# Patent

## Title
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.

## Background
This Nonprovisional application claims priority under 35 U.S.C. § 119(e) on U.S. Provisional Application No(s). 60/612,891; 60/613,134; and 60/637,174 filed on Sep. 23, 2004; Sep. 23, 2004 and Dec. 16, 2004, the entire contents of which are hereby incorporated by reference.
This application contains one (1) CDR submitted in duplicate (totaling 3 CDs), created using IBM-PC MS-Windows operating system. The CD-Rs contain the following file(s):












File Name
Date of Creation
File Size







Sequence Listing 2750-1619PUS2.txt
Sep. 23, 2005
64 KB











FIELD OF THE INVENTION
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
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.
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
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.
It is an object of the present invention to provide isolated polynucleotides that are promoter sequences. These promoter sequences comprise, for example,

    
    
        (1) a polynucleotide having a nucleotide sequence as set forth in the Sequence Listing or a fragment thereof;
        (2) a polynucleotide having a nucleotide sequence having at least 80% sequence identity to a sequence as set forth in the Sequence Listing or a fragment thereof; and
        (3) a polynucleotide having a nucleotide sequence which hybridizes to a sequence as set forth in the Sequence Listing under a condition establishing a Tm-20° C.
    
    


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,

    
    
        (1) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NOs: 1-46 or a fragment thereof;
        (2) a polynucleotide having a nucleotide sequence having at least 80% sequence identity to a sequence as set forth in SEQ ID NOs: 1-46 or fragment thereof; and
        (3) a polynucleotide having a nucleotide sequence which hybridizes to a sequence as set forth in SEQ ID NOs: 1-46 under a condition establishing a Tm-20° C.
    
    


Promoter or promoter control element sequences of the present invention are capable of modulating preferential transcription.
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.
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.
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.
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.
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-.
In yet another embodiment, the present host cell is a plant cell capable of regenerating into a plant.
It is yet another embodiment of the present invention to provide a plant comprising an isolated polynucleotide or vector described above.
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.
In another embodiment of the present method, the polynucleotide or vector preferentially modulates
(a) constitutive transcription,
(b) stress induced transcription,
(c) light induced transcription,
(d) dark induced transcription,
(e) leaf transcription,
(f) root transcription,
(g) stem or shoot transcription,
(h) silique transcription,
(i) callus transcription,
(j) flower transcription,
(k) immature bud and inflorescence specific transcription, or
(l) senescing induced transcription
(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 FIGURES
FIG. 1
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:

    Ori—the origin of replication used by an E. coli host
    RB—sequence for the right border of the T-DNA from pMOG800
    BstXI—restriction enzyme cleavage site used for cloning
    HAP1VP16—coding sequence for a fusion protein of the HAP1 and VP16 activation domains
    NOS—terminator region from the nopaline synthase gene
    HAP1UAS—the upstream activating sequence for HAP1
    5ERGFP—the green fluorescent protein gene that has been optimized for localization to the endoplasmic reticulum
    OCS2—the terminator sequence from the octopine synthase 2 gene
    OCS—the terminator sequence from the octopine synthase gene
    p28716 (a.k.a 28716 short)—promoter used to drive expression of the PAT (BAR) gene
    PAT (BAR)—a marker gene conferring herbicide resistance
    LB—sequence for the left border of the T-DNA from pMOG800
    Spec—a marker gene conferring spectinomycin resistance
    TrfA—transcription repression factor gene
    RK2-OriV—origin of replication for Agrobacterium 

FIG. 2



FIG. 2 is a schematic representation of the vector PT0678. The definitions of the abbreviations used in the vector map are as described above.
FIG. 3
FIG. 3 is a schematic of a gene.



DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
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.
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) 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.
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).
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.
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.
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.
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 T0 for the primary transgenic plant and T1 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.
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 (see FIG. 3). 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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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 and nucleotide sequences. Such sequences are publicly accessible, for example, on the BLAST databases on the NCBI FTP web site (accessible via the internet). 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).
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.
Related Sequences: “Related sequences” refer to either a polypeptide or a nucleotide sequence that exhibits some degree of sequence similarity with a reference sequence.
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”.
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 Tm, which is the temperature at which 50% of the complementary molecules in the hybridization are hybridized, in terms of a temperature differential from Tm. High stringency conditions are those providing a condition of Tm−5° C. to Tm−10° C. Medium or moderate stringency conditions are those providing Tm−20° C. to Tm−29° C. Low stringency conditions are those providing a condition of Tm−40° C. to Tm−48° C. The relationship of hybridization conditions to Tm (in ° C.) is expressed in the mathematical equation

Tm=81.5−16.6(log10[Na+])+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 Tm 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).

Tm=81.5+16.6 log {[Na+]/(1+0.7[Na+])}+0.41(% G+C)−500/L 0.63(% 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 Tm of equation (2) is affected by the nature of the hybrid; for DNA-RNA hybrids Tm is 10-15° C. higher than calculated, for RNA-RNA hybrids Tm is 20-25° C. higher. Because the Tm decreases about 1° 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.

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.
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° C. below Tm, medium or moderate stringency is 26-29° C. below Tm and low stringency is 45-48° C. below Tm.
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.
Suppressor: See “Enhancer/Suppressor”
TATA to start: “TATA to start” shall mean the distance, in number of nucleotides, between the primary TATA motif and the start of transcription.
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.
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.
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.
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.
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.
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
The polynucleotides of the invention comprise promoters and promoter control elements that are capable of modulating transcription.
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.
Specifically, promoters and control elements of the invention can be used to modulate transcription of a desired polynucleotide, which includes without limitation:

    
    
        (a) antisense;
        (b) ribozymes;
        (c) coding sequences; or
        (d) fragments thereof.

The promoter also can modulate transcription in a host genome in cis- or in trans-.

    
    


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
The following description of the present invention is outlined in the following table of contents.
A. Identifying and Isolating Promoter Sequences of the Invention

    
    
        (1) Cloning Methods
        (2) Chemical Synthesis
    
    


B. Isolating Related Promoter Sequences

    
    
        (1) Relatives Based on Nucleotide Sequence Identity
    
    


C. Promoter Control Elements of the Invention

    
    
        (1) Promoter Control Element Configuration
        (2) Those Identifiable by Bioinformatics
        (3) Those Identifiable by In Vitro and In Vivo Assays
        (4) Non-Natural Control Elements
    
    


D. Constructing Promoters and Control Elements

    
    
        (1) Combining Promoters and Promoter Control Elements
        (2) Number of Promoter Control Elements
        (3) Spacing Between Control Elements
        (4) Other Promoters
    
    


E. Vectors

    
    
        (1) Modification of Transcription by Promoters and Promoter Control Elements
        (2) Polynucleotide to be Transcribed
        (3) Other Regulatory Elements
        (4) Other Components of Vectors
    
    


F. Insertion of Polynucleotides and Vectors Into a Host Cell

    
    
        (1) Autonomous of the Host Genome
        (2) Integrated into the Host Genome
    
    


G. Utility
A. Identiying and Isolating Promoter Sequences of the Invention
The promoters and promoter control elements of the present invention are presented in SEQ ID NOs: 1-46 and were identified from Arabidopsis thaliana or Oryza sativa. Additional promoter sequences encompassed by the invention can be identified as described below.
(1) Cloning Methods
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.
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, 2nd Ed. (1989) Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), for example.
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.
(2) Chemical Synthesis
In addition, the promoters and promoter control elements described in SEQ ID NOs: 1-46 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.
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.
Synthetic RNA, including natural and/or analog building blocks, can be synthesized on the Biosearch 8600 machines, see above.
Oligonucleotides can be synthesized and then ligated together to construct the desired polynucleotide.
B. Isolating Related Promoter Sequences
Included in the present invention are promoter and promoter control elements that are related to those described in SEQ ID NOs: 1-46. Such a related sequence can be isolated utilizing
(a) nucleotide sequence identity;
(b) coding sequence identity; or
(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.
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.
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.
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.
(1) Relatives Based on Nucleotide Sequence Identity
Included in the present invention are promoters exhibiting nucleotide sequence identity to those described in SEQ ID NOs: 1-46.
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 SEQ ID NOs: 1-46. Such sequence identity can be calculated by the algorithms and computers programs described above.
Usually, such sequence identity is exhibited in an alignment region that is at least 75% of the length of a sequence shown in SEQ ID NOs: 1-46; 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”.
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 SEQ ID NOs: 1-46.
These related promoters may exhibit similar preferential transcription as those promoters described in Table 1 in the section entitled “The predicted promoter sequence”.
Construction of Polynucleotides
Naturally occurring promoters that exhibit nucleotide sequence identity to those shown in SEQ ID NOs: 1-46 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.
Non-natural promoter variants of those shown in SEQ ID NOs: 1-46 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.
Any related promoter showing sequence identity to those shown in Table can be chemically synthesized as described above.
Also, the present invention includes non-natural promoters that exhibit the above-sequence identity to those in SEQ ID NOs: 1-46.
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.
Testing of Polynucleotides
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.
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

    (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);
    (b) YAC: Burke et al., Science 236:806-812 (1987);
    (c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA. January;87(1):103-7 (1990);
    (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl Acids Res 23: 4850-4856 (1995);
    (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
    (g) Plasmid vectors: Sambrook et al., infra.


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 Fluroescent 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.
C. Promoter Control Elements of the Invention
The promoter control elements of the present invention include those that comprise a sequence shown in SEQ ID NOs: 1-46 and fragments thereof. The size of the fragments 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.
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.
(1) Promoter Control Element Configuration
A common configuration of the promoter control elements in RNA polymerase II promoters can be found in “Models for prediction and recognition of eukaryotic promoters”, T. Werner, Mammalian Genome, 10, 168-175 (1999).
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 TFIIB, TFIID, and TFIIE. Of these, TFIID 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.
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.
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.
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.
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).
(2) Those Identifiable by Bioinformatics
Promoter control elements from the promoters of the instant invention can be identified utilizing bioinformatic or computer driven techniques.
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 program identifies common sequence motifs in such genes. See, Roth et al., Nature Biotechnol. 16: 949-945 (1998); Tavazoie et al., Nat Genet 1999 Jul;22(3):281-5;
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.
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).
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.
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; MatInspector, Ghosh et al., Nucl. Acid Res. 21: 3117-3118 (1993) available via the internet and ConsInspector, Frech et al., Nucl. Acids Res. 21: 1655-1664 (1993), available via the internet; TFSearch; and TESS.
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.
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).
(3) Those Identifiable by In-Vitro and In-Vivo Assays
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.
In-Vitro Assays
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. (1989) Annu. Rev. Biochem. 58:799-839).
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)).
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 a., 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.
Cell-free transcription extracts can be prepared and used to directly assay in a reconstitutable system (Narayan et al., Biochemistry 39:818 (2000)).
In-Vivo Assays
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.
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.
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. (1979) Nature 277:324-325). 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. (1979) J. Bact. 140:400-407) and other bacterial hosts (Band, L. et al. (1983) Gene 26:313-315; Achen, M. G., Gene 45:45-49 (1986)), yeast (Goodey, A. R. et al. (1986) Mol. Gen. Genet. 204:505-511) and mammalian cells (Pater, M. M. et al. (1984) J. Mol. App. Gen. 2:363-371).
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 β-galatactosidase expression using the chromogenic substrate, X-gal.
A general review of tools for identifying transcriptional regulatory regions of genomic DNA is provided by J. W. Fickett et al. (2000) (Curr. Opn. Biotechnol. 11:19).
(4) Non-Natural Control Elements
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
(1) Combining Promoters and Promoter Control Elements
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.
Fragments, variants, as well as the full-length sequences of SEQ ID NOs: 1-46 and relatives are useful alone or in combination.
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.
(2) Number of Promoter Control Elements
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.
(3) Spacing Between Control Elements
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.
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.
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.
Typically, the spacing is no smaller than 5 bases; more typically, no smaller than 8; 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.
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.
Such spacing between promoter control elements can be determined using the techniques and assays described above.
(4) Other Promoters
The following are promoters 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).
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).
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 ACT111 (Arabidopsis; Huang et al. (1997) Plant Mol Biol 33:125-39) for inflorescence.
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 Biol 41:443-54), 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. (1995) 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
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.
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. (1985) Nature 317: 741-744; Gordon-Kamm et al. (1990) Plant Cell 2: 603-618; and Stalker et al. (1988) Science 242: 419-423). Other marker genes exist which provide hormone responsiveness.
(1) Modification of Transcription by Promoters and Promoter Control Elements
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.
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.
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).
(2) Polynucleotide to Be Transcribed
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.
Specific coding sequences may include, but are not limited to endogenous proteins or fragments thereof, or heterologous proteins including marker genes or fragments thereof.
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 (str1), 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.
(3) Other Regulatory Elements
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.
(4) Other Components of Vectors
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.
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.
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.
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 and 5,436,391; see also and Murray et al., (1989) Nucleic Acids Res. 17:477-498.
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.
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
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.
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.
(1) Polynucleotides Autonomous of the Host Genome
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.
Additionally, in some cases transient expression of a polynucleotide may be desired.
(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 10 Sprague et al. (Eds. pp. 345-387) American Society of Agronomy Inc. et al. 1988.
Methods of introducing polynucleotides into plant tissue include the direct infection or co-cultivation of plant cell with Agrobacterium tumefaciens, Horsch et al. (1985) Science, 227:1229. Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer provided by Gruber et al. supra.
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).
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 β-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.
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. (1998) Plant Mol. Biol. 38:393).
G. Utility
Common Uses
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.
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 (see Lindsey et al. (1993) Transgenic Research 2:3347 and Auch & Reth, et al. Nucleic Acids Research, 18: 674).
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.
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 IVET 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. (1993) Science 259:686-688, Mahan et al. (1995) PNAS USA 92:669-673, Heithoff et al. (1997) PNAS USA 94:934-939, and Wanget al. (1996) PNAS USA. 93:10434.
Constitutive Transcription
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.
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.
Typically, promoter or control elements that provide constitutive transcription produce transcription levels that are statistically similar in many tissues and environmental conditions observed.
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:



  
    
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where σ(S1 . . . S11, not including Sx)=the standard deviation of all sample intensities except Sx.
The P-value from the formula ranges from 1.0 to 0.0.
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 element is greater than 10−8; more usually, greater than 10−; even more usually, greater than 10−6; even more usually, greater than 10−5 or 10−4.
For up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Stress Induced Preferential Transcription
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.
Drought induction of genes, transcripts, and/or polypeptides is 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.
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 Mol Biol 1257-65), pyruvate decarboxylase (oxygen stress; rice; see Rivosal et al. (1997) Plant Physiol. 114(3): 1021-29) and chromoplast specific carotenoid gene (oxidative stress; capsicum; see Bouvier et al. (1998) Journal of Biological Chemistry 273:30651-59).
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.
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) Plant J 14(1): 137-42), hepatocyte growth factor activator inhibitor type 1 (HAI-1), which enhances tissue regeneration (tissue injury; human; Koono et al. (1999) J Histochem and Cytochem 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) and 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).
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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Light Induced Preferential Transcription
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

    
    
        (1) to increase the photosynthetic rate;
        (2) to increase storage of certain molecules in leaves or green parts only, e.g., silage with high protein or starch content;
        (3) to modulate production of exogenous compositions in green tissue, e.g., certain feed enzymes;
        (4) to induce growth or development, such as fruit development and maturity, during extended exposure to light;
        (5) to modulate guard cells to control the size of stomata in leaves to prevent water loss, or
        (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) Plant Cell 12: 35-52), asparagine synthetase (pea root nodules, see Tsai and Coruzzi (1990) EMBO J 9: 323-32), mdm2 gene (human tumor; see Saucedo et al. (1998) Cell Growth Differ 9: 119-30).

    
    


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.
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).
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Dark Induced Preferential Transcription
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,

    
    
        (1) to induce growth or development, such as fruit development and maturity, despite lack of light;
        (2) to modulate genes, transcripts, and/or polypeptide active at night or on cloudy days; or
        (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.

    
    


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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Leaf Preferential Transcription
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,
(1) to modulate leaf size, shape, and development;
(2) to modulate the number of leaves ; or
(3) to modulate energy or nutrient usage in relation to other organs and tissues
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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Root Preferential Transcription
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
(1) to modulate root size, shape, and development;
(2) to modulate the number of roots, or root hairs;
(3) to modulate mineral, fertilizer, or water uptake;
(4) to modulate transport of nutrients; or
(4) to modulate energy or nutrient usage in relation to other organs and tissues.
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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Stem/Shoot Preferential Transcription
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,
(1) to modulate stem/shoot size, shape, and development; or
(2) to modulate energy or nutrient usage in relation to other organs and tissues
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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Fruit and Seed Preferential Transcription
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

    
    
        (1) to modulate fruit size, shape, development, and maturity;
        (2) to modulate the number of fruit or seeds;
        (3) to modulate seed shattering;
        (4) to modulate components of seeds, such as, storage molecules, starch, protein, oil, vitamins, anti-nutritional components, such as phytic acid;
        (5) to modulate seed and/or seedling vigor or viability;
        (6) to incorporate exogenous compositions into a seed, such as lysine rich proteins;
        (7) to permit similar fruit maturity timing for early and late blooming flowers; or
        (8) to modulate energy or nutrient usage in relation to other organs and tissues.
    
    


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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Callus Preferential Transcription
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.
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.
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.
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 10−4; more usually, less than 10−5; even more usually, less than 10−6; even more usually, less than 10−7 or 10−8.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Flower Specific Transcription
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,
(1) to modulate petal color; or
(2) to modulate the fertility of pistil and/or stamen.
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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Immature Bud and Inflorescence Preferential Transcription
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,
(1) to modulate embryo development, size, and maturity;
(2) to modulate endosperm development, size, and composition;
(3) to modulate the number of seeds and fruits; or
(4) to modulate seed development and viability.
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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Senescence Preferential Transcription
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.
In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptides during senescencing is useful to modulate fruit ripening.
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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Germination Preferential Transcription
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,
(1) to modulate the emergence of they hypocotyls, cotyledons and radical; or
(2) to modulate shoot and primary root growth and development;
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.
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.
For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
GFP Experimental Procedures and Results
Procedures
The polynucleotide sequences of the present invention are tested for promoter activity using Green Fluorescent Protein (GFP) assays in the following manner.
Approximately 1-2 kb of genomic sequence occurring immediately upstream of the ATG translational start site of the gene of interest is isolated using appropriate primers tailed with BstXI restriction sites. Standard PCR reactions using these primers and genomic DNA are conducted. The resulting product is isolated, cleaved with BstXI and cloned into the BstXI site of an appropriate vector, such as pNewBin4-HAP1-GFP (see FIG. 1).
Transformation
The following procedure is used for transformation of plants
1. Seed Preparation and Plant Growth.
A homogeneous mixture of Arabidopsis thaliana seed in a 0.2% Phytagar solution is inclubated at 4° C. in the dark for 3 days. Seed is planted in 4 inch pots in a soil miture of Sunshine Mix, Vermiculite, Marathon and Osmocote. Pots are placed in flats, covered with plastic domes and subsequently subirrigated. After 3 to 4 days, the domes are removed.
Seven to ten days after planting, seedlings are thinned to 20 plants per pot. When 5-10 cm long bolts appear, they are clipped between the first node and the stem base to induce secondary bolts. Six to 7 days after clipping, the plants are transformed via dipping infiltration.
2. Preparation of Agrobacterium. 
Each 4 inch pot is inverted and the aerial portion of the plants submerged into a 16 oz. polypropylene container holding 200 mls of Agrobacterium tumefaciens (1×107 bacteria) in Infiltration media (2.2 g MS salts, 50 g sucrose, 110 μg BAP and 0.02% Silwet L-77 per liter). After 5 minutes, the Agrobacterium solution is removed while keeping the polypropylene containiner in place and the pots returned to an upright position. Pots are then placed in flats (10 pots per flat) containing approximately 1 inch of water and covered with shade cloth. After 24 hours, the shade cloth and polypropylene containers are removed.
After flowering, each pot is covered with a ciber plant sleeve. When plants are completely dry, seed is collected and stored.
3. High Throughput Screening—T1 Generation
Transformed seed are placed in pots containing a water saturated soil miture of Sunshine Mix, Vermiculite, Marathon and Osmocote. Pots are then placed in flats and stored in the dark at 4° C. for at least 2 days. After transferring the flats from the cooler to the greenhouse, they are covered with 55% shade cloth and propagation domes. When the cotyledons are fully expanded the cloth and domes are removed.
Plants are sprayed with a solution of 3 ml concentrated Finale in 48 oz water. Spraying is repeated every 3-4 days until only transformants remain. Transformants are thinned to a maximum of 5 plants per pot.
4. GFP Assay
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.













