WO2002022857A2 - Methods for the identification of modulators of magnesium chelatase expression or activity in plants - Google Patents
Methods for the identification of modulators of magnesium chelatase expression or activity in plants Download PDFInfo
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- WO2002022857A2 WO2002022857A2 PCT/US2001/028761 US0128761W WO0222857A2 WO 2002022857 A2 WO2002022857 A2 WO 2002022857A2 US 0128761 W US0128761 W US 0128761W WO 0222857 A2 WO0222857 A2 WO 0222857A2
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- chelatase
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
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- 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
- C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the invention relates generally to plant molecular biology.
- the invention relates to methods for the identification of compounds that regulate plant growth and development through the modulation of magnesium chelatase expression or activity.
- Magnesium chelatase is an enzyme involved in the synthesis of chlorophyll in plants and photosynthetic microorganisms, and of bacteriochlorophyll in photosynthetic bacteria. Specifically, Mg-chelatase catalyzes the ATP-dependent insertion of a magnesium ion into protoporphyrin LX, to form Mg- protoporphyrin LX. Protoporphyrin LX is the last common intermediate in chlorophyll, bacteriochlorophyll and heme biosynthesis. In all photosynthetic organisms studied to date, Mg-chelatase activity requires three subunits, which are commonly referred to as CHL D, CHL H and CHL I.
- the Mg-chelatase reaction is thought to occur in two steps, preactivation and catalysis. Grafe et al. ( 1999) Proc Natl Acad Sci 96:1941-1946. According to the current model, in the preactivation step, a CHL D dimer associates with two CHL I monomers in the presence of Mg 2+ and ATP. In the chelation step, which involves the hydrolysis of ATP, the CHL H subunits interact with the CHL D subunits, resulting in
- inhibitors of plant Mg-chelatases may have potential herbicidal activity (German patent application DE 197 17 656).
- herbicides that are known to act by modifying the activity of this enzyme. Accordingly, there is a need for assays that can be used to detect modulators of Mg- chelatase activity.
- Inhibitors of Mg-chelatase activity or expression have use as herbicides.
- Mg-chelatase is essential for the growth of Arabidopsis. Specifically, the inhibition of Mg-chelatase CHL H gene expression in Arabidopsis seedlings results in varying levels of chlorosis (yellowing), significantly reduced growth and developmental abnormalities. Thus, Arabidopsis Mg-chelatase can be used as a target for the identification of herbicides.
- the present invention provides methods for the identification of compounds that modulate Arabidopsis Mg-chelatase expression or activity, comprising: contacting a compound with Arabidopsis Mg-chelatase, or a subunit thereof, and detecting the presence and/or absence of binding between said compound and said Mg-chelatase, or detecting a change in Mg-chelatase expression or activity.
- the methods of the invention are useful for the identification of herbicides and other compounds that can modulate plant growth and development.
- the methods of the invention are useful for the identification of compounds that stimulate the expression or function of Mg-chelatase expression or function. Such compounds can be used to promote or manipulate plant growth and development.
- Figure 1 shows the chemical structures of protoporphyrinogen DC and Mg- protoporphyrinogen IX.
- Figure 2 is a digital image showing the effect of Mg-chelatase CHL H antisense expression on Arabidopsis thaliana seedlings.
- 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.
- herbicide refers to a chemical that may be used to kill or suppress the growth of at least one plant, plant cell, plant tissue or seed.
- herbicidally effective amount is meant an amount of a chemical or composition sufficient to kill a plant or decrease plant growth and/or viability by at least 20%. More preferably, the growth or viability will be decreased by 25%, 50%, 75%, 80%, 90% or more.
- inhibitor refers to a chemical substance that inactivates the enzymatic activity of Mg-chelatase.
- the inhibitor may function by interacting directly with the enzyme or a subunit thereof, 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.
- ligand refers to a molecule that will bind to a site on a polypeptide.
- Mg-chelatase is synonymous with “magnesium chelatase”, “protoporphyrinogen Mg-chelatase”, “protopo hyrinogen Mg-chelatase”, “protoporphyrinogen IX Mg-chelatase”, “Mg-protoporphyrinogen chelatase” and “Mg-protopo ⁇ hyrinogen IX chelatase”.
