WO2003040334A2

WO2003040334A2 - Methods for the identification of inhibitors of 4-coumarate-coa ligase expression or activity in plants

Info

Publication number: WO2003040334A2
Application number: PCT/US2002/035596
Authority: WO
Inventors: Keith Davis; Adel Zayed; Robert Ascenzi; Douglas Boyes; Rao Mulpuri; Neil Hoffman; Kenneth Phillips; Jorn Gorlach; Jeffrey Woessner; Carol Hamilton
Original assignee: Paradigm Genetics Inc.
Priority date: 2001-11-08
Filing date: 2002-11-06
Publication date: 2003-05-15
Also published as: WO2003040334A3; AU2002361590A1

Abstract

The present inventors have discovered that 4-coumarate-CoA ligase (4CL) is essential for plant growth. Specifically, the inhibition of 4CL gene expression in plant seedlings results in growth reduction and growth arrest. Thus, 4CL can be used as a target for the identification of herbicides. Accordingly, the present invention provides methods for the identification of compounds that inhibit 4CL expression or activity, comprising: contacting a compound with a 4CL and detecting the presence and/or absence of binding between said compound and said a 4CL, or detecting a decrease in 4CL expression or activity. The methods of the invention are useful for the identification of herbicides.

Description

METHODS FOR THE IDENTIFICATION OF INHIBITORS OF 4-COUMARATE- COA LIGASE EXPRESSION OR ACTIVITY IN PLANTS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/337,955, filed Nov. 08, 2001.

FIELD OF THE INVENTION The invention relates generally to plant molecular biology. In particular, the invention relates to methods for the identification of herbicides.

BACKGROUND OF THE INVENTION

The enzyme 4-coumarate-CoA ligase (4CL) plays a key role in channeling carbon flow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Three members of the 4CL family have been cloned from Arabidopsis thaliana. Ehlting et al. (1999) Plant J ^' 19:9-20. The present inventors have discovered that inhibition of the expression of an A. thaliana 4CL is detrimental to plant growth. Therefore, it would be desirable to determine the utility of this enzyme for evaluating plant growth regulators, especially herbicide compounds.

SUMMARY OF THE INVENTION The present inventors have discovered that antisense expression of a 4CL cDNA in Arabidopsis causes developmental abnormalities, including growth reduction and growth arrest. Thus, the present inventors have discovered that 4CL is essential for normal seed development and growth, and can be used as a target for the identification of herbicides. Accordingly, the present invention provides methods for the identification pf compounds that inhibit 4CL expression or activity, comprising: contacting a candidate compound with a 4CL and detecting the presence or absence of binding between said compound and said 4CL, or detecting a decrease in 4CL expression or activity. The methods of the invention are useful for the identification of herbicides.

BRIEF DESCRIPTION OF THE FIGURE

Fig. 1 shows the 4-coumarate-CoA ligase reaction.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term "4-coumarate-CoA ligase is synonymous with "4CL" and refers to an enzyme that catalyses the conversion of ATP, 4-coumaric acid, caffeic acid, and CoASH to AMP, 4-coumaroyl CoA, and caffeoyl CoA, as shown in Fig. 1, and as included herein as the protein of SEQ ID NO: 2 and/or its encoding cDNA, SEQ ID NO: 1.

The term "binding" refers to a noncovalent interaction that holds two molecules together. For example, two such molecules could be an enzyme and an inhibitor of that enzyme. Noncovalent interactions include hydrogen bonding, ionic interactions among charged groups, van der Waals interactions and hydrophobic interactions among nonpolar groups. One or more of these interactions can mediate the binding of two molecules to each other.

As used herein, the term "dl" means deionized.

As used herein, the term "ELISA" means enzyme-linked immunosorbent assay. As used herein, the term "GUS" means β-glucouronidase.

The term "herbicide", as used herein, refers to a compound that may be used to kill or suppress the growth of at least one plant, plant cell, plant tissue or seed.

As used herein, the term "HPLC" means high pressure liquid chromatography. The term "inhibitor", as used herein, refers to a chemical substance that inactivates the enzymatic activity of 4CL. The inhibitor may function by interacting directly with the enzyme, a cofactor of the enzyme, the substrate of the enzyme, or any combination thereof. A polynucleotide may be "introduced" into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection and the like. The introduced polynucleotide may be maintained in the cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosome. Alternatively, the introduced polynucleotide may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active.