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, primordial, chalaza,



miccropyle, 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, primordial, stomata, stipule, margin









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.
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 is 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° C. for 10-12 days. Cotyledons, roots, hypocotyls, petioles, leaves, and the shoot meristem region of individual seedlings are screened until two seedlings are observed to have the same pattern. Generally found the same expression pattern is found in the first two seedlings. However, up to 6 seedlings are screened before “no expression pattern” is recorded. All constructs are screened as T2 seedlings even if they did not have an expression pattern in the T1 generation.
T2 Mature: The T2 mature plants are screened in a similar manner to the T1 plants. The T2 seeds are planted in the greenhouse, exposed to selection and at least one plant screened to confirm the T1 expression pattern. In instances where there are any subtle changes in expression, multiple plants are examined and the changes noted in the tables.
T3 Seedling: This is done similar to the T2 seedlings except that only the plants for which we are trying to confirm the pattern are planted.
Image Data
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
An Inverted Leica DM IRB microscope is used with two Fluorescence filter blocks: (1) Blue excitation BP 450-490; long pass emission LP 515 and (2) Green excitation BP 515-560; long pass emission LP 590. The following objectives are used: HC PL FLUOTAR 5×/0.5, HCPL APO 10×/0.4 IMM water/glycerol/oil, HCPL APO 20×/0.7 IMM water/glycerol/oil and HCXL APO 63×/1.2 IMM water/glycerol/oil. A Leica TCS SP2 confocal scanner with a Spectral range of detector optics of 400-850 nm was used with a variable computer controlled pinhole diameter, an Optical zoom 1-32× and four simultaneous detectors: three channels for collection of fluorescence or reflected light and one channel for transmitted light detector. The laser sources are: (1) Blue Ar 458/5 mW, 476 nm/5 mW, 488 nm/20 mW, 514 nm/20 mW, (2) Green HeNe 543 mn/1.2 mW and (3) Red HeNe 633 nm/10 mW.
EXAMPLES
The Examples are organized by an Expression Report for each of the promoters of the invention. They provide the 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:

    
    
        T1: First generation transformant
        T2: Second generation transformant
        T3: Third generation transformant
        (L): low expression level
        (M): medium expression level
        (H): high expression level
    
    


Example 1












Promoter Expression Report #132


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
H pollen


Silique
H pollen tube H transmitting tissue


Primary Root
L epidermis







Observed expression pattern:


T1 mature: Highly specific GFP expression in mature pollen, hydration tube, attachment site and


germinating pollen tubes along placenta.


T2 seedling: Weak epidermal expression at root transition zone.


Expected expression pattern: Inducible promoter - induced by different forms of stress (e.g.,


drought, heat, cold).








Selection Criteria:
Microarray


Gene:
FAD-linked oxidoreductase family







GenBank: NM_101049 Arabidopsis thaliana FAD-linked oxidoreductase family (At1g11770)


mRNA, complete cds gi|18391313|ref|NM_101049.1|[18391313]








Source Promoter Organism:
Arabidopsis thaliana, Ws


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed.
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Cold
4 wks
T2
 2 Hr
2/0
No





24 Hr post 2 Hr 4 C
2/0
No


2. Heat
4 wks
T2
 2 Hr
2/0
No





 6 Hr
2/0
No





24 Hr post 2 Hr 42 C
2/0
No


3. Drought
4 wks
T2
 3 Hr air dry
2/0
No


4. Drought
4 wks
T2
 8 days no water
2/0
No







Inducible expression summary:


Treatment:  Time point induced:  Organs induced: Tissues induced:








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 3
  Events Expressing: n = 2







GFP expression as shown below








X Flower
pedicel receptacle nectary sepal petal filament anther H



pollen carpel style papillae vascular epidermis stomata



trichome



silique


X Silique
stigma style carpel septum placentae H transmitting tissue



vascular epidermis stomata abscission zone ovule H pollen



tube


Ovule
Pre-fertilization: inner integument outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack inner integument outer



integument endothelium 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


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-02: 2/6


Scheduled


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
L epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem








Promoter utility



Trait Area:
PG&D, water use and efficiency


Sub-trait Area:
Drought, heat, yield


Utility:
This promoter sequence can be used to modulate fertility



under heat or drought conditions.


Construct:
YP0390


Promoter candidate I.D:
11768778


cDNA I.D:
23495437


Lines expressing:
YP0390-05, -06









Example 2












Promoter Expression Report #176


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
   H pedicel H sepal H petal H filament M carpel M style H


epidermis M silique


Silique
M style M carpel H epidermis L ovule


Ovule
Post-fertilization: L outer integument


Embryo
M heart M torpedo M late


Stem
H vascular H pith


Hypocotyl
H epidermis


Cotyledon
H epidermis


Rosette Leaf
M epidermis


Primary Root
M epidermis L root hairs


Lateral root
M epidermis







Observed expression pattern:


T1 mature: GFP expressed throughout mature plant. In the flower, GFP is first expressed in


young buds at the pedicles and sepals, later in the mature flower, GFP expression extends to the


sepals, petals, and silique. GFP is not expressed in the anthers or stigma. High GFP expression is


observed in heart stage through mature embryos. Weak expression in outer integument of some


ovules. High GFP expression throughout vascular and pith regions of stem.


T2 seedling: High GFP expression throughout epidermal cells of seedling. No expression in root


caps or guard cells.








Expected expression pattern:
Shade Induced


Selection Criteria:
Microarray


Gene:
allergen V5/Tpx-1-related family protein







GenBank: NM_126057 Arabidopsis thaliana allergen V5/Tpx-1-related family protein


(At5g66590) mRNA, complete cds gi|30698237|ref|NM_126057.2|[30698237]








Source Promoter Organism:
Arabidopsis thaliana, Columbia ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  XT1 Mature XT2 Seedling T2 Mature T3 Seedling


Inductions completed.
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Far red
7 days
T2
 1 Hr
5/0
No


Far Red730 = 525 μW/cm2


24 Hr
5/0
No







Inducible expression summary:









Treatment:
Time point induced:
Organs induced:  Tissues induced:


1. Far red
1 Hr, 24 Hr
No differences observed.








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 6
  Events Expressing: n = 5







GFP Expression Detected








X Flower
H pedicel receptacle nectary H sepal H petal H filament anther



pollen M carpel M style papillae vascular H epidermis stomata



trichome



M silique


X Silique
stigma M style M carpel septum placentae transmitting tissue



vascular H epidermis stomata abscission zone L ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack inner



integument



L outer integument endothelium seed coat primordia chalaza



micropyle early endosperm mature endosperm embryo


X Embryo
suspensor preglobular globular M heart M torpedo M late



mature provascular hypophysis radicle cotyledons


X Stem
epidermis cortex H vascular xylem phloem H pith stomata



trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 4(6)


Event-02: 5(6)


GFP Expression Detected








X Hypocotyl
H epidermis cortex vascular xylem phloem stomata


X Cotyledon
mesophyll vascular H epidermis margin stomata hydathode


X Rosette Leaf
mesophyll vascular M epidermis trichome petiole primordia



stomata stipule margin hydathode


X Primary Root
M epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent columella root



cap



L root hairs


X Lateral root
M epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem








Promoter utility



Trait Area:
PG&D, nutrients, cold


Sub-trait Area:
Seed size, nitrogen use efficiency, cold tolerance


Utility:
This promoter sequence can be used to modulate seed size,







nitrogen use efficiency, and cold tolerance in seedlings.


Nitrogen inducible expression in broad range of tissues can


be useful for improving tolerance to low nitrogen.








Construct:
PT0681


Promoter candidate I.D:
15295979


cDNA I.D:
23541268 (12688858)


Lines expressing:
PT0681-01, -03, -04, -05, -06









Example 3












Promoter Expression Report #204


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Cotyledon
M hydathode


Primary Root
H epidermis H root hairs







Observed expression pattern:


T1 mature: No expression.


T2 seedling: GFP predominately expressed throughout root epidermal cells. Highest at GFP


expression near hypocotyl root transition zone decreasing toward root tip.








Expected expression pattern:
Shade Induced


Selection Criteria:
Microarray


Gene:
zinc finger (CCCH-type) family protein







GenBank: NM_123793 Arabidopsis thaliana zinc finger (CCCH-type) family protein


(At5g44260) mRNA, complete cds gi|30694483|ref|NM_123793.2|[30694483]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  XT1 Mature XT2 Seedling T2 Mature T3 Seedling


Inductions completed.
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Far red
7 days
T2
 1 Hr
2/0
No


Far Red730 = 525 μW/cm2


24 Hr
2/0
No







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Far red
No differences observed.


Far Red730 = 525 μW/cm2








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 3
  Events Expressing: n = 0


No GFP Expression Detected


T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 5/6


Event-02: 5/6


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


X Cotyledon
mesophyll vascular epidermis margin stomata



M hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap H root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem








Promoter utility



Trait Area:
Nutrients


Sub-trait Area:
Nitrogen


Utility:
This promoter sequence can be used to modulate tolerance to



high nitrogen.


Construct:
PT0675


Promoter candidate I.D:
15295952


cDNA I.D:
24418776 (OCKHAM3-CD)


Lines expressing:
PT0675-01, -02









Example 4












Promoter Expression Report #234


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Ovule
Post-fertilization: H chalaza


Hypocotyl
H vascular


Cotyledon
H vascular


Rosette Leaf
H vascular


Primary Root
H vascular







Observed expression pattern:


T1 mature: GFP expressed in vascular connective region of mature ovules.


T2 seedling: GFP expression throughout root and cotyledon vasculature.








Expected expression pattern:
Drought inducible - Up regulated In Drought Condition.


Selection Criteria:
Microarray


Gene:
Expressed protein







GenBank: NM_202299 Arabidopsis thaliana expressed protein (At1g54575) mRNA, complete


cds gi|42571874|ref|NM_202299.1|[42571874]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  X T1 Mature X T2 Seedling T2 Mature T3 Seedling


Inductions completed.
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
 7 days
T2
3 Hrs air dry
2/0
No


2. Drought
21 days
T2
8 days no water
2/0
No







Inducible expression summary:


Treatment:  Time point induced:  Organs induced:  Tissues induced:








1. Drought
No increased response observed.


2. Drought
No increased response observed under drought conditions. However,



additional GFP expression observed in epidermis and vasculature of petals



in flowers not observed in primary T1 mature aerial tissue screen.








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 6
  Events Expressing: n = 2


GFP Expression Detected








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata abscission zone ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia



H chalaza micropyle early endosperm mature endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons root meristem



shoot meristem


Stem
epidermis cortex interfascicular region vascular xylem phloem



pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 3
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-04: 0/6


Event-05: 5/6


Event-06: 4/6


GFP Expression Detected








X Hypocotyl
epidermis cortex H vascular xylem phloem stomata


X Cotyledon
mesophyll H vascular epidermis margin petiole



stomata



hydathode


X Rosette Leaf
mesophyll H vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem pericycle quiescent



columella



root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical meristem
shoot apical meristem


Promoter utility


Trait Area:
Source, PG&D, nutrients


Sub-trait Area:
Seed size, nutrient use efficiency


Utility:
This promoter sequence can be used to modulate:



seed size, and nutrient economy.








Construct:
PT0698


Promoter candidate I.D:
15371554


cDNA I.D:
23549209


Lines expressing:
PT0698-05, -06; Jun. 21, 2004









Example 5












Promoter Expression Report #235


Promoter Tested In: : Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Ovule
Post-fertilization: H mature endosperm H embryo


Embryo
H radicle







Observed expression pattern:


T1 mature: High specific expression in the cortical root cells of mature embryos and peripheral


endosperm of developing seed.


T2 seedling: No expression observed.


T2 mature: No expression observed in mature roots.








Expected expression pattern:
Drought - Up in drought not up anywhere else.


Selection Criteria:
Microarray







Gene: C2 domain-containing protein/GRAM domain-containing protein


GenBank: NM_124396 Arabidopsis thaliana C2 domain-containing protein/GRAM domain-


containing protein (At5g50170) mRNA, complete cdsgi|30695850|ref|NM_124396.2|








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  XT1 Mature XT2 Seedling XT2 Mature  T3 Seedling


Inductions completed.
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hrs air dry
2/0
No


2. Drought
4 wks
T2
8 days no water
2/0
No







Inducible expression summary:


Treatment:  Time point induced:  Organs induced: Tissues induced:








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 2
  Events Expressing: n = 2


GFP Expression Detected








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata abscission zone ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm H mature



endosperm H embryo


X Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis H radicle cotyledons hypocotyl


Stem
epidermis cortex interfascicular region vascular xylem phloem



pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








2 Seedling Expression
Tissues Screened


Events Screened: n = 2
Events Expressing: n = 0







Seedlings expressing/Seedlings screened


Event-01: 0/6


Event-02: 0/6


No GFP Expression Detected








Promoter utility



Trait Area:
PG&D, Nutrients


Sub-trait Area:
Seed size, seed establishment, nitrogen use efficiency


Utility:
This promoter sequence can be used to modulate seed size and







nutrient economy when coupled with other appropriate promoter gene constructs.


Notes: Nalefski EA, Falke JJ. 1996. The C2 domain calcium-binding motif: structural and


functional diversity. Protein Sci. 5: 2375-90. The C2 domain is a Ca(2+)-binding motif of


approximately 130 residues identified in the Ca(2+)-dependent isoforms of protein kinase C.


Single and multiple copies of C2 domains have been identified in a growing number of


eukaryotic signaling proteins that interact with cellular membranes and mediate a broad array


of critical intracellular processes, including membrane trafficking, the generation of lipid-


second messengers, activation of GTPases, and the control of protein phosphorylation. As a


group, C2 domains display the remarkable property of binding a variety of different ligands


and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular


proteins. Expanding this functional diversity is the fact that not all proteins containing C2


domains are regulated by Ca2+, suggesting that some C2 domains may play a purely


structural role or may have lost the ability to bind Ca2+.








Construct:
PT0708


Promoter candidate I.D:
15371629


cDNA I.D:
23509614


Lines expressing:
PT0708-01, -02









Example 6












Promoter Expression Report #236


Promoter Tested In:   Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
   M vascular


Silique
H vascular


Ovule
Post-fertilization: H embryo


Embryo
H cotyledons


Stem
H vascular H pith


Leaf
L vascular


Cotyledon
H vascular


Rosette Leaf
H mesophyll H vascular H epidermis


Primary Root
H vascular







Observed expression pattern:


T1 mature: GFP expression in vasculature of silique, petals, leaf and stem. In silique, GFP is


expressed in medial and lateral vasculature of carpels. Not observed in reproductive organs.


GFP expression specific to vascular and ground tissues of cotyledon in developing embryos.


T2 seedling: High GFP expression throughout emerging rosette leaves and in vasculature of


root and cotyledons.








Expected expression pattern:
Drought inducible - Up in drought not up anywhere



else.


Selection Criteria:
Microarray


Gene:
Expressed protein







GenBank: NM_121391 Arabidopsis thaliana expressed protein (At5g13880) mRNA,


complete cds gi|18417221|ref|NM_121391.1|[18417221]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  XT1 Mature XT2 Seedling T2 Mature T3 Seedling


Inductions completed.












Treatment:
Age:
Gen:
Time points:
Events Screened/Response
Response:


1. Drought
7 days
T2
3 air dry
2/1
Yes


2. Drought
4 wks
T2
8 days no water
2/1
Yes







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
3 air dry
Rosettes leaf
Epidermis


2. Drought
8 days no water
Inflorescence, Flowers
Pedicles,




Leaf
Vascular,





Guard cells,





Vascular, Guard cells








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 3
  Events Expressing: n = 2


GFP Expression Detected








X Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae M vascular epidermis stomata



trichome



silique


X Silique
stigma style carpel septum placentae funiculus



transmitting tissue H vascular epidermis stomata abscission



zone ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm H embryo


X Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle H cotyledons hypocotyl


X Stem
epidermis cortex interfascicular region H vascular xylem



phloem H pith stomata trichome


X Leaf
petiole mesophyll L vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 6/6


Event-02: 1/6


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


X Cotyledon
mesophyll H vascular epidermis margin petiole stomata



hydathode


X Rosette Leaf
H mesophyll H vascular H epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem


Promoter utility


Trait Area:
PG&D, water use efficiency


Sub-trait Area:
Seed size, drought tolerance


Utility:
This promoter sequence can be used to modulate seed size,



drought tolerance, and nutrient use efficiency.








Construct:
PT0710


Promoter candidate I.D:
15371635


cDNA I.D:
23500258


Lines expressing:
PT0710-01, -02









Example 7












Promoter Expression Report #237


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
   H pollen


Ovule
Pre-fertilization: H inner integument H outer integument H micropyle



Post-fertilization: H inner integument H outer integument H micropyle


Rosette Leaf
H mesophyll M epidermis


Primary Root
L epidermis







Observed expression pattern:


T1 mature: GFP expression specific to region of pollen and ovules. GFP appears to be expressed


in sperm cells and the vegetative nucleus of pollen cells. GFP is highly expressed in the distal


cells of the integuments in the region of the micropyle in developing ovules through mature seed.


T2 seedling: GFP expression in epidermal cells of emerging rosette leaves. Low GFP expression


in root epidermal cells near transition zone.








Expected expression pattern:
Drought inducible - Up in drought not anywhere else.


Selection Criteria:
Microarray


Gene:
Expressed protein







GenBank: NM_129154 Arabidopsis thaliana expressed protein (At2g35950) mRNA, complete


cds gi|18404005|ref|NM_129154.1|[18404005]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   X T1 Mature X T2 Seedling  T2 Mature  T3 Seedling


Inductions completed.
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 hours air dry
2/0
No


2. Drought
4 wks
T2
8 days no water
2/0
No







Inducible expression summary:


Treatment:  Time point induced:  Organs induced:  Tissues induced:








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 2
  Events Expressing: n = 6







GFP Expression Detected








X Flower
pedicel receptacle nectary sepal petal filament anther H



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata abscission zone ovule


X Ovule
Pre-fertilization: primordia H inner integument H outer integument



embryo sac funiculus chalaza H micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus H



inner integument H outer integument endothelium seed coat



primordia chalaza H micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


Stem
epidermis cortex interfascicular region vascular xylem phloem



pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-06: 2/6


Event-09: 4/6


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin petiole stomata



hydathode


X Rosette Leaf
H mesophyll vascular M epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
L epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem


Promoter utility


Trait Area:
PG&D


Sub-trait Area:
Seed size


Utility:
This promoter sequence can be used to modulate seed size.








Construct:
PT0721


Promoter candidate I.D:
15371677


cDNA I.D:
23516060


Lines expressing:
PT0721-06, -09









Example 8












Promoter Expression Report #238


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
   H pollen


Embryo
H provascular L radicle


Leaf
M vascular


Hypocotyl
L epidermis


Primary Root
L epidermis







Observed expression pattern:


T1 mature: High GFP specificity throughout vasculature of developing embryo. GFP


expression also in vasculature of mature leaves. GFP expressed throughout pollen cells.


T2 seedling: Low GFP expression in epidermal cells of hypocotyl.








