WO2015153964A1 - Glyphosate-inducible plant promoter and uses thereof - Google Patents

Abstract

The subject application pertains to polynucleotides, compositions thereof, and methods for regulating gene transcription in a plant. Glyphosate. Thus, various embodiments provide polynucleotide comprising the sequence of SEQ ID NO: 1, complement thereof, fragments thereof (e.g., SEQ ID NO: 40), or homo logs thereof that are capable of driving the transcription of an operably linked heterologous nucleic acid sequence in plant cells or plants only when an inducer, such as glyphosate, is present in the plant cells or the plants.

Description

DESCRIPTION

GLYPHOSATE -INDUCIBLE PLANT PROMOTER AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application Serial No.

61/975,493, filed April 4, 2014, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled "Seq-List.txt" which was created on April 3, 2015 and is 29 KB. The entire contents of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Recent advances in plant genetic engineering have enabled the engineering of plants having improved characteristics or traits, such as disease resistance, insect resistance, herbicide resistance, enhanced stability, increased shelf-life of the ultimate consumer product obtained from the plants, and improvement of the nutritional quality of the edible portions of the plant. Thus, one or more desired genes from a source different than the plant, i.e. heterologous genes, engineered to impart different or improved characteristics or qualities to the plant, can be incorporated into the plant's genome. One or more new genes can then be expressed in the plant cell to exhibit the desired phenotype such as a new trait or characteristic.

Promoter is an important czs-regulatory element for gene expression and plays an important role in the process of plant gene expression and regulation. Constitutive promoters, such as CaMV35S promoter, are used to drive heterologous gene expression in most transgenic engineering. Although constitutive promoters can increase the expression of candidate genes greatly, over- and/or constitutive-expression of heterologous genes have been shown to cause stunted growth and reduced yield in transgenic plants. Also, the constitutive promoters could lead transgenic approach unsuccessful when they are used to increase (overexpression) or reduce (RNAi knockdown) expression of certain vital genes for which inappropriate expression results in highly deformed or lethal plants. Additionally, with the development of plant biotechnology, more and more heterologous genes are desired to be expressed in a specific manner, for example, in a specific tissue, at a specific growth stage, or at a specific time. Some examples of such specific expressions include conditional expression of stress resistant or herbicide resistant genes, lack of expression of pesticides resistant gene in grains and fruits, induction of conditional male or female sterility for molecular breeding.

These specific expressions of heterologous genes are important for achieving various goals, for example, providing better biosafety of the transgenic plants. Therefore, inducible plant promoters which are expressed only when an inducer is present will substantially benefit plant genetic engineering.

BRIEF SUMMARY OF THE INVENTION

The subject application provides polynucleotides, compositions thereof, and methods for regulating gene expression in a plant cell or a plant. Polynucleotides disclosed herein comprise novel plant promoter sequences that are capable of driving/initiating transcription of an operably linked heterologous polynucleotide in a plant cell or a plant. Polynucleotides disclosed herein also provide novel plant promoters that are capable of driving/initiating transcription of an operably linked heterologous polynucleotide in a plant cell or a plant only when an inducer is present in the plant cell or in the plant. An example of an inducer is glyphosate.

Thus, various embodiments provide nucleotides having sequence of SEQ ID NO: 1, complements thereof, or fragments thereof that are capable of driving the transcription of an operably linked heterologous nucleic acid sequence in a plant cell or a plant only when an inducer is present in the plant cell or in the plant. Other polynucleotides disclosed herein provide nucleotide sequences having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 1 or fragments thereof. These polynucleotides are capable of driving the transcription of an operably linked heterologous polynucleotides in a plant cell or a plant. Polynucleotides complementary to such polynucleotides (polynucleotide sequences having at least 70%) sequence identity to SEQ ID NO: 1 or fragments thereof) are also provided by the subject application.

Other aspects provide DNA constructs (sometimes referred to as nucleotide constructs) comprising promoters comprising SEQ ID NO: 1 or complement thereof, fragments thereof, or homologs thereof operably linked to a heterologous polynucleotide, wherein said promoters are capable of driving transcription of the heterologous polynucleotide in a plant cell or a plant. Certain aspects of the invention provide plants, seed, gametophytes, spores, zygotes, or plant cells containing the DNA constructs as disclosed herein. Further aspects of the invention provide plants, seed, gametophytes, spores, zygotes, or plant cells having a DNA construct as disclosed herein incorporated into their genomes.

Methods of expressing a heterologous polynucleotide in a plant are also provided, the method comprising transforming a plant cell with a DNA construct as disclosed herein and, optionally, regenerating a transformed plant from said plant cell. The DNA constructs comprise a promoter comprising SEQ ID NO: 1 or a complement thereof, a fragment thereof, or a homolog thereof operably linked to a heterologous polynucleotide, wherein the promoter is capable of driving/initiating transcription of said heterologous polynucleotide in the plant. Thus, the promoters disclosed herein are useful for controlling the expression of operably linked heterologous polynucleotides in a plant.

Downstream from, and under the transcriptional initiation regulation of the promoter is a heterologous polynucleotide, expression of which provides for modification of the phenotype of the plant. Such modification caused by the heterologous polynucleotide includes modulating amount or relative distribution of an endogenous product or the production of an exogenous product, to provide for a novel function or product in the plant. For example, a heterologous polynucleotide that encodes a gene product that confers pathogen, antibiotic, herbicide, salt, cold, drought, or insect resistance can be operably linked to a promoter as disclosed herein. Additional embodiments of heterologous polynucleotides that can modify a phenotype in a plant can be designed by a person of ordinary skill in the art and such embodiments are within the purview of the current invention.

Further aspects of the invention provide a method for modulating expression of a heterologous polynucleotide in a stably transformed plant, the method comprising the steps of:

(a) transforming a plant cell with a DNA construct comprising a disclosed promoter or a fragment thereof operably linked to a heterologous polynucleotide, wherein the promoter or the fragment thereof is capable of driving the transcription of the operably linked heterologous nucleic acid sequence in the plant cell only when an inducer is present in the plant cell;

(b) growing the plant cell; and

(c) regenerating a stably transformed plant from the plant cell wherein the expression of the operably linked heterologous polynucleotide alters the phenotype of the plant. BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, with color drawing(s), will be provided by the Office upon request and payment of the necessary fee.

Figure 1 shows frequency distribution of horseweed GS-FLX 454 sequence raw read lengths.

Figures 2A-2B show the characteristics of assembled horseweed GS-FLX 454 contigs; (a) length frequency distribution of assembled contigs; (b) average coverage frequency distribution of assembled contigs.

Figures 3A-3C show the summary of GO annotation of 454 unique sequences. Annotated sequences were classified into A, "Biological Process"; B, "Molecular Function"; and C, "Cellular Component" groups, and 45 subgroups.

Figure 4 shows expression levels of 17 ABC transporters genes in young leaves of glyphosate treated TN-R biotype horseweed plants relative to an internal control actin gene using real-time RT-PCR. Data are presented as mean ± SE of three technical replicates for each biotype-treatment combination (one pooled sample each).

Figure 5 shows relative expression profiles (compared to expression level in Tennessee-susceptible (TN-S) control plants referred to as (SC)) of 17 ABC transporter genes in young horseweed leaves from the following plant-treatment combinations: Tennessee- susceptible glyphosate-sprayed (SG); Tennessee-resistant untreated control (RC); and Tennessee-resistant glyphosate sprayed (RG). Data are presented as mean ± SE of three independent real-time RT-PCR analyses. Each RNA sample was isolated from leaves of six individual plants grown under the same conditions for each biotype and treatment and pooled to give one sample each.

Figure 6 shows an example of one unique sequence annotated by a similarity search of a custom plant protein database via NCBI Standalone Blast program.

Figure 7 shows an example of tabular annotation information of unique sequences. XML format BlastX results were parsed out with Query ID, hit accession number, annotated protein name, E-value, and score bits.

Figure 8 shows the number of contigs that have hits in the Arabidopsis protein database at various E-value thresholds.

Figure 9 shows structure of M10 gene. Figures lOA-lOC show expression profiles of M10. 10A: expression level of M10 in different tissues of TN-R horseweed plants; 10B: response of M10 and Mi l to selected treatments; IOC: comparative transcriptome analysis of M10 mRNA abundant in different samples (TNSC, TN-S untreated control; TNSG, TN-S glyphosate treated; TNRC, TN-R untreated control; TNRG, TN-R glyphosate treated).

Figure 11 shows the results of Glyphosate concentration assay. RoundUp WeatherMAX™ (540g/L) was diluted with water 100, 1000, 10000, and 100000 folds. 2 ml of each dilution was smeared with a brush on to the leaves of tobacco planted in 10cm* 10cm plate. Same amount of water smeared was as control. Tobacco plants were treated with RoundUp™ for one week.

Figures 12A-12B show transient expression of GUS and GFP. The promoter analysis constructs were transformed into young leaves of five week old tobacco via infiltration method. After infiltrated for two days, the leaves were treated with different amount RoundUp WeatherMAX™ (0.108, 0.0108, 0.0054, 0.00108 kg/ha ae) or water as control. The transient expression of GUS reporter gene was observed after additional two or five days (Figure 12 A); the transient expression of GFP reporter gene was observed after additional three days.

Figures 13A-13B shows the ability of the promoter to drive expression of GUS was also examined in transgenic tobacco plants. A: GUS staining of leaf disc of transgenic tobacco plants before or after RoundUp™ treatment shown the promoter was induced by glyphosate treatment in stable transgenic tobacco plants. B: GUS gene expression was quantified by using real time RT-PCR (12 hpi is shown as the first bar in each series; 24 hpi is shown as the middle bar in each series; 72 hpi is shown as the last bar in each series).

Figures 14A-14C shows the relative activity of glyphosate inducible promoters in stable transgenic tobacco plants (GFP as reporter gene). A: GFP expression in Ml IP transgenic lines; B: GFP expression in Ml OP transgenic lines; C: GFP expression level was quantified with a fluorescence spectroscopy, the excitation peak was 490 nm and the emission peak was 509 nm (0 dpi is shown as the first bar in each series; 3 dpi is shown as the middle bar in each series; 5 dpi is shown as the last bar in each series).

