.
5. Stratify the Seeds.
[0729] [0730] After sowing the seed for all the flats, place them
into a dark 4.degree. C. cooler. [0731] Keep the flats in the
cooler for 2 nights for WS seed. Other ecotypes may take longer.
This cold treatment will help promote uniform germination of the
seed. 6. Remove Flats From Cooler and Cover With Shade Cloth.
(Shade cloth is only needed in the greenhouse) [0732] After the
appropriate time, remove the flats from the cooler and place onto
growth racks or benches. [0733] Cover the entire set of flats with
55% shade cloth. The cloth is necessary to cut down the light
intensity during the delicate germination period. [0734] The cloth
and domes should remain on the flats until the cotyledons have
fully expanded. This usually takes about 4-5 days under standard
greenhouse conditions.
7. Remove 55% Shade Cloth and Propagation Domes.
[0734] [0735] After the cotyledons have fully expanded, remove both
the 55% shade cloth and propagation domes. 8. Spray Plants With
Finale Mixture. Wear gloves and protective clothing at all times.
[0736] Prepare working Finale mixture by mixing 3 ml concentrated
Finale in 48 oz of water in the Poly-TEK sprayer. [0737] Completely
and evenly spray plants with a fine mist of the Finale mixture.
[0738] Repeat Finale spraying every 3-4 days until only
transformants remain. (Approximately 3 applications are necessary.)
[0739] When satisfied that only transformants remain, discontinue
Finale spraying.
9. Weed Out Excess Transformants.
[0740] Weed out excess transformants such that a maximum number of
five plants per pot exist evenly spaced throughout the pot.
[0741] GFP Assay
[0742] Tissues are dissected by eye or under magnification using
INOX 5 grade forceps and placed on a slide with water and
coversliped. An attempt is made to record images of observed
expression patterns at earliest and latest stages of development of
tissues listed below. Specific tissues will be preceded with High
(H), Medium (M), Low (L) designations.
TABLE-US-00002 Flower pedicel receptacle nectary sepal petal
filament anther pollen carpel style papillae vascular epidermis
stomata trichome Silique stigma style carpel septum placentae
transmitting tissue vascular epidermis stomata abscission zone
ovule Ovule Pre-fertilization: inner integument outer integument
embryo sac funiculus chalaza micropyle gametophyte
Post-fertilization: zygote inner integument outer integument seed
coat primordia chalaza micropyle early endosperm mature endosperm
embryo Embryo suspensor preglobular globular heart torpedo late
mature provascular hypophysis radicle cotyledons hypocotyl Stem
epidermis cortex vascular xylem phloem pith stomata trichome Leaf
petiole mesophyll vascular epidermis trichome primordia stomata
stipule margin
[0743] T1 Mature: These are the T1 plants resulting from
independent transformation events. These are screened between stage
6.50-6.90 (means the plant is flowering and that 50-90% of the
flowers that the plant will make have developed) which is 4-6 weeks
of age. At this stage the mature plant possesses flowers, siliques
at all stages of development, and fully expanded leaves. We do not
generally differentiate between 6.50 and 6.90 in the report but
rather just indicate 6.50. The plants are initially imaged under UV
with a Leica Confocal microscope. This allows examination of the
plants on a global level. If expression is present, they are imaged
using scanning laser confocal micsrocopy.
[0744] T2 Seedling: Progeny are collected from the T1 plants giving
the same expression pattern and the progeny (T2) are sterilized and
plated on agar-solidified medium containing M&S salts. In the
event that there was no expression in the T1 plants, T2 seeds are
planted from all lines. The seedlings are grown in Percival
incubators under continuous light at 22.degree. C. for 10-12 days.
Cotyledons, roots, hypocotyls, petioles, leaves, and the shoot
meristem region of individual seedlings were screened until two
seedlings were observed to have the same pattern. Generally found
the same expression pattern was found in the first two seedlings.
However, up to 6 seedlings were screened before "no expression
pattern" was recorded. All constructs are screened as T2 seedlings
even if they did not have an expression pattern in the T1
generation.
[0745] T2 Mature: The T2 mature plants were screened in a similar
manner to the T1 plants. The T2 seeds were planted in the
greenhouse, exposed to selection and at least one plant screened to
confirm the T1 expression pattern. In instances where there were
any subtle changes in expression, multiple plants were examined and
the changes noted in the tables.
[0746] T3 Seedling: This was done similar to the T2 seedlings
except that only the plants for which we are trying to confirm the
pattern are planted.
Image Data:
[0747] Images are collected by scanning laser confocal microscopy.
Scanned images are taken as 2-D optical sections or 3-D images
generated by stacking the 2-D optical sections collected in series.
All scanned images are saved as TIFF files by imaging software,
edited in Adobe Photoshop, and labeled in Powerpoint specifying
organ and specific expressing tissues.
Instrumentation:
Microscope
Inverted Leica DM IRB
[0748] Fluorescence filter blocks: Blue excitation BP 450-490; long
pass emission LP 515. Green excitation BP 515-560; long pass
emission LP 590
Objectives
HCPL FLUOTAR 5.times./0.5
[0749] HCPL APO 10.times./0.4 IMM water/glycerol/oil HCPL APO
20.times./0.7 IMM water/glycerol/oil HCXL APO 63.times./1.2 IMM
water/glycerol/oil
Leica TCS SP2 Confocal Scanner
[0750] Spectral range of detector optics 400-850 nm Variable
computer controlled pinhole diameter. Optical zoom 1-32.times..
Four simultaneous detectors: Three channels for collection of
fluorescence or reflected light. One channel for transmitted light
detector. Laser sources: Blue Ar 458/5 mW, 476 nm15 mW, 488 nm/20
mW, 514 nm/20 mW. Green HeNe 543 nm/1.2 mW Red HeNe 633 nm/10
mW
Results
[0751] The section in Table 1 entitled "The spatial expression of
the promoter-marker-vector" presents the results of the GFP assays
as reported by their corresponding cDNA ID number, construct number
and line number. Unlike the microarray results, which measure the
difference in expression of the endogenous cDNA under various
conditions, the GFP data gives the location of expression that is
visible under the imaging parameters. Table 3 summarizes the
results of the spatial expression results for each promoter.
Explanation of Table 1
[0752] Table 1 includes various information about each promoter or
promoter control element of the invention including the nucleotid
sequence, the spatial expression promoted by each promoter, and the
corresponding results from different expression experiments.
TABLE-US-00003 Lengthy table referenced here
US20130117881A1-20130509-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-00004 Lengthy table referenced here
US20130117881A1-20130509-T00002 Please refer to the end of the
specification for access instructions.
TABLE-US-00005 Lengthy table referenced here
US20130117881A1-20130509-T00003 Please refer to the end of the
specification for access instructions.
[0753] The invention being thus described, it will be apparent to
one of ordinary skill in the art that various modifications of the
materials and methods for practicing the invention can be made.
Such modifications are to be considered within the scope of the
invention as defined by the following claims.
[0754] Each of the references from the patent and periodical
literature cited herein is hereby expressly incorporated in its
entirety by such citation.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130117881A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
TABLE-US-00006 MEGA
* * * * *
References