Expected expression pattern:
Drought inducible - Loudest in drought


Selection Criteria:
Microarray


Gene:
Em-like protein GEA1 (EM1)







GenBank: NM_115040 Arabidopsis thaliana Em-like protein GEA1 (EM1) (At3g51810)


mRNA, complete cds gi|30693538|ref|NM_115040.21|[30693538]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature X T2 Seedling  T2 Mature  T3 Seedling


Inductions completed.
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hrs air dry
2/0
No


2. Drought
4 wks
T2
8 days no water
2/0
No







Inducible expression summary:


Treatment:  Time point induced:  Organs induced:  Tissues induced:








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 3
  Events Expressing: n = 2







GFP Expression Detected








X Flower
pedicel receptacle nectary sepal petal filament anther H



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae funiculus



transmitting tissue vascular epidermis stomata abscission zone



ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


X Embryo
suspensor preglobular globular heart torpedo late mature



H provascular hypophysis L radicle cotyledons hypocotyl


Stem
epidermis cortex interfascicular region vascular xylem



phloem pith stomata trichome


X Leaf
petiole mesophyll M vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 3/6


Event-02: 4/7


GFP Expression Detected








X Hypocotyl
L epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin petiole stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
L epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem


Promoter utility


Trait Area:
PG&D, water use efficiency


Sub-trait Area:
drought tolerance


Utility:
This promoter sequence can be used to modulate:



reproductive drought







Notes: Vicient CM, Bies-Etheve N, Delseny M. 2000. Changes in gene expression in the


leafy cotyledon1 (lec1) and fusca3 (fus3) mutants of Arabidopsis thaliana L. J Exp Bot.


5: 995-1003. In lec1-1 seeds, the AtEm1 gene was expressed at a higher level than in the wild


type and earlier in development. Transgenic analysis using 5′-promoter deletions


demonstrated that at least two regions of AtEm1 gene promoter interact with the LEC1-


dependent transcriptional regulatory pathway. In spite of expression of the AtEm1 promoter


and accumulation of AtEm1 mRNA, the corresponding Em1 protein does not accumulate in


lec1-1 seeds. The ABA inducibility of the AtEm1 promoter was not affected by the lec1


mutation. [Ceres does not see ABA inducibility in our TxP data]. Gaubier P, Raynal M, Hull G,


Huestis GM, Grellet F, Arenas C, Pages M, Delseny M. 1993. Two different Em-like


genes are expressed in Arabidopsis thaliana seeds during maturation Mol Gen Genet.


238: 409-18. Using a radish cDNA probe, we have isolated and characterized two genomic


clones from Arabidopsis thaliana (GEA1 and GEA6) encoding two different proteins that are


homologous to the “Early methionine-labelled” (Em) protein of wheat. GEA1 differs from


GEA6 and Em clones of wheat in that a sequence coding for 20 amino acid residues is


tandemly repeated 4 times. These two genomic clones correspond to two genes named AtEm1


and AtEm6. GEA1 appears in immature seeds and is maximum in dry seeds. No expression


of either gene could be detected in leaf, stem, or floral buds. Expression of both genes could


be detected in immature seeds when the siliques were incubated with abscisic acid (ABA),


demonstrating that both genes are ABA responsive. However, examination of the 5′ upstream


region does not reveal any extensive homology, suggesting that regulation of the two genes


differs. In situ hybridization with a GEA1 probe demonstrated that the expression of this gene


is essentially located in the provascular tissues of the cotyledons and axis of the dry seed as


well as in the epiderm and outer layers of the cortex in the embryo axis.








Construct:
PT0740


Promoter candidate I.D:
15371779


cDNA I.D:
23635276


Lines expressing:
PT0740-11, -12









Example 9












Promoter Expression Report #111


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
   L pedicel L epidermis


Stem
L epidermis


Hypocotyl
H epidermis


Cotyledon
H mesophyll H vascular H epidermis H petiole


Rosette Leaf
H epidermis H petiole


Primary Root
H epidermis


Lateral root
H lateral root cap







Observed expression pattern:


T1 mature: Low epidermal expression in stem and pedicles near inflorescence apical meristem.


T2 seedling: High epidermal expression in cotyledons, petioles of emerging rosette leaves,


hypocotyl, and root. Expression observed in vascular and mesophyll cells of cotyledons.








Expected expression pattern:
Drought inducible


Selection Criteria:
Ceres expression data


Gene:
Hypothetical protein







GenBank: NM_102758 Arabidopsis thaliana hypothetical protein (At1g30190) mRNA,


complete cds gi|18397396|ref|NM_102758.1|[18397396]








Source Promoter Organism:
Arabidopsis thaliana, WS ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response:
Response:


1. Drought
7 days
T2
3 Hrs Air dry
2/0
No


2. Drought
4 weeks
T2
10-12 day No H20
2/2
Yes







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


2. Drought
10-12 day No H20
Flowers
Pedicel, Epidermis




Siliques
Epidermis




Leaf
Epidermis, Vascular




Stem
Epidermis








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 6
  Events Expressing: n = 2







GFP Expression Detected








X Flower
L pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular L epidermis stomata



trichome



silique


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 embryo sack inner integument outer



integument endothelium seed coat primordia chalaza micropyle



early endosperm mature endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
L epidermis cortex vascular xylem phloem pith stomata



trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-04: 6/6


Event-06: 4/6


Scheduled


GFP Expression Detected








X Hypocotyl
H epidermis cortex vascular xylem phloem stomata


X Cotyledon
H mesophyll H vascular H epidermis margin H petiole



stomata



hydathode


X Rosette Leaf
mesophyll vascular H epidermis trichome H petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


X Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular H lateral root cap


Shoot apical meristem
Shoot apical meristem


Promoter utility


Trait Area:
Water use efficiency


Sub-trait Area:
Drought


Utility:
This promoter sequence can be used to improve:



Modulation growth and development. Modulation of nutrient



uptake and loading. Expression of nitrate transports and water



pumps. Modulation of drought responses, including modulation of



water uptake and transport under drought conditions.


Notes:
Candidate to drive genes involved in osmotic stresses such as



NCED. Endogenous promoter induced under drought.


Construct:
YP0286


Promoter candidate I.D:
11768589


cDNA I.D:
12669548 (OCKHAM3-C)


Lines expressing:
YP0286-04, -06; Jul. 3, 2003









Example 10












Promoter Expression Report #112


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
   L anther


Ovule
Post-fertilization: L endothelium


Cotyledon
H epidermis H petiole


Rosette Leaf
H trichome


Primary Root
H epidermis H root hairs







Observed expression pattern


T1 mature: Low GFP expression in endothelium cells of mature ovules and stomium region of


developing and dehiscencing anthers. Endothelium expression is very weak and may not have


been detected by standard screen. Only tissue with visible GFP expression is analyzed by confocal


microscopy. This may account for the expressing/screened ratio. Not expressed in pollen. T2


seedling: High GFP expression specific to epidermal tissues of cotyledons, root and trichomes of


rosette leaves.








Expected expression pattern:
Drought-inducible


Selection Criteria:
Ceres expression data


Gene:
phi-1-related protein







GenBank: NM_125822 Arabidopsis thaliana phi-1-related protein (At5g64260) mRNA,


complete cds gi|30697983|ref|NM_125822.2|[30697983] “phosphate-responsive 1 family protein”








Source Promoter Organism:
Arabidopsis thaliana, WS ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hrs air dry
2/0
No


2. Drought
4 weeks
T2
10-12 days no H20
2/0
No







Inducible expression summary:


Treatment:  Time point induced:  Organs induced:  Tissues induced:


1. Drought - No differences observed. Images not shown.


2. Drought - No differences observed. Images not shown.








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 9
  Events Expressing: n = 3







GFP Expression Detected








X Flower
pedicel receptacle nectary sepal petal filament L anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone ovule


X Ovule
Pre-fertilization: inner integument outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack inner integument outer



integument L endothelium 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


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 9
  Events Expressing: n = 3







Seedlings expressing/Seedlings screened


Event-01: 5/6    Event-02: 3/6    Event-03: Not tested


Scheduled


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


X Cotyledon
mesophyll vascular H epidermis margin H petiole stomata



hydathode


X Rosette Leaf
mesophyll vascular epidermis H trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap H root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Induction Screens


1. Drought - No differences observed.


2. Drought - No differences observed.








Promoter utility



Trait Area:
PG&D


Sub-trait Area:
Yield, Germination and mobilization of nutrient reserves


Utility:
This promoter sequence can be used to improve:



Modulation seed size and seed shape. Modulation of pollen



development and dehiscence and engineering of male sterility.



Engineering of plant responses to insects and production and



loading of volatiles into trichomes and other epidermal cells.



Alteration in loading and transport of metabolites from the soil and



environment. Protection against insects and microbes. Modulation



of root signaling. Drive CDS that can play a role in shade



avoidance.


Notes:
Endogenous promoter is up-regulated in far red light.


Construct:
YP0289


Promoter candidate I.D:
11768596


cDNA I.D:
12326995


Lines expressing:
YP0289-03; Jun. 4, 2003, -10, -13









Example 11












Promoter Expression Report #125


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
    H pedicel H petal H epidermis


Silique
H stigma L style L carpel L septum L epidermis


Ovule
Post-fertilization: H outer integument


Stem
H epidermis H stomata


Hypocotyl
H epidermis


Cotyledon
H epidermis


Rosette Leaf
H epidermis H trichome







Observed expression pattern:


T1 mature: GFP expression specific to epidermal cell types. High GFP expression in


epidermis of stem decreasing toward pedicles and inflorescence apex. In the flower, high


expression observed in epidermal cells of petals and stigma, and lower expression in carpels.


High expression in outer integuments of maturing ovules. High expression throughout


epidermal cells of mature lower stem.


T2 seedling: GFP expression specific to epidermal cell types. High expression in epidermis


of hypocotyl, cotyledon, and trichomes of rosette leaves. Not detected in root.








Expected expression pattern:
Drought inducible


Selection Criteria:
Public reference; Mol Gen Genet. April 1993; 238 (1-2): 17-25.


Gene:
Dehydration-induced protein RD22







GenBank: NM_122472 Arabidopsis thaliana dehydration-induced protein RD22


(At5g25610) mRNA, complete cds gi|30689960|ref|NM_122472.2|[30689960]








Source Promoter Organism:
Arabidopsis thaliana, WS ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
 7 days
T2
3 Hr air dry
2/2
Yes


2. ABA
10 days
T2
2 Hr
2/0
No


100 uM


6 Hr
2/0


3. Drought
 4 weeks
T2
10-12 days no
2/2
Yes





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
3 Hr air dry
Rosette leaf
Epidermal, Vascular


3. Drought
10-12 days no H20
Silique, Leaf, Stem
Epidermal







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 2     Events Expressing:  n = 2


GFP Expression Detected








X Flower
H pedicel receptacle nectary sepal H petal filament anther



pollen carpel style papillae vascular H epidermis stomata



trichome



silique


X Silique
H stigma L style L carpel L septum placentae transmitting tissue



vascular L epidermis stomata abscission zone ovule


X Ovule
Pre-fertilization: inner integument outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack inner integument H outer



integument endothelium seed coat primordia chalaza micropyle



early endosperm mature endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
H epidermis cortex vascular xylem phloem pith H stomata



trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression     Tissues Screened


Events Screened: n = 2     Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 1/6


Event-04: 4/6


Scheduled


GFP Expression Detected








X Hypocotyl
H epidermis cortex vascular xylem phloem stomata


X Cotyledon
mesophyll vascular H epidermis margin stomata



hydathode


X Rosette Leaf
mesophyll vascular H epidermis H trichome petiole



primordia stomata stipule margin hydathode


Primary Root
epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
PG&D, Stress, Nutrients


Sub-trait Area:
Drought, heat and cold


Utility:
This promoter sequence can be used to improve:



Modulation of compatibility/incompatibility. Modulation seed size



and seed shape. Modulation of stem size and shape. Protection



against insects and microbes. Modulation of ROS signaling. Uptake



of nutrients. Modulation of drought responses including leaf and



flower wilting, ovule abortion, infertility and seed abortion.



Alteration of seed development and shape, attention of seed



dormancy and germination.







Notes: Goh CH, Nam HG, Park YS.


Stress memory in plants: a negative regulation of stomatal response and transient induction of


rd22 gene to light in abscisic acid-entrained Arabidopsis plants.


Plant J. October 2003; 36 (2): 240-55.


Iwasaki T, Yamaguchi-Shinozaki K, Shinozaki K.


Identification of a cis-regulatory region of a gene in Arabidopsis thaliana whose induction by


dehydration is mediated by abscisic acid and requires protein synthesis.


Mol Gen Genet. May 20, 1995; 247 (4): 391-8.


Yamaguchi-Shinozaki K, Shinozaki K.


The plant hormone abscisic acid mediates the drought-induced expression but not the seed-


specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana.


Mol Gen Genet. April 1993; 238 (1-2): 17-25.


Endogenous promoter is down-regulated in roots and only mildly and inconsistently up-regulated


in drought.








Construct:
YP0356


Promoter candidate I.D:
11768602


cDNA I.D:
12394809


Lines expressing:
YP0356-01, -04; Oct. 16, 2003









Example 12












Promoter Expression Report #126


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Ovule
Pre-fertilization:   H outer integument



Post-fertilization:  L outer integument L chalaza


Hypocotyl
L vascular


Primary Root
L epidermis M vascular M pericycle







Observed expression pattern:


T1 mature: GFP expressed in outer integument of developing ovule primordium. Higher


integument expression at chalazal pole observed through maturity.


T2 seedling: Medium to low expression in root vascular bundles weakening toward hypocotyl.


Weak expression in epidermal cells at root transition zone.








Expected expression pattern:
Drought inducible


Selection Criteria:
Ceres, Inc. Expression data


Gene:
Putative cytochrome P450







GenBank: NM_112814 Arabidopsis thaliana cytochrome P450, putative (At3g19270) mRNA,


complete cds gi|18402178|ref|NM_112814.1|[18402178]








Source Promoter Organism:
Arabidopsis thaliana, Columbia ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed: Drought, Heat












Treatment:
Age:
Gen:
Time points:
Events Screened/Response
Response:


1. Drought
4 wks
T2
10-12 d. No H20
2/0
No


2. Drought
7 days
T2
 3 hrs Air Dry
2/0
No


3. Heat
7 days
T2
 1 Hr
2/0
No


42 C


 4 Hr
2/0
No





24 Hr post-
2/1
Yes





treatment







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


3. Heat
24 Hr post-treatment
Rosette leaf
Epidermis Vascular







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 2     Events Expressing:  n = 2


GFP Expression Detected








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone ovule


X Ovule
Pre-fertilization: inner integument H outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack inner integument L outer



integument endothelium seed coat primordia L 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


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression    Tissues Screened


Events Screened: n = 2     Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 3/5


Event-02: 1/6


Scheduled


GFP Expression Detected








X Hypocotyl
epidermis cortex L vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
L epidermis trichoblast atrichoblast cortex endodermis



M vascular xylem phloem M pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
Seed Biology, Stress


Sub-trait Area:
Seed fill, seed size, stress during seed fill, stress protection,


endosperm



production


Utility:
This promoter sequence can be used to improve:



Protection against ovule and seed abortion. Modulation seed size,



seed shape. Modulation of endosperm growth and development.



Modulation of heat responses and protection against heat stress.



Modulation of water and mineral ion uptake and transport. Loading



and transport of metabolites into seeds. Modulation of breeding



system.


Notes:
Heat stress is a major determinant of seed and fruit yield in many



crops, and protection against transient heat stress is a primary goal



of modern agriculture. This promoter induces in leaves at



approximately 24 hour after heat stress. Few promoters of this kind



are available at this time. The leaf induction could protect against



water loss from the leaf, offering a possibility of protection against



heat and drought conditions, which often coincide.



Promoter is mildly induced by drought and drought-like conditions.








Construct:
YP0374


Promoter candidate I.D:
11768817


cDNA I.D:
12370888


Lines expressing:
YP0374-01, -02









Example 13












Promoter Expression Report #127


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
  M sepal M petal M epidermis


Hypocotyl
L epidermis L vascular H stomata


Cotyledon
M vascular L epidermis


Primary Root
M epidermis M vascular M root hairs







Observed expression pattern:


T1 mature: Expressed in epidermal cells of sepals and petals in developing flowers.


T2 seedling: Medium to low expression in epidermal and vascular cells of hypocotyls and


cotyledons. Epidermal and vascular expression at root transition zone decreasing toward root tip.








Expected expression pattern:
PEG or Osmotic stress-inducible


Selection Criteria:
Ceres expression data


Gene:
product = “glycine-rich protein”, note: unknown protein







GenBank: NM_100587 Arabidopsis thaliana glycine-rich protein (At1g07135) mRNA, complete


cds gi|22329385|ref|NM_100587.2|[22329385]








Source Promoter Organism:
Arabidopsis thaliana, WS ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hrs airy dry
2/2
Yes


2. Drought
4 weeks
T2
10-12 days no
2/2
Decrease





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
3 Hrs airy dry
Cotyledons
Epidermis







Impeded expression summary:










Treatment:
Time point impeded:
Organs impeded:
Tissues impeded:


2. Drought
10-12 days no H20
Inflorescence meristem
Sepals




Sepals
Epidermis, Cortex, Vascular




Stem
Epidermis, Cortex




Silique
Epidermis, Cortex







Notes: Decreased levels of GFP were observed in the primary inflorescence shoot meritem. Stems


and siliques were taken from top third of mature plants. No expression observed in stems and


siliques from bottom half of secondary inflorescence of mature plants. No differences observed


between primary and secondary inflorescence meritems.


T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 2     Events Expressing:  n = 2


GFP Expression Detected








X Flower
pedicel receptacle nectary M sepal M petal filament anther



pollen carpel style papillae vascular M epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone funiculus ovule


Ovule
Pre-fertilization: inner integument outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack inner integument outer



integument funiculus endothelium 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


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression    Tissues Screened


Events Screened: n = 2     Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-02: 4/6


Event-06: 5/6


Scheduled


GFP Expression Detected








X Hypocotyl
L epidermis cortex L vascular xylem phloem H stomata


X Cotyledon
mesophyll M vascular L epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
M epidermis trichoblast atrichoblast cortex endodermis



M vascular xylem phloem pericycle quiescent



columella root cap M root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
Nutrients, Stress


Sub-trait Area:
Drought, Nitrogen uptake


Utility:
This promoter sequence can be used to improve:



Modulation of water and nutrient uptake and seedling



establishment. Modulation of plant-microbe interactions. Changes



to flower development and structure. Modulation of responses to



drought, especially in the flower and inflorescence and in the



stigma. Changes in pollination biology and incompatibility upon



drought.


Notes:
Endogenous promoter is up-regulated under PEG treatment but not



with ABA and drought.








Construct:
YP0377


Promoter candidate I.D:
11768593


cDNA I.D:
13613778


Lines expressing:
YP0377-02, -06; Oct. 16, 2003









Example 14












Promoter Expression Report #128


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
  H pedicel H receptacle H sepal H petal H filament H anther H


carpel



H style   H stigma H epidermis H stomata H silique


Silique
H stigma H style H carpel H septum H placentae H epidermis


Stem
L epidermis L cortex H stomata


Leaf
H mesophyll H epidermis H trichome H stomata


Hypocotyl
H epidermis H stomata


Cotyledon
H mesophyll H epidermis


Rosette Leaf
H mesophyll H epidermis


Primary Root
H epidermis







Observed expression pattern:


T1 mature: Vegetative expression. Not expressed in shoot apical meristem, early flower


primordia, pollen and ovules. High expression throughout floral organs. High expression in stem


guard cells and cortex cells surrounding stomal chamber (see Table 1. FIG. P). Expression


throughout placenta and funiculus but not in any ovule tissue.


T2 seedling: Expressed in all tissues near seedling apex increasing toward root. High root


epidermis expression. Not expressed in cotyledon and rosette leaf guard cells (Table. 2. G, H.).