Figures 15A-15E show the activity of glyphosate inducible promoter Ml OP in reproductive organ of transgenic tobacco plants (GUS as reporter gene). A: GUS expression in flowers, with (Right) and without RoundUp™ treatment (Left); B & C: inside the flower with RoundUp™ treatment; D: GUS expression in young seeds with and without RoundUp¹ treatment; E: GUS expression in pollen with and without RoundUp™ treatment.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1: Promoter sequence of M10 gene.

SEQ ID NO: 2: Nucleotide sequence coding for M10 protein.

SEQ ID NO: 3: Amino acid sequence of M10 protein.

SEQ ID NO: 4: Forward primer for real-time RT-PCR analysis of actin.

SEQ ID NO: 5: Reverse primer for real-time RT-PCR analysis of actin.

SEQ ID NO: 6: Forward primer for real-time RT-PCR analysis of Ml gene.

SEQ ID NO: 7: Reverse primer for real-time RT-PCR analysis of Ml gene.

SEQ ID NO: 8: Forward primer for real-time RT-PCR analysis of M2 gene.

SEQ ID NO: 9: Reverse primer for real-time RT-PCR analysis of M2 gene.

SEQ ID NO: 10: Forward primer for real-time RT-PCR analysis of M3 gene. SEQ ID NO: 11: Reverse primer for real-time RT-PCR analysis of M3 gene.

SEQ ID NO: 12: Forward primer for real-time RT-PCR analysis of M4 gene. SEQ ID NO: 13: Reverse primer for real-time RT-PCR analysis of M4 gene.

SEQ ID NO: 14: Forward primer for real-time RT-PCR analysis of M5 gene. SEQ ID NO: 15: Reverse primer for real-time RT-PCR analysis of M5 gene.

SEQ ID NO: 16: Forward primer for real-time RT-PCR analysis of M6 gene. SEQ ID NO: 17: Reverse primer for real-time RT-PCR analysis of M6 gene.

SEQ ID NO: 18: Forward primer for real-time RT-PCR analysis of M7 gene. SEQ ID NO: 19: Reverse primer for real-time RT-PCR analysis of M7 gene.

SEQ ID NO: 20: Forward primer for real-time RT-PCR analysis of M8 gene. SEQ ID NO: 21: Reverse primer for real-time RT-PCR analysis of M8 gene.

SEQ ID NO: 22: Forward primer for real-time RT-PCR analysis of M9 gene. SEQ ID NO: 23: Reverse primer for real-time RT-PCR analysis of M9 gene.

SEQ ID NO: 24: Forward primer for real-time RT-PCR analysis of M10 gene. SEQ ID NO: 25: Reverse primer for real-time RT-PCR analysis of M10 gene. SEQ ID NO: 26: Forward primer for real-time RT-PCR analysis of Ml 1 gene. SEQ ID NO: 27: Reverse primer for real-time RT-PCR analysis of Ml 1 gene. SEQ ID NO: 28: Forward primer for real-time RT-PCR analysis of PI gene.

SEQ ID NO: 29: Reverse primer for real-time RT-PCR analysis of PI gene. SEQ ID NO: 30: Forward primer for real-time RT-PCR analysis of P2 gene,

SEQ ID NO: 31: Reverse primer for real-time RT-PCR analysis of P2 gene.

SEQ ID NO: 32: Forward primer for real-time RT-PCR analysis of P3 gene,

SEQ ID NO: 33: Reverse primer for real-time RT-PCR analysis of P3 gene.

SEQ ID NO: 34: Forward primer for real-time RT-PCR analysis of P4 gene,

SEQ ID NO: 35: Reverse primer for real-time RT-PCR analysis of P4 gene.

SEQ ID NO: 36: Forward primer for real-time RT-PCR analysis of P5 gene,

SEQ ID NO: 37: Reverse primer for real-time RT-PCR analysis of P5 gene.

SEQ ID NO: 38: Forward primer for real-time RT-PCR analysis of P6 gene,

SEQ ID NO: 39: Reverse primer for real-time RT-PCR analysis of P6 gene.

SEQ ID NO: 40: Putative core promoter sequence.

DETAILED DISCLOSURE OF THE INVENTION

The term "about" is used in this patent application to describe some quantitative aspects of the invention, for example, concentration of an inducer or percent identity between nucleotide sequences. It should be understood that absolute accuracy is not required with respect to those aspects for the invention to operate. When the term "about" is used to describe a quantitative aspect of the invention the relevant aspect may be varied by ±10%.

The promoter sequences disclosed herein are useful for expressing operably linked heterologous polynucleotides. As disclosed herein, SEQ ID NO: 1, a complement thereof, or a fragment thereof is a promoter capable of driving transcription of an operably linked polynucleotide sequence in a plant cell or a plant only when an inducer is present in the plant cell or in the plant.

The term "promoter" refers to a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter can additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. It is recognized that having identified the nucleotide sequences for the promoter regions disclosed herein, it is within the state of the art to isolate and identify further regulatory elements in the 5' untranslated region upstream from the particular promoter regions identified herein. Promoter elements that enable transcription, can be identified, isolated, and used with core promoters (for example, a fragment of SEQ ID NO: 1) to confer transcription. In this aspect of the invention, a "core promoter" is intended to mean a promoter without promoter elements generally found upstream and/or downstream of the core promoter (the minimal portion of the promoter required to properly initiating transcription which includes a Transcription Start Site (TSS) a binding site for RNA polymerase and general transcription factors binding sites). Core promoters can be identified by various methods, including bioinformatics (see Solovyev et al., 2010, Methods Mol Biol. 674: 57-83) or deletion analysis of the promoter region identified herein (according to methods known in the art).

An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. The inducer can either be a chemical agent, such as a metabolite, growth regulator, herbicide, or a phenolic compound, or a physiological stress imposed on the plant, such as cold, heat, drought, or flooding. For a well-controlled expression of a target gene, it is highly desirable to express the gene using tightly regulated stress or chemically-inducible promoters. A gene under control of a tightly regulated inducible promoter is not transcribed at all or has a very low level of transcription in the absence of an inducer whereas the gene is transcribed at a high level only when the inducer is present.

For the purpose of this invention, the term "when the inducer is present" indicates that the inducer is present in the plant cell or the plant at sufficient concentration to induce the expression of the gene under the control of the inducible promoter. In the case of the promoter of SEQ ID NO: 1, the inducer is glyphosate and sufficient concentration of glyphosate which can induce the transcription of the genes under the control of SEQ ID NO: 1 and fragments or homo logs thereof is about 0.0011 kg/ha ae to about 0.11 kg/ha ae, about 0.0055 kg/ha ae to about 0.011 kg/ha ae. The inducer can be applied to a plant via foliar application, seed soaking, or through roots from applying the inducer to soil. Additional techniques of applying an inducer to a plant are well known to a person of ordinary skill in the art and such methods are within the purview of the current invention.

The term "regulatory element" also refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a structural gene, which includes sequences which control the transcription of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site. An example of a regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter element. A promoter element comprises a core promoter element, responsible for the initiation of transcription, as well as other regulatory elements (as discussed elsewhere in this application) that modify gene transcription. It is to be understood that nucleotide sequences, located within introns, or 3' of the coding region sequence can also contribute to the regulation of transcription of a coding region of interest. A regulatory element can also include those elements located downstream (3') to the site of transcription initiation, or within transcribed regions, or both. In the context of this disclosure, a post-transcriptional regulatory element can include elements that are active following transcription initiation, for example translational and transcriptional enhancers, translational and transcriptional repressors, and mR A stability determinants.

The regulatory elements, or fragments thereof, can be operatively associated with heterologous regulatory elements or promoters in order to modulate the activity of the heterologous regulatory element. Such modulation includes enhancing transcriptional activity of the heterologous regulatory element, repressing transcriptional activity of the heterologous regulatory element, modulating post-transcriptional events, enhancing transcriptional activity of the heterologous regulatory element and modulating post- transcriptional events, or repressing transcriptional activity of the heterologous regulatory element and modulating post-transcriptional events.

The promoter sequences disclosed herein, when assembled within a DNA construct such that the promoter is operably linked to a heterologous polynucleotide, enable transcription of the heterologous polynucleotide only when an inducer is present in a plant cell or a plant transformed with this DNA construct. The term "operably linked" is intended to mean that the transcription or translation of the heterologous polynucleotide is under the influence of the promoter sequence. "Operably linked" is also intended to mean the joining of two nucleotide sequences such that the coding sequence of each DNA fragment remain in the proper reading frame. In this manner, the nucleotide sequences for the promoters are provided in DNA constructs along with a heterologous polynucleotide for expression in a plant cell or a plant of interest. The term "heterologous polynucleotide" is intended to mean a sequence that is not naturally operably linked with the promoter sequence. While this nucleotide sequence is heterologous to the promoter sequence, it can be homologous, or native; or heterologous, or foreign, to the host plant cell or host plant.

For the purpose of this invention, transcription of a heterologous polynucleotide in a plant cell or a plant only when an inducer is present means that the heterologous polynucleotide operably linked to a promoter sequence as disclosed herein is transcribed only when the inducer is present in a plant cell or a plant and the heterologous gene is not transcribed or is transcribed at a low level when the inducer is absent.

Certain aspects provide fragments of SEQ ID NO: 1 or polynucleotides having at least 70% sequence identity with SEQ ID NO: 1 (sequence homo logs). These fragments and sequence homologs are capable of driving/initiating transcription of an operably linked heterologous polynucleotide in a plant cell or a plant. In certain embodiments these fragments and sequence homologs are capable of driving/initiating transcription of an operably linked heterologous polynucleotide in a plant cell or a plant only when an inducer is present in the plant cell or in the plant.

The fragments and sequence homologs of SEQ ID NO: 1 that are capable of driving/initiating transcription are designed based on sequence analysis of SEQ ID NO: 1 to identify transcription regulatory elements. Certain sites on a promoter are important in driving/initiating transcription, for example, transcription factor binding sites. Identification of such sites can be performed in silico based on a number of promoter analysis software programs available to a skilled artisan. Examples of such software program include, but are not limited to, AGRIS (Arabidopsis Gene Regulatory Information Server), AthaMap {Arabidopsis thaliana map), AtProbe (Arabidopsis thaliana Promoter Binding Element Database), DoOP (Databases of Orthologous Promoters), PlantCare (Plant Cis Acting Regulatory Elements), PlantProm DB (Plant Promoter DataBase), Place (Plant Czs-acting Regulatory DNA Elements), Promoter 2.0 Prediction Server, TSSP (Prediction of PLANT Promoters Using RegSite Plant DB), NSITE-PL (Recognition of PLANT Regulatory motifs with statistics) and Transfac. Such software programs can be used to identify specific portions of SEQ ID NO: 1 that drive/initiate transcription, for example, transcription binding sites and to identify portions that do not drive/initiate transcription.

