Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the invention relates generally to plant molecular biology.
• the invention relates to methods for the identification of herbicides.
• Flavanone 3-hydroxylase (EC 1.14.11.9) (“FHT”) is commonly referred to as the following: naringenin 3-dioxygenase; flavanone 3-hydroxylase; flavanone 3- dioxygenase; flavanone 3b-hydroxylase; flavanone synthase I; (2S)-fiavanone 3- hydroxylase.
• cDNA clones for this gene have been isolated from a number of plants and the enzyme has been purified from plants.
• a genomic clone encoding flavanone 3- hydroxylase was isolated from Arabidopsis thaliana. Pelletier et al., Ill PLANT PHYSIOL. 339-45 (1996) (PMID: 8685272)).
• the present inventors have discovered that antisense expression of a FHT cDNA in Arabidopsis causes developmental abnormalities, and small and chlorotic plant seedlings.
• FHT is essential for normal seed development and growth, and can be used as a target for the identification of herbicides.
• the present invention provides methods for the identification of compounds that inhibit FHT expression or activity, comprising: contacting a candidate compound with a FHT and detecting the presence or absence of binding between the compound and the FHT, or detecting a decrease in FHT expression or activity.
• the methods of the invention are useful for the identification of herbicides.
• Fig. 1 shows the Flavanone 3-hydroxylase reaction.
• binding refers to a noncovalent interaction that holds two molecules together.
• 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.
• NAD+ /"NADH refer to the coenzyme “nicotinamide adenine dinucleotide,” a participant in many redox reactions in biological systems.
• the nicotinamide ring of NAD+ accepts a hydrogen atom plus an electron (a hydride ion, H-) forming NADH.
• H- an electron
• the ring is no longer stable, and therefore the added hydride ion is easily transferred to other molecules.
• herbicide 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.
• inhibitor refers to a chemical substance that inactivates the enzymatic activity of FHT. 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.
• the introduced polynucleotide may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active.
• 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.
• plant organs and tissues e.g., stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores and the like
• 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.
• binding refers to an interaction between FHT and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of FHT.
• the present inventors have discovered that inhibition of FHT gene expression strongly inhibits the growth and development of plant seedlings. Thus, the inventors are the first to demonstrate that FHT is a target for herbicides.
• the invention provides methods for identifying compounds that inhibit FHT gene expression or activity. Such methods include ligand binding assays, assays for enzyme activity and assays for FHT gene expression. Any compound that is a ligand for FHT, other than its substrates, naringenin, 2-oxoglutarate, and O , may have herbicidal activity.
• 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.
• the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a FHT with the compound; and b) detecting the presence and/or absence of binding between the compound and the FHT, wherein binding indicates that the compound is a candidate for a herbicide.
• FHT is meant any enzyme that catalyzes the interconversion of naringenin, 2-oxoglutarate, and O with 3-dihydrokaempferol, succinate, and CO .
• the FHT may have the amino acid sequence of a naturally occurring FHT found in a plant, animal or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence.
• the FHT is a plant FHT.
• the cDNA (SEQ ID NO: 1) encoding the FHT protein or polypeptide (SEQ ID NO:2) can be found herein as well as in the TIGR database at locus F24M12_280.
• plant FHT is meant an enzyme that can be found in at least one plant, and which catalyzes the interconversion of naringenin, 2-oxoglutarate, and O with 3- dihydrokaempferol, succinate, and CO 2 .
• the FHT may be from any plant, including both monocots and dicots.
• the FHT is an Arabidopsis FHT.
• 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.
• the Arabidopsis FHT is from Arabidopsis thaliana.
• the FHT can be from barnyard grass (Echinochloa crus- galli), crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis), perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa), nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L.), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexus, Sida spinosa, Xanthium strumarium and the like. Fragments of a FHT polypeptide may be used in the methods of the invention.
• the fragments comprise at least 10 consecutive amino acids of a FHT.
• 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 FHT.
• the fragment is from an Arabidopsis FHT.