Expected expression pattern:
Drought inducible


Selection Criteria:
Ceres, Inc. microarray


Gene:
Responsive to Dehydration 20







GenBank: NM_128898 Arabidopsis thaliana RD20 protein (At2g33380) mRNA, complete cds


gi|30685670|ref|NM_128898.2|[30685670]








Source Promoter Organism:
Arabidopsis thaliana, WS ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 hours air dry
2/2
Yes


2. Drought
7 days
T2
At Wilt
2/2
Yes


3. ABA
7 days
T2
6 Hrs
2/2
Yes


100 uM


4. Drought
4 weeks
T2
10-12 days no
2/2
Yes





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
3 hours air dry
Cotyledons
Epidermis




Hypocotyl
Guard cells


2. Drought
At Wilt
Cotyledons
Epidermis




Root
Epidermis


3. ABA 100 uM
6 Hrs
Cotyledons, Rosette leaf
Epidermis, Guard cells





Mesophyll,





Vasculature




Roots
Epidermis


4. Drought
10-12 days no H20
Flower
Silique, Abscission zone




Silique
Epidermis




Leaf
Epidermis, mesophyll




Stem
Cortex, Epidermis







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 3     Events Expressing:  n = 3


GFP Expression Detected








X Flower
H pedicel H receptacle nectary H sepal H petal H filament H anther



pollen H carpel H style H stigma vascular H epidermis H stomata



trichome H silique


X Silique
H stigma H style H carpel H septum H placentae funiculus



transmitting tissue vascular H epidermis stomata abscission zone



ovule


Ovule
Pre-fertilization: inner integument outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack inner integument outer



integument funiculus endothelium seed coat primordia chalaza



micropyle early endosperm mature endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
L epidermis L cortex vascular xylem phloem pith H stomata



trichome


X Leaf
petiole H mesophyll vascular H epidermis H trichome primordia



H stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression    Tissues Screened


Events Screened: n = 2      Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-04: 5/6


Scheduled


GFP Expression Detected








X Hypocotyl
H epidermis cortex vascular xylem phloem H stomata


X Cotyledon
H mesophyll vascular H epidermis margin stomata



hydathode


X Rosette Leaf
H mesophyll vascular H epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
Water stress, Seed Biology, PG&D


Sub-trait Area:
Drought, Seed fill


Utility:
This promoter sequence can be used to improve:



Valuable as drought-inducible promoter for yield protection in



drought or heat stress conditions. Modulation of plant growth rate



and architecture. Modulation of growth and development in absence



of effects on reproductive cells. Enhancement of source strength



and seed filling. Engineering of male sterility.







Notes: Takahashi S, Katagiri T, Yamaguchi-Shinozaki K, Shinozaki K. An Arabidopsis gene


encoding a Ca2+-binding protein is induced by abscisic acid during dehydration.Plant Cell


Physiol. July 2000; 41(7): 898-903. Yamaguchi-Shinozaki K, Shinozaki K. The plant hormone


abscisic acid mediates the drought-induced expression but not the seed-specific expression of


rd22, a gene responsive to dehydration stress in Arabidopsis thaliana. Mol Gen Genet.


April 1993; 238 (1-2): 17-25.


Endogenous gene is up-regulated under drought and ABA.








Construct:
YP0380


Promoter candidate I.D:
11768580


cDNA I.D:
12462179 (OCKHAM3-CD)


Lines expressing:
YP0380-01, -04, -02; Oct. 16, 2003









Example 15












Promoter Expression Report #129


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
L pedicel H nectary L epidermis


Hypocotyl
L vascular


Primary Root
H vascular







Observed expression pattern:


T1 mature: High expression in nectary glands of flowers. Low expression in epidermis of pedicels


of developing flowers.


T2 seedling: GFP expressed in root and hypocotyl vasculature.








Expected expression pattern:
Drought inducible


Selection Criteria:
Ceres, Inc. microarray data


Gene:
Unknown expressed protein







GenBank: NM_113878 Arabidopsis thaliana expressed protein (At3g29575) mRNA, complete cds


gi|30689672|ref|NM_113878.3|[30689672]








Source Promoter Organism:
Arabidopsis thaliana, Ws ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 hours air dry
2/0
No


2. ABA 100 uM
7 days
T2
6 Hrs
2/1
Yes


3. Drought
4 weeks
T2
10-12 days no
2/2
Yes





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


2. ABA 100 uM*
6 Hrs
Root
Vasculature


3. Drought
10-12 days no H20
Flower
Sepals, Petals, Silques, Pedicles,





Nectaries, Abscission zone




Silique
Carpels, Epidermis, Vascular




Leaf
Epidermis, Mesophyll, Vascular




Stem
Vascular, Pith







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 2     Events Expressing:  n = 2


GFP Expression Detected








X Flower
L pedicel receptacle H nectary sepal petal filament anther



pollen carpel style papillae vascular L epidermis stomata



trichome



silique


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 embryo sack inner integument outer



integument endothelium 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


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression    Tissues Screened


Events Screened: n = 2      Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-05: 1/6


Event-06: 6/6


Scheduled


GFP Expression Detected








X Hypocotyl
epidermis cortex L vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem







Promoter utility








Trait Area:
PG&D


Sub-trait Area:
Breeding biology


Utility:
This promoter sequence can be used to improve: Modulation of



plant secondary products including volatiles or fragrances. Useful to



drive genes important in insect attraction for stimulation of



outbreeding and hybridization. Useful for making flowerless plants.



Modulation of drought stress tolerance. Modulation of heat stress



tolerance.







Notes: Baum SF, Eshed Y, Bowman JL. The Arabidopsis nectary is an ABC-independent floral


structure.Development. November 2001; 128(22): 4657-67.









Endogenous promoter is up-regulated in drought, ABA and far red



light.








Construct:
YP0381


Promoter candidate I.D:
11768582


cDNA I.D:
12736859 (OCKHAM3-CD)


Lines expressing:
YP0381-05, -06; Oct. 16, 2003









Example 16












Promoter Expression Report #130


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
  H nectary M sepal M vascular


Hypocotyl
L vascular


Cotyledon
L vascular


Rosette Leaf
L vascular


Primary Root
H epidermis H root cap L root hairs







Observed expression pattern:


T1 mature: Expressed in nectary glands of flowers and vasculature of sepals.


T2 seedling: High GFP expression in root epidermal cells through to root cap. Low GFP


expression in vasculature aerial organs.








Expected expression pattern:
PEG or Osmotic stress-inducible


Selection Criteria:
Ceres, Inc. Expression data


Gene:
product = “expressed protein”







GenBank: NM_129727 Arabidopsis thaliana expressed protein (At2g41640) mRNA,


complete cds gi|30688728|ref|NM_129727.2|[30688728]








Source Promoter Organism:
Arabidopsis thaliana, Ws ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed.
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hr air dry
2/0
No


2. Drought
4 weeks
T2
10-12 days no
2/0
No





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:








1. Drought
No differences observed. Images not shown.


2. Drought
No differences observed. Images not shown.







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 2     Events Expressing:  n = 2


GFP Expression Detected








X Flower
pedicel receptacle H nectary M sepal petal filament anther



pollen carpel style papillae M vascular epidermis stomata



trichome



silique


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 embryo sack inner integument outer



integument endothelium 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


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression    Tissues Screened


Events Screened: n = 2      Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-04: 5/6


Event-05: 4/6


Scheduled


GFP Expression Detected








X Hypocotyl
epidermis cortex L vascular xylem phloem stomata


X Cotyledon
mesophyll L vascular epidermis margin stomata



hydathode


X Rosette Leaf
mesophyll L vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella H root cap L root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Induction Screens








1. Drought
No differences observed.


2. Drought
No differences observed.







Promoter utility








Trait Area:
PGD, Stress


Sub-trait Area:
Drought, Root Architecture, Nutrient Uptake


Utility:
This promoter sequence can be used to improve:



Modification of growth and development, especially of flower



development. Improvements to nectary tissue and nectar



production, and improvements in insect and pollination



(outcrossing) biology. Modulation of water and nutrient uptake,



loading and transport.


Notes:
Nectaries are secretory structures that produce nectar, a solution



composed mainly of sugars. Carpels, stamen, sepals, and



nectary vasculature are derived from vascular bundles of the



receptacle.



Endogenous promoter is down-regulated in shoots, siliques and



flowers.








Construct:
YP0382


Promoter candidate I.D:
11768592


cDNA I.D:
12735575 (OCKHAM3-CD)


Lines expressing:
-04, -05 (Oct. 16, 2004)









Example 17












Promoter Expression Report #131


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
  H filament H anther H stomata


Silique
H ovule


Ovule
Post-fertilization: H outer integument H seed coat H chalaza


Leaf
L vascular H stomata


Primary Root
H epidermis







Observed expression pattern:


T1 mature: Very high GFP expression levels in stamens of developing flowers. Low expression


in vasculature of leaves and guard cells throughout plant. High expression in outer integument of


ovules and in seed coats. High incidence of aborted ovules.


T2 seedling: Low expression in root epidermal cells.








Expected expression pattern:
Drought inducible


Selection Criteria:
Ceres, Inc. Expression data


Gene:
product = “protein phosphatase 2C (PP2C), putative”







GenBank: NM_125312 Arabidopsis thaliana protein phosphatase 2C (PP2C), putative


(At5g59220) mRNA, complete cds gi|30697191|ref|NM_125312.2|[30697191]








Source Promoter Organism:
Arabidopsis thaliana, Ws


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:











Treatment:
Gen:
Time points:
Events Screened/Response
Response:


1. ABA
T2
 6 Hr
2/1
Yes


100 uM

16 Hr post-treatment
2/2
Yes


2. Drought
T2
 3 Hr air dry
2/0
No


3. Drought
T2
10-12 days no H20
2/2
Yes







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. ABA 100 uM
 6 Hr
Leaf
Vasculature



16 Hr post-treatment
Cotyledons
Vasculature, Guard cells




Hypocotyl
Epidermis, Vasculature




Leaf
Epidermis, Vasculature,





Guard cells


3. Drought
10-12 days no H20
Flowers
Sepals, Epidermis,





Guard cells




Leaf
Epidermis, Mesophyll




Stem
Epidermis







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 4     Events Expressing:  n = 2


GFP Expression Detected








X Flower
pedicel receptacle nectary sepal petal H filament H anther



pollen carpel style papillae vascular epidermis H stomata



trichome



silique


X Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone H ovule


X Ovule
Pre-fertilization: inner integument outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack funiculus inner



integument



H outer integument endothelium H seed coat primordia H 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


X Leaf
petiole mesophyll L vascular epidermis trichome primordia



H stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression   Tissues Screened


Events Screened: n = 2     Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-02: 4/5


Scheduled


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
PG&D, Nutrients, Seed Biology


Sub-trait Area:
Seed size, heat tolerance, drought


Utility:
This promoter sequence can be used to improve:



Modulation of seed size and seed shape by transgene-induced



alteration of endosperm loading and/or integument growth.



Modulation of flower structure and development especially stamen



and anther development. Modulation of root growth and improved



water and nutrient loading and transport. Engineering of male



sterility and manipulation of stamen and anther development.



Improved drought and heat stress tolerance. Improved seedling



performance in drought and heat stress conditions. Making longer



filaments to improve fertility.







Notes: Sulpice R, Tsukaya H, Nonaka H, Mustardy L, Chen TH, Murata N.


Enhanced formation of flowers in salt-stressed Arabidopsis after genetic engineering of the


synthesis of glycine betaine. Plant J. October 2003; 36(2): 165-76.


Saez A, Apostolova N, Gonzalez-Guzman M, Gonzalez-Garcia MP, Nicolas C, Lorenzo O,


Rodriguez PL.


Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2C HAB1 reveal its


role as a negative regulator of abscisic acid signalling. Plant J. February 2004; 37(3): 354-369.


Tahtiharju S, Palva T. Antisense inhibition of protein phosphatase 2C accelerates cold


acclimation in Arabidopsis thaliana. Plant J. May 2001; 26(4): 461-70.


Endogenous promoter is induced in drought and drought-like conditions.








Construct:
YP0388


Promoter candidate I.D:
11768590


cDNA I.D:
13593066 (OCKHAM3-CD)


Lines expressing:
YP0388-01, -05; Oct. 24, 2003









Example 18












Promoter Expression Report # 133


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
  H sepal H petal H anther H style


Silique
H style H ovule


Ovule
Prefertilization: H outer integument



Postfertilization: H outer integument L seed coat


Leaf
H vascular


Primary Root
H epidermis







Observed expression pattern:


T1 mature: High GFP expression in the style, sepals, petals, and anthers in flowers.


Expressed in outer integuments of ovule primordia through developing seed stages and in


remnants of aborted ovules. High vasculature expression in leaf


T2 seedling: Medium to low root epidermal expression at root transition zone decreasing toward


root tip. Specific to epidermal cells flanking lateral roots.








Expected expression pattern:
Drought-inducible


Selection Criteria:
Ceres expression data


Gene:
PAR-related protein







GenBank: NM_124618 Arabidopsis thaliana photoassimilate-responsive protein PAR-related


protein (At5g52390) mRNA, complete cds gi|30696178|ref|NM_124618.2|[30696178]








Source Promoter Organism:
Arabidopsis thaliana, Ws ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hrs air dry
2/0
No


2. Drought
4 weeks
T2
10-12 days no
2/2
Yes





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


2. Drought
10-12 days no H20
Flowers
Sepals, Petals,





Anthers, Silique




Silique
Carpels, Epidermis,





Cortex




Leaf
Epidermis, Mesophyll







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 4     Events Expressing:  n = 3


GFP Expression Detected








X Flower
pedicel receptacle nectary H sepal H petal filament H anther



pollen carpel H style papillae vascular epidermis stomata



trichome



silique


X Silique
stigma H style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata abscission zone H ovule


X Ovule
Pre-fertilization: inner integument H outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack inner integument H outer



integument funiculus endothelium L 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


X Leaf
petiole mesophyll H vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression    Tissues Screened


Events Screened: n = 2     Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 2/4


Event-02: 5/5


Scheduled


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
Abiotic stresses, PG&D


Sub-trait Area:
Drought, Root architecture


Utility:
This promoter sequence can be used to improve: Modulation



self-incompatability including gametophytic incompatibility.



Modulation of flower development and structure. Modulation



of seed size and shape. Altered seed uptake of water and



mineral ions and an altered seed dormancy and germination.



Modulation of drought responses including leaf and flower



wilting, ovule abortion, infertility and seed abortion.







Notes: Fujita K, Okada M, Lei K, Ito J, Ohkura K, Adu-Gyamfi JJ, Mohapatra PK.


Effect of P-deficiency on photoassimilate partitioning and rhythmic changes in fruit and stem


diameter of tomato (Lycopersicon esculentum) during fruit growth.


J Exp Bot. November 2003; 54(392): 2519-28.


Murillo I, Roca R, Bortolotti C, Segundo BS.


Engineering photoassimilate partitioning in tobacco plants improves growth and productivity


and provides pathogen resistance.


Plant J. November 2003; 36(3): 330-41.


The endogenous gene is consistently induced under drought conditions but not surrogate


drought treatments.








Construct:
YP0396


Promoter candidate I.D:
11768788


cDNA I.D:
12646726


Lines expressing:
YP0396-02, -03; Oct. 24, 2003









Example 19












Promoter Expression Report #143


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Primary Root
L epidermis L trichoblast L atrichoblast L root hair







Observed expression pattern:


T1 mature: No expression.


T2 seedling: Low expression in root epidermal cells at transition zone decreasing to expression in


single cells at mid root.








Expected expression pattern:
Drought inducible.


Selection Criteria:
Ceres, Inc. Expression data


Gene:
Unknown protein.







GenBank: NM_101546 Arabidopsis thaliana expressed protein (At1g16850) mRNA, complete


cds gi|18394408|ref|NM_101546.1|[18394408]








Source Promoter Organism:
Arabidopsis thaliana, WS ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:












Treatment:
Age:
Gen.
Time points:
Events Screened/Response
Response:


1. Drought
4 wks
T2
10-12 d. No H20
3/3
Yes


2. Drought
7 days
T2
 3 Hr. Air Dry
2/0
No


3. Cold 4 C
7 days
T2
 2 Hr
0/3
No





 6 Hr.
0/3
No





24 Hr post-
3/2
Yes





treatment


4. ABA
7 days
T2
 6 Hr.
2/2
Yes


100 um







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
10-12 d. No H20:
Flowers
Sepals Petals Guard cells


3. Cold 4 C
24 Hr post-treatment:
Cotyledons
Epidermis Vascular




Root
Epidermis Vascular


4. ABA 100 um
6 Hr
Cotyledons
Vascular







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 3     Events Expressing:  n = 0


No GFP Expression Detected


T2 Seedling Expression    Tissues Screened


Events Screened:  n = 3     Events Expressing:  n = 3


Seedlings expressing/Seedlings screened


Event-01: 3/6


Event-02: 3/6


Event-03: 2/6


Scheduled


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
L epidermis L trichoblast L atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap L root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
PG&D, Nutrients


Sub-trait Area:
Drought, Nutrient uptake


Utility:
This promoter sequence can be used to improve: Modulation



of root growth rate, size and shape. Modulation of leaf growth,



size and shape. Modulation of water and nutrient uptake.



Modulation of plant-microbe including plant rhizobium



interactions. Useful for making insecticidal proteins for



protection against root worm. Modulation of drought responses



and protection against drought and desiccation. Modulation of



cold stress responses and protection against cold stress.


Notes:
This promoter can have multiple utilities, both in plant growth



and development and in protection against plant stress. Few



promoters of this kind are available at this time. In stress, this



promoter can be useful for protecting against drought and cold



which are sometimes associated (at high latitude and/or high



altitude) and sometimes not associated (in temperate regions),



suggesting potentially very versatile utility or protection against



abiotic stress. The cold-induction could be very valuable since



roots are very sensitive to cold stress. The drought induction in



the leaf could protect against water loss through the leaf.



Endogenous promoter induced in heat and drought as well as



surrogate drought treatments.








Construct:
YP0337


Promoter candidate I.D:
11768598


cDNA I.D:
12326510


Lines expressing:
YP0337-01, -02, -03; Oct. 16, 2003









Example 20












Promoter Expression Report #144


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Primary Root
H epidermis H trichoblast H atrichoblast







Observed expression pattern:


T1 mature: No expression.


T2 seedling: Root specific GFP expression. High expression throughout root epidermal cells.








Expected expression pattern:
Drought inducible


Selection Criteria:
Ceres expression data


Gene:
Heat shock transcription factor family







GenBank: NM_113182 Arabidopsis thaliana heat shock transcription factor family (At3g22830)








Source Promoter Organism:
Arabidopsis thaliana, WS ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:   XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling


Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hrs air dry
2/0
No


2. Drought
4 weeks
T2
10-12 days no
2/2
Yes





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
3 Hrs air dry
None detected.


2. Drought
10-12 days no H20
Leaf
Epidermis, Vascular







T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 3     Events Expressing:  n = 0


No GFP Expression Detected


T2 Seedling Expression   Tissues Screened


Events Screened:  n = 2     Events Expressing:  n = 2


Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-02: 3/4


Scheduled


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis H trichoblast H atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
PG&D, Nutrients, Stress, Pest Protection


Sub-trait Area:
Drought, Nutrient uptake


Utility:
This promoter sequence can be used to improve: Modulation of



root growth rate, size and shape. Modulation of water and nutrient



uptake. Modulation of plant-microbe including plant rhizobium



interactions. Useful for making insecticidal proteins for protection



against root worm. Protection against drought stress conditions.



Protection against heat stress conditions.


Notes:
Endogenous promoter up-regulated under drought, heat and PEG.








Construct:
YP0384


Promoter candidate I.D:
11768599


cDNA I.D:
12730108 (OCKHAM3-CD)


Lines expressing:
YP0384-01, -02; Oct. 24, 2003









Example 21












Promoter Expression Report #146


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
   L receptacle


Silique
L abscission zone


Primary Root
H epidermis







Observed expression pattern:


T1 mature: Weak GFP expression specific to abscission zone of mature flowers.


T2 seedling: Weak expression in root epidermal cells. Expression rapidly decreases from root


transition zone to mid root.








