| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Enhancers of net photosynthesis and methods of enhancing net photosynthesis |
| PATENT ABSTRACT |
The present invention provides novel enhancers of photosynthesis and methods of using same. In one embodiment, the enhancer is derived from a microorganism. In alternative embodiments, the compounds are microbial extracts, a microbial secretion products, and microbially generated natural products. The invention also provides a method for increasing net photosynthesis in a plant by applying an agent comprising triacetylphloroglucinol, diacetylphloroglucinol or monoacetylphloroglucinol to the plant in an amount effective in increasing net photosynthesis in the plant. -------------------------------------------------------------------------------- |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | November 17, 1998 |
| PATENT REFERENCES CITED |
Strzelczyk, et al., "Synthesis of Auxins from Tryptophan and Tryptophan-precursors by Fungi Isolated from Mycorrhizae of Pine (Pinus silvestris L.)"; ACTA Microbiologica Polonica, vol. 26, No. 3, pp. 255-264 (1977). Ryle, et al., "The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover"; Journal of Experimental Botany, vol. 30, No. 114, pp. 145-153 (Feb, 1979). DeJong, et al., "Nitrogen Stress and Apparent Photosynthesis in symbiotically Grown Pisum sativum L.", Plant Physiol. vol. 68, pp 309-313 (1981). M.E. Brown, "Plant Growth Substances Produced by Micro-organisms of Soil and Rhizosphere"; J. appl. Bast., 35:443-451 (1972). Volpin, et al., "Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots"; Plant Physiol., 116:777-783 (1998). Keel, et al., "Pseudomonads as Antagonists of Plant Pathogens in the Rhizosphere: Role of the Antibiotic 2,4-Diacetylphloroglucinol in the Suppression of Black Root Rot of Tobacco"; Symbiosis, 9:327-341 (1990). Raaijmakers, et al., "Natural Plant Protection by 2,4-Diacetylphloroglucinol-Producing Pseudomonas ssp. in Take-All Decline Soils"; Molecular Plant-Microbe Interactions, vol. 11, No. 2, pp. 144-152 (1998). Zhang, et al., "Phosphoenolpyruvate Carboxylase Protein Kinase from Soybean Root Nodules: Partial Purification, Characterization, and Up/Down-Regulation by Photosynthate Supply from the Shoots"; Archives of Biochemistry and Biophysics, vol. 343, No. 2, pp. 260-268 (Jul. 1997). J.J. Oertli, "Exogenous application of vitamins as regulators for growth and development of plants--a review";Z. Pflanzenernahr. Bodenk., 150, 375-391 (1987). Lugtenberg, et al., "Microbial stimulation of plant growth and protection from disease"; Current Opinion in Biotechnology, 2:457-464, (1991). Barea, et al., "Impact on Arbuscular Mycorrhiza Formation of Pseudomonas Strains Used as Inoculants for Biocontrol of Soil-Borne Fungal Plant Pathogens"; Applied and Environmental Microbiology, vol 64, No. 6, pp. 2304-2307, (1998). Naseby, et al., "Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea"; Molecular Ecology, 7:617-625 (1998). Brimecombe, et al., "Effect of genetically modified Pseudomonas fluorescens strains on the uptake of nitrogen by pea from .sup.15 N enriched organic residues"; Letters in Applied Microbiology 26:155-160 (1998). Fenton, et al., "Exploitation of Gene(s) Involved in 2,4-Diacetylphloroglucinol Biosynthesis to Confer a New Biocontrol Capability to a Pseudomonas Strain"; Applied and Environmental Microbiology, vol. 58, No. 12 pp. 3873-3878 (Dec. 1992). Boadbent, et al., "C-Acetylphloroglucinols From Pseudomonas fluorescens"; Phytochemistry, vol. 15, pp. 1785 (1976). Shanahan, et al., "Isolation, trace enrichment and liquid chromatographic analysis of diacetylphloroglucinol in culture and soil samples using UV and amperometric detection"; Journal of Chromatography, 606:171-177 (1992). O.P. Jones, "Effect of phloridzin and phloroglucinol on apple shoots"; Nature vol. 262 pp. 592 (Jul. 1976). Grochowska, "Chromatographic Degradation of Phloridzin"; Plant Physiol. 41:432-436, (1966). Marre, et al., "Azione Di Stimolo Della Florizina Sulla Fotosintesi E Sulla Fotolisi Da Parte Di Cloroplasti Isolati"; Nuovo Giornale Botanico Italiano, vol. LXII, p. 566, (1955). Shanahan, et al., "Liquid chromatographic assay of microbially derived phloroglucinol antibiotics for establishing the biosynthetic route to production, and the factors affecting their regulation"; Analytica Chimica Acta, 272:271-277 (1993). Primary |