Fragments of SEQ ID NO: 1 that retain the portions of SEQ ID NO: 1 that facilitate driving/initiating transcription of an operably linked heterologous polynucleotide are also provided, but these fragments can lack some portions of SEQ ID NO: 1. In certain embodiments, the fragments of SEQ ID NO: 1 are capable of driving/initiating transcription of an operably linked heterologous polynucleotide only when an inducer is present in the plant cell or in the plant. A person of ordinary skill in the art can design such fragments based on the sequence analysis of SEQ ID NO: 1 and such fragments are within the purview of the current invention. A fragment or a homo log of the promoter of SEQ ID NO: 1 can be prepared by designing the fragment or the homo log of SEQ ID NO: 1 for example, based on sequence analysis of SEQ ID NO: 1, and assessing the activity of that fragment or homo log in driving/initiating the transcription of an operably linked heterologous polynucleotide, such as a reporter gene in a plant cell or a plant only when an inducer is present in the plant cell or in the plant. Nucleic acid molecules that are fragments of a promoter nucleotide sequence comprise at least 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or up to one nucleotide fewer than the total number of nucleotides in SEQ ID NO: 1.

Such fragments can be obtained by use of restriction enzymes to cleave the naturally occurring promoter nucleotide sequence disclosed herein; by synthesizing a nucleotide sequence from the naturally occurring sequence of the promoter DNA sequence; or through the use of PCR technology. See particularly, Mullis et al. (1987) Methods Enzymol. 155:335- 350, and Erlich, ed. (1989) PCR Technology (Stockton Press, New York). Variants of these promoter fragments, such as those resulting from site-directed mutagenesis and a procedure, such as DNA "shuffling", are also encompassed by the instant disclosure.

The present invention further comprises fragments of the polynucleotide sequences of the instant invention. Representative fragments of the polynucleotide sequences according to the invention will be understood to mean any nucleotide fragment having at least 5 successive nucleotides, preferably at least 12 successive nucleotides, and still more preferably at least 15, 18, or at least 20 successive nucleotides of SEQ ID NO: 1. The upper limit for such fragments is the total number of nucleotides found in the full-length sequence of SEQ ID NO: 1. The term "successive" can be interchanged with the term "consecutive" or the phrase "contiguous span". Thus, in some embodiments, a polynucleotide fragment of SEQ ID NO: 1 can be referred to as "a contiguous span of at least X nucleotides", wherein X is any integer value between 5 and 1504.

In some embodiments, the subject invention provides fragments of SEQ ID NO: 1 that are capable of hybridizing under various conditions of stringency {e.g., high, intermediate, or low stringency) with a nucleotide sequence of SEQ ID NO: 1. Fragments that are capable of hybridizing with a nucleotide sequence of the subject invention can be, optionally, labeled as set forth below. Similarly, polynucleotides having at least 70% sequence identity to SEQ ID NO: 1 that are capable of driving transcription of an operably linked heterologous polynucleotide can be designed by mutating, deleting, or inserting nucleotides in regions identified as important in driving/initiating transcription. Such polynucleotides retain the transcription binding sites that facilitate transcription of an operably linked heterologous polynucleotide.

The current invention provides sequence homo logs of SEQ ID NO: 1 that retain the sequence identity in portions of SEQ ID NO: 1 that facilitate driving/initiating transcription of an operably linked heterologous polynucleotide, for example, transcription binding sites; but carry modifications in the portions of SEQ ID NO: 1 that are not identified as important in facilitating driving/initiating transcription. In certain embodiments, the homologs of SEQ ID NO: 1 are capable of driving/initiating transcription of an operably linked heterologous polynucleotide in a plant. In additional embodiments, the homologs of SEQ ID NO: 1 are capable of driving/initiating transcription of an operably linked heterologous polynucleotide in a plant cell or a plant only when an inducer, for example, glyphosate, is present in the plant cell or in the plant. A person of ordinary skill in the art can design such homologs based on sequence analysis of SEQ ID NO: 1 and such homologs are within the purview of the current invention.

Naturally occurring homologs of SEQ ID NO: 1 can be identified with the use of well-known molecular biology techniques, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Homologs also include synthetically derived nucleotide sequences, for example, polynucleotides generated by using site-directed mutagenesis. Promoter activity of the homologs can be measured by using techniques such as Northern blot analysis, reporter activity measurements taken from transcriptional fusions, and the like. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter "Sambrook," herein incorporated by reference. Alternatively, levels of a reporter gene, such as green fluorescent protein (GFP) or the like produced under the control of a promoter fragment or homolog, can be measured. See, for example, U.S. Patent No. 6,072,050, herein incorporated by reference. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Homologs also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different promoter sequences can be manipulated to create a new promoter possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related polynucleotide sequences comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391 :288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The promoter sequence disclosed herein, as well as homologs and fragments thereof, are useful for genetic engineering of plants, e.g. for the production of a transformed or transgenic plant, to express a phenotype of interest. As used herein, the terms "transformed plant" and "transgenic plant" refer to a plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome of a transgenic or transformed plant such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. It is to be understood that as used herein the term "transgenic" includes any cell, cell line, callus, tissue, plant part, or plant the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross- fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non- recombinant transposition, or spontaneous mutation.

A transgenic "event" is produced by transformation of plant cells with a heterologous DNA construct, including a nucleic acid DNA construct that comprises a transgene of interest, the regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion into a particular genome location. An event is characterized phenotypically by the transcription of the transgene in the plant in a tissue specific manner. At the genetic level, an event is part of the genetic makeup of a plant. The term "event" also refers to progeny produced by a sexual outcross between the transformant and another variety that include the heterologous DNA.

As used herein, the term "plant" includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of same. Parts of transgenic plants are to be understood within the scope of the invention comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, fruits, ovules, leaves, or roots originating in transgenic plants or their progeny previously transformed with a DNA molecule of the invention, and therefore consisting at least in part of transgenic cells. As used herein, the term "plant cell" includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, crown, buds, apex, stems, shoots, gametophytes, sporophytes, pollen, and microspores. Monocotyledonous plants, dicotyledonous plants, and ferns can be transformed with a promoter or DNA construct as disclosed herein.

The promoter sequences and methods disclosed herein are useful in regulating expression of any heterologous polynucleotide in a plant cell or a plant. Thus, the heterologous polynucleotide operably linked to the promoters disclosed herein can be a structural gene encoding a protein of interest. Genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include: genes that provide for decreased lignin content, increased sucrose/cellulose content, increased biomass yield, etc.; genes encoding proteins conferring resistance to abiotic stress, such as drought, flooding, temperature (heat or cold), salinity, and toxins such as pesticides and herbicides, or to biotic stress, such as attacks by fungi, viruses, bacteria, insects, and nematodes, and development of diseases associated with these organisms. Various changes in phenotype are of interest including modifying expression of a gene in only when the inducer is present, altering a plant's pathogen or insect defense mechanism, increasing the plant's tolerance to herbicides, altering tissue development to respond to environmental stress, altering biomass and cellulose content and/or lignin content in a plant cell or a plant. The results can be achieved by providing transcription of heterologous or increased transcription of endogenous products in plant cells or plants only when an inducer is present in the plant cells or plants. Thus, a DNA construct comprising a gene of interest, such as those described below, to create plants having a desired phenotype (e.g., disease, herbicide or insect resistance), to create heat or cold tolerance in a plant, or to create or enhance resistance to drought or flood conditions in a plant.

Disease resistance and insect resistance genes such as lysozymes, cecropins, maganins, or thionins for antibacterial protection, or the pathogenesis-related (PR) proteins such as glucanases and chitinases for anti-fungal protection, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, and glycosidases for controlling nematodes or insects are all examples of useful gene products. Pathogens include, but are not limited to, viruses or viroids, bacteria, insects, nematodes, fungi, and the like. Viruses include tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Nematodes include parasitic nematodes such as root knot, cyst, and lesion nematodes, etc.

Genes encoding disease resistance traits include detoxification genes, such as against fumonisin (U.S. Pat. No. 5,792,931) avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089); and the like. Insect resistance genes can encode resistance to pests that have great yield drag such as rootworm, cutworm, European corn borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48: 109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); and the like.

Herbicide resistance traits can be introduced into plants by genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides {e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or Basta® (glufosinate) {e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide Basta®, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.

Commercial traits can also be encoded on a gene or genes that could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321. Genes such as β-ketothiolase, PHBase (polyhydroxybutyrate synthase), and acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs).

Agronomically important traits that affect quality of grain, such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, levels of cellulose, starch, and protein content can be genetically altered. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and modifying starch. Hordothionin protein modifications in corn are described in U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802 and 5,703,049; herein incorporated by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley, Williamson et al. (1987) Eur. J. Biochem. 165:99- 106, the disclosures of which are herein incorporated by reference.

Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. Examples of other applicable genes and their associated phenotype include genes that confer viral resistance; genes that confer fungal resistance; genes that confer insect resistance; genes that promote yield improvement; and genes that provide for resistance to stress, such as dehydration resulting from heat and salinity, toxic metal or trace elements, or the like.

In one embodiment, DNA constructs will comprise a transcriptional initiation region comprising a promoter sequence, as disclosed herein, or homologs or fragments thereof, operably linked to a heterologous polynucleotide whose transcription is to be controlled by the promoter in a plant. In additional embodiments, DNA constructs will comprise a transcriptional initiation region comprising a promoter sequence, as disclosed herein, or homologs or fragments thereof, operably linked to a heterologous polynucleotide whose transcription is to be controlled by the promoter in a plant cell or a plant only when an inducer, for example, glyphosate, is present in the plant cell or in the plant. Such DNA constructs are provided with a plurality of restriction sites for insertion of the nucleotide sequence to be under the transcriptional regulation of the regulatory regions. The DNA constructs can additionally contain selectable marker genes.