Expected expression pattern:
Drought inducible


Selection Criteria:
Ceres expression data


Gene:
Neoxanthin cleavage enzyme







GenBank: NM_112304 Arabidopsis thaliana 9-cis-epoxycarotenoid dioxygenase [neoxanthin


cleavage enzyme](NC1)(NCED1), putative (At3g14440) mRNA, complete cds








Source Promoter Organism:
Arabidopsis thaliana, WS ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
XT1 Mature
XT2 Seedling
T2 Mature
T3 Seedling







Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hr air dry
2/2
Yes


2. ABA 100 uM
7 days
T2
6 Hr
4/1
Yes


3. Drought
4 weeks
T2
10-12 days no
2/0
No





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
3 Hr air dry
Hypocotyl, Cotyledon,
Epidermis, vascular




Rosette Leaf, Primary Root


2. ABA 100 uM
6 Hr
Cotyledon
Epidermis


3. Drought
10-12 days no H20
No differences observed.








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 6
   Events Expressing: n = 3


GFP Expression Detected








X Flower
pedicel L receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


X Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata L abscission zone ovule


Ovule
Pre-fertilization: inner integument outer integument embryo sac



funiculus chalaza micropyle gametophyte



Post-fertilization: zygote embryo sack inner integument outer



integument endothelium 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


Shoot apical
shoot apical meristem flower primordium


meristem


T2 Seedling Expression
Tissues Screened


Events Screened: n = 4
   Events Expressing: n = 3







Seedlings expressing/Seedlings screened


Event-01: 5/6


Event-02: 5/6


Scheduled


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem


Promoter utility








Trait Area:
PG&D, Stress


Sub-trait Area:
Flower abscission, Drought


Utility:
This promoter sequence can be used to improve: Delaying or



accelerating flower abscission in canola. Delaying or



accelerating fruit drop. Making flowerless plants. Modulation



of water and mineral nutrient uptake. Useful to drive genes



important in corn root worm resistance and other interactions



involving roots and pests or microbes. Enhanced tolerance of



drought conditions. Enhanced tolerance of heat conditions.







Notes: Milborrow BV. The pathway of biosynthesis of abscisic acid in vascular plants: a


review of the present state of knowledge of ABA biosynthesis. J Exp Bot. June 2001;


52(359): 1145-64. Review. Qin X. Zeevaart JA. The 9-cis-epoxycarotenoid cleavage


reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean. Proc


Natl Acad Sci U S A. Dec. 21, 1999; 96(26): 15354-









Endogenous promoter generally up-regulated under drought.


Construct:
YP0385


Promoter candidate I.D:
11768579


cDNA I.D:
12658348 (OCKHAM3-C)


Lines expressing:
YP0385-02, -03, -06; Oct. 24, 2004









Example 22












Promoter Expression Report #153


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
   L pedicel L sepal M style M silique


Silique
M style M septum


Stem
L epidermis L cortex H pith


Hypocotyl
L epidermis


Cotyledon
L mesophyll L epidermis


Primary Root
L epidermis L cortex







Observed expression pattern:


T1 mature: GFP expression in vegetative tissues. Highly expressed in ground tissues of leaf and


stem. Not detected in vascular tissue. In flowers, expressed in epidermal cells of sepals and in


style-valve margin region of silique. Not expressed in carpels, placenta or ovules. Specific


expression localized to group of cells at the junction of the stem and pedicle.


T2 seedling: Expressed throughout epidermal cells of seedling. Also expressed in mesophyll


cells of cotyledon, and cortex cells in root.








Expected expression pattern:
Drought Inducible


Selection Criteria:
Public


Gene:
RD29A







GenBank: NM_124610 Arabidopsis thaliana low-temperature-responsive protein 78 (LTI78)/


desiccation-responsive protein 29A (RD29A) (At5g52310) mRNA, complete cds gi|306961








Source Promoter Organism:
Arabidopsis thaliana, Columbia ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
XT1 Mature
XT2 Seedling
T2 Mature
T3 Seedling







Inductions completed:
















Events Screened/



Treatment:
Age:
Gen:
Time points:
Response
Response:


1. Drought
7 days
T2
3 Hrs air dry
2/2
Yes


2. Drought
4 weeks
T2
10-12 days no
2/2
Yes





H20







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
3 Hrs air dry
Hypocotyl
Epidermis, Vascular




Rosette Leaf
Trichomes, petiole, vascular


2. Drought
10-12 days no H20
Inflorescence
SAM, Flowers, Pedicels




meristem




Flowers
Sepals, Siliques




Sepals
Epidermis




Silique
Epidermis, Septum




Stem
Pith, Vascular




Leaf
Epidermis, Mesophyll,





Vascular








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 4
   Events Expressing: n = 4







GFP Expression Detected








X Flower
L pedicel receptacle nectary L sepal petal filament anther



pollen carpel M style papillae vascular epidermis stomata



trichome



M silique


X Silique
stigma M style carpel M septum placentae funiculus



transmitting tissue vascular epidermis stomata abscission zone



ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack inner



integument outer integument funiculus endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
L epidermis L cortex vascular xylem phloem H pith stomata



trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
   Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-02: 4/6


GFP Expression Detected








X Hypocotyl
L epidermis cortex vascular xylem phloem stomata


X Cotyledon
L mesophyll vascular L epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
L epidermis trichoblast atrichoblast L cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem


Promoter utility








Trait Area:
PG&D, Stress, Nutrients


Sub-trait Area:
Plant architecture, drought, nitrogen uptake


Utility:
This promoter sequence can be used to improve: General



alterations in growth and development, including modulation of leaf



growth and expansion, root growth elongation, branching patterns



and phyllotaxy. Useful in controlling silique shatter and seed



dispersal. Modulation of incompatibility, especially gametophytic



incompatibility. Protection against drought stress conditions.



Protection against heat stress conditions. Modulation of plant



responses to abiotic stress.


Notes:
Endogenous promoter generally induced under drought and cold.


Construct:
PT0633


Promoter candidate I.D:
11768604


cDNA I.D:
12328867 (OCKHAM3-CD)


Lines expressing:
PT0633 01, 02, 04, 06.









Example 23












Promoter Expression Report #156


Promoter Tested In: Arabidopsis thaliana, Wassilewskija WS ecotype


Spatial expression summary:








Flower
H sepal H petal H filament H epidermis L stomata


Silique
L stomata


Stem
L stomata


Hypocotyl
L stomata


Cotyledon
L stomata


Rosette Leaf
L stomata


Primary Root
H epidermis H vascular







Observed expression pattern:


T1 mature: Expressed in second and third whorls of floral organs. High GFP expression in


epidermal and vascular tissues of petals and stamens in flowers with expression restricted


vasculature in sepals. GFP expression in stamens is localized to filaments and connective


tissue between locules of anthers. Guard cell expression throughout floral organs and stem.


Expression in siliques restricted to guard cells.


T2 seedling: High GFP expression throughout vasculature and epidermal cells near root


transition zone of seedling root. Guard cell expression throughout seedlings.


Expected expression pattern: Shoot apex including leaf primordia and parts of leaves


Selection Criteria: Ceres expression data. Greater than 5x up in stm microarray.


Gene: Cys/Met metabolism pyridoxal-phosphate-dependent enzyme family protein


GenBank: NM_105141 Arabidopsis thaliana Cys/Met metabolism pyridoxal-phosphate-


dependent enzyme family protein (At1g64660) mRNA, complete cds








Source Promoter Organism:
Arabidopsis thaliana, Columbia ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
X T1 Mature
XT2 Seedling
T2 Mature
T3 Seedling








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 2
   Events Expressing: n = 2







GFP Expression Detected








X Flower
pedicel receptacle nectary H sepal H petal H filament anther



pollen carpel style papillae vascular H epidermis L stomata



trichome silique


X Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis L stomata abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack inner integument



outer integument endothelium seed coat primordia chalaza



micropyle early endosperm mature endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
epidermis cortex vascular xylem phloem pith L stomata



trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
   Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 1/6


Event-02: 6/6


Scheduled


GFP Expression Detected








X Hypocotyl
epidermis cortex vascular xylem phloem L stomata


X Cotyledon
mesophyll vascular epidermis margin L stomata



hydathode


X Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia L stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem








Promoter utility



Trait Area:
Nutrients, Stress


Sub-trait Area:
Nitrogen uptake, drought


Utility:
This promoter sequence can be used to improve:



Modulation of flower development and structure. Modulation



of drought responses. Modulation of nutrient and water uptake.


Notes:
Up-regulated in drought, ABA and far red light treatments.


Construct:
PT0564


Promoter candidate I.D:
11768766


cDNA I.D:
12348328


Lines expressing:
PT0564-01, -02; Jan. 12, 2004.









Example 24












Promoter Expression Report #158








Promoter Tested In:
Arabidopsis thaliana, Wassilewskija WS ecotype







Spatial expression summary:








Silique
H ovule H gametophyte


Hypocotyl
L vascular


Primary Root
H vascular H pericycle







Observed expression pattern:


T1 mature: Expression specific to cell types within the developing female gametophyte of the


pre-fertilized ovule. GFP levels decrease post-fertilization with degenerating GFP appearing in


suspensor cells of embryo and micropylar region in ovules.


T2 seedling: High expression in vascular bundle of root with decreasing expression within


hypocotyl.


Expected expression pattern: Expressed primarily in the vascular cylinder of root.


Selection Criteria: Public reference


Gene: Leucine-rich repeat protein kinase


GenBank: NM_126243 Arabidopsis thaliana leucine-rich repeat protein kinase, putative


(At2g01820) mRNA, complete cds gi|42568868|ref|NM_126243.2|[42568868]








Source Promoter Organism:
Arabidopsis thaliana, Columbia ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
X T1 Mature
X T2 Seedling
T2 Mature
T3 Seedling








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 2
   Events Expressing: n = 2


GFP Expression Detected








X Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


X Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone H ovule


H Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle H gametophyte



Post-fertilization: zygote suspensor embryo sac inner



integument outer integument endothelium 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


Shoot apical
Shoot apical meristem Flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 3/6


Event-02: 5/6


Scheduled


GFP Expression Detected








X Hypocotyl
epidermis cortex L vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem H pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem


Promoter utility








Trait Area:
Seeds, PG&D, Nutrients, Stress


Sub-trait Area:
Seed number and size, nitrogen uptake


Utility:
This promoter sequence can be used to improve:



Modulation of fertility control. Modulation of nitrogen and water



transport. Modulation of seed fill and seed size.








Construct:
PT0569


Promoter candidate I.D:
11768780


cDNA I.D:
13578506 (OCKHAM3-CD)


Lines expressing:
-01, -03; Jan. 12, 2004









Example 25












Promoter Expression Report #168


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Ovule
Post-fertilization: H outer integument, H mature endosperm


Embryo
H mature


Primary Root
H cortex H endodermis







Observed expression pattern:


T1 mature: GFP expression in cellularizing endosperm of pre-torpedo stage ovule and mature


embryo and outer integument of developing seed.


T2 seedling: GFP expression in cortex and endodermis cells of roots. Not observed in vascular


bundle as described by Haseloff characterization of line H002536.


Expected expression pattern: Vascular exp in root after elongation zone


Selection Criteria: Haseloff two component line HAP1_ID: H002536


Gene: “transketolase family protein”


GenBank: NM_129013 Arabidopsis thaliana transketolase family protein (At2g34590) mRNA,


complete cds gi|30686124|ref|NM_129013.2








Source Promoter Organism:
Arabidopsis thaliana, Columbia ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
XT1 Mature
XT2 Seedling
T2 Mature
T3 Seedling








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 6
   Events Expressing: n = 3








GFP Expression Detected



Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sac inner



integument



H outer integument endothelium seed coat primordia chalaza



micropyle early endosperm H mature endosperm embryo


X Embryo
suspensor preglobular globular heart torpedo late H 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


Shoot apical
Shoot apical meristem Flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
   Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-03: 1/2


Event-04: 2/3


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast H cortex H endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem


Promoter utility








Trait Area:
PG&D, nutrients, Seeds


Sub-trait Area:
Root architecture, drought, seed size


Utility:
This promoter sequence can be used to improve: Alteration in



seed composition. Enhanced desiccation tolerance and seed



germination. Modulation endosperm development and composition.



Enhanced seedling vigor. Modulation of drought tolerance and



protection against drought stress. Modulation of seed size.


Notes:
Endosperm and embryo expression is not a common expression



pattern. This promoter can be unusual and valuable for



manipulation of an entire seed.



Endogenous promoter induced by nitrate, heat, and UV-B



treatments. Also induced in germinating seeds and root tips.


Construct:
PT0650


Promoter candidate I.D:
15224209


cDNA I.D:
13602857


Lines expressing:
PT0650-03, -04, -06









Example 26












Promoter Expression Report # 172








Promoter Tested In:
Arabidopsis thaliana, WS ecotype







Spatial expression summary:








Embryo
H radicle


Primary Root
H root cap


Lateral root
H lateral root cap







Observed expression pattern:


T1 mature: High GFP expression and specificity to root cap of primary and lateral roots.


Expression in root cap observed from mid-globular to mature embryo. Low expression in


mature endosperm.


T2 seedling: High GFP expression and specificity to root cap of main and lateral roots.


Expected expression pattern: Haseloff characterization of line H000737. Expression in


epidermis and cortex of primary and secondary roots. Not in root tip. Expression main-


tained throughout elongation but lost soon after elongation is complete


Selection Criteria: Two component line HAP1_ID: H000737


Gene: “Scarecrow transcription factor family protein”


GenBank: NM_100626 Arabidopsis thaliana scarecrow transcription factor family protein


(At1g07520) mRNA, complete cds gi|42561783|ref|NM_100626.2|[42561783]








Source Promoter Organism:
Arabidopsis thaliana, Columbia


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  XT1 Mature XT2 Seedling T2 Mature T3 Seedling








T1 Mature Plant Expression
Organs/Tissues screened







Events Screened: n = 5        Events Expressing: n = 3


GFP Expression Detected








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sac inner



integument outer integument endothelium seed coat primordia



chalaza micropyle early endosperm mature endosperm embryo


X Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis H radicle cotyledons hypocotyl


Stem
epidermis cortex vascular xylem phloem pith stomata



trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
Shoot apical meristem Flower primordium


meristem







T2 Seedling Expression    Tissues Screened


Events Screened: n = 2     Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 3/4


Event-02: 3/6


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella H root cap root hairs


X Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular H lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
Nutrients, PG&D, Stress


Sub-trait Area:
Nitrogen uptake, drought, root growth


Utility:
This promoter sequence can be used to improve:



Modulation of root growth. Modulation of nutrient uptake.



Modulation of drought stress. Modulation of biotic stress



tolerance







Notes: Sabatini S, Heidstra R, Wildwater M, Scheres B. SCARECROW is involved in


positioning the stem cell niche in the Arabidopsis root meristem. Genes Dev. Feb. 1, 2003;


17(3): 354-8. PMID: 12569126


Long JA, Woody S, Poethig S, Meyerowitz EM, Barton MK. Transformation of shoots into


roots in Arabidopsis embryos mutant at the TOPLESS locus. Development. June 2002;


129(12): 2797-806. PMID:








Construct:
PT0659


Promoter candidate I.D:
15224254


cDNA I.D:
12661578 (OCKHAM3-CD)


Lines expressing:
PT0659-02, -05, -04; Apr. 26, 2004









Example 27












Promoter Expression Report #179








Promoter Tested In:
Arabidopsis thaliana, Wassilewskija (WS) ecotype







Spatial expression summary:








Silique
H funiculus


Hypocotyl
M vascular


Cotyledon
L vascular


Primary Root
H vascular







Observed expression pattern:


T1 mature: GFP expression specific to funiculus of developing seed, decreasing in maturing


seed. No expression observed in funiculus of prefertilized ovules.


T2 seedling: GFP expression observed in vascular tissues of root, hypocotyls, petioles and


cotyledons.


Expected expression pattern: H004797: Seems to be expressed in the quiescent center and in


all the initials around. Faint expression in vasculature later in the root.


Selection Criteria: Two component line HAP1_ID: H004797


Gene: “adenylylsulfate kinase 1 (AKN1)”


GenBank: NM_127039 Arabidopsis thaliana adenylylsulfate kinase 1 (AKN1) (At2g14750)


mRNA, complete cds gi|30679236|ref|NM_127039.2








Source Promoter Organism:
Arabidopsis thaliana, Col


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  XT1 Mature X T2 Seedling T2 Mature T3 Seedling


T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 3     Events Expressing: n = 2


GFP Expression Detected








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


X Silique
stigma style carpel septum placentae H funiculus transmitting



tissue vascular epidermis stomata abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sac inner



integument outer integument endothelium 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


Shoot apical
Shoot apical meristem Flower primordium


meristem







T2 Seedling Expression   Tissues Screened


Events Screened: n = 2     Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 3/6


Event-02: 3/6


GFP Expression Detected








X Hypocotyl
epidermis cortex M vascular xylem phloem stomata


X Cotyledon
mesophyll L vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem







Promoter utility








Trait Area:
PG&D, Nutrients


Sub-trait Area:
Seed fill, nutrient uptake


Utility:
This promoter sequence can be used to improve:



Modulation of nitrogen uptake and transport. Modulation of seed



size.


Notes:
Endogenous promoter is up-regulated by nitrates and UV-B and



down-regulated in siliques.


Construct:
PT0655


Promoter candidate I.D:
15224239


cDNA I.D:
13616568


Lines expressing:
PT0655-02, -04; Apr. 26, 2004









Example 28












Promoter Expression Report #180










Promoter Tested In: Arabidopsis thaliana, Wassilewskija WS ecotype


Spatial expression summary:








Ovule
H embryo sac H gametophyte H early endosperm


Primary Root
L epidermis M cortex H endodermis L root cap







Observed expression pattern:


T1 mature: Low expressivity or penetrance of GFP within endosperm of


developing ovules. Expressed in gametophyte of prefertilized through


early cellularization stage fertilized ovules. Observed in few ovules per


silique.


T2 seedling: GFP expression throughout ground tissues of seedling roots.


Not observed in vascular tissues.


Expected expression pattern: Expression in several layers of the root. In


colette, primary and secondary root. A few cells of root tip. Very strong


expression. Also some patches in leaves and occasionally in the axils of


the stem.


Selection Criteria: Two component line HAP1_ID: H003816


Gene: The DER1 locus encodes ACTIN2 (ACT2), a major actin of


the vegetative tissue. Arabidopsis mutant, deformed root hairs 1 (der1),


that is impaired in root hair development.


GenBank: NM_119078 Arabidopsis thaliana Der1-like family


protein/degradation in the ER-like family protein (At4g29330) mRNA,


complete cdsgi|30688288|ref|NM_119078.3


Source Promoter Organism: Arabidopsis thaliana, Columbia


Vector: pNewbin4-HAP1-GFP


Marker Type: GFP-ER


Generation Screened: X T1 Mature XT2 Seedling T2 Mature T3 Seedling











T1 Mature Plant Expression
Organs/Tissues screened





Events Screened: n = 6
Events Expressing: n = 2


GFP Expression Detected


Flower
pedicel receptacle nectary sepal petal



filament anther pollen carpel style



papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae



transmitting tissue vascular epidermis



stomata abscission zone ovule


X Ovule
Pre-fertilization: primordia inner integument



outer integument



H embryo sac funiculus chalaza micropyle



H gametophyte



Post-fertilization: zygote suspensor embryo



sac inner integument outer integument



endothelium seed coat primordia chalaza



micropyle H 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


Shoot apical
shoot apical meristem flower primordium


meristem





T2 Seedling Expression
Tissues Screened





Events Screened: n = 3
Events Expressing: n = 3







Seedlings expressing/Seedlings screened


Event-01: 1/6


Event-02: 1/6


Event-03: 3/6


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem



stomata


Cotyledon
mesophyll vascular epidermis margin



stomata hydathode


Rosette Leaf
mesophyll vascular epidermis trichome



petiole primordia stomata stipule



margin hydathode


X Primary Root
L epidermis trichoblast atrichoblast M cortex



H endodermis



vascular xylem phloem pericycle quiescent



columella L root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex



endodermis initials flanking cells vascular



lateral root cap


Shoot apical meristem
Shoot apical meristem










Promoter utility











Trait Area:
Nutrients, Drought, Seeds


Sub-trait Area:
Nitrogen uptake, drought


Utility:
This promoter sequence can be used to



improve:



Modulation of fertility control. Modulation of drought



tolerance.



Modulation of nitrogen uptake and transport.