| PATENT GOVERNMENT INTERESTS |
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant No. IBN-9722988, awarded by the National Science Foundation. The Government may have certain rights in this invention. |
| PATENT CLAIMS |
What is claimed is: 1. A method for increasing net photosynthesis in a plant, the method comprising applying to a root of the plant a composition comprising an agent selected from the group consisting of triacetylphloroglucinol, diacetylphloroglucinol and monoacetylphloroglucinol in an amount effective for increasing net photosynthesis in the plant. 2. The method of claim 1, wherein said composition thus applied comprises a bacterium capable of generating the agent. 3. The method of claim 2, wherein said bacterium is selected from the group consisting of Sinorhizobium meliloti, or Pseudomonas fluorescens. 4. The method of claim 2, wherein said bacterium is applied in a concentration of about 10.sup.7 to about 10.sup.8 bacteria per mL. 5. The method of claim 1, wherein said composition thus applied comprises a bacteria culture media. 6. The method of claim 1, wherein said composition thus applied comprises an isolated bacterial product. 7. The method of claim 6, wherein said bacterial product is isolated by chromatography. 8. The method of claim 1, wherein said composition thus applied comprises a diacetylphloroglucinol. 9. The method of claim 8, wherein said diacetylphloroglucinol is 2,4,-diacetylphloroglucinol. 10. The method of claim 1, wherein said agent is in a concentration of about 50 to about 100 nM and the composition is applied to the root of said plant. 11. The method of claim 1, comprising applying said composition to said plant in multiple applications. 12. The method of claim 1, comprising applying said composition to said plant about every 24 to 48 hours. 13. The method of claim 1, wherein said plant is an angiosperm. 14. The method of claim 13, wherein said angiosperm selected from the group consisting of monocotyledonous plants and dicotyledonous plants. 15. The method of claim 14, wherein said dicotyledonous plant is a legume. 16. The method of claim 15, wherein said legume is alfalfa. 17. A method for increasing net photosynthesis in a plant, the method comprising applying to said plant a composition comprising a bacterium, wherein the composition is applied in an amount effective to increase net photosynthesis in the plant, wherein the bacterium is selected from the group consisting of Sinorhizobium meliloti and Pseudomonas fluorescens. 18. The method of claim 17, wherein the bacterium is Sinorhizobium meliloti. 19. The method of claim 18, wherein the bacterium is Pseudomonas fluorescens. 20. The method of claim 1, wherein the plant is of a genus selected from the group consisting of Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea. 21. A method for increasing net photosynthesis in a plant, the method comprising applying to said plant a composition comprising an agent selected from the group consisting of triacetylphloroglucinol, diacetylphloroglucinol and monoacetylphloroglucinol to the plant in an amount effective for increasing net photosynthesis in the plant, with the proviso that the plant is not from the genus Nicotiana. |
| PATENT DESCRIPTION |
INTRODUCTION The present invention provides a novel enhancers of net photosynthesis and methods of enhancing net photosynthesis in plants. Due to the ever increasing need to grow crops more efficiently, the ability to increase a plant's growth rate would be of great social and economic value. An attractive biochemical target to accomplish this is the plant's ability to fix carbon dioxide by photosynthesis. The ability to enhance net photosynthesis will allow the grower to generate a plant that is faster growing and/or has increased biomass in, for example, storage products, such as starch (polysaccharides), protein or fats. Researchers have been investigating means to boost a plant's photosynthetic rate in a variety of ways. Some examples include exposing a plant to light with the same wavelengths as sunlight (see, e.g., JP04166017); generating transgenic plants with exogenous genes involved in photosynthesis (see, e.g., WO 96/21737); application of various chemicals to increase the rate of photosynthesis (see, e.g., JP03109304, U.S. Pat. Nos. 4,704,161; 