Where appropriate, the heterologous polynucleotide whose expression is to be under the control of the promoter sequence disclosed herein can be optimized for increased expression in the transformed plant. In other embodiments, the heterologous polynucleotide whose expression is to be under the control of the promoter sequence disclosed herein can be optimized for increased expression in the transformed plant in a tissue specific manner, for example, specifically in green tissue. For optimization for increased expression in a plant, the heterologous nucleotides can be synthesized using plant preferred codons for improved expression. Methods are available in the art for synthesizing plant-preferred nucleotide sequences. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Reporter genes or selectable marker genes can be included in the DNA constructs. Examples of suitable reporter genes known in the art can be found in, for example, Jefferson et al. (1991) in Plant Molecular Biology Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al. (1995) BioTechniques 19:650-655; and Chiu et al. (1996) Current Biology 6:325- 330. Selectable marker genes for selection of transformed cells or tissues can include genes that confer antibiotic resistance or resistance to herbicides. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella et al. (1983) Nature 303:209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al. (1985) Plant Mol. Biol. 5: 103-108; Zhijian et al. (1995) Plant Science 108:219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5: 131- 137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15: 127-136); bromoxynil (Stalker et al. (1988) Science 242:419- 423); glyphosate (Shaw et al. (1986) Science 233:478-481); phosphinothricin (DeBlock et al. (1987) EMBO J. 6:2513- 2518). Other genes that could serve utility in the recovery of transgenic events but might not be required in the final product would include, but are not limited to, examples such as GUS (β-glucuronidase; Jefferson (1987) Plant Mol. Biol. Rep. 5:387), GFP (green florescence protein; Chalfie et al. (1994) Science 263:802), luciferase (Riggs et al. (1987) Nucleic Acids Res. 15(19): 8115 and Luehrsen et al. (1992) Methods Enzymol. 216:397-414), and the maize genes encoding for anthocyanin production (Ludwig et al. (1990) Science 247:449).

Thus, the subject invention provides a nucleic acid molecule comprising a promoter operably linked to a heterologous polynucleotide, wherein the promoter is: (a) the sequence set forth in SEQ ID NO: 1 or a complement thereof;

(b) a fragment of the sequence set forth in SEQ ID NO: 1 or a complement thereof; or

(c) a polynucleotide having at least about 70% to about 99.99% sequence identity to the sequence set forth in SEQ ID NO: l, a fragment thereof, or a complement thereof; and wherein the promoter or fragment thereof is capable of initiating/driving transcription of the heterologous polynucleotide in a plant cell or in a plant.

Further, aspects of the application provide a polynucleotide that hybridizes under low, intermediate or high stringency with the polynucleotide sequences as set forth in (a), (b) or (c). Furthermore, a probe comprising the polynucleotide according to (a), (b) or (c) and, optionally, a label or a marker is provided.

Sequence homology and sequence identity can also be determined by hybridization studies under high stringency, intermediate stringency, and/or low stringency. Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity required for duplex formation. Severity of conditions can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under low, intermediate, or high stringency conditions by techniques well known in the art, as described, for example, in Keller, G.H., M.M. Manak [1987] DNA Probes, Stockton Press, New York, NY., pp. 169-170.

For example, hybridization of immobilized DNA on Southern blots with ³²P-labeled gene-specific probes can be performed by standard methods (Maniatis et al. [1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In general, hybridization and subsequent washes can be carried out under intermediate to high stringency conditions that allow for detection of target sequences with homology to the exemplified polynucleotide sequence. For double-stranded DNA gene probes, hybridization can be carried out overnight at 20-25 °C below the melting temperature (T_m) of the DNA hybrid in 6X SSPE, 5X Denhardfs solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz et al. [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285).

Tm = 81.5°C + 16.6 Log[Na⁺] + 0.41(%G+C) - 0.61(% formamide) - 600/length of duplex in base pairs.

Examples of low, intermediate, and high stringency washes are: (1) twice at room temperature for 15 minutes in IX SSPE, 0.1% SDS (low stringency wash);

(2) once at (T_m - 20°C) for 15 minutes in 0.2X SSPE, 0.1% SDS (intermediate stringency wash).

(3) Twice at (T_m - 15°C) for 15 minutes in 0.1X SSPE, 0.1% SDS (high stringency wash).

For oligonucleotide probes, hybridization can be carried out overnight at 10-20°C below the melting temperature (T_m) of the hybrid in 6X SSPE, 5X Denhardt's solution, 0.1 %> SDS, 0.1 mg/ml denatured DNA. T_m for oligonucleotide probes can be determined by the following formula:

T_m(°C) = 2(number T/A base pairs) + 4(number G/C base pairs) (Suggs et al. [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D.D. Brown [ed.], Academic Press, New York, 23:683-693).

Washes can be carried out as follows:

(1) twice at room temperature for 15 minutes IX SSPE, 0.1%> SDS (low stringency wash);

2) once at the hybridization temperature for 15 minutes in IX SSPE, 0.1% SDS (intermediate stringency wash).

(3) Twice at (T_m - 15°C) for 15 minutes in 0.1X SSPE, 0.1% SDS (high stringency wash).

In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment >70 or so bases in length, the following conditions can be used:

Low: 1 or 2X SSPE, room temperature

Low: 1 or 2X SSPE, 42°C

Intermediate: 0.2X or IX SSPE, 65°C

High: 0. IX SSPE, 65°C.

By way of another non-limiting example, procedures using conditions of high stringency can also be performed as follows: Pre-hybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% FicoU, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C, the preferred hybridization temperature, in pre-hybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 x 10⁶ cpm of ³²P-labeled probe. Alternatively, the hybridization step can be performed at 65°C in the presence of SSC buffer, IX SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1X SSC at 50°C for 45 min. Alternatively, filter washes can be performed in a solution containing 2X SSC and 0.1% SDS, or 0.5X SSC and 0.1% SDS, or 0.1X SSC and 0.1% SDS at 68°C for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which can be used are well known in the art and as cited in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. are incorporated herein in their entirety.

Another non-limiting example of procedures using conditions of intermediate stringency are as follows: Filters containing DNA are pre-hybridized, and then hybridized at a temperature of 60°C in the presence of a 5X SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2X SSC at 50°C and the hybridized probes are detectable by autoradiography. Other conditions of intermediate stringency which can be used are well known in the art and as cited in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47- 9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. are incorporated herein in their entirety.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to a person of ordinary skill in the art.

Various further aspects provide a DNA construct comprising the nucleic acid molecule as set forth in (a)-(c) above and certain embodiments provide a host cell comprising the disclosed DNA construct. The DNA construct can be present in the host plant cell in a transient manner or a stable manner. In transiently transfected host plant cell, the DNA construct is only present in the host plant cell for a limited period of time; whereas, in the stably transfected host cell, the DNA construct is permanently present in the plant cell. In the stably transfected plant cell, the DNA construct is incorporated into the genome of the plant cell. The plant cell can be from a monocot plant, a dicot plant, or a fern. In another embodiment, the cell can be a spore, a gametophyte, or a zygote.

A further embodiment of the invention provides a plant having stably incorporated into its genome the DNA construct as disclosed herein and the plant can be a monocot, a dicot, or a fern.

A method for expressing a heterologous polynucleotide in a plant cell or a plant is also provided, said method comprising introducing a DNA construct into the plant cell or the plant, said DNA construct comprising a promoter operably linked to the heterologous polynucleotide, wherein the promoter comprises:

(a) the sequence set forth in SEQ ID NO: lor a complement thereof;

(b) a fragment of the sequence set forth in SEQ ID NO: 1 or a complement thereof; or

(c) a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 1 or a complement thereof, or a fragment thereof;

and wherein the promoter set forth in (a), (b) or (c) is capable of initiating/driving transcription of the heterologous polynucleotide in the plant cell or the plant. In certain embodiments of the invention, the promoter set forth in (a), (b) or (c) is capable of initiating/driving transcription of the heterologous polynucleotide in the plant cell or the plant only when glyphosate is present in the plant cell or in the plant. The plant cell can be from a dicot, a monocot, or a fern. Similarly, the plant can be a monocot, dicot, or a fern.

A further embodiment of the invention provides a method for introducing a DNA construct into a plant cell or in a plant, wherein the DNA construct comprises a promoter operably linked to a heterologous polynucleotide, and wherein the promoter is a polynucleotide comprising:

(a) the sequence set forth in SEQ ID NO: lor a complement thereof;

(b) a fragment of the sequence set forth in SEQ ID NO: 1 or a complement thereof;

(c) the sequence which is at least 70% identical to the sequence set forth in SEQ ID NO: 1 or a complement thereof, or a fragment thereof;

and wherein the promoter set forth in (a), (b) or (c) is capable of initiating/driving transcription of the operably linked heterologous polynucleotide in the plant cell or in the plant. In certain embodiments of the invention, the promoter set forth in (a), (b) or (c) is capable of initiating/driving transcription of the heterologous polynucleotide in the plant cell or the plant only when an inducer (e.g., glyphosate) is present in the plant cell or in the plant.

"Nucleotide sequence", "polynucleotide" or "nucleic acid" can be used interchangeably and are understood to mean, according to the present invention, either a double-stranded DNA, a single-stranded DNA or products of transcription of the said DNAs (e.g., RNA molecules). The nucleic acid, polynucleotide, or nucleotide sequences of the invention can be isolated, purified (or partially purified), by separation methods including, but not limited to, ion-exchange chromatography, molecular size exclusion chromatography, or by genetic engineering methods such as amplification, subtractive hybridization, cloning, subcloning or chemical synthesis, or combinations of these genetic engineering methods.

A homologous polynucleotide for the purposes of the present invention, encompasses a sequence having a percentage identity with the polynucleotide sequences, set forth herein, of between at least (or at least about) 70.00% to 99.99% (inclusive). The aforementioned range of percent identity is to be taken as including, and providing written description and support for, any fractional percentage, in intervals of 0.01%, between 70.00%) and, up to, including 99.99%>. These percentages are purely statistical and differences between two nucleic acid sequences can be distributed randomly and over the entire sequence length. For example, homologous sequences can exhibit a percent identity of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent with the sequences of the instant invention. Typically, the percent identity is calculated with reference to the full length, native, and/or naturally occurring polynucleotide. The terms "identical" or percent "identity", in the context of two or more polynucleotide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection. In certain aspects of the invention, homologous sequence to SEQ ID NO: 1 or a fragment thereof have at least 70% sequence identity to SEQ ID NO: 1 over its full length or over the full length of a given fragment of SEQ ID NO: 1.