Notes:


Ringli C. Baumberger N, Diet A, Frey B, Keller B. ACTIN2 is essential


for bulge site selection and tip growth during root hair development of


Arabidopsis.Plant Physiol.August 2002;129(4):1464-72. PMID:


12177460. Knop M, Finger A, Braun T, Hellmuth K, Wolf DH.


Der1, a novel protein specifically required for endoplasmic reticulum


degradation in yeast.


EMBO J. Feb. 15, 1996; 15(4):753-63.













Construct:
PT0656



Promoter candidate I.D:
15224245



cDNA I.D:
13593536 (OCKHAM3-CD)



Lines expressing:
PT0656 -01, -02, -03










Example 29












Promoter Expression Report #181


Promoter Tested In: Arabidopsis thaliana, Wassilewskija WS ecotype


Spatial expression summary:








Flower
H anther H silique


Silique
H ovule


Ovule
Post-fertilization: H zygote H suspensor H outer integument H



seed coat



H embryo


Embryo
H suspensor H heart H torpedo M late L mature


Stem
L vascular


Shoot apical
L Shoot apical meristem L Flower primordium


Meristem


Hypocotyl
M epidermis M vascular


Cotyledon
M vascular M epidermis


Rosette Leaf
H mesophyll M vascular H epidermis H petiole


Primary Root
H vascular H root cap


Lateral root
H initials







Observed expression pattern:


T1 mature: Low GFP expression in shoot apical meristem and floral primordia. In flowers,


expression observed in the carpels of the developing silique, tapetum tissue of developing


anthers, nectarines, and ovules. GFP is expressed in the outer integuments and globular to


mature embryos from ovules to mature seed. Expression in embryos is preferentially located


in the suspensor cells and embryonic root meristmatic cells. GFP expression in vasculature of


stem.


T2 seedling: GFP expression throughout vasculature, mesophyll and epidermis. In root,


expression restricted to vascular bundle, lateral root primordia and root cap.


Expected expression pattern: Strong and nice expression in root tip and in the elongation


zone. Expression fades out quickly. Also expression in the initials of lateral root (low


expression).


Selection Criteria: Two component line HAP1_ID: H004467


Gene: product = “nascent polypeptide-associated complex (NAC)domain-containing protein/


BTF3b-like transcription factor, putative”


GenBank: NM_101651 Arabidopsis thaliana nascent polypeptide-associated complex


(NAC) domain-containing protein/BTF3b-like transcription factor, putative (At1g17880)


mRNA








Source Promoter Organism:
Arabidopsis thaliana, Columbia


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
XT1 Mature
XT2 Seedling
T2 Mature
T3 Seedling








T1 Mature Plant Expression
Organs/Tissues screened








Events Screened: n = 4
Events Expressing: n = 4


GFP Expression Detected








X Flower
pedicel receptacle nectary sepal petal filament H anther



pollen H carpel style papillae vascular epidermis stomata



trichome



H silique


X Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone H ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: H zygote H suspensor embryo sack inner



integument H outer integument endothelium H seed coat



primordia chalaza micropyle early endosperm mature



endosperm H embryo


X Embryo
H suspensor preglobular globular H heart H torpedo M late L



mature provascular hypophysis radicle cotyledons hypocotyl


X Stem
epidermis cortex L vascular xylem phloem pith stomata



trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


X Shoot apical
L Shoot apical meristem L Flower primordium


meristem








T2 Seedling Expression
Tissues Screened


Events Screened: n = 3
  Events Expressing: n = 3







Seedlings expressing/Seedlings screened


Event-01: 4/4


Event-02: 3/6


Event-03: 2/3


GFP Expression Detected








X Hypocotyl
M epidermis cortex M vascular xylem phloem stomata


X Cotyledon
mesophyll M vascular M epidermis margin stomata



hydathode


X Rosette Leaf
H mesophyll M vascular H epidermis trichome H petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem H pericycle quiescent



columella H root cap root hairs


X Lateral root
epidermis trichoblast atrichoblast cortex endodermis



H initials flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem








Promoter utility



Trait Area:
PG&D, Nutrients, Stress, Seeds


Sub-trait Area:
Drought, nitrogen uptake, Seed size, seed fill


Utility:
This promoter sequence can be used to improve:



Modulation of drought responses. Modulation of nitrogen and water



uptake. Modulation of seed size and seed fill. Modulation of leaf



angle.







Notes: Nascent Polypeptide-associated Complex Stimulates Protein Import into Yeast


Mitochondria. Funfschilling U, Rospert S. Mol Biol Cell. Oct. 1, 1999; 10(10): 3289-3299.


Down-regulated in germinating seeds and up-regulated in root tips.








Construct:
PT0657


Promoter candidate I.D:
15224248


cDNA I.D:
13604449 (OCKHAM3-CD)


Lines expressing:
PT0657-01, -02, -03, -04; Apr. 26, 2004









Example 30












Promoter Expression Report #186


Promoter Tested In: Arabidopsis thaliana, Wassilewskija WS ecotype


Spatial expression summary:








Primary Root
L cortex L vascular L pericycle







Observed expression pattern:


T1 mature: No expression observed.


T2 seedling: Low expression in cortex and pericycle cells throughout root.


Expected expression pattern: Expression in vascular or pericycle.


Selection Criteria: Two-component line HAP1_ID: H006530


Gene: transcriptional factor B3 family protein


GenBank: NM_112785 Arabidopsis thaliana transcriptional factor B3 family protein


(At3g18990) mRNA, complete cds gi|42564995|ref|NM_112785.3|[42564995]








Source Promoter Organism:
Arabidopsis thaliana, Columbia


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
XT1 Mature
XT2 Seedling
T2 Mature
Seedling








T2 Seedling Expression
Tissues Screened


Events Screened: n = 3
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened








Event-01: 1/3



Event-02: 2/6


Event-03: 0/6


GFP Expression Detected


Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast L cortex endodermis



L vascular xylem phloem L pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem


Promoter utility


Trait Area:
Stress, Nutrients


Sub-trait Area:
Drought, Nitrogen uptake


Utility:
This promoter sequence can be used to improve:



Modulation of drought tolerance. Modulation of nutrient uptake



and transport.


Construct:
PT0660


Promoter candidate I.D:
15224257


cDNA I.D:
13614201


Lines expressing:
PT0660-01, -02; Apr. 12, 2004









Example 31












Promoter Expression Report #203


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Hypocotyl
L stomata


Cotyledon
L stomata


Rosette Leaf
L stomata


Primary Root
L epidermis H endodermis







Observed expression pattern:


T1 mature: No expression observed.


T2 seedling: Primary GFP expression in root endodermal cell layer with weak expression in


epidermal cells near transition zone. Guard cell expression throughout aerial tissue.








Expected expression pattern:
Shade Induced


Selection Criteria:
Ceres expression data


Gene:
Expressed protein







GenBank: NM_125622 Arabidopsis thaliana expressed protein (At5g62280) mRNA, complete


cds gi|30697652|ref|NM_125622.2|[30697652]








Source Promoter Organism:
Arabidopsis thaliana, Columbia


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
XT1 Mature
XT2 Seedling
T2 Mature
T3 Seedling







Inductions completed:












Treatment:
Age:
Gen:
Time points:
Events Screened/Response
Response:


1. Far red
7 days
T2
 1 Hr
2/0
No


Far Red730 =


24 Hr
2/0
No


525 μW/cm2







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:









1. Far red
 1 Hr
No differences observed.


Far Red730 = 525 μW/cm2
24 Hr
No differences observed.







Observation note: No differences were observed between control and experimental seedlings


under far red conditions. Difference in expression levels compared to original T2 seedling screen.


Higher expression can be seen in root epidermal cells and no GFP in guard cells can be detected.








T1 Mature Plant Expression
Organs/Tissues screened








Events Screened: n = 3
Events Expressing: n = 0







No GFP Expression Detected








T2 Seedling Expression
Tissues Screened








Events Screened: n = 2
Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 2/6


Event-04: 2/6








GFP Expression Detected



X Hypocotyl
epidermis cortex vascular xylem phloem L stomata


X Cotyledon
mesophyll vascular epidermis margin L stomata



hydathode


X Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia L stomata stipule margin hydathode


X Primary Root
L epidermis trichoblast atrichoblast cortex H endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical meristem
shoot apical meristem


Promoter utility


Trait Area:
Stress, nutrients


Sub-trait Area:
Drought, nitrogen uptake


Utility:
This promoter sequence can be used to improve: Modulation of



all responses to drought and heat stress. Protection against drought



stress. Protection against heat stress.


Notes:
Endogenous promoter is down-regulated in heat and drought, up-



regulated in far red light and circadian rhythm.


Construct:
PT0679


Promoter candidate I.D:
15295967


cDNA I.D:
12732583 (OCKHAM3-CD)


Lines expressin:
PT0679-01









Example 32












Promoter Expression Report #205


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Silique
L funiculus H ovule


Ovule
Post-fertilization: L funiculus H outer integument


Hypocotyl
L epidermis


Cotyledon
H epidermis H petiole


Rosette Leaf
H epidermis H petiole


Primary Root
H endodermis







Observed expression pattern:


T1 mature: GFP expression highly specific to second cell layer of the outer integument in


developing seed. Also expressed at ovule connective site of funiculus. No expression observed in


pre-fertilized ovules.


T2 seedling: GFP exhibits expression in a highly polar fashion in cotyledons and rosette leaves.


GFP is expressed proximal-distal on the abaxial surface of the petioles of cotyledons and rosette


leaves with respect to the shoot apical meristem. Low GFP expression observed on the adaxial


surface of petioles of cotyledons and rosette leaves. GFP expression in epidermal cells of


hypocotyl at the root transition zone decreases toward apex. Highly specific expression of GFP in


endodermis cells of the root.








Expected expression pattern:
Shade Induced


Selection Criteria:
Ceres expression data


Gene:
Acyl CoA reductase, putative







GenBank: NM_122155 Arabidopsis thaliana acyl CoA reductase, putative/male-sterility


protein, putative (At5g22500) mRNA, complete cds gi|30688503|ref|NM_122155.2|[30688503]








Source Promoter Organism:
Arabidopsis thaliana, Columbia ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
XT1 Mature
XT2 Seedling
T2 Mature
T3 Seedling


Inductions completed.












Treatment:
Age:
Gen:
Time points:
Events Screened/Response
Response:


1. Far red
7 days
T2
1 Hr
2/0
No


Far Red730 = 525 μW/cm2


24 Hr
2/0
No







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Far red
No differences observed.








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 3
  Events Expressing: n = 3








GFP Expression Detected



Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


X Silique
stigma style carpel septum placentae L funiculus transmitting



tissue vascular epidermis stomata abscission zone H ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



funiculus embryo sac chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack L funiculus



inner integument H outer integument endothelium 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


Shoot apical
shoot apical meristem flower primordium


meristem


T2 Seedling Expression
Tissues Screened


Events Screened: n = 2
  Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 2(6)


Event-02: 2(6)








GFP Expression Detected



X Hypocotyl
L epidermis cortex vascular xylem phloem stomata


X Cotyledon
mesophyll vascular H epidermis margin H petiole stomata



hydathode


X Rosette Leaf
mesophyll vascular H epidermis trichome H petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex H endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem


Induction Screens








1. Far red
No differences observed.


Promoter utility


Trait Area:
PG&D, nutrients


Sub-trait Area:
Nitrogen use efficiency, seed size


Utility:
This promoter sequence can be used to modulate seed growth



and development, seed dormancy and germination. Also, useful



for enhancement of leaf and root growth, resulting in increased



source capacity and water and nutrient loading. Useful in



procedures and technologies aimed at improving source-sink



relationships and seed filling and yield.







Notes: Lardizabal KD, Metz JG, Sakamoto T, Hutton WC, Pollard MR, Lassner MW.


Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high


levels of wax in seeds of transgenic arabidopsis. Plant Physiol. March 2000; 122(3): 645-55.


Metz JG, Pollard MR, Anderson L, Hayes TR, Lassner MW. Purification of a jojoba embryo


fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed.


Plant Physiol. 2000 122: 635-44.








Construct:
PT0676


Promoter candidate I.D:
15295958


cDNA I.D:
23658955


Lines expressing:
PT0676-01, -02; Apr. 12, 2004









Example 33












Promoter Expression Report #177


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
H pedicel H petal M filament M anther H epidermis H carpel


Silique
H carpel H epidermis


Stem
L endodermis H pith


Rosette Leaf
L epidermis H trichome H petiole







Observed expression pattern:


T1 mature: Vegetatative expression. GFP expressed in epidermis of stem and pedicels of the


inflorescence meristem near the shoot apex. In the Flower, GFP expression is specific to stamen


and carpels of siliques. GFP highly expressed in parenchyma cells of stem. No expression in


ovules.


T2 seedling: High specific GFP expression in cells at the base of developing trichomes and


petioles of rosette leaves. Expression in adventitious trichome cells of the hypocotyls.








Expected expression pattern:
Shade Induced


Selection Criteria:
Ceres expression data


Gene:
expressed protein; “/product = “unknown protein”







GenBank: NM_113862 expressed protein (At3g29370) mRNA, complete








Source Promoter Organism:
Arabidopsis thaliana, Columbia ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER











Generation Screened:
XT1 Mature
XT2 Seedling
T2 Mature
T3 Seedling


Inductions completed.















Time
Events Screened/



Treatment:
Age:
Gen:
points:
Response
Response:


1. Far red
7 days
T2
 1 Hr
2/0
No


Far Red730 = 525 μW/cm2


24 Hr
2/1
Yes







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Far red
24 Hr
Petiole
Epidermis, Cortex,


Far Red730 = 525 μW/cm2


Vascular








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened: n = 6
  Events Expressing: n = 2








GFP Expression Detected



X Flower
H pedicel receptacle nectary sepal H petal M filament M



anther pollen H carpel style papillae vascular H epidermis



stomata trichome silique


X Silique
stigma style H carpel septum placentae transmitting tissue



vascular H epidermis stomata abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
epidermis cortex L endodermis vascular xylem phloem H



pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
Shoot apical meristem Flower primordium


meristem


T2 Seedling Expression
  Tissues Screened


Events Screened: n = 2
    Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 4(6)


Event-05: 3(6)








GFP Expression Detected



Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


X Rosette Leaf
mesophyll vascular L epidermis H trichome H petiole



primordia stomata stipule margin hydathode


Primary Root
epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical meristem
Shoot apical meristem


Promoter utility


Utility:
This promoter sequence can be used to improve: Modulation of



flower and inflorescence structure, especially numbers of flowers



per inflorescence and therefore seeds per inflorescence.



Modulation of carpel symmetry and number of valves per carpel



and number of seeds per carpel. Modulation of shade avoidance



responses, especially petiole and leaf elongation under shade.



Enhanced seedling and plant performance under shade



conditions. Combinational effects or growth, development,



fertility and responses to shade.


Construct:
PT0684


Promoter candidate I.D:
15295997


cDNA I.D:
13486695


Lines expressing:
PT0684-01-05; Apr. 26, 2004









Example 34












Promoter Expression Report #178


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
H stomata


Ovule
Post-fertilization: M endothelium


Stem
H stomata


Leaf
H stomata


Cotyledon
H stomata


Rosette Leaf
H stomata


Primary Root
M epidermis H pericycle H root hairs


Lateral root
H initials H lateral root cap







Observed expression pattern:


T1 mature: High GFP expression in guard cells throughout mature plant aerial tissue and


endothelium cell layer of developing seed.


T2 seedling: GFP expression in trichomes and guard cells of cotyledons and rosette leaves.


Primary expression in root localized to pericycle cells and lateral root initials and later in


mature lateral root cap. Weak epidermal and root hair expression.








Expected expression pattern:
Shade Induced


Selection Criteria:
Ceres expression data


Gene:
short-chain dehydrogenase/reductase (SDR) family



protein, oxidoreductase activity







GenBank: NM_120332 Arabidopsis thaliana short-chain dehydrogenase/reductase (SDR)


family protein (At5g02540) mRNA, complete cds gi|30679675|ref|NM_120332.2|[30679675]








Source Promoter Organism:
Arabidopsis thaliana, Columbia


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling







Inductions completed:















Time
Events Screened/



Treatment:
Age:
Gen:
points:
Response
Response:


1. Far red
7 days
T2
 1 Hr
2/0
No


Far Red730 = 525 μW/cm2


24 Hr
2/2
Yes







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Far red
 1 Hr
No differences observed.


Far Red730 = 525 μW/cm2
24 Hr
Cotyledon
Stomata




Rosette Leaf
Stomata







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 4    Events Expressing: n = 2


GFP Expression Detected








X Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis H stomata



trichome



silique


Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack inner



integument outer integument M endothelium seed coat primordia



chalaza micropyle early endosperm mature endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
epidermis cortex vascular xylem phloem pith H stomata



trichome


X Leaf
petiole mesophyll vascular epidermis trichome primordia H



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression  Tissues Screened


Events Screened: n = 2   Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-03: 6(6)


Event-04: 5(6)


GFP Expression Detected








Hypocotyl
epidermis cortex vascular xylem phloem stomata


X Cotyledon
mesophyll vascular epidermis margin H stomata



hydathode


X Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia H stomata stipule margin hydathode


X Primary Root
M epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem H pericycle quiescent



columella root cap H root hairs


X Lateral root
epidermis trichoblast atrichoblast cortex endodermis



H initials flanking cells vascular H lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
Stress, Nitrogen, PG&D


Sub-trait Area:
Drought, drought, nitrogen uptake, root architecture


Utility:
This promoter sequence can be used to improve: Tolerance



to drought conditions. Tolerance to heat conditions. Modulation



of responses to abiotic stress. Modulation of plant interactions



with insects and protection against insects. Modulation of



production and loading of volatiles into trichomes and other



epidermal cells. Enhanced root rate and root size. Modulation



of water and mineral ion uptake. Modulation of lateral root



initiation and growth and root architecture.


Notes:
Endogenous promoter is up-regulated in shoots and siliques and



under far red light.


Construct:
 PT0678


Promoter candidate
 15295964


I.D:


cDNA I.D:
 12712683 (OCKHAM3-CD)


Lines expressing:
 PT0678-03, -04









Example 35












Promoter Expression Report #161


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
H pedicel H sepal H petal H filament H anther H carpel H style



H epidermis H stomata H silique


Silique
H style H carpel H transmitting tissue H epidermis H ovule


Ovule
Pre-fertilization: L funiculus L outer integument



Post-fertilization: H funiculus H outer integument H seed coat


Stem
L vascular L phloem


Leaf
L vascular


Hypocotyl
H epidermis L vascular


Cotyledon
L epidermis


Rosette Leaf
L vascular L epidermis


Primary Root
H epidermis H cortex H endodermis H vascular H quiescent H root cap



H root hairs







Observed expression pattern:


T1 mature: GFP expression in sepals, petals, stamens and siliques of developing floral buds


through to mature flowers. GFP expression throughout all tissues of stamen excluding pollen.


GFP expression throughout all tissues of silique excluding stigma. Within ovules, highest GFP


expression is at funiculus, outer integument and mature seed coat. GFP expression in vascular


tissues of flowers, stems and leaves. In stem, expression in phloem cells within vascular bundle.


Expression in guard cells throughout plant.


T2 seedling: High GFP expression throughout epidermal tissues of seedlings. High GFP


expression throughout all root cell types decreasing toward elongation zone. GFP is expressed in


root cap and meristem cells.