5,597,400); and, applying chemicals to suppress photorespiration, resulting in an increase in the efficiency of photosynthesis (see, e.g., JP01086822, JP60115501, JP55136205, JP55036437). However, prior to this invention, no enhancers of net photosynthesis have been identified. Furthermore, the present invention taps a rich, yet to date, uptapped source, of enhancers of net photosynthesis from microorganisms, particularly, bacteria. Identification of enhancers of net photosynthesis from microorganisms would offer many benefits. For example, the reagent, as a natural product, can be generated on a large, industrial scale using conventional techniques. This eliminates the need to structurally analyze and/or synthetically produce the bioactive component of the microbial extract. Furthermore, if the microorganism can grow in soil or can colonize plant roots, application of the microbe to the soil could be equivalent to applying the bacterial natural product or extract itself. If the genetic mechanism responsible for producing the bioactive agent is found, transgenic microorganisms can be constructed. Thus, any soil-borne or plant (e.g., root, leaf)-colonizing microorganism can be recombinantly manipulated to generate an enhancer of net photosynthesis of the invention. The present invention, by providing novel enhancers of net photosynthesis, particularly enhancers that can be synthesized by microbes and isolated from microbial extracts, fulfills these, and other, needs. SUMMARY OF THE INVENTION The present invention provides novel enhancers of net photosynthesis and methods of using same. In alternative embodiments, the enhancer is a microorganism, or, is an isolated or purified composition derived from a microorganism. In alternative embodiments, the compounds are microbial extracts, a microbial secretion products, and microbially generated natural products. The invention also provides methods for increasing net photosynthesis in a plant by applying an agent comprising triacetylphloroglucinol, diacetylphloroglucinol or monoacetylphloroglucinol to the plant in an amount effective in increasing net photosynthesis in the plant. The triacetylphloroglucinol, diacetylphloroglucinol (DAPG) or monoacetylphloroglucinol can be natural products derived from, or purified from (e.g., a bacterial extract) a microorganism, or they can be generated by organic synthesis (in fact, DAPG can be purchased from commercial sources). In a preferred embodiment, the agent thus applied is 2,4,-diacetylphloroglucinol. In various embodiments, the enhancer of net photosynthesis can be applied to any part of the plant, including, e.g., the root of the plant. In the methods of the invention, the net photosynthesis-enhancing agent thus applied can comprise a bacterium, which can be, e.g., in preferred embodiments, Sinorhizobium meliloti, or Pseudomonas fluorescens. The bacterium can be applied in a concentration of about 10.sup.7 to about 10.sup.8 bacteria per mL. Alternatively, the agent can comprise a bacteria culture media or an isolated bacterial product. Bacterial products can be isolated by any means, including, e.g., various chromatographic techniques, such as HPLC. While the net photosynthesis-enhancing agents of the invention can be applied in any concentration (or estimated concentration or level of purity, if the agent is an extract from a microorganisms), in alternative embodiments, the agent is applied in a concentration of about 1 nM to about 1.0 M, from about 10 nM to about 100 mM, and 50 nM to about 100 nM to the root of said plant. As discussed below, the amount of exact amount of agent necessary to increase net photosynthesis in a given situation under a specific set of conditions can be determined by the skilled artisan using routine testing, as described herein. In the methods of the invention, the photosynthesis-enhancing agent can be applied to the plant in multiple applications, for example, every 24 to 48 hours. The invention provides methods for increasing net photosynthesis in any plant; in various embodiments, the plants can be angiosperms, which can be monocotyledonous plants or dicotyledonous plants. The dicotyledonous plant can be a legume, which can be an alfalfa plant. The invention further provides a method for increasing net photosynthesis in a plant by applying an agent comprising a bacterium, wherein the bacterium