Nucleic acid sequence homologies can be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 55^:2444-2448; Altschul et al, 1990, J. Mol. Biol. 275(0:403-410; Thompson et al, 1994, Nucleic Acids Res. 220:4673-4680; Higgins et al, 1996, Methods Enzymol. 266:383-402; Altschul et al, 1990, J. Mol. Biol. 2750:403-410; Altschul et al, 1993, Nature Genetics 5:266-272). Sequence comparisons are, typically, conducted using default parameters provided by the vendor or using those parameters set forth in the above-identified references, which are hereby incorporated by reference in their entireties.

A "complementary" polynucleotide sequence, as used herein, generally refers to a sequence arising from the hydrogen bonding between a particular purine and a particular pyrimidine in double-stranded nucleic acid molecules (DNA-DNA, DNA-R A, or RNA- R A). The major specific pairings are guanine with cytosine and adenine with thymine or uracil. A "complementary" polynucleotide sequence can also be referred to as an "antisense" polynucleotide sequence or an "antisense sequence". In various aspects of the invention, sequences are "fully complementary" to a reference sequence {e.g., SEQ ID NO: 1). The phrase "fully complementary" refers to sequences containing no mismatches in their base pairing.

Thus, the subject invention also provides detection probes {e.g., fragments of the disclosed polynucleotide sequences) for hybridization with a target sequence or the amplicon generated from the target sequence. Such a detection probe will comprise a contiguous/consecutive span of at least 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides of SEQ ID NO: 1. Labeled probes or primers are labeled with a radioactive compound or with another type of label, e.g., 1) radioactive labels, 2) enzyme labels, 3) chemiluminescent labels, 4) fluorescent labels, or 5) magnetic labels). Alternatively, non-labeled nucleotide sequences can be used directly as probes or primers; however, the sequences are generally labeled with a radioactive element ( 32 P, 35 S, 3 H, 125 I) or with a molecule such as biotin, acetylaminofluorene, digoxigenin, 5-bromo-deoxyuridine, or fluorescein to provide probes that can be used in numerous applications.

The nucleic acid molecules disclosed herein are useful in methods of expressing a nucleotide sequence in a plant. This can be accomplished by transforming a plant cell of interest with a DNA construct comprising a promoter identified herein, operably linked to a heterologous polynucleotide, and regenerating a stably transformed plant from said plant cell.

Plant species suitable for transformation include, but are not limited to, corn {Zea cans), Brassica spp. {e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Other plants suitable for transformation with a promoter as disclosed herein include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp , tulips (Tulipa spp , daffodils (Narcissus spp , petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.

Additionally, ferns and monocots, such as maize, rice, barley, oats, wheat, sorghum, rye, sugarcane, ferns, mosses, grasses, switchgrass, pineapple, yams, onion, banana, coconut, Miscanthus (grass), Brachypodium distachyon (grass), cowpea, poplar, Physcomitrella patens (moss), Pteris vittata (fern), Arabidopsis thaliana, and dates can be transformed with a promoter as disclosed herein.

As used herein, "vector" refers to a DNA molecule such as a plasmid, cosmid, or bacterial phage for introducing a nucleotide construct, for example, a DNA construct, into a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance, or ampicillin resistance.

Various methods disclosed herein include introducing a nucleotide (DNA) construct into a plant cell or a plant. The term "introducing" is used herein to mean presenting to the plant cell or the plant the nucleotide construct in such a manner that the construct gains access to the interior of the plant cell or a cell of the plant. These methods do not depend on a particular method for introducing a nucleotide construct to a plant, only that the nucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plant cells or plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

By "stable transformation" is intended that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof. By "transient transformation" is intended that a nucleotide construct introduced into a plant does not integrate into the genome of the plant. The nucleotide constructs disclosed herein can be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct within a viral DNA or RNA molecule. Methods for introducing nucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931; herein incorporated by reference.

Transformation protocols as well as protocols for introducing nucleotide sequences into plants can be modified depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,981,840 and 5,563,055), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio /Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91 :440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311 :763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

The cells that have been transformed can be grown into plants according to methods known in the art. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown, and either pollinated with the same transformed plant variety or different varieties, and the resulting hybrid having a desired phenotypic characteristic. Two or more generations can be grown to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved. Thus as used herein, "transformed seeds" refers to seeds that contain the nucleotide construct stably integrated into the plant genome.

There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, (1988) In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., San Diego, Calif). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. The regenerated plants are generally self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.

MATERIALS AND METHODS

Horseweed samples for 454 and Illumina transcriptome sequencing

Horseweed 454 transcriptome sequencing was performed as described in Peng et al. (2010). The horseweed Illumina deep transcriptome sequencing is described below. Horseweed plants were grown in potting media in a greenhouse at the University of Tennessee, Knoxville, TN, USA under a 16 hour photoperiod at ambient temperatures (25±2°C). Plants were watered and fertilized as necessary with Osmocote slow-release fertilizer. Young leaves and meristematic tissue were harvested at the rosette stage from plants that were approximately 3 months old and 6 to 8 cm in diameter. Total RNA was isolated separately from the following four samples represented by six plants each: untreated (water-sprayed) and treated after 24 h glyphosate-sprayed with the field rate of Round-up WeatherMAX™ (0.84 kg/ha ae, Monsanto, St. Louis, MO, USA) of TN-susceptible (TNS) horseweed (from Knoxville, TN, USA), and untreated and treated TN-resistant (TNR) biotype from western Tennessee (Lauderdale County, TN, USA), respectively.²⁴ RNA extraction was done using TriReagent according to the manufacturer's protocol (MRC, Cincinnati, OH, USA). DNA samples were also isolated from the same plant material for promoter cloning. The RNA samples were used to generate double-stranded cDNA using SMART™ cDNA Library Construction Kit (Clontech, Mountain View, CA, USA). The cDNA samples were then fractionated into smaller pieces (300-500 bp). The ends of these fragments were subsequently polished by treating with DNA polymerase to fill in or remove any unpaired bases. The short Illumina sequencing adaptors contain unique bar coding sequences were then ligated on to each resulting fragment, which provided priming sequences for both emulsion PCR amplification and pyrosequencing, forming the basis of the single-stranded template library. Multiplex pyrosequencing using Illumina HiSeq 2000 was performed at UT-ORNL Joint Institute of Biological Science (JIBS) sequencing facility as described previously.¹²' ²⁵ Comparative transcriptome data analysis for glyphosate response genes Horseweed 454 transcriptome data analysis was described in Peng et al. (2010). The comparative transcriptome analysis on Illumina HiSeq 2000 data were processed using CLC genomic workbench 5.5. Reference transcriptome sequences were annotated by similarity search (NCBI Standalone Blast program, ftp://ftp.ncbi.nih.gov/blast/) of three protein databases: Arabidopsis all proteins database (AGIallAA.gz, 130,814 protein sequences; ftp://ftp.arabidopsis.org/home/tair/Sequences/blast_datasets/other_datasets/12-18-07/), UniProtKB/Swiss-Prot annotated protein database (353,658 protein sequences, world wide website: uniprot.org/downloads), and a custom protein database, in which included all green plants proteins from GenBank (677,422 protein sequences). The best five protein hits for each query sequences were parsed out to create annotated tables, which included available information, such as taxonomy, key words, protein function, accession number and/or gene ontology (GO) terms.

Expression analysis of selected ABC transporter genes using real-time RT-PCR Plants were grown, harvested, and total RNA extracted as described above. Four combinations of plant biotypes and treatments were made: TN-S and TN-R biotypes that were glyphosate-treated and untreated were compared for gene expression differences. Young leaves of six individual plants were used for total RNA extraction for each biotype- treatment combination. Therefore, the four combinations were represented by one sample each. The residual genomic DNA in the total RNA extract was removed by several treatments with RNase-free DNase I (Invitrogen, Carlsbad, CA, USA). First strand cDNA was synthesized using: 2 μg of total RNA, 0.5 μg oligo(dT)i₈ and Superscript® III reverse transcriptase, according to the manufacturer's instructions (Invitrogen) employing a Eppendorf MasterCycler (Eppendorf, Hamburg, GER). The cDNAs were diluted to 100 μΐ with sterile water of which 2 μΐ was used per real-time PCR sample. Real-time PCR was carried out in an ABI-7000 thermal cycling system using a real-time PCR Power Mix Kit (ABI, Foster City, CA, USA). The reaction mixture (25 μΐ) contained 2 μΐ of first strand cDNA, 0.5 μΜ of each of the forward and reverse primers and appropriate amounts of other components as recommended by the manufacturer (ABI). ABI-7000 thermal cycler was programmed as follows: 2 min at 95°C for pre-denature; 40 cycles of 15 s at 94°C, 15 s at 55°C, 20 s at 72°C. Data were collected during the extension step. The cDNA samples were tested by using three independent repetitions in the same condition. For control reactions, either no sample was added or RNA alone was added without reverse transcription to test if the RNA sample was contaminated with genomic DNA. An actin-like housekeeping gene (contig9305, 916 bp) was used as a reference gene. The absolute expression level of this actin-like gene was relatively invariant (average ±0.31 Ct value, within 10% variation) using equal amounts of cDNA samples from glyphosate treated plants in this study. Furthermore, abiotic stresses (salt, drought and cold; data not shown) did not change its expression. The relative expression of target genes to the actin control was calculated using the efficiency adjusted AACt method as described by Yuan et al.²⁹ The oligonucleotide primers (Table 1) were designed with the Primer Express 2.0 software (ABI). To test the suitability of these primer sets, the specificity and identity of the RT-PCR products were monitored by a melting curve analysis (65-99°C, 5°C s^"1) of the reaction products, which can distinguish the gene- specific PCR products from the nonspecific PCR products. All primers were synthesized by Integrated DNA Technologies (IDT, Iowa City, IA, USA). The expression profiles of M10 and Mi l response to selected factors, salt (100 mM NaCl), drought (10% PEG-3350), cold (4°C), and pathogen (P. syringae pv. Tomato) in TN-R horseweed plants after 24 h treatment were tested using qRT-PCR as described above.