- Mg-chelatase catalyzes the insertion of Mg 2+ into protopo ⁇ hyrinogen IX to yield Mg-protopo ⁇ hyrinogen IX.
- Three subunits, CHL D, CHL H and CHL I are required for Mg-chelatase activity.
- Mg-chelatase refers to a composition comprising these three subunits.
- the "percent (%) sequence identity" between two polynucleotide or two polypeptide sequences is 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) JMol Biol 275:403-410) in the Wisconsin Genetics Software Package (Devererreux et al. (1984) Nucl Acid Res 12:387), Genetics Computer Group (GCG), Madison, Wisconsin.
- 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 Mg-chelatase or a subunit thereof, and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of Mg-chelatase and/or the Mg-chelatase subunit.
- the present inventors have discovered that inhibition of the Mg-chelatase subunit CHL H gene expression strongly inhibits the growth and development of Arabidopsis plant seedlings. Thus, the inventors are the first to show that Arabidopsis Mg-chelatase is a target for herbicides.
- the invention provides methods for identifying compounds that modulate Arabidopsis Mg-chelatase gene expression or activity.
- Such methods include ligand binding assays, assays for enzyme activity and assays for Arabidopsis Mg-chelatase gene expression.
- Any compound that is a ligand for Arabidopsis Mg- chelatase or a subunit thereof, other than its substrate or cofactors i.e., protopo ⁇ hyrinogen IX, Mg 2+ and ATP (adenosine 5'-triphosphate)
- the compounds identified by the methods of the invention are useful for the modulation of plant growth and development.
- the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting an Arabidopsis Mg-chelatase, or at least one subunit thereof, with said compound; and b) detecting the presence and/or absence of binding between said compound and said Arabidopsis Mg-chelatase or subunit thereof; wherein binding indicates that said compound is a candidate for a herbicide.
- an Arabidopsis Mg-chelatase refers to a composition containing the CHL D, CHL H and CHL I Mg- chelatase subunits from an species of Arabidopsis.
- the Mg-chelatase subunits may have the amino acid sequences of naturally occuring Arabidopsis Mg-chelatase subunits, or may have at least 90% amino acid sequence identity with naturally occuring Arabidopsis Mg-chelatase subunit sequences.
- the sequence identity is at least 92%, more preferably the identity is at least 95%, most preferably the sequence identity is at least 98%.
- the Mg-chelatases useful in the methods of the invention can be from any species of Arabidopsis.
- Arabidopsis species include, but are not limited to, Arabidopsis arenosa, Arabidopsis bursifolia, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis grtffithiana, Arabidopsis halleri, Arabidopsis himalaica, Arabidopsis korshinskyi, Arabidopsis lyrata, Arabidopsis neglecta, Arabidopsis pumila, Arabidopsis suecica, Arabidopsis thaliana and Arabidopsis wallichii.
- the Mg-chelatase is from Arabidopsis thaliana, most preferably from Arabidopsis thaliana strain Columbia.
- the amino acid sequences of the Arabidopsis thaliana Mg-chelatase subunits are publicly available. See, for example, GenBank Accession No. AF083555 (A. thaliana CHL D precursor amino acid sequence and mRNA); GenBank accession No. Z68495 and EMBase accession No. S71288 (A. thaliana CHL H amino acid sequence and mRNA); and EMBL Accession No. X51799 and Swiss-Prot Accession No. PI 6127 (A. thaliana CHL I amino acid sequence and mRNA).
- Fragments of an Arabidopsis Mg-chelatase may be used in the methods of the invention.
- the fragments comprise at least 20 consecutive amino acids of an Arabidopsis Mg-chelatase subunit.
- the f agment comprises at least 25, 30, 35 or at least 40 consecutive amino acids residues of an Arabidopsis Mg-chelatase subunit.
- the fragment comprises at least 20 consecutive amino acids of a conserved and/or functional region of a Mg-chelatase subunit.
- 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: an amino acid sequence comprising at least 20 consecutive amino acids of an. Arabidopsis Mg-chelatase subunit and an amino acid sequence having at least 90% sequence identity with an Arabidopsis Mg-chelatase subunit; 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.