As used herein, the term "LB" means Luria-Bertani media.

As used herein, the term "Ni" refers to nickel.

As used herein, the term "Ni-NTA" refers to nickel sepharose. As used herein, the term "PCR" means polymerase chain reaction.

The "percent (%) sequence identity" between two polynucleotide or two polypeptide sequences can be determined according to the either the BLAST program (Basic Local Alignment Search Tool; Altschul and Gish (1996) Meth Enzymol 265:460-480 and Altschul (1990) JMolBiol 275:403-410) in the Wisconsin Genetics Software Package (Devererreux et al. (1984) Nucl Acid Res 72:387), Genetics Computer Group (GCG), Madison, Wisconsin. (NCBI, Version 2.0.11, default settings) or using Smith Waterman Alignment (Smith and Waterman (1981) AdvAppl Math 2:482) as incorporated into GeneMatcher Plus™ (Paracel, Inc., using the default settings and the version current at the time of filing). It is understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymine nucleotide is equivalent to a uracil nucleotide.

"Plant" refers to whole plants, plant organs and tissues (e.g., stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores and the like) seeds, plant cells and the progeny thereof.

By "polypeptide" is meant a chain of at least four amino acids joined by peptide bonds. The chain may be linear, branched, circular or combinations thereof. The polypeptides may contain amino acid analogs and other modifications, including, but not limited to glycosylated or phosphorylated residues. As used herein, the term "SDS" means sodium dodecyl sulfate.

As used herein, the term "SDS-PAGE" means sodium dodecyl sulfate - polyacrylimide gel electrophoresis.

The term "specific binding" refers to an interaction between 4CL and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of 4CL.

As used herein, the term "TATA box" refers to a sequence of nucleotides that serves as the main recognition site for the attachment of RNA polymerase in the promoter region of eukaryotic genes. Located at around 25 nucleotides before the start of transcription, it consists of the seven-base consensus sequence TATAAAA, and is analogous to the Pribnow box in prokaryotic promoters.

As used herein, the term "TLC" means thin layer chromatography.

Embodiments of the Invention The present inventors have discovered that inhibition of 4CL gene expression strongly inhibits the growth and development of plant seedlings. Thus, the inventors are the first to demonstrate that 4CL is a target for herbicides.

Accordingly, the invention provides methods for identifying compounds that inhibit 4CL gene expression or activity. Such methods include ligand binding assays, assays for enzyme activity and assays for 4CL gene expression. Any compound that is a ligand for 4CL, other than its substrates ATP, 4-coumaric acid, caffeic acid, and CoASH, may have herbicidal activity. For the purposes of the invention, "ligand" refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the invention are useful as herbicides.

Thus, in one embodiment, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a 4CL with said compound; and b) detecting the presence and/or absence of binding between said compound and said 4CL; wherein binding indicates that said compound is a candidate for a herbicide. By "4CL" is meant any enzyme that catalyzes the interconversion of ATP, 4- coumaric acid, caffeic acid, and CoASH with AMP, 4-coumaroyl CoA, and caffeoyl CoA. The 4CL may have the amino acid sequence of a naturally occurring 4CL found in a plant, animal or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence. Preferably the 4CL is a plant 4CL. The cDNA (SEQ ID NO:l) encoding the 4CL protein or polypeptide (SEQ ID NO:2) can be found herein as well as in the TIGR database at locus At3g48990.

By "plant 4CL" is meant an enzyme that can be found in at least one plant, and which catalyzes the interconversion of ATP, 4-coumaric acid, caffeic acid, and CoASH with AMP, 4-coumaroyl CoA, and caffeoyl CoA. The 4CL may be from any plant, including both monocots and dicots.