is applied in an amount effective to increase net photosynthesis in the plant, wherein the bacterium is selected from the group consisting of Sinorhizobium meliloti and Pseudomonas fluorescens. Also provided is a method for increasing net photosynthesis in a plant comprising applying to the plant an agent comprising an isolated bacterial product, wherein the agent is applied in an amount effective to increase net photosynthesis in the plant, wherein the bacterium is selected from the group consisting of Sinorhizobium meliloti and Pseudomonas fluorescens. The isolated bacterial product thus applied can comprise a Sinorhizobium meliloti bacterial product whose UV visible absorbance analysis contains maxima at about 216 nm, about 260 nm, about 351 nm, and about 390 nm in methanol/water. In alternative embodiments of the methods, the isolated bacterial product thus applied can comprise a Sinorhizobium meliloti bacterial product whose UV visible absorbance analysis contains maxima at about 222 nm, about 266 nm, about 370 nm, and about 445 nm in methanol/water; or whose proton nuclear magnetic resonance (NMR) analysis contains signals consistent with the presence of two separate and distinct protons at about 7.75 to about 8.1 ppm for every two aromatic methyl groups, wherein the presence of the two proton signals around 8.0 indicate the protons are part of a heterocylic structure. The NMR analysis can further contain an additional proton signal between about 8 ppm and about 9 ppm, which is consistent with a proton on an aromatic N atom; or, can further contain signals consistent with the presence of several aromatic moieties linked together. In another embodiment of the methods, the Sinorhizobium meliloti bacterial product can have a molecular weight (MW) of about 770 amu as estimated by unit resolution mass spectrometry analysis; or, a MW of about 752 amu as estimated by unit resolution mass spectrometry analysis. The invention also provides isolated bacterial products for increasing net photosynthesis in a plant. The bacterial products can be derived from Sinorhizobium meliloti and have a UV visible absorbance analysis containing maxima at about 216 nm, about 260 nm, about 351 nm, and about 390 nm in methanol/water; or, the product can have a UV visible absorbance analysis containing maxima at about 222 nm, about 266 nm, about 370 nm, and about 445 nm in methanol/water. In one embodiment, the Sinorhizobium meliloti bacterial product can have a proton NMR analysis containing signals consistent with the presence of two separate and distinct aromatic protons at about 7.75 to about 8.1 ppm for every two aromatic methyl groups, wherein the presence of the two proton signals around 8.0 indicates the protons are part of a heterocylic structure. The proton NMR analysis can further contain an additional proton signal at approximately 8.5 ppm, which is consistent with a proton on an aromatic N atom. The composition's proton NMR analysis can further contain signals consistent with the presence of several aromatic moieties linked together. The isolated Sinorhizobium meliloti bacterial product can also have a MW of about 770 amu as estimated by unit resolution mass spectrometry analysis; or, it can have MW of about 752 amu as estimated by unit resolution mass spectrometry analysis. A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, the figures and claims. All publications, GenBank Accession references (sequences), ATCC Deposits, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes. DETAILED DESCRIPTION OF THE INVENTION This invention is a first description of novel, bacterially derived, enhancers of net photosynthesis. Use of enhancers of net photosynthesis from microbial sources confers many advantages, especially in the production of the reagents and their application to plants and crops. As natural products, the photosynthetic enhancers can be produced on a large, economical scale using conventional industrial techniques. The invention provides various concentrations and levels of purity of these novel enhancers of net photosynthesis. For example, the bacteria known to produce the enhancers of the invention can be directly applied to a plant. Alternatively, the enhancer can be isolated (term defined below) before applying to the plant. As used herein, such isolation includes substantially pure enhancing