- the ligand and target are combined in a buffer.
- Polypeptides and proteins that can reduce non-specific binding, such as BSA or protein extracts from cells that do not produce the target, may be included in binding assay.
- Mg 2+ and ATP may also be included in the binding assay.
- an array of immobilized candidate ligands is provided.
- the immobilized ligands are contacted with one or more Mg-chelatase subunits or variants thereof, the unbound protein is removed and the bound Mg-chelatase subunit(s) is detected.
- bound Mg-chelatase is detected using a labeled binding partner, such as a labeled antibody.
- a Mg-chelatase subunit 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.
- a compound Once a compound is identified as a candidate for a herbicide, it can be tested for the ability to inhibit or otherwise modulate Mg-chelatase enzyme activity.
- the compounds can be tested using either in vitro or cell based enzyme assays.
- 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.
- 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.
- decrease in growth is meant that the herbicide candidate causes at least a 20% 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 25%. 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.
- Mg-chelatase catalyzes the insertion of Mg 2+ into protopo ⁇ hyrinogen IX to yield Mg- protopo ⁇ hyrinogen IX.
- the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting protopo ⁇ hyrinogen IX with Mg-chelatase in the absence of said compound; b) contacting protopo ⁇ hyrinogen IX with Mg-chelatase in the presence of said compound; and c) determining the concentration of protopo ⁇ hyrinogen IX and/or Mg-protopo ⁇ hyrinogen IX after the contacting of steps (a) and (b); wherein said Mg-chelatase is from Arabidopsis and said contacting is performed in the presence of Mg 2+ and ATP.
- a candidate compound inhibits Mg-chelatase activity, a higher concentration of the substrate (protopo ⁇ hyrinogen IX) and a lower level of the product (Mg- protopo ⁇ hyrinogen IX) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
- the contacting should occur under conditions suitable for Mg-chelatase activity.
- Methods for measuring protopo ⁇ hyrinogen IX and Mg-protopo ⁇ hyrinogen IX and Mg-chelatase activity are known in the art. Such methods include, but are not limited to, fluorometry, spectrophotometry, HPLC, TLC and mass spectroscopy. See, for example, Hansson et al. (1999) Proc NatlAcad Sci 96: 1744- 1749; Grafe et al. (1999) Proc NatlAcad Sci 96:1941-1946; Petersen et al.
- protopo ⁇ hyrinogen IX and Mg-protopo ⁇ hyrinogen IX are detected by fluorometry.
- the characteristic fluorescence maxima of protopo ⁇ hyrinogen IX and Mg-protopo ⁇ hyrinogen LX are 633 and 595 nm, respectively.
- Mg-protopo ⁇ hyrin is concentration is determined using a fluorescence detector with excitation at approximately 400-430 nm and emission at 580-605 nm.
- Protopo ⁇ hyrin IX and ATP are available through a variety of chemical suppliers, including Sigma.
- 100 ml buffer containing 50 n M Tricine/NaOH pH 7.9, 12 mM MgCl 2 , 4 mM ATP, 6 ⁇ M protopo ⁇ hyrinogen IX, 50 ng CHL D, 200 ng CHL I and 10 ⁇ g CHL H are incubated at 34°C for 30 minutes.
- the reaction is stopped by the addition of 900 ⁇ l acetone/water/32% ammonia (80:20:1, vol/vol/vol) and centrifuged at 15,000 g for 5 minutes.
- the aqueous phase is analyzed by spectroflurometry with an excitation wavelength of 420 nm and recordation of the emission spectrum between 550 and 650 nm. See Gibson et al. (1999) Biochem J337:243-251.
- Mg-chelatase subunits are added to 6 ⁇ M deuteropo ⁇ hyrin IX, 5 mM ATP, 25 mM creatine kinase, 25 MgCl , 2.5 units creatine kinase, 20 mM Tris pH 9.0 in a volume of 50 ⁇ l, and incubated for 20 minutes at 30°C.
- the reaction is stopped by the addition of 1 ml acetone/water/25% ammonia (80:20: 1 , vol/vol/vol) and 200 ⁇ l hexane.