In one embodiment, the 4CL is an Arabidopsis ACL. Arabidopsis species include, but are not limited to, Arabidopsis arenosa, Arabidopsis bursifolia, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis griffithiana, Arabidopsis halleri, Arabidopsis himalaica, Arabidopsis korshinskyi, Arabidopsis lyrata, Arabidopsis neglecta, Arabidopsis pumila, Arabidopsis suecica, Arabidopsis thaliana and Arabidopsis wallichii. Preferably, the Arabidopsis 4CL is from Arabidopsis thaliana. In various embodiments, the 4CL can be from barnyard grass (Echinochloa crus-galli), crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis), perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilos ), nightshade (Solarium nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium alburn £.), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexus, Sida spinosa, Xanthium strumarium and the like. Fragments of a 4CL polypeptide may be used in the methods of the invention. The fragments comprise at least 10 consecutive amino acids of a 4CL. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or at least 100 consecutive amino acids residues of a 4CL. In one embodiment, the fragment is from an Arabidopsis ACL. Preferably, the fragment contains an amino acid sequence conserved among plant 4-coumarate-CoA ligases. Such conserved fragments are identified in Gri a-Pettenuti et al. (1993) Plant Mol Biol 27:1085-1095 and Taveres et al. (2000), supra. Those skilled in the art could identify additional conserved fragments using sequence comparison software.

Polypeptides having at least 80% sequence identity with a plant 4CL are also useful in the methods of the invention. Preferably, the sequence identity is at least 85%, more preferably the identity is at least 90%, most preferably the sequence identity is at least 95% or 99%. In addition, it is preferred that the polypeptide has at least 50% of the activity of a plant 4CL. More preferably, the polypeptide has at least 60%, at least 70%, at least 80%_> or at least 90% of the activity of a plant 4CL. Most preferably, the polypeptide has at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the activity of the A. thaliana ACL protein.

Thus, in another embodiment, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with at least one polypeptide selected from the group consisting of: a plant 4CL, a polypeptide comprising at least ten consecutive amino acids of a plant 4CL, a polypeptide having at least 85% sequence identity with a plant 4CL, and a polypeptide having at least 80% sequence identity with a plant 4CL and at least 50% of the activity thereof; and b) detecting the presence and/or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide.

Any technique for detecting the binding of a ligand to its target may be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand- target complex or the detection of a change in the properties of a target when it is bound to a ligand. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a 4CL protein or a fragment or variant thereof, the unbound protein is removed and the bound 4CL is detected. In a preferred embodiment, bound 4CL is detected using a labeled binding partner, such as a labeled antibody. In a variation of this assay, 4CL is labeled prior to contacting the immobilized candidate ligands. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods (Evotec Biosystems). Once a compound is identified as a candidate for a herbicide, it can be tested for the ability to inhibit 4CL enzyme activity. The compounds can be tested using either in vitro or cell based enzyme assays. Alternatively, a compound can be tested by applying it directly to a plant or plant cell, or expressing it therein, and monitoring the plant or plant cell for changes or decreases in growth, development, viability or alterations in gene expression.

Thus, in one embodiment, the invention provides a method for determining whether a compound identified as a herbicide candidate by an above method has herbicidal activity, comprising: contacting a plant or plant cells with said herbicide candidate and detecting the presence or absence of a decrease in the growth or viability of said plant or plant cells.

By decrease in growth, is meant that the herbicide candidate causes at least a 10% decrease in the growth of the plant or plant cells, as compared to the growth of the plants or plant cells in the absence of the herbicide candidate. By a decrease in viability is meant that at least 20% of the plants cells, or portion of the plant contacted with the herbicide candidate are nonviable. Preferably, the growth or viability will be at decreased by at least 40%. More preferably, the growth or viability will be decreased by at least 50%, 75% or at least 90% or more. Methods for measuring plant growth and cell viability are known to those skilled in the art. It is possible that a candidate compound may have herbicidal activity only for certain plants or certain plant species.