reagents, relatively crude preparations, and any variation thereof, as long as the preparation has the ability to enhance photosynthesis. If the enhancer is secreted into a microbial culture medium, the medium itself can be applied, or, it may only be necessary to concentrate the medium. Thus, significant quantities of active reagent can be produced and used without the need for substantial purification or structural characterization of the active chemical entity in the identified natural product. The level of purity or concentration of an enhancer of net photosynthesis of the invention needed for a particular situation may vary depending on, e.g., what plants are being treated, where and how the plants are treated (e.g., spraying on leaves, liquid application to soil, hydroponic cultures (e.g., Wang (1996) Biol. Trace Elem. Res. 55:147-62; Ling (1993) J Chromatogr. 643:351-5), "aeroponic" growth on moistened filter paper in Petri dishes (e.g., Tari (1990) Acta Biol Hung 41:387-97) growth in nutrient solution-circulating growth chambers (e.g., Shima (1997) Mutat Res 1997 Dec 12;395(2-3): 199-208), injections into the plant) and the like, which can be determined by the skilled artisan with routine screening and testing. Being bacterial natural products, significant quantities of an enhancer of the invention can be identified, produced and used without the need for purification to homogeneity or structural characterization. Furthermore, if the microorganism producing an enhancer of the invention can grow in soil or can colonize the plant (e.g., roots, leaves), application of the microbe to the soil would be sufficient to enhance net photosynthesis. Alternatively, if the active reagent is purified and/structurally identified, biosynthetic and genetic mechanisms responsible for producing the bioactive agent by the microbe can also be found. Using this information, transgenic microorganisms capable of synthesizing the enhancers of the invention can then be constructed. Thus, any soil-borne or plant (e.g., root, leaf)-colonizing microorganism can be recombinantly manipulated to generate an enhancer of net photosynthesis of the invention. In an alternative embodiment, the invention provides a novel method for increasing net photosynthesis in a plant by applying an agent containing triacetylphloroglucinol, diacetylphloroglucinol, or monoacetylphloroglucinol to the plant in an amount effective for increasing net photosynthesis in the plant. These enhancers of net photosynthesis can also be synthesized as natural products by bacterial and used with the advantages described above. However, it is not necessary that the enhancers of the invention be natural products of microbes. In one embodiment, the enhancers of net photosynthesis are organically synthesized, as described below. DEFINITIONS To facilitate understanding the invention, a number of terms are defined below. As used herein, the terms "isolated" or "isolate," when referring to a molecule or compound, such as, e.g., an extract or isolate from a microorganism, such as a bacterium, means that the molecule or composition is separated from at least one other compound, such as a protein, a sugar, a lipid, a nucleic acid (e.g., RNA, DNA), or other contaminants with which it is associated in vivo or in its naturally occurring state. Thus, an enhancer of net photosynthesis of the invention is considered isolated when it has been separated from at least one other component with which it is associated in vivo or in vitro, e.g., cell membrane, as in a cell extract, or from a culture media into which the compound had been secreted by a microbe. For example, enhancers of net photosynthesis as bacterial products are isolated to varying degrees of purity using column chromatography and high performance liquid chromatography (HPLC), as described below. Furthermore, an isolated enhancer of the invention can also be substantially pure. An isolated composition can be in a homogeneous state and can be in a dry or an aqueous solution. Purity and homogeneity can be determined, e.g., using analytical chemistry techniques such as polyacrylamide gel electrophoresis (SDS-PAGE) or HPLC. As used herein, the terms "bacterium" and "bacteria" incorporate their common usage, and includes, e.g., all genera and species from the Prokaryotae (Monera) Kingdom (e.g., bacteria, including Eubacteria