- the phases are then separated by brief centrifugation, and the emission spectrum of the bottom acetone phase is determined form 550 to 650 with an excitation wavelength of 408 nm.
- Mg-chelatase and subunits thereof may be purified from Arabidopsis, expressed in an in vitro transcription/translation system, or may be recombinantly produced in and purified from a plant, bacteria, or eukaryotic cell culture.
- His-tagged Mg-chelatase subunits have been over expressed in E. coli and purified over Ni 2+ -agarose affinity columns.
- Petersen et al. (1998) JBacteriol 180:699-704.
- Hansson et al. (1999) Proc NatlAcad Sci 96:1744- 1749 describe a method for purifying Mg chelatase subunits from plant leaves. Additional methods for the purification of Mg-chelatase subunits are known to those skilled in the art.
- these proteins are produced using a baculovirus or yeast expression system.
- the invention also provides plant and plant cell based assays.
- the invention provides a method for identifying a chemical as a candidate for a herbicide, comprising: a) measuring the expression of Mg-chelatase in an Arabidopsis plant or plant cell in the absence of said chemical; b) contacting an Arabidopsis plant or plant cell with said chemical and measuring the expression of Mg-chelatase in said plant or plant cell; c) comparing the expression of Mg-chelatase in steps (a) and (b).
- a reduction in Mg-chelatase expression indicates that the compound is a herbicide candidate.
- the plant or plant cell is an Arabidopsis thaliana plant or plant cell.
- Mg-chelatase can be measured by detecting Mg-chelatase primary transcripts or rnRNAs, Mg-chelatase enzyme or subunits or Mg-chelatase 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 Mg-chelatase 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, bDNA assays and microarray assays.
- amplification assays such as quantitative PCR
- hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, 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 ELIS A assays, polyacrylamide gel electrophoresis, mass spectroscopy and enzymatic assays.
- any reporter gene system may be used to detect Mg-chelatase protein expression.
- a polynucleotide encoding a reporter protein is fused in frame with a Mg-chelatase subunit, 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.
- Chemicals, compounds or compositions identified by the above methods as modulators of Mg-chelatase 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.
- such compounds may be applied to or expressed in a particular plant tissue or organ so as to modulate growth of that tissue or organ.
- 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.
- undesired plants include, but are not limited to 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 iheophrasti), 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
- 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 "2 s "1 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 he ⁇ es simplex virus protein VP16 (amino acid residues 413-490). Schwechheimer et al. (1998) Plant Mol Biol 56:195-204.
- This chimeric driver is a rranscriptional activator specific for promoters having GAL4 binding sites. Expression of the driver is controlled by two tandem copies of the constitutive CaMV 35S promoter.
- the driver expression cassette was introduced into Arabidopsis thaliana by agroinfection. Transgenic plants that stably expressed the driver transcription factor were obtained.
- a DNA corresponding to a 1128 nuclotide fragment of the A. thaliana Mg- chelatase CHL H subunit cDNA was ligated into the Pad /Ascl sites of the E.coli/Agrobacterium binary vector PGT3.2 in the antisense orientation. This placed transcription of the Mg-chelatase CHL H 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. pPG315 and pPG316 express Mg-chelatase antisense RNA. The antisense expression cassette and a constitutive barnase expression cassette are located between right and left T-DNA borders. Thus, this DNA can be transferred into a recipient plant cell by agroinfection.
- pPG315 and pPG316 were transformed into Agrobacterium tumefaciens by electroporation. Transformed Agrobacterium colonies were isolated using Basta selection. Basta resistant colonies were purified. DNA was prepared from such clones and the inserts were amplified by PCR and sequenced to confirm the sequence and orientation. The clones were was stored as a frozen glycerol stock.
- the Mg-chelatase antisense expression cassettes (pPG315 and pPG316) were introduced into Arabidopsis thaliana wild type plants by the following agroinfection 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 pPG234.
- the cultures were incubated overnight at 28°C at 250 ⁇ m 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 ⁇ m.