Promoter cloning and functional analysis

Gene specific primers were designed based on the transcriptome data. The upstream promoter region of M10 was cloned by using genome walking method. Then the promoter was cloned into the pCR8/GW/TOPO vector, and then subcloned into the pMDC164 plant transformation vector upstream of the GUS reporter gene, and also used to replace the 35S promoter in pBIN-mGFP5er vector with GFP as reporter gene. The recombinant binary vectors were introduced into Agrobacterium tumefaciens GV3850 strain by freeze/thaw method. The constructs were transformed into young leaves of five week old tobacco via infiltration method for transient expression analysis. The stable transgenic plants were also been generated via leaf disc and floral dip methods via agrobacterium-mediated transformation.

A glyphosate concentration assay was also performed. RoundUp WeatherMAX™ (540g/L) was diluted with water for 100, 1000, 10000, 100000 folds, respectively. Then, 2 ml of each was smeared on the leaves of tobacco planted in 10cm* 10cm plate with a brush. Same amount of water smeared was as control. Tobacco plants treated with RoundUp™ for one week and the results are illustrated.

The ability of the tested promoters to drive expression of GUS and GFP were also examined in transgenic tobacco plants. The promoter (GUS as reporter gene) was induced by RoundUp™ treatment in stable transgenic tobacco plants (multiple plants from the same independent transgene line) were observed by GUS staining and quantified by using real time RT-PCR. GFP expression was observed and also quantified using fluorescence spectroscopy.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 - ROCHE GS-FLX SEQUENCING AND ASSEMBLY Normalized cDNA was used to reduce oversampling of high abundance transcripts and obtain sufficient coverage of low abundance transcripts. Two sequencing runs (1.5 plates) plus a titration run yielded a total of 411,962 raw reads. The average length of each read was 233 bp (Table 2) with 79.2% distributed between 200 bp and 300 bp, and a total data size was 95.8 Mb (Fig. 1). The sequence yield was somewhat lower compared with genomic DNA 454 sequencing, but was higher than other de novo transcriptome sequencing projects for non-model plant species.³⁰' ³¹ The difference resulted from shorter DNA fragments from the transcriptome preparation or other input effects compared with those data from genome studies. Compared with Sanger EST library sequencing methods, cDNA molecules needed to be fractionated into smaller pieces and size-scanned rather than fully cloned into vectors. Shotgun 454 sequences are located evenly across the cDNA of a given gene,³² which resulted in multiple fragments per single gene, requiring further analysis to assess their relationships.

Initial quality filtering of the 454 reads was performed at the machine level before base-calling. These sequences were subsequently trimmed as described in Materials and Methods. Ninety- four percent of sequences (379,152) passed the quality-control filter for assembly into unique sequences. A total of 363,471 high quality clean sequences resulted in 7.05 Mb representing 16,102 contigs. After assembly, 55% of contigs (8,817) were longer than 300 bp and 19.5% of contigs (3,145) were longer than 600 bp (Fig. 2). Of these contigs, 15,681 high-quality clean sequences (3.8%) remained as singletons (coverage depth = 1) with data size totaling 3.3 Mb. This resulted in 10.35 Mb of new horseweed transcriptome sequencing data representing 31,783 unique sequences (Table 3). Further quality-trimmed 2,016 unique transcripts (average length, 689 bp; total size, 1.39 Mb) obtained from horseweed cDNA libraries using traditional Sanger sequencing techniques⁵ were used to gauge the quality of the 454 sequencing and assembly.

The most challenging aspect of de novo assembly is obtaining abundant coverage of sequences. In this study, 95.9% of the high quality trimmed sequences were assembled into contigs with an average length of 438 bp. However, this average length was still shorter than the average length of Sanger ESTs. Given that the average coverage depth for each contig and each nucleotide position was ~ 22-fold and ~ 12-fold, respectively, this high coverage depth of contigs ensured the 454 sequences were likely more accurate than traditional Sanger sequences that rely on a single or very few reads.

EXAMPLE 2 - QUALITY AND PERFORMANCE OF THE 454 ASSEMBLY To test the quality and performance of the sequence assembly, we aligned contigs against themselves and the singletons using the NCBI Blastn program. 4,405 contigs (27.1%) had best Blast hits {i.e., had significantly similar sequences based on a bitscore > 45, E-value < 0.0001 produced by the BlastN program) with > 95% identity with other contigs and singletons, but in no case did these alignments extend over the entire length of either the Blast subjects or queries. These perfect match alignments averaged 92 bp and 73 bp in length for contig vs. contigs and contigs vs. singleton hits, respectively. Also, the average coverage of the match alignments were 18.5% and 11.5% of the length of the queried contigs in the cases of contigs vs. contigs and contigs vs. singletons, respectively. 2,768 of these contigs had Blastx hits (bitscore > 45) against the all green plants protein database, and only 285 (1.8%) of those Blastn-paired contigs had same best Blastx hits in the protein database (Table 4). Considering conserved motifs in different genes widely exist in the genome and different transcripts resulting from alternative splicing of single genes occurs frequently,³³'³⁴ our assembly appropriately partitioned these gene regions that produced high identity but short coverage alignments into different contigs.

To estimate the error rate of 454 sequencing and the quality of assembly, 2,016 high- quality trimmed Sanger-sequenced ESTs⁵ were aligned with the 454 contigs and singletons. Of these, 1,540 (76.4%) had strong Blast hits to 454 sequences (Table 5). Nucleotide alignments of Sanger vs. 454 sequences were 95.8%> identical for all alignments, 97.3% for those alignments involving 454 contigs and 99.3% for those alignments with bitscore of more than 100. The average number of gaps for alignments involving 454 singletons was 0.22 and 7 per 1,000 aligned bases. The average number of gaps for alignments involving 454 contigs was 0.04 and 1.4 per 1,000 aligned bases, which was less than that for 454 singletons. This comparison might overestimate the real 454 sequencing error rates since they include base mismatches caused by polymorphisms, possible gaps created by alternative splicing, and alignments with end regions of Sanger sequences, which are known to have decreased accuracy. The horseweed genotypes between Sanger and 454 sequencing were not the same. However, these results indicated a sufficient coverage depth could efficiently reduce the error rate in 454 sequencing and it is reasonable to suggest that it could be more accurate than traditional Sanger sequences on the basis of depth of coverage.

Table 5. Summary Blast data for assembled Sanger sequences against FLX-454 contigs and singletons. All Blast results refer to hits with bitscores greater than or equal to 45. Alignment lengths refer to nucleotides.

Number of Sanger sequences 2,016

Number of Sanger sequences with at least one Blast hit against 454 1,540 sequences

Percent Sanger sequences with a Blast hit against 454 sequence 76.4%

Mean percent identity of Sanger vs. all 454 contig Blast hit alignments 95.8%

Mean percent identity of Sanger vs. all 454 Blast hit alignment 97.3%

Mean percent identity of Sanger vs. 454 Blast hit alignment (bitscore>100) 99.3%

Mean number of gaps within Sanger vs. all 454 contig Blast hit alignments 0.04

Median number of gaps within Sanger vs. all 454 contig Blast hit 0 alignments

Mean number of gaps within Sanger vs. all 454 singleton Blast hit 0.22 alignments

Median number of gaps within Sanger vs. 454 singleton Blast hit 0 alignments

EXAMPLE 3 - FUNCTIONAL ANNOTATION OF 454 UNIQUE SEQUENCES

All unique sequences (contigs and singletons) were used as queries to search annotated protein databases and were assigned a gene description and/or a GO term (Figures 6 and 7). A number of factors, especially the E-value, affect the reliability of results when searching databases for similarities. The E-value is the probability, due to chance, that there is another alignment with a similarity greater than the given bitscore. In short, a lower E-value set translates to higher confidence in the search results. A total of 10,698 contigs had hits to the protein database with the E-value threshold set at 0.1, which was 1,438 (-16%) more than that with the E-value threshold set at 0.0001 (Figure 8). The database was enlarged to allow maximal functional searching for gene discovery in this de novo transcriptome sequencing project. The number of contigs that had hits to different protein databases was counted based on the 'best 5 hits' of Blastx search (E-value < 0.0001, score bits > 45). The greatest yield of protein counts was obtained when searching the all green plant protein database, which hit 629 more contigs than searching the Arabidopsis protein database; about 20%> more putative proteins were identified. Thus, a total 16,306 unique sequences were annotated. Of these, 13,708 (84.1%) were associated with Biological Process GO classification and were divided into 14 subgroups, 12,404 (76.1%) were associated with "cellular components" and were further divided into 16 subgroups, and 7,364 (45.2%) were associated with "molecular function" and were divided into 15 subgroups (Fig. 3).

Only 39.8%) of singletons found hits in our custom protein database and could be annotated, while 61.5% of the contigs could be annotated, possibly the result of low coverage depth and short average length of the singletons. The average length of annotated contigs was 526 bp with a 30.3 average coverage, while non-annotated contigs averaged 297 bp with only 9.6 coverage. Similarly, the average length of annotated singletons was 30 bp longer than that of non-annotated singletons. In 15,477 non-annotated unique sequences, -2.8% of these (431) had hits in plant microR A database; - 0.9% of them (134) had hits with non- plant proteins (Table 6).

EXAMPLE 4 - IDENTIFYING CANDIDATE GENES INVOLVED IN HERBICIDE

RESISTANCE

The effectiveness of this horseweed 454 transcriptome sequencing for identifying gene candidates involved in herbicide resistance was estimated by comparing with waterhemp data²⁶. Eleven herbicide target- site genes/gene families and four non-target gene families were identified from unique horseweed and waterhemp sequences (Table 7). In ten gene families, more resistance-gene candidates were identified in waterhemp compared with horseweed. In the remaining five gene families, more resistance gene candidates were identified in horseweed than waterhemp. About 430 unique sequences were identified which might be involved in the evolution of herbicide resistance. These findings demonstrate the enormous value of 454 transcriptome sequencing for gene discovery in an important weedy plant with scant sequence data. The utility of the horseweed transcriptome data by exploring a non-target glyphosate resistance hypothesis in this species is illustrated below.⁵ Specifically, the transcriptome data enabled expression analysis of ABC transporter genes based on real time RT-PCR experiments. Table 7. Comparison of the number of hits (contigs - H singletons) to herbicide target-site genes and gene families and non-targ et gene families from transcriptome 454 sequencing of horseweed and water lemp.