and Archeabacteria), as described in further detail below. The methods of the invention include application of any microorganism that can generate a net photosynthesis-enhancing agent of the invention, including, e.g., Sinorhizobium meliloti (also known as Rhizobium meliloti), and Pseudomonas fluorescens (see Examples, below). As used herein, the term "bacterial product" incorporates its common usage, and includes, e.g., any and all compositions associated with (e.g., generated or synthesized by) a bacteria, internal or external or secreted, including the entire bacterium. As used herein, the terms "monoacetylphloroglucinol," "diacetylphloroglucinol," and "triacetylphloroglucinol," incorporate their broadest chemical meaning. Diacetylphloroglucinol has the chemical formula C.sub.10 H.sub.10 O.sub.5. One form of diacetylphloroglucinol is 2,4-diacetylphloroglucinol, also known as 2,4-diacetyl-1,3,5-benzenetriol. As used herein, the term "increase in net photosynthesis" means that, over a time period, more photosynthesis than respiration (including the sum of photorespiration and dark respiration) occurs in the plant as a whole. A variety of means are available to the skilled artisan for evaluating and measuring a net increase in photosynthesis in a plant in practicing the methods of the invention. For example, if there is an increase in net photosynthesis, then the increase in the amount of oxygen gas released from the plant is greater than the increase in the amount of carbon dioxide gas released (which is, as discussed below, detectable and quantifiable). Furthermore, if there is an increase in net photosynthesis, the plant normally increases its biomass, which can be determined by, e.g., measuring an accumulation of photosynthates or an increase in plant "dry weight." However, the increase in biomass generated by the net increase in the photosynthetic reaction may not be uniform throughout the plant or may be transient. For example, when increased amounts of photosynthate (generated by net increase in photosynthesis) are transported to the roots and used to drive an increase rate of dark respiration in the root, the photosynthates are consumed in the dark respiration reaction; they are not used to increase the dry weight (biomass) of the root. Thus, the term "enhancer of net photosynthesis" indicates that a composition, an extract, a microorganism, can increase net photosynthesis in a plant, as can be determined by methods described herein. The compositions of the invention can be identified and evaluated by determining their ability to increase net photosynthesis in a plant using these routine tests. As used herein, the term "plant" includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), plant protoplasts, seeds and plant cells and progeny of same. The class of plants which can be used in the method of the invention includes angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous (see discussion, infra). Determining Net Photosynthesis in a Plant The invention provides novel methods and compositions for increasing net photosynthesis in a plant. These methods comprise applying to the plant agents that include triacetylphloroglucinol, diacetylphloroglucinol and monoacetylphloroglucinol, and novel isolated bacterial products of the invention, as described herein. These agents are applied to the plant in amounts effective for increasing net photosynthesis in the plant. To evaluate the levels of purity of an agent or bacterial isolate, the effective amounts of agent needed for particular application, the sites and modes of delivery, the frequency of applications needed, and the like, any means of measuring rates and levels of net photosynthesis can be used. Such routine testing means are well known in the art and include, e.g., increases in, e.g., dry weight; carbon dioxide uptake, oxygen generation, carbon assimilation, plant storage products, such as, e.g., polysaccharides (e.g., starches), lipids, proteins and the like (depending on the plant type or stage in life cycle). In practicing the methods of the invention, enhancement of net photosynthesis can also be evaluated by analysis of various steps in the photosynthetic biochemical mechanism. For example, the oxidation (redox) state of photosynthetic metalloproteins can be determined by, e.g., UV absorbance changes. Measuring Net Increase in Carbon Dioxide Uptake by a Plant An agent or bacterial isolate of the invention can be evaluated by determining the level of net increase in carbon dioxide (CO.sub.2) gas uptake by the plant over a measured time period. Any means can be used to measure a net increase in carbon dioxide uptake by the plant, such as by gas chromatograph, as described in detail below. See also, e.g., Borjesson (1992) Appl. Environ. Microbiol. 