Expected expression pattern:
High in siliques


Selection Criteria:
Microarray data







Gene: Chlorophyll A-B binding family protein/early light-induced protein


GenBank: NM_113183 Arabidopsis thaliana chlorophyll A-B binding family protein/early


light-induced protein (ELIP) (At3g22840) mRNA, complete cds gi|30686801|








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
X T1 Mature X T2 Seedling  T2 Mature  T3 Seedling












Treatment:
Age:
Gen:
Time points:
Events Screened/Response:
Response:


1. Drought
4 wks
T2
8 days no water
2/0
No


2. Heat
7 days
T2
2 Hr 42 C.
3/0
No





6 Hr 42 C.
3/0
No





16 Hrs - Post 42 C.
3/0
No







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 6   Events Expressing: n = 3


GFP Expression








X Flower
H pedicel receptacle nectary H sepal H petal H filament H anther



pollen H carpel H style papillae vascular H epidermis H



stomata trichome H silique


X Silique
stigma H style H carpel septum placentae H transmitting tissue



vascular H epidermis stomata abscission zone H ovule


X Ovule
Pre-fertilization: primordia inner integument L outer integument



embryo sac L funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sac H funiculus



inner integument H outer integument endothelium H seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
epidermis cortex L vascular xylem L phloem pith stomata



trichome


X Leaf
petiole mesophyll L vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression  Tissues Screened


Events Screened: n = 3   Events Expressing: n = 3


Seedlings expressing/Seedlings screened


Event-01: 3/4


Event-05: 3/6


Event-06: 4/6








X Hypocotyl
H epidermis cortex L vascular xylem phloem stomata


X Cotyledon
mesophyll vascular L epidermis margin stomata



hydathode


X Rosette Leaf
mesophyll L vascular L epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast H cortex H endodermis



H vascular xylem phloem pericycle H quiescent



columella H root cap H root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
Nutrients, seed yield, water use efficiency


Sub-trait Area:
Nitrogen use efficiency, ovule/seed abortion, endosperm cell



number/size, endosperm granule number/size, seed



enhancement, seed number, harvest index, heat, water potential,



drought, moisture stress at seed set








Utility:
This promoter sequence can be used to improve: Nitrogen use



efficiency, ovule/seed abortion, endosperm cell number/size,



endosperm granule number/size, seed enhancement, seed number,



harvest index, heat, water potential, drought, moisture stress at seed



set







Notes: This promoter is strongly differentially regulated under drought conditions and in a


number of tissues.








Construct:
PT0623


Promoter candidate I.D:
11768718


cDNA I.D:
23644072


Lines expressing:
PT0623-01, -05, -06









Example 36












Promoter Expression Report #163


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
H pollen


Silique
M ovule


Ovule
Post-fertilization: M inner integument M endothelium M embryo


Embryo
M mature embryo H root tip L radicle


Stem
L vascular


Primary Root
H epidermis H cortex L root cap H root hairs







Observed expression pattern:


T1 mature: High GFP expression in endothelium cell layer of ovules and root cap of embryos.


GFP expressed in pollen. Low GFP expression in stem vascular tissue.


T2 seedling: High GFP expression specific to epidermal cells and root cap. GFP expression in


epidermal cells decreases toward root elongation zone where no expression is observed.








Expected expression pattern:
Flowers and buds


Selection Criteria:
Microarray data







Gene: product = “expressed protein”/note = “similar to myo-inositol oxygenase


GenBank: NM_127538 Arabidopsis thaliana expressed protein (At2g19800) mRNA, complete








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling












Treatment:
Age:
Gen:
Time points:
Events Screened/Response:
Response:


1. Drought
4 wks
T2
8 days no water
5/0
No


2. ABA 100 uM
7 days
T2
2 Hr
4/0
No





6 Hr
4/0
No







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 4    Events Expressing:  n = 2








X Flower
pedicel receptacle nectary sepal petal filament anther H



pollen carpel style papillae vascular epidermis stomata



trichome



silique


X Silique
stigma style carpel septum placentae transmitting tissue



vascular epidermis stomata abscission zone M ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sac M inner



integument outer integument M endothelium seed coat primordia



chalaza micropyle early endosperm mature endosperm M embryo


X Embryo
suspensor preglobular globular heart torpedo late M mature



provascular hypophysis H root tip L radicle cotyledons



hypocotyl


X Stem
epidermis cortex L vascular xylem phloem pith stomata



trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
shoot apical meristem flower primordium


meristem







T2 Seedling Expression  Tissues Screened


Events Screened: n = 2   Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 5/6


Event-02: 4/6








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast H cortex endodermis



vascular xylem phloem pericycle quiescent



columella L root cap H root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







Promoter utility








Trait Area:
 Seed, yield, nutrients


Sub-trait Area:
 Seed enhancement, harvest index, nitrogen use efficiency


Utility:
This promoter sequence can be used to improve: Seed



enahancement, nutrient uptake and thus yield.







Notes: Lorence A, Chevone BI, Mendes P, Nessler CL. myo-inositol oxygenase offers a


possible entry point into plant ascorbate biosynthesis. Plant Physiol. March 2004; 134(3): 1200-5.


Epub Feb. 19, 2004.








Construct:
 PT0613


Promoter candidate
 13148297


I.D:


cDNA I.D:
 23555688


Lines expressing:
 PT0613-01, -04; Feb. 20, 2004









Example 37












Promoter Expression Report #190


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
H pedicel H carpel H style H epidermis H silique


Silique
H style H carpel H epidermis H cortex H abscission zone


Stem
H epidermis H cortex


Leaf
H epidermis


Hypocotyl
L epidermis L stomata


Cotyledon
L epidermis L stomata







Observed expression pattern:


T1 mature: Vegetative GFP expression. GFP expressed in walls of carpels and not expressed in


placenta, ovules or embryo. GFP expression in carpels of developing siliques beginning at floral


stage 12 and decreases in mature siliques. GFP also highly expressed in pedicels and abscission


zone. High GFP expression in epidermis of leaf. GFP highly expressed throughout epidermis and


cortex cells of stem.


T2 seedling: GFP is expressed in epidermis of hypocotyls and cotyledons. Not observed in rosette


leaf or root.








Expected expression pattern:
Drought inducible - Up regulated under drought


Selection Criteria:
Microarray


Gene:
expressed protein







GenBank: NM_124750 Arabidopsis thaliana expressed protein (At5g53710) mRNA, complete


cds gi|18423543|ref|NM_124750.1|[18423543]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
X T1 Mature X T2 Seedling  T2 Mature  T3 Seedling












Treatment:
Age:
Gen:
Time points:
Events Screened/Response:
Response:


1. Drought
7 days
T2
3 hours air dry
2/1
Yes


2. Drought
4 wks
T2
8 days no water
2/1
Yes







Inducible expression summary:










Treatment:
Time point induced:
Organs induced:
Tissues induced:


1. Drought
3 hours air dry
Hypocotyl
L Epidermis


2. Drought
8 days no water
Stem
H Epidermis




Siliques
H Epidermis







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 4    Events Expressing:  n = 4








X Flower
H pedicel receptacle nectary sepal petal filament anther



pollen H carpel H style papillae vascular H epidermis stomata



trichome



H silique


X Silique
stigma H style H carpel septum placentae funiculus



transmitting tissue vascular H epidermis H cortex stomata H



abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons hypocotyl


X Stem
H epidermis H cortex interfascicular region vascular xylem



phloem pith stomata trichome


X Leaf
H epidermis petiole mesophyll vascular trichome primordia



stomata stipule margin


Shoot apical
Shoot apical meristem Flower primordium


meristem







T2 Seedling Expression   Tissues Screened


Events Screened: n = 2    Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-02: 5/6








X Hypocotyl
L epidermis cortex vascular xylem phloem L stomata


X Cotyledon
mesophyll vascular L epidermis margin petiole L stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole primordia



stomata stipule margin hydathode


Primary Root
epidermis trichoblast atrichoblast cortex endodermis vascular



xylem phloem pericycle quiescent columella root cap root



hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis initials



flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem







Induction Screens


1. Drought Seedling


Increase GFP expression relative to control is observed in the epidermal tissues of the hypocotyl


after 3 Hr air dried seedlings are scanned.


2. Drought Mature


Events of line PT0848 under drought and controlled conditions. Increase in GFP expression


relative to control line observed in stems and siliques of event 01.


Promoter utility








Trait Area:
  PG&D, Drought


Sub-trait Area:
 Fruit size, desiccation tolerance, recovery from drought


Utility:
Among other uses this promoter sequence can be used to improve:



Fruit size, desiccation tolerance, recovery from drought







Notes: Smyth DR, Bowman JL, Meyerowitz EM. Early flower development in Arabidopsis.Plant


Cell. August 1990; 2(8): 755-67.








Construct:
PT0848


Promoter candidate
15371545


I.D:


cDNA I.D:
23541050


Lines expressing:
PT0848 01-04









Example 38












Promoter Expression Report #192.PT0852


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
H pedicel H nectary H sepal H petal H anther H epidermis


Silique
H abscission zone


Ovule
Post-fertilization: L inner integument L seed coat


Stem
L epidermis


Inflorescence
L shoot apical meristem


Meristem


Hypocotyl
L epidermis


Cotyledon
L epidermis H petiole


Rosette Leaf
H epidermis H petiole


Primary Root
H epidermis







Observed expression pattern:


T1 Mature expression: High GFP expression levels in the inflorescence meristem. GFP is


expressed throughout stem and pedicles near the inflorescence meristem and in sepals, petals and


stamens of flowers. High GFP expression at the abscission zone of mature flowers and


senescencing siliques. Low GFP expression detected at shoot apical meristem. Increased


expression of GFP observed in inner integument cells at micropylar-chalazal poles of ovules and


throughout endothelium of developing and mature seed. Low GFP expression throughout stem


with increased GFP expression in the trichome producing epidermal base cells.


T2 Seedling expression: High GFP expression in pedicles of cotyledons and rosette leaves and


the entire apical region of seedlings. Increased GFP expression in the trichome producing


epidermal base cells. High GFP expression in epidermal cells in the hypocotyl-root transition


zone and root.








Expected expression pattern:
Shade-induced


Selection Criteria:
Microarray data







Gene: Long hypocotyl in far-red 1 (HFR1)/reduced phytochrome signaling (REP1)/basic helix-


loop-helix FBI1 protein (FBI1)/reduced sensitivity to far-red light (RSF1)/bHLH protein 26


(BHLH026) (BHLH26).


GenBank: NM_100115 Arabidopsis thaliana long hypocotyls in far red1 (HFR1)/reduced


phytochrome signaling (REP1)/basic helix-loop-helix FBI1 protein (FBI1) (At1g02340) mRNA.








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
XT1 Mature XT2 Seedling  T2 Mature  T3 Seedling







Inductions completed.












Treatment:
Age:
Gen:
Time points:
Events Screened/Response
Response:


1. Far red
7 d.
T2
 1 Hr
3/0
No


Far Red730 = 525 μW/cm2


24 Hr
3/0
No







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 5    Events Expressing:  n = 3








X Flower
H pedicel receptacle H nectary H sepal H petal filament H anther



pollen carpel style papillae vascular H epidermis stomata



trichome



silique


X Silique
stigma style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata H abscission zone ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus L



inner integument outer integument endothelium L seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons root meristem



shoot meristem


X Stem
L epidermis cortex interfascicular region vascular xylem



phloem pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


X Inflorescence
L shoot apical meristem flower primordium


meristem







T2 Seedling Expression  Tissues Screened


Events Screened: n = 3   Events Expressing: n = 3


Seedlings expressing/Seedlings screened


Event-01: 3/5


Event-02: 2/7


Event-03: 4/5








X Hypocotyl
L epidermis cortex vascular xylem phloem stomata


X Cotyledon
mesophyll vascular L epidermis margin H petiole



stomata



hydathode


X Rosette Leaf
mesophyll vascular H epidermis trichome H petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials primordia flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem


Promoter utility



Trait Area:
Plant Growth and Development


Sub-trait Area:
Growth Control, flowering and abscission


Utility:
Among other uses this promoter sequence can be used to


improve:
growth rate, plant size, branching, stature, seedling



establishment and growth rate response to shade and low light,



flower and fruit lifetime and abscission, water and nutrient uptake



and resistance to root nematodes







Notes:


1. FIN219, an auxin-regulated gene, defines a link between phytochrome A and the downstream


regulator COP1 in light control of Arabidopsis development. Hsieh HL, Okamoto H, Wang M,


Ang LH, Matsui M, Goodman H, Deng XW.Genes Dev. Aug. 1, 2000; 14(15): 1958-1970.


PMCID: 316819


2. SPINDLY and GIGANTEA Interact and Act in Arabidopsis thaliana Pathways Involved in


Light Responses, Flowering, and Rhythms in Cotyledon Movements.Tseng TS, Salomé PA,


McClung CR, Olszewski NE. Plant Cell. June 2004; 16(6): 1550-1563.PMCID: 490045.


3. Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in Blue-Light-Induced Shrinking


of Arabidopsis Hypocotyl Protoplasts.Wang X, Iino M. Plant Physiol. Aug. 1, 1998; 117(4): 1265-1279.


PMCID: 34890


4. shl, a New Set of Arabidopsis Mutants with Exaggerated Developmental Responses to


Available Red, Far-Red, and Blue Light. Pepper AE, Seong-Kim MS, Hebst SM, Ivey KN, Kwak


SJ, Broyles DE.Plant Physiol. Sep. 1, 2001; 127(1): 295-304. PMCID: 117985








Construct:
PT0852


Promoter candidate
15295946


I.D:


cDNA I.D:
23516971


Events expressing:
02, 03, 05









Example 39












Promoter Expression Report #200.PT0857


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Mature Root
H mature root


Hypocotyl
H epidermis H cortex H vascular


Cotyledon
H petiole


Primary Root
H vascular L pericycle


Lateral Root
H pericycle H initials H flanking cells H vascular







Observed expression pattern:


T1 mature: GFP expressed in roots of mature plants. No expression observed in aerial organs.


T2 seedling: High GFP expression throughout hypocotyl and petioles of cotyledons. In root, GFP


is highly expressed throughout vasculature and pericycle cells in regions of lateral root


formations.


T2 mature: GFP expressed in roots of mature plants. *Observed in aerial tissues in 1 of 6 events


screened








Expected expression pattern:
Up in roots only


Selection Criteria:
Arabidopsis thaliana, Microarray








Gene:
Proton-dependent oligopeptide transport (POT) family protein







GenBank: NM_114439 Arabidopsis thaliana proton-dependent oligopeptide transport (POT)


family protein (At3g45700) mRNA, complete cds gi|30692627|ref|NM_114439.2 |[30692627]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
XT1 Mature XT2 Seedling  XT2 Mature  T3 Seedling







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 3    Events Expressing: n ≧ 1








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons root meristem



shoot meristem


Stem
epidermis cortex interfascicular region vascular xylem phloem



pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Shoot apical
Shoot apical meristem Flower primordium


meristem


X Mature Root
H mature root







T2 Seedling Expression   Tissues Screened


Events Screened: n = 3  Events Expressing: n = 3


Seedlings expressing/Seedlings screened


Event-01: 3/6


Event-02: 4/6


Event-03: 4/6








X Hypocotyl
H epidermis H cortex H vascular xylem phloem stomata


X Cotyledon
mesophyll vascular epidermis margin H petiole stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem L pericycle quiescent



columella root cap root hairs


X Lateral root
epidermis trichoblast atrichoblast cortex endodermis H



pericycle



H initials H flanking cells H vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem







T2 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 6  Events Expressing:  n = 5








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons root meristem



shoot meristem


Stem
epidermis cortex interfascicular region vascular xylem phloem



pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


X Aerial organs
H inflorescence meristem shoot apical meristem flower primordium



H flowers silique ovule embryo L stem leaf


X Mature root
H Mature root







High GFP expression specific in roots for 5 of 6 events scanned. Event 04: GFP expression in


aerial organs in addition to root.








Promoter utility



Trait Area:
Nutrients, PG&D


Sub-trait Area:
Nutrient uptake, growth rate


Utility:
Among other uses this promoter sequence can be used to


improve:
Nutrient uptake, seedling establishment


Construct:
 PT0857


Promoter candidate
 15371818


I.D:


cDNA I.D:
 23507609


Events expressing:
 PT0857-01, -02, -03, -04, -06









Example 40












Promoter Expression Report #255.PT0757


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Primary Root
H epidermis H cortex







Observed expression pattern:


T1 mature: No expression observed.


T2 seedling: GFP specific to root epidermis and cortex cells. Highest expression near root


transition zone decreasing near root elongation zone. No expression in lateral root.


T2 Mature: No expression observed.








Expected expression pattern:
Up in roots only


Selection Criteria:
Arabidopsis thaliana, Microarray


Gene:
2-oxoglutarate-dependent dioxygenase, putative







GenBank: NM_128637 Arabidopsis thaliana 2-oxoglutarate-dependent dioxygenase, putative


(At2g30840) mRNA, complete cds gi|42569490|ref|NM_128637.3|[42569490]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened: X T1 Mature X T2 Seedling  X T2 Mature  T3 Seedling


T1 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 3    Events Expressing: n = 0


No GFP Expression Detected


T2 Seedling Expression  Tissues Screened


Events Screened:  n = 3   Events Expressing: n = 2


Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-02: 3/6








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin petiole stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast H cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral Root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







T2 Mature Plant Expression  Organs/Tissues screened


Events Screened: n = 6     Events Expressing: n = 0


No GFP Expression Detected








Promoter utility



Trait Area:
Nutrients


Sub-trait Area:
low nitrogen tolerance,


Utility:
Among other uses this promoter sequence can be used to


improve:
low nitrogen tolerance and resistance to root insects



and nematodes


Construct:
 PT0757


Promoter candidate
 15371857


I.D:


cDNA I.D:
 23496046


Events expressing:
 PT0757-01, -02









Example 41












Promoter Expression Report #256.PT0764


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Primary Root
H epidermis







Observed expression pattern:


T1 mature: No expression observed.


T2 seedling: High GFP expression specific to root epidermis cells.


T2 mature: Weak GFP expression detected in roots.








Expected expression pattern:
Up in roots only


Selection Criteria:
Arabidopsis thaliana, Microarray


Gene:
Short-chain dehydrogenase/reductase (SDR) family protein







GenBank: NM_202867 Arabidopsis thaliana short-chain dehydrogenase/reductase (SDR) family


protein (At4g23420) mRNA, complete cds gi|42572998|ref|NM_202867.1|[42572998]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
X T1 Mature X T2 Seedling  X T2 Mature  T3 Seedling







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened: n = 3     Events Expressing:  n = 0


No GFP Expression Detected


T2 Seedling Expression  Tissues Screened


Events Screened:  n = 2   Events Expressing:  n = 3


Seedlings expressing/Seedlings screened


Event-01: 3/6


Event-02: 5/6








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin petiole stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
H epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral Root
epidermis trichoblast atrichoblast cortex endodermis



initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







T2 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 6   Events Expressing: n = 2








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons root meristem



shoot meristem


Stem
epidermis cortex interfascicular region vascular xylem phloem



pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Aerial organs
inflorescence meristem shoot apical meristem flower primordium



silique ovule embryo



stem leaf


X Mature Root
L mature root







Low GFP expression detected in roots of events -02 and -05.








Promoter utility



Trait Area:
Nutrients, Biotic stress


Sub-trait Area:
low nitrogen tolerance, nematode resistance


Utility:
Among other uses this promoter sequence can be used to



improve: Tolerance to low nutrients, nematode resistance


Construct:
 PT0764


Promoter candidate
 15371890


I.D:


cDNA I.D:
 1819818


Events expressing:
 PT0764-01, -02, -05









Example 42












Promoter Expression Report #257.PT0767


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Lateral Root
H cortex H initials H flanking cells H lateral root cap







Observed expression pattern:


T1 mature: No expression observed.


T2 seedling: High GFP expression specific to lateral root primordial and cortex cells overlaying


lateral root initiation sites.


T2 mature: No expression detected.








Expected expression pattern:
Up in roots only


Selection Criteria:
Arabidopsis thaliana, Microarray


Gene:
expressed protein







GenBank: NM_129494 Arabidopsis thaliana expressed protein (At2g39370) mRNA, complete


cds gi|18405026|ref|NM_129494.1|[18405026]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
X T1 Mature X T2 Seedling  X T2 Mature  T3 Seedling







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened: n = 3    Events Expressing: n = 0


No GFP Expression Detected


Table 2 T2 Seedling Expression  Tissues Screened


Events Screened:  n = 3   Events Expressing:  n = 2


Seedlings expressing/Seedlings screened


Event-02: 2/6


Event-03: 3/6








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin petiole stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


Primary Root
epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


X Lateral Root
epidermis trichoblast atrichoblast H cortex endodermis



H initials H flanking cells vascular H lateral root cap


Shoot apical
shoot apical meristem


meristem







T2 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 6    Events Expressing:  n = 0


No GFP Expression Detected









Promoter utility


Trait Area:
Nutrients, PG&D


Sub-trait Area:
low nitrogen tolerance, seedling establishment


Utility:
Among other uses this promoter sequence can be used to



improve: Low nitrogen tolerance, seedling establishment, root



worm and nematode resistance


Construct:
 PT0767


Promoter candidate
 15371902


I.D:


cDNA I.D:
 23519162


Events expressing:
 PT0767-02, -03









Example 43












Promoter Expression Report #259.PT0776


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Mature Root
L mature root


Primary Root
H pericycle


Lateral Root
H initials







Observed expression pattern:


T1 mature: No expression observed.