The ability of a compound to inhibit 4CL activity can be detected using in vitro enzymatic assays in which the disappearance of a substrate or the appearance of a product is directly or indirectly detected. 4CL catalyzes the irreversible or reversible reaction of ATP, 4-coumaric acid, caffeic acid, and CoASH to AMP, 4-coumaroyl CoA, and caffeoyl CoA. Methods for detection of ATP, 4-coumaric acid, caffeic acid, and CoASH, and/or AMP, 4-coumaroyl CoA, and caffeoyl CoA, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

Thus, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a ATP, 4-coumaric acid, caffeic acid, and CoASH with 4CL; b) contacting said ATP, 4-coumaric acid, caffeic acid, and

CoASH with 4CL and said candidate compound; and c) determining the concentration of AMP, 4-coumaroyl CoA, and caffeoyl CoA after the contacting of steps (a) and (b). If a candidate compound inhibits 4CL activity, a higher concentration of the substrate (ATP, 4-coumaric acid, caffeic acid, and CoASH) and a lower level of the product (AMP, 4-coumaroyl CoA, and caffeoyl CoA) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a). Preferably the 4CL is a plant 4CL. Enzymatically active fragments of a plant

4CL are also useful in the methods of the invention. For example, a polypeptide comprising at least 100 consecutive amino acid residues of a plant 4CL may be used in the methods of the invention, hi addition, a polypeptide having at least 80%, 85%, 90%, 95%, 98% or at least 99%) sequence identity with a plant 4CL may be used in the methods of the invention. Preferably, the polypeptide has at least 80% sequence identity with a plant 4CL and at least 50%, 75%, 90% or at least 95% of the activity thereof.

Thus, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting ATP, 4-coumaric acid, caffeic acid, and CoASH with a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant 4CL, a polypeptide having at least 80% sequence identity with a plant 4CL and at least 50% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a plant 4CL; b) contacting said ATP, 4-coumaric acid, caffeic acid, and CoASH with said polypeptide and said compound; and c) determining the concentration of AMP, 4-coumaroyl CoA, and caffeoyl CoA after the contacting of steps (a) and (b).

Again, if a candidate compound inhibits 4CL activity, a higher concentration of the substrate (ATP, 4-coumaric acid, caffeic acid, and CoASH) and a lower level of the product (AMP, 4-coumaroyl CoA, and caffeoyl CoA) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).

For the in vitro enzymatic assays, 4CL protein and derivatives thereof may be purified from a plant or may be recombinantly produced in and purified from a plant, bacteria, or eukaryotic cell culture. Preferably these proteins are produced using a baculovirus or E. coli expression system. Methods for purifying 4CL may be found in Knoblock and Hahlbrock (1977) Arch Biochem Biophys 184:237-248. Other methods for the purification of 4CL proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the invention also provides plant and plant cell based assays. In one embodiment, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) measuring the expression of 4CL in a plant or plant cell in the absence of said compound; b) contacting a plant or plant cell with said compound and measuring the expression of 4CL in said plant or plant cell; c) comparing the expression of 4CL in steps (a) and (b).

A reduction in 4CL expression indicates that the compound is a herbicide candidate. In one embodiment, the plant or plant cell is an Arabidopsis thaliana plant or plant cell.

Expression of 4CL can be measured by detecting the 4CL primary transcript or mRNA, 4CL polypeptide or 4CL enzymatic activity. Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al. , eds., Greene Publishing and Wiley-Interscience, New York, 1995. The method of detection is not critical to the invention. Methods for detecting 4CL RNA include, but are not limited to amplification assays such as quantitative PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using a 4CL promoter fused to a reporter gene, bDNA assays and microarray assays. Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, His Tag and ELISA assays, polyacrylamide gel electrophoresis, mass spectroscopy and enzymatic assays. Also, any reporter gene system may be used to detect 4CL protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with 4CL, so as to produce a chimeric polypeptide. Methods for using reporter systems are known to those skilled in the art. Examples of reporter genes include, but are not limited to, chloramphenicol acetyltransferase (Gorman et al. (1982) Mol Cell Biol :1104; Prost et al. (1986) Gene 45:107-111), β-galactosidase (Nolan et al. (1988) Proc NatlAcad Sci USA 55:2603-2607), alkaline phosphatase (Berger et α/. (1988) Gene 66:10), luciferase (De Wet et al. (1987) Mol Cell Biol 7:725-737), β-glucuronidase (GUS), fluorescent proteins, chromogenic proteins and the like. Methods for detecting 4CL activity are described above. Chemicals, compounds or compositions identified by the above methods as modulators of 4CL expression or activity can then be used to control plant growth. For example, compounds that inhibit plant growth can be applied to a plant or expressed in a plant, in order to prevent plant growth. Thus, the invention provides a method for inhibiting plant growth, comprising contacting a plant with a compound identified by the methods of the invention as having herbicidal activity.