58:2599-2605. Measuring Net Increase in Oxygen Generated by a Plant An agent or bacterial isolate of the invention can be evaluated by determining the level of net increase in oxygen gas (O.sub.2) generated by the plant over a measured time period. Any means can be used to measure a net increase in oxygen generated by the plant, such as by gas chromatograph, as described in detail below (see Example 1, below). See also, e.g., use of the "Barcroft manometer" (Umbreit, W. W., R. H. Burris, J. F. Stauffer. 1972. Manometric & Biochemical Techniques, p. 111-125); and, Greenbaum, U.S. Pat. No. 4,789,436, describing methods and apparatus for nondestructive in vivo measurement of photosynthesis in plants using oxygen electrodes. Measuring Net Increase in Radioactive Carbon Uptake by a Plant An agent or bacterial isolate of the invention can be evaluated by determining the level of net increase in carbon assimilation by the plant over a measured time period. Any means can be used to measure a net increase in carbon assimilation by the plant. In a preferred embodiment, a net increase in carbon assimilation is measured by a net increase in radioactive carbon (radioisotope .sup.14 C ) uptake by a plant, as described, e.g., in the isotopic assay for measuring net photosynthesis with .sup.14 CO.sub.2 in Sheikholeslam (1980) Botanical Gazette 141:48-52(see Example 1, below). See also, e.g., Andralojc (1994) Biochem J 304:781-6. Measuring Net Increase in Plant Photosynthates Another preferred means to determine of there is a net increase in carbon assimilation of the plant over the measured time period is to measure a net increase in plant photosynthate levels. As defined above, plant photosynthates include, e.g., polysaccharides (e.g., starch, carbohydrates), oligosaccharides and monosaccharides. Any means can be used to measure an increase in the different classes and levels of photosynthates. See, e.g., Zhang (1997) Arch Biochem Biophys 343:260-8; Zhang (1997) FEBS Lett 410:126-30. Measuring Net Increase in Dry Weight of a Plant An agent or bacterial isolate of the invention can be evaluated by determining the level of net increase in dry weight by the plant over a measured time period. Any means can be used to measure a net increase in dry weight of the plant, see, e.g., Almazan (1997) Plant Foods Hum Nutr 50:259-68. Any individual part of the plant (e.g., roots, leaves, stems, flowers, fruits) can be individually analyzed for which various individual constituents accumulate after a net increase in photosynthesis. For example, dry matter, protein, fat, ash, minerals (Ca, Fe, K, Mg, Na, Zn), vitamins (carotene, ascorbic acid, thiamin), and various other chemicals (e.g., acids) can be measured. This data will provide additional information to aid in the use of the enhancers of net photosynthesis of the invention by determining which bioconversion processes are mobilized by the reagent for biomass conversion into food or forms suitable for crop production. See, e.g., Almazan (1997) supra; Hung (1997) Chemosphere 35:959-77. Measuring Photosynthesis Photosynthesis can be monitored concurrently with any of the above means to measure an increase in net photosynthesis in the plant. Photosynthesis can be measured both before, during and after application of the agent or bacterial isolate of the invention to the plant. Any means known in the art can be used. For example, photosynthesis can be evaluated by measuring the redox state of a photosystem membrane (see, e.g., Stirbet (1998) Theor. Biol. 193(1): 131-51). Means to measure the redox state of a photosystem membrane of a plant are well known, e.g., using electron paramagnetic resonance (EPR) and flash photolysis (see, e.g., Hoshida (1997) Biochemistry. 