• the fragment contains an amino acid sequence conserved among plant Flavanone 3-hydroxylases. Such conserved fragments are identified in Grima-Pettenuti et al., 21 PLANT MOL. BiOL. 1085-1095 (1993). Those skilled in the art could identify additional conserved fragments using sequence comparison software.
• Polypeptides having at least 80% sequence identity with a plant FHT are also useful in the methods of the invention.
• 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%.
• the polypeptide has at least 50% of the activity of a plant FHT. More preferably, the polypeptide has at least 60%, at least 70%, at least 80% or at least 90% of the activity of a plant FHT. 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 FHT protein.
• the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting the compound with at least one polypeptide selected from the group consisting of: a plant FHT, a polypeptide comprising at least ten consecutive amino acids of a plant FHT, a polypeptide having at least 85% sequence identity with a plant FHT, and a polypeptide having at least 80% sequence identity with a plant FHT and at least 50% of the activity thereof; and b) detecting the presence and/or absence of binding between the compound and the polypeptide, wherein binding indicates that the compound is a candidate for a herbicide.
• at least one polypeptide selected from the group consisting of: a plant FHT, a polypeptide comprising at least ten consecutive amino acids of a plant FHT, a polypeptide having at least 85% sequence identity with a plant FHT, and a polypeptide having at least 80% sequence identity with a plant FHT and at least 50% of the activity thereof.
• any technique for detecting the binding of a ligand to its target may be used in the methods of the invention.
• 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.
• an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a FHT protein or a fragment or variant thereof, the unbound protein is removed and the bound FHT is detected.
• bound FHT is detected using a labeled binding partner, such as a labeled antibody.
• FHT is labeled prior to contacting the immobilized candidate ligands.
• Preferred labels include fluorescent or radioactive moieties.
• Preferred detection methods include fluorescence correlation specfroscopy (FCS) and FCS-related confocal nanofluorimetric methods.
• 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 the herbicide candidate and detecting the presence or absence of a decrease in the growth or viability of the plant or plant cells.
• 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.
• 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.
• 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.
• FHT catalyzes the irreversible or reversible reaction of naringenin, 2-oxoglutarate, and O 2 to 3-dihydrokaempferol, succinate, and CO 2 .
• Methods for detection of naringenin, 2-oxoglutarate, and O 2 , and/or 3- dihydrokaempferol, succinate, and CO 2 include spectrophotomefry, mass specfroscopy, thin layer chromatography (TLC) and reverse phase HPLC.
• the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a naringenin, 2-oxoglutarate, and O with FHT; b) contacting the naringenin, 2-oxoglutarate, and O 2 with FHT and the candidate compound; and c) determining the concentration of 3-dihydrokaempferol, succinate, and/or CO 2 after the contacting of steps (a) and (b).
• a candidate compound inhibits FHT activity, a higher concentration of the substrates (naringenin, 2-oxoglutarate, and O 2 ) and a lower level of the products (3- dihydrokaempferol, succinate, and CO 2 ) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
• the FHT is a plant FHT.
• Enzymatically active fragments of a plant FHT are also useful in the methods of the invention.
• a polypeptide comprising at least 100 consecutive amino acid residues of a plant FHT may be used in the methods of the invention.
• a polypeptide having at least 80%, 85%, 90%, 95%, 98% or at least 99% sequence identity with a plant FHT may be used in the methods of the invention.
• the polypeptide has at least 80% sequence identity with a plant FHT and at least 50%, 75%, 90% or at least 95% of the activity thereof.
• the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting naringenin, 2-oxoglutarate, and O 2 with a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant FHT, a polypeptide having at least 80% sequence identity with a plant FHT and at least 50% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a plant FHT; b) contacting the naringenin, 2-oxoglutarate, and O 2 with the polypeptide and the compound; and c) determining the concentration of 3-dihydrokaempferol, succinate, and/or CO 2 after the contacting of steps (a) and (b).
• FHT 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.
• the invention also provides plant and plant cell based assays.