- the cells were pelleted by centrifugation at 8000 ⁇ m 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 ⁇ l/L Silwet L-77TM (84% polyalkyleneoxide modified heptamethyltrisiloxane and 16% allyloxypolyethyleneglycol methyl ether), and transferred to a one liter beaker.
- 500 ml infiltration medium autoclaved 5% sucrose
- Silwet L-77TM 84% polyalkyleneoxide modified heptamethyltrisiloxane and 16% allyloxypolyethyleneglycol methyl ether
- 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 cover 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 using glufosinate treatment. 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.
- the surface of the seeds was sterilized using the chlorine gas method. Briefly, the open vials were placed in a vacuum desiccator in a safety hood. A glass beaker containing 200 ml 5.25% sodium hypochlorite solution was placed in the desiccator. Two ml concentrated HC1 was added to the hypochlorite solution and the cover was placed on the desiccator. Vacuum was applied briefly to seal the dessicator, and the seeds were left in the desiccator overnight. One vial of sterilized 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.
- the flat was sprayed uniformly with a 1:3000 dilution of LibertyTM (AgrEvo; 11.3% glufosinate) in water, 0.005% Silwet (50 ⁇ l/L) until the leaves were completely wetted. The spraying was repeated for the following two days.
- Tl plants 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 represent stably transformed Tl plants. Two Mg-chelatase target plant lines were obtained for each of the pPG315 and pPG316 constructs. Mature Tl plants were then dried and harvested for T2 seeds.
- the Mg-chelatase target plants from three 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 daily for growth and development. During this period, approximately half of seedlings derived from three Arabidopsis Mg-chelatase antisense target lines developed varying levels of chlorosis (yellowing), abnormal development and significantly impaired growth.
- Figure 2 shows the effect of Mg-chelatase CHL H antisense expression on Arabidopsis seedlings. The results are summarized in Table 1.
- Table 1 Phenotypes of plants expressing Mg-chelatase antisense RNA
Abstract
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PCT/US2001/028761 WO2002022857A2 (en) | 2000-09-12 | 2001-09-14 | Methods for the identification of modulators of magnesium chelatase expression or activity in plants |
AU2001290927A AU2001290927A1 (en) | 2000-09-12 | 2001-09-14 | Methods for the identification of modulators of magnesium chelatase expression or activity in plants |
EP01970987A EP1444524A2 (en) | 2001-09-14 | 2001-09-14 | Methods for the identification of modulators of magnesium chelatase expression or activity in plants |
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Cited By (3)
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US7005255B2 (en) | 2000-04-14 | 2006-02-28 | Metabolon, Inc. | Methods for drug discovery, disease treatment, and diagnosis using metabolomics |
US7329489B2 (en) | 2000-04-14 | 2008-02-12 | Matabolon, Inc. | Methods for drug discovery, disease treatment, and diagnosis using metabolomics |
US8849577B2 (en) | 2006-09-15 | 2014-09-30 | Metabolon, Inc. | Methods of identifying biochemical pathways |
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DE19717656A1 (en) * | 1997-04-25 | 1998-10-29 | Hoechst Schering Agrevo Gmbh | DNA sequences coding for the subunit CHLD of plant magnesium chelatases, as well as methods for determining the activity of plant magnesium chelatases |
WO2000075340A2 (en) * | 1999-06-04 | 2000-12-14 | E.I. Du Pont De Nemours And Company | Magnesium chelatase |
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2001
- 2001-09-14 WO PCT/US2001/028761 patent/WO2002022857A2/en not_active Application Discontinuation
- 2001-09-14 EP EP01970987A patent/EP1444524A2/en not_active Withdrawn
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DE19717656A1 (en) * | 1997-04-25 | 1998-10-29 | Hoechst Schering Agrevo Gmbh | DNA sequences coding for the subunit CHLD of plant magnesium chelatases, as well as methods for determining the activity of plant magnesium chelatases |
WO2000075340A2 (en) * | 1999-06-04 | 2000-12-14 | E.I. Du Pont De Nemours And Company | Magnesium chelatase |
Non-Patent Citations (2)
Title |
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WO2002022857A8 (en) | 2004-06-17 |
WO2002022857A3 (en) | 2003-06-19 |
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