T2 seedling: High GFP expression specific to pericycle cells and lateral root initials.


T2 mature: Low GFP expression in roots.








Expected expression pattern:
Up in roots only


Selection Criteria:
Arabidopsis thaliana, Microarray


Gene:
CLAVATA1 receptor kinase (CLV1)







GenBank: NM_100732 Arabidopsis thaliana CLAVATA1 receptor kinase (CLV1) (At1g08590)


mRNA, complete cds gi|30680710|ref|NM_100732.2|[30680710]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER








Generation Screened:
X T1 Mature X T2 Seedling  XT2 Mature T3 Seedling







T1 Mature Plant Expression  Organs/Tissues screened


Events Screened: n = 0  Events Expressing: n = 3


No GFP Expression Detected


T2 Seedling Expression  Tissues Screened


Events Screened:  n = 3    Events Expressing:  n = 2


Seedlings expressing/Seedlings screened


Event-02: 1/6


Event-03: 2/6








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin petiole stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem H pericycle quiescent



columella root cap root hairs


X Lateral Root
epidermis trichoblast atrichoblast cortex endodermis



H initials flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







T2 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 5   Events Expressing:  n = 4








Flower
pedicel receptacle nectary sepal petal filament anther



pollen carpel style papillae vascular epidermis stomata



trichome



silique


Silique
stigma style carpel septum placentae funiculus transmitting



tissue vascular epidermis stomata abscission zone ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons root meristem



shoot meristem


Stem
epidermis cortex interfascicular region vascular xylem phloem



pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


Aerial organs
inflorescence meristem shoot apical meristem flower primordium



silique ovule embryo



stem leaf


X Mature Root
L mature root








Promoter utility



Trait Area:
Nutrients, Drought, PG&D


Sub-trait Area:
Low nutrient uptake, drought tolerance, seedling establishment


Utility:
Among other uses this promoter sequence can be used to



improve: Drought tolerance, low nitrogen tolerance, seedling



establishment, root insect and nematode resistance.


Construct:
 PT0776


Promoter candidate
 15371953


I.D:


cDNA I.D:
 23515451


Events expressing:
 PT0776-02, -03, -04, -05









Example 44












Promoter Expression Report #260.PT0889








Promoter Tested In:
Arabidopsis thaliana, Wassilewskija (WS) ecotype







Spatial expression summary:








Lateral Root
H initials H primordia







Observed expression pattern:


T1 mature: No expression observed.


T2 seedling: GFP expression specific to lateral root initials and primordia.


T2 mature: No expression detected.








Expected expression pattern:
Up in roots only


Selection Criteria:
Arabidopsis thaliana, Microarray


Gene:
basic helix-loop-helix (bHLH) family protein







GenBank: NM_179235 Arabidopsis thaliana basic helix-loop-helix (bHLH) family protein


(At4g36060) mRNA, complete cds gi|30690567|ref|NM_179235.1|[30690567]








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  X T1 Mature X T2 Seedling X T2 Mature  T3 Seedling








T1 Mature Plant Expression
Organs/Tissues screened


Events Screened:  n = 3
Events Expressing: n = 0


No GFP Expression Detected


T2 Seedling Expression
Tissues Screened








Events Screened: n = 3
Events Expressing: n = 2







Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-02: 3/6








Hypocotyl
epidermis cortex vascular xylem phloem stomata


Cotyledon
mesophyll vascular epidermis margin petiole stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


Primary Root
epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


X Lateral Root
epidermis trichoblast atrichoblast cortex endodermis



H initials H primordia flanking cells vascular lateral root cap


Shoot apical
shoot apical meristem


meristem







T2 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 5     Events Expressing: n = 0


No GFP Expression Detected


Promoter utility








Trait Area:
PG&D


Sub-trait Area:
Growth rate, root biomass


Utility:
Among other uses this promoter sequence can be used to



improve: root biomass, seedling establishment, drought



tolerance, tolerance to low nutrients.








Construct:
PT0889


Promoter candidate I.D:
15371968


cDNA I.D:
23504527


Events expressing:
PT0889-01, -02









Example 45












Promoter Expression Report #267.PT0585








Promoter Tested In:
Arabidopsis thaliana, Wassilewskija (WS) ecotype







Spatial expression summary:








Flower
H pedicel H sepal H carpel H epidermis H silique


Silique
H style H carpel H septum H epidermis


Stem
H epidermis H cortex


Leaf
H mesophyll H vascular H epidermis


Hypocotyl
L epidermis


Cotyledon
H mesophyll H epidermis







Observed expression pattern:


T1 Mature expression: GFP broadly expressed throughout vegetative organs. No expression in


stamen, placenta or ovules of flowers. High GFP expression in pedicels and sepals of developing


flowers and in siliques of abscised flowers. No expression in silique of early stage and developing


flower. High GFP expression in epidermis, vascular, and mesophyll cells of leaf. High GFP


expression in epidermis and cortex cells of stem.


T2 Seedling expression: High GFP expression in epidermis and mesophyll cells of cotyledons


with low expression in hypocotyls. No expression in root.








Expected expression pattern:
Arabidopsis knockout line gametophytic lethal


Selection Criteria:
Mutant lines


Gene:
glutamine synthetase, putative







GenBank:  NM_122954 glutamine synthetase (GS2) (At5g35630) mRNA, complete cds








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  X T1 Mature X T2 Seedling  T2 Mature  T3 Seedling


T1 Mature Plant Expression  Organs/Tissues screened


Events Screened:  n = 3     Events Expressing: n = 2


GFP expression as shown below








X Flower
H pedicel receptacle nectary H sepal petal filament anther



pollen H carpel style papillae vascular H epidermis stomata



trichome



H silique


X Silique
stigma H style H carpel H septum placentae funiculus



transmitting tissue vascular H epidermis stomata abscission zone



ovule


Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument outer integument endothelium seed coat



primordia chalaza micropyle early endosperm mature



endosperm embryo


Embryo
suspensor preglobular globular heart torpedo late mature



provascular hypophysis radicle cotyledons root meristem



shoot meristem


X Stem
H epidermis H cortex interfascicular region vascular xylem



phloem pith stomata trichome


X Leaf
petiole H mesophyll H vascular H epidermis trichome primordia



stomata stipule margin


Shoot apical
Shoot apical meristem Flower primordium


meristem







T2 Seedling Expression    Tissues Screened


Events Screened: n = 6       Events Expressing: n = 5


Seedlings expressing/Seedlings screened


Event-01: 4/6


Event-02: 5/6








X Hypocotyl
L epidermis cortex vascular xylem phloem stomata


X Cotyledon
H mesophyll vascular H epidermis margin petiole stomata



hydathode


Rosette Leaf
mesophyll vascular epidermis trichome petiole



primordia stomata stipule margin hydathode


Primary Root
epidermis trichoblast atrichoblast cortex endodermis



vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral Root
epidermis trichoblast atrichoblast cortex endodermis



initials primordia flanking cells vascular lateral root cap


Shoot apical
Shoot apical meristem


meristem







Scan of seedlings from 6 events of line PT0585. GFP expression was observed in 5 of 6 events (7


days old). Events-01, -02, -04, -05, -06 showed expression, while -03 showed no expression


T2 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 6     Events Expressing: n = 6








X Aerial organs
H inflorescence meristem shoot apical meristem flower primordium



H silique ovule embryo H stem H leaf


X Mature root
H mature root


Promoter utility








Trait Area:
Plant growth and development


Sub-trait Area:
Source Strength


Utility:
Among other uses this promoter sequence can be used to



improve: plant biomass, size, stature, xylem and mechanical



properties. Also seed filling, seed size, seed yield, seed



composition. Manipulation of dehiscence and seed scatter.








Construct:
PT0585


Promoter candidate I.D:
11768829


cDNA I.D:
23531789


Events expressing:
PT0585-01, -02, -04, -05, -06









Example 46












Promoter Expression Report #268.PT0565


Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype


Spatial expression summary:








Flower
H pedicel H sepal H petal H anther H carpel H style H epidermis H silique


Silique
H carpel H septum H epidermis H ovule


Ovule
Post-fertilization: H outer integument H seed coat H embryo


Embryo
H torpedo H late H mature H radicle H cotyledons


Stem
H epidermis H cortex H vascular H xylem H phloem L pith


Shoot apical
L shoot apical meristem


meristem


Mature Root
H mature root H vascular


Hypocotyl
L vascular


Cotyledon
H mesophyll H epidermis


Rosette Leaf
H mesophyll H epidermis


Primary Root
H vascular







Observed expression pattern:


T1 Mature expression: High GFP expression in flowers, root and stem. GFP expressed


throughout all organs of the flower with highest expression in developing and mature siliques.


High GFP expression in developing seed coats and embryos. GFP is expressed in epidermis,


cortex, pith, and vascular bundles of stem. High GFP in vasculature of roots of mature plants.


T2 Seedling expression: High GFP expression in epidermis and mesophyll of expanding


cotyledons and rosette leaves. GFP also expressed in vasculature of root.








Expected expression pattern:
Dividing cells


Selection Criteria:
Public reference


Gene:
cyclin, putative







GenBank: NM_103537 Arabidopsis thaliana cyclin, putative (At1g44110) mRNA, complete cds








Source Promoter Organism:
Arabidopsis thaliana, Columbia (Col) ecotype


Vector:
pNewbin4-HAP1-GFP


Marker Type:
GFP-ER







Generation Screened:  X T1 Mature X T2 Seedling  T2 Mature T3 Seedling


T1 Mature Plant Expression   Organs/Tissues screened


Events Screened:  n = 3      Events Expressing: n = 2








X Flower
H pedicel receptacle nectary H sepal H petal filament H anther



pollen



H carpel H style papillae vascular H epidermis stomata trichome



H silique


X Silique
stigma style H carpel H septum placentae funiculus transmitting



tissue vascular H epidermis stomata abscission zone H ovule


X Ovule
Pre-fertilization: primordia inner integument outer integument



embryo sac funiculus chalaza micropyle gametophyte



Post-fertilization: zygote suspensor embryo sack funiculus



inner integument H outer integument endothelium H seed coat



primordia chalaza micropyle early endosperm mature



endosperm H embryo


X Embryo
suspensor preglobular globular heart H torpedo H late H mature



provascular hypophysis H radicle H cotyledons root meristem



shoot meristem


X Stem
H epidermis H cortex interfascicular region H vascular H xylem H



phloem L pith stomata trichome


Leaf
petiole mesophyll vascular epidermis trichome primordia



stomata stipule margin


X Shoot apical
L shoot apical meristem Flower primordium


meristem


X Mature Root
H mature root H vascular







T2 Seedling Expression    Tissues Screened


Events Screened: n = 6      Events Expressing: n = 4


Seedlings expressing/Seedlings screened


Event-01: 3/6


Event-02: 5/6


Event-05: 4/6


Event-06: 4/6








X Hypocotyl
epidermis cortex L vascular xylem phloem stomata


X Cotyledon
H mesophyll vascular H epidermis margin petiole



stomata



hydathode


X Rosette Leaf
H mesophyll vascular H epidermis trichome petiole



primordia stomata stipule margin hydathode


X Primary Root
epidermis trichoblast atrichoblast cortex endodermis



H vascular xylem phloem pericycle quiescent



columella root cap root hairs


Lateral root
epidermis trichoblast atrichoblast cortex endodermis



initials primordia flanking cells vascular lateral root cap


Shoot apical meristem
Shoot apical meristem







Scan of seedlings from six Events of line PT0565. GFP expression (+) observed in 4 of 6 events.


Events-01, -02, -05, -06 showed expression, while events -03, and -04 did not show expression.








Promoter utility



Trait Area:
Plant growth and development


Sub-trait Area:
Size and source capacity


Utility:
Among other uses this promoter sequence can be used to



improve: plant size and architecture, growth rate, seedling



establishment, responses to shade and low light, responses to



drought and cold, source capacity and sucrose loading, seed filling,



seed size and plant yield.







Notes: Similar to mitotic cyclin a2-type [Glycine max] GI: 857397, cyclin A-like protein


[Nicotiana tabacum] GI: 1064927; contains Pfam profiles PF00134: Cyclin, N-terminal domain,


PF02984: Cyclin, C-terminal domain product cyclin, putative protein_id NP_175077.1


CLUSTAL MUSCLE (3.52) multiple sequence alignment


>Lycopersicon esculentum var. cerasiforme mRNA for protein kinase WEE1 (wee1 gene)


>Arabidopsis thaliana cyclin, putative (At1g44110) mRNA, complete cds








Coding sequence alignment:
Percentage of identity: 59.9%


Protein sequence alignment:
Percentage of identity: 17.0%







Gonzalez N, Hernould M, Delmas F, Gevaudant F, Duffe P, Causse M, Mouras A, Chevalier C.


Molecular characterization of a WEE1 gene homologue in tomato (Lycopersicon esculentum


Mill.).Plant Mol Biol. December 2004; 56(6): 849-61. Epub Apr. 7, 2005 PMID: 15821985


[PubMed - in process]


Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds in two distinct


phases of growth that comprise cell division and cell expansion, respectively. In pericarp and the


jelly like locular tissue of tomato fruit, the transition between cell division to cell expansion is


characterized by the arrest of mitotic activity, numerous rounds of nuclear DNA


endoreduplication and the inhibition of Cyclin-Dependent Kinase A (CDKA) activity. To


investigate whether the WEE1 kinase may play a role during the endoreduplication process, we


isolated and characterized the tomato homologue for WEE1. The LeWEE1 gene consisted of 10


exons with a predicted 510 amino acid-long protein. The accumulation of the corresponding


transcripts was associated with mitotically active organs: developing fruits, seeds and roots.


Interestingly, LeWEE1was expressed in the jelly like locular tissue concomitant with


endoreduplication during fruit development. Using tobacco BY-2 synchronized cells, we showed


that the WEE1 gene expression is cell-cycle regulated with a maximum transcript accumulation at


S phase. Our data indicate the putative dual contribution of LeWEE1 in the classical cell cycle


and the endocycle.








Construct:
PT0565


Promoter candidate I.D:
11768774


cDNA I.D:
23521098


Events expressing:
PT0565-01, -02, -05, -06









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.
Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation.

## Claims

1. A plant cell comprising a nucleic acid sequence which is a promoter as set forth in SEQ ID NO: 37, wherein said promoter is capable of causing transcription in the vegetative tissue of a plant, and wherein said promoter is operably linked to an exogenous sequence.

2. A plant cell comprising a vector construct, wherein said vector construct comprises:
(a) a first nucleic acid molecule which is a promoter that have the sequence set forth in SEQ ID NO: 37; and
(b) a second nucleic acid molecule to be transcribed,
Wherein said promoter and said second nucleic acid molecule are operably linked and heterologous to each other, and wherein said promoter is capable of causing transcription of said second nucleic acid molecule in the vegetative tissue of a plant.

3. A method of causing transcription in a plant comprising:
(a) operably linking (i) a first nucleic acid molecule which is a promoter as set forth in SEQ ID NO: 37 and (ii) a second nucleic acid molecule to be transcribed;
(b) inserting said operably linked promoter and transcribed nucleic acid molecule into a plant cell; and
(c) regenerating said plant cell into a plant,
wherein the first nicleic acid molcule is heterologous to the second nucleic acid molecule, and
wherein said promoter is capable of causing transcription of said second nucleic acid molecule in the vegetative tissue of a plant.

4. The method according to claim 3, wherein said promoter is capable of causing increased levels of transcription under drought conditions.

5. A plant comprising the plant cell of claim 2.

6. A method of introducing a vector construct into a host cell comprising:
(a) providing said isolated vector construct; and
(b) contacting said isolated vector construct with said host cell under conditions that permit insertion of said vector into said host cell,
wherein said vector construct comprises
(i) a first nucleic acid molecule which is a promoter as set forth in SEQ ID NO: 37; and
(ii) a second nucleic acid molecule to be transcribed, and
wherein the first nucleic acid molecule is heterologous to the second nucleic acid molecule, and
wherein said promoter and said second nucleic acid molecule are operably linked, and said promoter is capable of causing transcription of said second nucleic acid molecule in the vegetative tissue of a plant.

7. A plant cell, plant material or seed of a plant that comprises a nucleic acid molecule , wherein said nucleic acid molecule is a promoter as set forth in SEQ ID NO: 37, and wherein said promoter is operably linked to a heteroloaous plant nucleic acid molecule, and wherein said promoter is capable of causing transcription in the vegetative tissue of said plant nucleic acid molecule, and wherein said promoter is exogenous or heterologous to said plant or plant cell.

8. A plant cell, plant material or seed of a plant which comprises a vector construct, wherein said vector construct comprises:
(a) a first nucleic acid molecule which is a promoter as set forth in SEQ ID NO: 37; and
(b) a second nucleic acid molecule to be transcribed,
wherein the first nucleic acid molecule is heterologous to the second nucleic acid molecule, and
wherein said promoter and said second nucleic acid molecule are operably linked, and wherein said promoter is capable of causing transcription of said second nucleic acid molecule in the vegetative tissue of a plant.

9. A plant that has been regenerated from a plant cell or seed according to claim 7 or 8.

10. A plant, plant cell, plant material or seed of a plant which comprises:
(a) a first nucleic acid sequence which is a promoter as set forth in SEQ ID NO: 37,
(b) a second nucleic acid sequence to be transcribed,
wherein said promoter and said second nucleic acid sequence are operably linked, and wherein said promoter is exogenous to said plant or plant cell and heterologous to said second nucleic acid molecule, and wherein said promoter causes increased transcription of said second nucleic acid molecule under drought conditions as compared to an untransformed plant cultivated under the same conditions.

11. A plant cell comprising:
(a) a first nucleic acid sequence which is a promoter asset forth in SEQ ID NO: 37, and
(b) a second nucleic sequence to be transcribed,
wherein the first nucleic acid molecule is heterologous to the second nucleic acid molecule, and
wherein said promoter causes increased transcription of said second nucleic acid molecule under drought conditions as compared to an untransformed plant cultivated under the same conditions, and wherein said promoter is flanked by exogenous sequence.

12. A plant cell comprising a vector construct, wherein said vector construct comprises:
(a) a first nucleic acid molecule which is a promoter as set forth in SEQ ID NO: 37; and
(b) a second nucleic acid molecule to be transcribed,
wherein said promoter and said second nucleic acid molecule are operably linked, and wherein said promoter is exogenous plant and heterologous to said second nucleic acid sequence, and wherein said promoter causes increased transcription of said second nucleic acid molecule under drought conditions as compared to an untransformed plant cultivated under the same conditions.

13. The plant cell of claim 1 or 2, wherein said promoter is capable of causing increased levels of transcription under drought conditions.

14. The plant cell of claim 1, 2, 10 or 11, wherein said promoter is capable of causing transcription in at least one member selected from the group of plant tissues and/or organs consisting of the walls of carpels, the pedicels, the abseission zone, the leaf epidermis and the cortex cells of stem.

15. The plant cell of claim 1, 2, 10 or 11, wherein said promoter is further capable of causing transcription in at least on member selected from the group of seedling tissues and organs consisting of the epidermis of hypocotyls, and the epidermis of cotyledons.

16. The plant cell of claim 1, 2, 10 or 11, wherein said promoter is the sequence set forth in SEQ ID NO: 37.

17. The plant cell of claim 1, 2, 10 or 11, wherein said promoter is the sequence set forth in SEQ ID NO: 37.

18. The plant cell of claim 1, 2, 3, 10, 11 or 12 wherein said promoter has the sequence set forth in SEQ ID NO: 37.