• the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) measuring the expression of FHT in a plant or plant cell in the absence of the compound; b) contacting a plant or plant cell with the compound and measuring the expression of FHT in the plant or plant cell; c) comparing the expression of FHT in steps (a) and (b).
• a reduction in FHT expression indicates that the compound is a herbicide candidate.
• the plant or plant cell is an Arabidopsis thaliana plant or plant cell.
• FHT FHT protein kinase
• RNA and proteins are known to those skilled in the art. See e.g., Current Protocols in Molecular Biology, (Ausubel et al., eds., Greene Publishing and Wiley- Interscience) (1995). The method of detection is not critical to the invention.
• Methods for detecting FHT 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 FHT promoter fused to a reporter gene, bDNA assays and microarray assays.
• amplification assays such as quantitative PCR
• hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using a FHT 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 specfroscopy and enzymatic assays.
• any reporter gene system may be used to detect FHT protein expression.
• a polynucleotide encoding a reporter protein is fused in frame with FHT, 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., 2 MOL. CELL BIOL.
• Chemicals, compounds, or compositions identified by the above methods as modulators of FHT expression or activity can then be used to control plant growth.
• compounds that inhibit plant growth can be applied to a plant or expressed in a plant, in order to prevent plant growth.
• 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
• 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 ⁇ -E m " s " supplied over 16 hour day period.
• the "Driver” is an artificial transcription factor comprising a chimera of the DNA-binding domain of the yeast GAL4 protein (amino acid residues 147) fused to two tandem activation domains of herpes simplex virus protein VP 16 (amino acid residues 413-490). Schwechheimer et al. (1998) Plant MolBiol 3(5:195-204. This chimeric driver is a franscriptional 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.
• a fragment, fragment or variant of an Arabidopsis thaliana cDNA corresponding to SEQ ID NO:l was ligated 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:l. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus F24M12_280. The coding sequence for this locus is shown as SEQ ID NO:l. The protein encoded by these mRNAs 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.
• the antisense expression cassettes can be transferred into a recipient plant cell by agroinfection.
• 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.
• 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.
• 200 ml LB in a 500 ml flask was inoculated with 500 ⁇ l 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.
• Transgenic Arabidopsis TI 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 ⁇ l/L) until the leaves were completely wetted. The spraying was repeated for the following two days.
• the TI antisense target plants from the transformed plant lines obtained in Example 4 were crossed with the Arabidopsis transgenic driver line described above.
• the resulting FI seeds were then subjected to a 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.
• the following protocol may be employed to obtain the purified FHT protein.
• FHT gene can be cloned into E. coli (pET 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:
• the enzymatic activity of FHT 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:
• Radiochemical assay This assay is based on the conversion of 2-oxo[l- 14 C]glutarate in the enzyme assay to dihydrokaempferol and 14 CO 2 , which are formed in a molar ratio of 1:1, as described by Britsch. Britsch & Grisebach ,156 EUR. J. BlOCHEM 569-77 (1986).
• NADH assay is based on the conversion of 2-oxo[l- 14 C]glutarate in the enzyme assay to dihydrokaempferol and 14 CO 2 , which are formed in a molar ratio of 1:1, as described by Britsch. Britsch & Grisebach ,156 EUR. J. BlOCHEM 569-77 (1986).
• the product of the forward reaction, succinate is itself a substrate for the enzyme Succinate Semialdehyde Dehydrogenase (E.C. 1.2.1.24).
• This enzyme is capable of converting succinate to Succinic semialdehyde in an NADH dependant manner.
• the loss of NADH can be monitored by either absorbance at 340nm or flourescence at ex.340/em.460 to assay for enzymatic activity.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Analytical Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Biotechnology (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Physics & Mathematics (AREA)
• Mycology (AREA)
• Botany (AREA)
• Immunology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=23111769

Family Applications (1)

Country Status (2)

Family Cites Families (3)

• 2002
  • 2002-05-08 WO PCT/US2002/014694 patent/WO2002090598A2/en not_active Application Discontinuation
  • 2002-05-08 EP EP02726854A patent/EP1419269A2/de not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events