Herbicide target gene family Horseweed Waterhemp

Acetolactate synthase 6 2

Dl protein (plastidic gene) 4 2

Tubulin 29 33

Protoporphyrinogen oxidase 2 8

Phytoene desaturase 5 1

Glutamine synthetase 9 7

1 -deoxy-D-xylulose-5 -phosphate 6 1

synthase

4-hydroxyphenylpyruvate dioxygenase 1 2

Acetyl-CoA carboxylase 6 8

Dihydropteroate synthase 1 2

5 -enolpyruvylshikimate-3 -phosphate 2 3

synthase

Non-target gene family

Glutathione S -transferase 7 22

Cytochrome P450 monooxygenases 125 191

Glycosyltransferases 76 84

ABC transporter genes 151 192

EXAMPLE 5 - EXPRESSION ANALYSIS OF ABC TRANSPORTER-LIKE GENES ABC transporters are transmembrane proteins that utilize the energy of ATP hydrolysis to transport a wide of variety substrates across extra- and intra-cellular membranes, including metabolic products, lipids and sterols, and drugs.³⁵' ³⁶ A number of ABC transporter genes were shown to be upregulated in our previous microarray analysis that suggested one or more might contribute to the glyphosate resistance in TN-R horseweed plants.⁵ One model for non-target resistance is glyphosate sequestration into vacuoles via

5 10 37 38

active transport of glyphosate by ABC transporters; ' ' ' therefore overexpression of ABC transporters may account for glyphosate resistance. In fact, some gene families that might be involved in glyphosate resistance in horseweed³⁹ were found in the dataset, which included ABC transporters, glutathione S-transferases (GSTs), glycosyltransferases and P450s (Table 7). In the case of ABC transporters, we identified 67 unique sequences belonging to the subfamilies of multidrug resistance protein/multidrug resistance-associated protein (MRP) and pleiotropic drug resistance (PDR). Members of these subfamilies were shown to be up- regulated at a high frequency by glyphosate in our previous heterologous microarray study.⁵ We therefore performed a preliminary gene -by-gene transcription analysis of 17 ABC- transporter genes (Ml to Mi l from the MRP-like subfamily; PI to P6 from the PDR-like subfamily). The most abundant transcript of these 17 ABC transporters, Mi l (contig9470, 2120 bp of determined sequence), was found in glyphosate-treated TN-R horseweed plants and was 1.4 times higher than the expression of the actin housekeeping gene that we used as an internal control (Fig. 4). This AtMRP3-like ABC transporter was the highest up-regulated gene, with a fold change of 29.6, from our previous heterologous microarray study in horseweed.⁵ Also, the identity of Mi l with the 70 bp Arabidopsis probe sequence was ~ 90%. All other ABC transporter genes had much lower absolute abundance: Ml, M2, M3, M8, M9, and P4 were among those with moderate abundance levels, while the others can be classified as low abundance transcripts, but were still detectable by real-time RT-PCR (Fig. 4)·

Compared with TN-S plants, M2 and PI had lower expression levels in TN-R horseweed plants. Ml and M8 had the same expression level in both biotypes, while the remainder of ABC-transporters had higher expression levels in TN-R horseweed plants. The responses of these ABC transporter genes to 24 h glyphosate treatment varied as shown in Fig. 5. Ml, M2, M8, M9, M10, Mi l, P4, and P5 were shown to be upregulated in both TN-S and TN-R biotypes. However, Ml and M2 had higher expression levels in TN-S plants. M9, M10, and Mi l had higher expression levels in TN-R plants. The expression levels of M8, P4, and P5 were comparative between the two biotypes. M3, M6, M7, and P3 were shown to be upregulated in TN-S horseweed whereas there was almost no response in TN-R plants. M5 and P6 were shown to be upregulated in TN-S horseweed but downregulated in TN-R plants. PI was downregulated in TN-S horseweed, whereas there was little response in TN-R plants. M4 and P2 had almost no response in both TN-S and TN-R biotype horseweed plants (Fig. 5). M6, M7, M10, Mi l, and P3 are more likely to be involved in the glyphosate resistance since their expression levels are always higher in resistant lines than in the susceptible lines.

M10 and Mi l transcription exhibited strong response to glyphosate. M10 had a low expression level, ~ 6x 10^"5 in TN-S and ~1.2x l0^~3 in TN-R plants, respectively, compared with the actin control gene. M10 was upregulated by nearly 300-fold in treated TN-S plants, and 16-fold in treated TN-R plants, compared with their untreated controls; however, TN-R plants had the highest expression level (Fig 4). Ml 1 was upregulated by 60-fold and 45-fold in treated TN-S plants and TN-R plants, respectively. Therefore, Mi l promoter can be used as a potential glyphosate sensor. Both M10 and Mi l had a higher expression level in TN-R treated plants than TN-S treated plants.

There are several features of Mi l that are intriguing with regards to a potential non- target glyphosate resistance candidate. These features include its high levels of absolute transcription, up-regulation by glyphosate, which is also highest in resistant plants, and its putative tonoplast localization. Its orthologue in Arabidopsis is, tonoplast targeted.⁴⁰ Thus, Mi l could play a very important role in glyphosate transport into vacuoles, thereby resulting in the glyphosate resistance in TN-R horseweed. M10 has the highest expression level in mature leaves, while a relative lower expression in other tissues (Fig 10A). Furthermore, M10 was specifically up-regulated by glyphosate but not by other selected factors in TN-R Conyza— 24 h post treatment (Fig 10B). Recently, the response of M10 to glyphosate treatment was further confirmed by a comparative transcriptome analysis (Fig IOC).

EXAMPLE 6 - FUNCTIONAL ANALYSIS OF M10 PROMOTER IN TRANSGENIC

PLANTS

A glyphosate concentration assay was performed before the inducible experiments. RoundUp WeatherMAX™ (540g/L) was diluted with water for 100, 1000, 10000, 100000 folds. 2 ml of each dilution was smeared on to the leaves of tobacco planted in 10cm* 10cm plate with a brush. Same amount of water smeared was as control. Tobacco plants were treated with RoundUp™ for one week and the results are illustrated in Figure 11. Plants died at the dosage of 1.08 and 0.108 kg/ha ae; while survived at dosage lower than 0.0108.

The ability of the promoter to drive expression of GUS and GFP were examined in transgenic tobacco plants. The constructs were transformed into young leaves of five week old tobacco via infiltration method. After being infiltrated for two days, the leaves were treated with different amounts RoundUp WeatherMAX™ (0.108, 0.0108, 0.0054, 0.00108 kg/ha ae) or water as control. The transient expression of GUS reporter gene was observed after additional two (Figure 12 A) or five days (Figure 12B). The expression of gus gene could be induced by dosage as low as 0.00108 kg/ha ae.

The ability of the promoter to drive expression of GUS and GFP were also examined in transgenic tobacco plants. The promoter (GUS as reporter gene) was induced by RoundUp™ treatment in stable transgenic tobacco plants (multiple plants from the same independent transgene line) were observed by GUS staining and quantified by using real time RT-PCR (Figure 13). Mllp has higher basal expression than Ml Op, therefore, the fold change after induction were less. Also the GFP expression was observed and also quantified using fluorescence spectroscopy (Figure 14). The activity and inducible expression of GUS in flower tissues, young seeds, and pollens were also inspected. The results suggested these promoters can be used to produce conditional male and female sterility system for molecular breeding or to prevent gene flow of transgenic event.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

REFERENCES Dill GM, Cajacob CA and Padgette SR, Glyphosate-resistant crops: adoption, use and future consideration. Pest Manag Sci 64:326-331(2008).

Duke SO and Powles SB, Glyphosate: a once-in-a-century herbicide. Pest Manag Sci 64:319-325 (2008).

Heap I, The International Survey of Herbicide Resistant Weeds. Available online at world wide website: weedscience.com (2010).

Van Gessel MJ, Glyphosate-resistant horseweed from Delaware. Weed Sci 49:703- 705 (2001).

Yuan JS, Good LG, Cao Y, Halfhill MD, Zhou X, Peng Y, Hu J, Rao MR, Heck GR, Larosa TJ et ah Functional genomics analysis of glyphosate resistance in Conyza canadensis (horseweed). Weed Sci in press (2010).

Weaver SE, The biology of Canadian weeds. 115. Conyza canadensis. Can. J. Plant Sci 81 :867-875 (2001).

Basu C, Halfhill MD, Mueller TC, and Stewart CN, Jr, Weed genomics: new tools to understand weed biology. Trends Plant Sci 9:391-398 (2004).

Stewart CN, Tranel PJ, Horvath DP, Anderson JV, Rieseberg LH, Westwood JH, Mallory-Smith CA, Zapiola ML and Dlugosch KM, Evolution of weediness and invasiveness: charting the course for weed genomics. Weed Sci 57: 451-462 (2009). Halfhill MD, Good LL, Basu C, Burris J, Main CL, Mueller TC and Stewart CN, Transformation and segregation of GFP fluorescence and glyphosate resistance in horseweed {Conyza canadensis) hybrids. Plant Cell Rep 26: 303-311 (2007).

Feng PCC, Tran M, Chiu T, Sammons RD, Heck GR and Cajacob CA, Investigations into glyphosate resistant horseweed {Conyza canadensis): retention, uptake, translocation, and metabolism. Weed Sci. 52: 498-505 (2004).

Zelaya IA, Owen MDK, and VanGessel MJ, Inheritance of evolved glyphosate resistance in Conyza canadensis (L.) Cronq. Theor. Appl. Genet 110:58-57 (2004). Margulies M, Egholm E, Altman WE et al., Genome sequencing in micro fabricated high-density picolitre reactors. Nature 437:376-380 (2005).

Morozova O and Marra MA, Applications of next-generation sequencing technologies in functional genomics. Genomics 92:255-264 (2008). Rothberg JM and Leamon JH,. The development and impact of 454 sequencing. Nat Biotechnol 26: 1117-1124 (2008).

Emrich SJ, Barbazuk WB, Li L and Schnable PS, Gene discovery and annotation using LCM-454 transcriptome sequencing. Genome Res 17:69-73 (2007).

Korbel JO, Urban AE, Affourtit JP, Godwin B, Grubert F, Simons JF, Kim PM, Palejev D, Carriero NJ, Du L, Taillon BE, Chen Z, Tanzer A, Saunders ACE, Chi J, Yang F, Carter NP, Hurles ME, Weissman SM, Harkins TT, Gerstein MB, Egholm M, and Michael Snyder, Paired-end mapping reveals extensive structural variation in the human genome. Science 318: 420-426 (2007).