Herbicides and herbicide candidates identified by the methods of the invention can be used to control the growth of undesired plants, including both monocots and dicots. Examples of undesired plants include, but are not limited to barnyard grass (Echinochloa crus-gallϊ), crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis), perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa), nightshade (Solarium nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L.), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Arnaranthus retroflexus, Sida spinosa, Xanthium strumarium and the like.

EXPERIMENTAL

Plant Growth Conditions Unless, otherwise indicated, all plants are grown in Scotts Metro-Mix™ soil

(the Scotts Company) or a similar soil mixture in an environmental growth room at 22°C, 65% humidity, 65% humidity and a light intensity of ~100 μ-E m^"2 s^"1 supplied over 16 hour day period.

Seed Sterilization

All seeds are surface sterilized before sowing onto phytagel plates using the following protocol. 1. Place approximately 20-30 seeds into a labeled 1.5 ml conical screw cap tube. Perform all remaining steps in a sterile hood using sterile technique.

2. Fill each tube with 1ml 70% ethanol and place on rotisserie for 5 minutes.

3. Carefully remove ethanol from each tube using a sterile plastic dropper; avoid removing any seeds.

4. Fill each tube with 1ml of 30% Clorox and 0.5% SDS solution and place on rotisserie for 10 minutes.

5. Carefully remove bleach/SDS solution.

6. Fill each tube with 1ml sterile dl H₂0; seeds should be stirred up by pipetting of water into tube. Carefully remove water. Repeat 3 to 5 times to ensure removal of

Clorox/SDS solution.

7. Fill each tube with enough sterile dl H₂0 for seed plating (-200-400 μl). Cap tube until ready to begin seed plating.

Plate Growth Assays

Surface sterilized seeds are sown onto plate containing 40 ml half strength sterile MS (Murashige and Skoog, no sucrose) medium and 1 %> Phytagel using the following protocol:

1. Using pipette man and 200 μl tip, carefully fill tip with seed solution. Place 10 seeds across the top of the plate, about V in down from the top edge of the plate.

2. Place plate lid % of the way over the plate and allow to dry for 10 minutes.

3. Using sterile micropore tape, seal the edge of the plate where the top and bottom meet.

4. Place plates stored in a vertical rack in the dark at 4°C for three days. 5. Three days after sowing, the plates transferred into a growth chamber with a day and night temperature of 22 and 20°C, respectively, 65% humidity and a light intensity of ~100 μ-E m^" s^" supplied over 16 hour day period. 6. Beginning on day 3, daily measurements are carried out to track the seedlings development until day 14. Seedlings are harvested on day 14 (or when root length reaches 6 cm) for root and rosette analysis.

Example 1 Construction of a Transgenic Plant expressing the Driver The "Driver" is an artificial transcription factor comprising a chimera of the DNA-binding domain of the yeast GAL4 protein (amino acid residues 1-147) fused to two tandem activation domains of herpes simplex virus protein VP16 (amino acid residues 413-490). Schwechheimer et al. (1998) Plant MolBiol 3(5:195-204. This chimeric driver is a transcriptional activator specific for promoters having GAL4 binding sites. Expression of the driver is controlled by two tandem copies of the constitutive CaMV 35 S promoter.

The driver expression cassette was introduced into Arabidopsis thaliana by agroinfection. Transgenic plants that stably expressed the driver transcription factor were obtained.

Example 2 Construction of Antisense Expression Cassettes in a Binary Vector

A fragment, fragment or variant of an Arabidopsis thaliana cDNA corresponding to SEQ ID NO:l was li gated into the Pacl/Ascl sites of an E.coli/Agrobacterium binary vector in the antisense orientation. This placed transcription of the antisense RNA under the control of an artificial promoter that is active only in the presence of the driver transcription factor described above. The artificial promoter contains four contiguous binding sites for the GAL4 transcriptional activator upstream of a minimal promoter comprising a TATA box.

The ligated DNA was transformed into E.coli. Kanamycin resistant clones were selected and purified. DNA was isolated from each clone and characterized by PCR and sequence analysis. The DNA was inserted in a vector that expresses the A. thaliana antisense RNA, which is complementary to a portion of the DNA of SEQ ID NO: 1. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus At3g48990. The coding sequence for this locus is shown as SEQ ID NO: 1. The protein encoded by this mRNA is shown as SEQ ID NO: 2. The antisense expression cassette and a constitutive chemical resistance expression cassette are located between right and left T-DNA borders. Thus, the antisense expression cassettes can be transferred into a recipient plant cell by agroinfection. Example 3 Transformation of Agrobacterium with the Antisense Expression Cassette

The vector was transforrhed into Agrobacterium turnefaciens by electroporation. Transformed Agrobacterium colonies were isolated using chemical selection. DNA was prepared from purified resistant colonies and the inserts were amplified by PCR and sequenced to confirm sequence and orientation.

Example 4 Construction of an Arabidopsis Antisense Target Plants

The antisense expression cassette was introduced into Arabidopsis thaliana wild-type plants by the following method. Five days prior to agroinfection, the primary inflorescence of Arabidopsis thaliana plants grown in 2.5 inch pots were clipped in order enhance the emergence of secondary bolts.

At two days prior to agroinfection, 5 ml LB broth (10 g/L Peptone, 5 g/L Yeast extract, 5 g/L NaCl, pH 7.0 plus 25 mg/L kanamycin added prior to use) was inoculated with a clonal glycerol stock of Agrobacterium carrying the desired DNA. The cultures were incubated overnight at 28°C at 250 rpm until the cells reached stationary phase. The following morning, 200 ml LB in a 500 ml flask was inoculated with 500 μl of the overnight culture and the cells were grown to stationary phase by overnight incubation at 28°C at 250 rpm. The cells were pelleted by centrifugation at 8000 rpm for 5 minutes. The supernatant was removed and excess media was removed by setting the centrifuge bottles upside down on a paper towel for several minutes. The cells were then resuspended in 500 ml infiltration medium (autoclaved 5% sucrose) and 250 μl/L Silwet L-77™ (84% polyalkyleneoxide modified heptamethyltrisiloxane and 16% allyloxypolyethyleneglycol methyl ether), and transferred to a one liter beaker.

The previously clipped Arabidopsis plants were dipped into the Agrobacterium suspension so that all above ground parts were immersed and agitated gently for 10 seconds. The dipped plants were then covered with a tall clear plastic dome in order to maintain the humidity, and returned to the growth room. The following day, the dome was removed and the plants were grown under normal light conditions until mature seeds were produced. Mature seeds were collected and stored desiccated at 4 °C.

Transgenic Arabidopsis Tl seedlings were selected. Approximately 70 mg seeds from an agrotransformed plant were mixed approximately 4:1 with sand and placed in a 2 ml screw cap cryo vial.

One vial of seeds was then sown in a cell of an 8 cell flat. The flat was covered with a dome, stored at 4°C for 3 days, and then transferred to a growth room. The domes were removed when the seedlings first emerged. After the emergence of the first primary leaves, the flat was sprayed uniformly with a herbicide corresponding to the chemical resistance marker plus 0.005%> Silwet (50 μl/L) until the leaves were completely wetted. The spraying was repeated for the following two days.

Ten days after the first spraying resistant plants were transplanted to 2.5 inch round pots containing moistened sterile potting soil. The transplants were then sprayed with herbicide and returned to the growth room. These herbicide resistant plants represented stably transformed Tl plants.

Example 5 Effect of Antisense Expression in Arabidopsis Seedlings

The Tl antisense target plants from the transformed plant lines obtained in Example 4 were crossed with the Arabidopsis transgenic driver line described above. The resulting Fl seeds were then subjected to a Paradigm Genetics, Inc. (PGI) plate assay to observe seedling growth over a 2-week period. Seedlings were inspected for growth and development. The transgenic plant line containing the antisense construct exhibited significant developmental abnormalities during early development. Four of the seven seedlings examined from the transgenic line exhibited growth reduction, and in three of those four, the growth reduction was of such a severe nature that it represented arrest of the growth process. Therefore, this gene represents an essential gene for normal plant growth and development.