Holt KE, Parkhill J, Mazzoni CJ, Roumagnac P, Weill FX, Goodhead I, Ranee R, Baker S, Maskell DJ, Wain J, Dolecek C, Achtman M and Dougan G, High- throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi. Nat Genet O: 987-993 (2008).

Vera JC, Wheat CW, Fescemyer HW, Frilander MJ, Crawford DL, Hanski I and Marden JH, Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing. MolEcol 17: 1636-47 (2008).

Mao C, Evans C, Jensen RV and Sobral BW, Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system. BMC Microbiol 8:72 (2008).

Alagna F, DAgostino N, Torchia L, Servili M, Rao R, Pietrella M, Giuliano G, Chiusano ML, Baldoni L and Perrotta G, Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development. BMC Genomics 10:399 (2009).

Droege M and Hill B, The genome sequencer FLX™ system- longer reads, more applications, straight forward bioinformatics and more complete datasets. J Biotech 136: 3-10 (2008).

Nyren P, Pettersson B and Uhlen M, Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay. Anal. Biochem 208: 171-175 (1993).

Ronaghi M, Karamohamed S, Pettersson B, Uhlen M, and Nyren P, Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem 242:84-89 (1996). Mueller TC, Massey JH, Hayes RM, Main CL and Stewart CN Jr, Shikimate accumulates in both glyphosate-sensitive and glyphosate-resistant horseweed (Conyza canadensis L. Cronq.). J Agr Food Chem 51 : 680-684 (2003).

Dassanayake M, Haas JS, Bohnert HJ and Cheeseman JM, Shedding light on an extremophile lifestyle through transcriptomics. New Phytol. 183:764-775 (2009). Riggins CW, Peng Y, Stewart CN Jr and Tranel PJ, Characterization of waterhemp transcriptome using 454 pyrosequencing and its application for studies of herbicide target-site genes. Pest Management Science (accepted)

Chou HH and Holmes MH, DNA sequencing quality trimming and vector removal. Bioinformatics 17: 1093-1104 (2001)

Huang X and Madan A, CAP3: A DNA sequence assembly program. Genome Res 9:868-877 (1999).

Yuan JS, Wang D and Stewart CN, Statistical methods for efficiency adjusted realtime PCR analysis. Biotechnology Journal. 3: 112-123 (2008).

Alagna F, DAgostino N, Torchia L, Servili M, Rao R, Pietrella M, Giuliano G, Chiusano ML, Baldoni L and Perrotta G, Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development. BMC Genomics. 10:399 (2009).

Wang W, Wang Y, Zhang Q, Qi Y and Guo D, Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing. BMC Genomics. 10:465 (2009).

Weber AP, Weber KL, Carr K, Wilkerson C and Ohlrogge JB, Sampling the Arabidopsis transcriptome with massively parallel pyrosequencing. Plant Physiol 144:32-42 (2007).

Black DL, Mechanisms of alternative pre-messenger RNA splicing. Ann Rev of Biochem 72: 291-336 (2003).

Yuan Y, Chung JD, Fu X, Johnson VE, Ranjan P, Booth SL, Harding SA and Tsaia CJ, Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis. Proc Natl Acad Sci U S A. 106:22020-22025 (2009)

Rea PA, Plant ATP -binding cassette transporters. Ann Rev Plant Biol 58:347-375 (2007). Verrier PJ, Bird D, Burla B, Dassa E, Forestier C, Geisler M, Klein M, Kolukisaoglu U, Lee Y, Martinoia E, Murphy A, Rea PA, Samuels L, Schulz B, Spalding EJ, Yazaki K and Theodoulou FL, Plant ABC proteins - a unified nomenclature and updated inventory. Trends Plant Sci 13: 151-159 (2008).

Shaner DL, The role of translocation as a mechanism of resistance to glyphosate. Weed Sci 57: 118-123 (2009).

Ge X, dAvignon DA, Ackerman JJH and Sammons RD, Rapid vacuolar sequestration: the horseweed glyphosate resistance mechanism. Pest Management Sci. Online first. DOI: 10.1002/ps.1911 (2010).

Yuan JS, Tranel PJ, and Stewart CN Jr, Non-target site herbicide resistance: a family business. Trends Plant Sci 12:6-13 (2007).

Dunkley TP, Hester S, Shadforth IP, Runions J, Weimar T, Hanton SL, Griffin JL, Bessant C, Brandizzi F, Hawes C, Watson RB, Dupree P and Lilley KS, Mapping the Arabidopsis organelle proteome. Proc Natl Acad Sci USA. 103:6518-6523 (2006). Gressel J, Arabidopsis is not a weed, and mostly not a good model for weed genomics; there is no good model for weed genomics, in Weedy and Invasive Plant Genomics, ed. by Stewart CN, Jr.,Wiley-Blackwell, Ames, Iowa, pp.25-32 (2009). Lee, RM, Thimmapuram, J, Thinglum, KA, Gong, G, Hernandez, AG, Wright, CL, Kim, RW, Mikel, M and Tranel, PJ, Sampling the waterhemp (Amaranthus tuberculatus) genome using pyrosequencing technology. Weed Sci. 57: 463-469 (2009).

Anderson JV, Horvath DP, and Chao WS, Foley ME., Hernandez AG, Thimmapuram J, Liu L, Gong GL, Band M, Kim R, and. Mikel MA, Characterization of an EST database for the perennial weed leafy spurge: an important resource for weed biology research. Weed Sci 55: 193-203 (2007).

Lokko Y, Anderson JV, Rudd S, Raji AAJ, Horvath D, Mikel MA, Kim R, Liu L, Hernandez A, Dixon AGO and Ingelbrecht IL, Characterization of an 18,166 EST dataset for cassava (Manihot esculenta Crantz) enriched for drought-responsive genes. Plant Cell Rep 26: 1605-1618 (2007).

Claims

CLAIMS We claim:

1. A nucleic acid molecule comprising a promoter operably linked to a heterologous polynucleotide, wherein the promoter is a polynucleotide comprising:

(a) the sequence set forth in SEQ ID NO: l or SEQ ID NO: 40 or a complement thereof;

(b) a fragment of the sequence set forth in SEQ ID NO: 1 or a complement thereof; or

(c) a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:l, a fragment thereof, or a complement thereof;

and wherein the promoter set forth in (a), (b) or (c) is capable of initiating/driving transcription of the heterologous polynucleotide in a plant cell or in a plant.

2. The nucleic acid molecule of claim 1, wherein the promoter is capable of initiating/driving transcription of the heterologous polynucleotide in the plant cell or in the plant only when an inducer is present in the plant cell or in the plant.

3. The nucleic acid molecule of claim 2, wherein the inducer is glyphosate.

4. A DNA construct comprising the nucleic acid molecule according to claim 1.

5. A vector comprising the DNA construct of claim 4.

6. A plant cell having stably incorporated into its genome the DNA construct of claim 4.

7. The plant cell of claim 6, wherein the plant cell is from a monocot.

8. The plant cell of claim 6, wherein the plant cell is from a dicot.

9. The plant cell of claim 6, wherein the plant cell is from a fern.

10. A plant having stably incorporated into its genome the DNA construct of claim 4.

11. The plant of claim 10, wherein the plant is the monocot.

12. The plant of claim 10, wherein the plant is the dicot.

13. The plant of claim 10, wherein the plant is the fern.

14. The plant of claim 10, wherein the heterologous polynucleotide encodes a gene product is a marker or a gene product that confers resistance to a disease, antibiotic, herbicide, salt, heat, cold, flood, drought, pathogen, or insects.

15. A transgenic seed comprising the DNA construct of claim 4.

16. A transgenic spore, gametophyte or zygote comprising the DNA construct according to claim 4.

17. A method for expressing a heterologous polynucleotide in a plant cell or a plant, the method comprising introducing a DNA construct into the plant cell or the plant, the DNA construct comprising a promoter operably linked to the heterologous polynucleotide, wherein the promoter comprises:

(a) the sequence set forth in SEQ ID NO: l or SEQ ID NO: 40 or a complement thereof;

(b) a fragment of the sequence set forth in SEQ ID NO: 1 or a complement thereof; or

(c) a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 1 or a complement thereof, or a fragment thereof;

and wherein the promoter set forth in (a), (b) or (c) is capable of initiating/driving transcription of the heterologous polynucleotide in the plant cell or the plant only when an inducer is present in the plant cell or in the plant.

18. The method of claim 17, wherein the inducer is glyphosate.

19. The method of claim 17, wherein said plant cell is from a dicot or the plant is the dicot.

20. The method of claim 17, wherein said plant cell is from a monocot or the plant is the monocot.

21. The method of claim 17, wherein said plant cell is from a fern or the plant is the fern.

22. The method of claim 17, wherein the heterologous polynucleotide encodes a gene product is a marker or a gene product that confers resistance to a disease, antibiotic, herbicide, salt, heat, cold, flood, drought, pathogen, or insects.

23. A method for introducing a DNA construct into a plant cell or a plant, wherein the DNA construct comprises a promoter operably linked to a heterologous polynucleotide, and wherein the promoter is a polynucleotide comprising:

(a) the sequence set forth in SEQ ID NO: l or SEQ ID NO:40 or a complement thereof;

(b) a fragment of the sequence set forth in SEQ ID NO: 1 or a complement thereof;

(c) the sequence which is at least 70% identical to the sequence set forth in SEQ ID NO: 1 or a complement thereof, or a fragment thereof;

and wherein the promoter set forth in (a), (b) or (c) is capable of initiating/driving transcription of the operably linked heterologous polynucleotide in the plant cell or in the plant only in the presence of an inducer.

24. The method of claim 23, wherein the inducer is glyphosate.

25. The method of claim 23, wherein the plant cell is from a monocot or the plant is the monocot.

26. The method of claim 23, wherein the plant cell is from a dicot or the plant is the dicot.

27. The method of claim 23, wherein the plant cell is from a fern or the plant is the fern.

28. The nucleic acid molecule of claim 1, wherein the heterologous polynucleotide encodes a gene product is a marker or a gene product that confers resistance to a disease, antibiotic, herbicide, salt, heat, cold, flood, drought, pathogen, or insects.