Example 6. Cloning and Expression Strategies, Extraction and Purification of the 4CL protein. The following protocol may be employed to obtain the purified 4CL protein.

Cloning and expression strategies:

An 4CL gene can be cloned into E. coli (pΕT vectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags. Evaluate the expression of recombinant protein by SDS-PAGE and Western blot analysis.

Extraction: Extract recombinant protein from 250 ml cell pellet in 3 mL of extraction buffer

By sonicating 6 times, with 6 sec pulses at 4°C. Centrifuge extract at 15000xg for 10 min and collect supernatant. Assess biological activity of the recombinant protein by activity assay.

Purification:

Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen). Purification protocol: perform all steps at 4oC:

• Use 3 ml Ni-beads (Qiagen) • Equilibrate column with the buffer

• Load protein extract

• Wash with the equilibration buffer

• Elute bound protein with 0.5 M imidazole

Example 7

Assays for Testing Inhibitors or Candidates for Inhibition of 4CL Activity

The enzymatic activity of 4CL may be determined in the presence and absence of candidate inhibitors in a suitable reaction mixture, such as described by any of the following known assay protocols:

A. Spectrophoto etric assay:

Add 25 μl of a standard assay buffer containing one of the substrates (either 4-coumarate or caffeic acid). Add 25 μl of the 4CL enzyme in assay buffer. Incubate at thirty degrees Celcius for one hour, then determine optical density at either 333 run (if substrate is 4-coumarate) or 363 run (if substrate is caffeic acid).

B. Luminescence assay: This assay can be used to measure the ATP remaining after completion of the 4CL reaction. The reaction may begin with 25 μl of a standard Hepes assay buffer (a suitable buffer may be obtained from Packard BioScience) containing a 4CL substrate. Add 25 μl of the 4CL enzyme in the assay buffer. Incubate the mixture at thirty degrees Celsius for one hour. Add 50 μl of a luciferase reaction mixture. Read the luminescence after ten minutes. The luciferase reaction may be performed as either a flash reaction or a long-lived glow signal by using Packard Bioscience ATPLite reaction mix (with a half-life of five hours).

While the foregoing describes certain embodiments of the invention, it will be understood by those skilled in the art that variations and modifications may be made and still fall within the scope of the invention.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a 4CL with said compound; and b) detecting the presence and/or absence of binding between said compound and said 4CL; wherein binding indicates that said compound is a candidate for a herbicide.

2. The method of claim 1 , wherein said 4CL is a plant 4CL.

3. The method of claim 2, wherein said 4CL is an Arabidopsis ACL.

A. The method of claim 3, wherein said 4CL is SEQ ID. NO: 2.

5. A method for determining whether a compound identified as a herbicide candidate by the method of claim 1 has herbicidal activity, comprising: contacting a plant or plant cells with said herbicide candidate and detecting the presence or absence of a decrease in growth or viability of said plant or plant cells.

6. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with at least one polypeptide selected from the group consisting of: i) the polypeptide of SEQ ID NO:2; and ii) a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2; and b) detecting the presence and/or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide.

7. A method for determining whether a compound identified as a herbicide candidate by the method of claim 6 has herbicidal activity, comprising: contacting a plant or plant cells with said herbicide candidate and detecting the presence or absence of a decrease in growth or viability of said plant or plant cells.

8. The method of claim 6, wherein said polypeptide is the polypeptide of SEQ ID NO:2.

9. The method of claim 6, wherein said polypeptide has at least 90% sequence identity with the polypeptide of SEQ ED NO:2.

10. The method of claim 6, wherein said polypeptide has at least 95% sequence identity with the polypeptide of SEQ ED NO:2.

11. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting an ATP, 4-coumaric acid, caffeic acid, and CoASH with 4CL; b) contacting said ATP, 4-coumaric acid, caffeic acid, and CoASH with 4CL and said candidate compound; and c) determining the concentration of at least one of ATP, 4- coumaric acid, caffeic acid, and CoASH, and/or AMP, 4-coumaroyl CoA, and caffeoyl CoA after the contacting of steps (a) and (b), wherein a higher concentration of a substrate (ATP, 4-coumaric acid, caffeic acid, and CoASH) and/or a lower level of a product (AMP, 4-coumaroyl CoA, and caffeoyl CoA) detected in the presence of the candidate compound (step b) than in the absence of the compound (step a) indicates that said compound is a candidate for a herbicide.

12. The method of claim 11 , wherein said 4CL is a plant 4CL.

13. The method of claim 11, wherein said 4CL is an Arabidopsis ACL.

14. The method of claim 13, wherein said 4CL is SEQ ID. NO: 2.

15. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting an ATP, 4-coumaric acid, caffeic acid, and CoASH with a polypeptide selected from the group consisting of: i) the polypeptide of SEQ ID NO:2; and ii) a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2; b) contacting said ATP, 4-coumaric acid, caffeic acid, and CoASH with said polypeptide and said compound; and b) determining the concentration of at least one of ATP, 4- coumaric acid, caffeic acid, and CoASH, and or AMP, 4- coumaroyl CoA, and caffeoyl CoA after the contacting of steps

(a) and (b) wherein a higher concentration of a substrate (ATP, 4-coumaric acid, caffeic acid, and CoASH) and/or a lower level of a product (AMP, 4-coumaroyl CoA, and caffeoyl CoA) detected in the presence of the candidate compound (step b) than in the absence of the compound (step a) indicates that said compound is a candidate for a herbicide.

16. The method of claim 15, wherein said polypeptide is the polypeptide of SEQ ID NO:2.

17. The method of claim 15, wherein said polypeptide has at least 90% sequence identity with the polypeptide of SEQ ID NO:2.

18. The method of claim 15, wherein said polypeptide has at least 95% sequence identity with the polypeptide of SEQ ID NO:2.

19. A method for identifying a compound as a candidate for a herbicide, comprising: a) measuring the expression of a 4CL in a plant or plant cell in the absence of said compound; b) contacting a plant or plant cell with said compound and measuring the expression of said 4CL in said plant or plant cell; c) comparing the expression of 4CL in steps (a) and (b), wherein a lower level of 4CL expression indicates that said compound is a candidate for a herbicide.

20. The method of claim 19 wherein said plant or plant cell is an Arabidopsis plant or plant cell.

21. The method of claim 20, wherein said 4CL is SEQ ID NO: 2.

22. The method of claim 19, wherein the expression of 4CL is measured by detecting 4CL mRNA.

23. The method of claim 19, wherein the expression of 4CL is measured by detecting 4CL polypeptide.

24. A method for modulating plant growth and/or development comprising: a) introducing into a plant or plant cell at least one RNA polynucleotide, said RNA polynucleotide selected from the group consisting of: i) an RNA complementary to SEQ ID NO : 1 ; ii) an RNA complementary to at least 20 consecutive nucleotides of SEQ ID NO: 1; iii) an RNA complementary to a nucleic acid having at least 80% sequence identity with SEQ ID NO:l; iv) an RNA complementary to at least 30 consecutive nucleotides of a nucleic acid encoding SEQ ID NO:2; v) an RNA complementary to a nucleic acid encoding a polypeptide having at least 80%> sequence identity with SEQ ID NO:2; vi) a ribozyme specific for a nucleic acid encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2; vii) a dsRNA specific for a nucleic acid encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2; viii) an RNA having at least 80%> sequence identity with SEQ ED

NO:l; ix) an RNA encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2; and selecting said plant or plant cell expressing said RNA polynucleotide; wherein said plant growth and/or development is decreased or altered.

Figure 1. The 4-coumarate-CoA ligase (4CL) reaction.

Pathway:

ATP, 4-coumaric acid, caffeic acid, and CoASH

AMP, 4-coumaroyl CoA, and caffeoyl CoA

Substrates:

ATP, 4-coumaric acid, caffeic acid, and CoASH

Products:

AMP, 4-coumaroyl CoA, and caffeoyl CoA

Function:

The 4CL enzyme is involved in the conversion of ATP, 4-coumaric acid, caffeic acid, and CoASH to AMP, 4-coumaroyl CoA, and caffeoyl CoA in plants.