36:12053-61; Gourovskaya (1997) FEBS Lett 414:193-6; Yruela (1996) Biochemistry 35:9469-74. Selecting and Using Microorganisms The methods of the invention include applying to a plant any microbe, e.g., any bacterium, capable of generating an enhancer of net photosynthesis of the invention. The bacterium, as defined above, can be, e.g., any member of the Prokaryotae (Monera) kingdom. While, without limitation, any bacteria can be used, preferred embodiments use and application of Sinorhizobium meliloti, Pseudomonas fluorescens, and species within the genera Rhizobium or Bradyrhizobium. The agents and bacterial isolates of the invention can be applied as preparations purified to varying degrees, as extracts, or as secretion products of a microorganism. In one embodiment, a microorganism (which can generate a net photosynthesis-enhancing agent of the invention, either as a natural product, or because it has been recombinantly manipulated to secrete the agent) can be applied directly to the plant, e.g., the root, shoot, stem, leaf. In one embodiment, the agent or bacterial isolate, whether a microorganism, a composition derived from the microorganism, or a synthetic version of the agent, is applied to the soil or directly around or into the root of the plant. Soil Dwelling or Root Colonizing Microorganisms In a preferred embodiment, microorganisms that generate a net photosynthesis-enhancing agent of the invention which can live in soil or can colonize roots (e.g., in root rhizospheres) are used. For example, all microorganisms which are known to colonize roots are preferred embodiments to be used in the methods of the invention. The skilled artisan can further isolate root-colonizing microbes by, e.g., isolating root nodules and rhizospheres and determining rhizobial root microorganism populations (see, e.g., Petersen (1996) FEMS Microbiol Lett 142:271-276; Simons (1996) Mol. Plant Microbe Interact. 9:600-607). See also, Kloepper, et. al., U.S. Pat. Nos. 5,503,651, and 5,503,652, describing means to isolate bacterial strains from the rhizosphere. Exemplary bacterial species, include, e.g., Rhizobium meliloti, which invade alfalfa root nodules to establish an effective nitrogen-fixing symbiosis (see, e.g., Cheng (1998) J Bacteriol. 180:5183-5191). Pseudomonas fluorescens are known to colonize the root tips of, e.g., alfalfa, tomato, radish, and wheat (see, e.g., Dekkers (1998) Mol. Plant Microbe Interact. 11:763-771. Agrobacterium tumefaciens is known to infect a variety of plant roots and other cell types (see, e.g., Matthysse (1998) Appl. Environ. Microbiol. 64:2341-2345). Azospirillum brasilense and Pseudomonas aureofaciens are known to infect the roots (rhizospheres) of, e.g., wheat (see, e.g., Pereg-Gerk (1998) Mol. Plant Microbe Interact. 11:177-187; Wood (1997) J. Bacteriol. 179:7663-7670). Exemplary fungal root-colonizing species include, e.g., arbuscular mycorrhizal fungi, such as Glomus mosseae, G. intraradices, Gigaspora rosea, Scutellospora castanea (see, e.g., Burleigh (1998) Gene 216:47-53; van Tuinen (1998) Mol. Ecol. 7:879-887). Preparing Net Photosynthesis-Enhancing Agents Microorganisms that Generate Net Photosynthesis-Enhancing Agents In one embodiment, the methods of the invention involve applying a microorganism that generates a net photosynthesis-enhancing agent of the invention, or a secretion product of that microorganism. Methodologies for culturing microorganisms, particularly in a large scale, are well known in the art, see, e.g., Moir (1990) Bioprocess Technol 9:67-94; Gailliot (1990) Biotechnol Prog 6:370-5. Bacterial Extracts As Net Photosynthesis-Enhancing Agents In various embodiments, the net photosynthesis-enhancing agents and bacterial extracts are isolated and purified from a microorganism or a microbial secretion. The isolation of the net photosynthesis enhancing agent or bacterial isolate can be accomplished using any methodology; for general information relating to standard purification procedures, including. e.g., selective precipitation with such substances as ammonium sulfate; electrophoresis, immunopurification, chromatography (such as HPLC, see Examples, below), and others, see, e.g., Scopes, PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, Springer-Verlag: New York (1982); VanBogelen (1995) Biotechnol Annu Rev 1:69-103; Evans (1995) Biotechnology 13:46-52; Perkins (1991) J Chromatogr 540:239-56; Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. Greene Publishing and Wiley-Interscience, New York (1987). See also, e.g., Afeyan, et. al., U.S. Pat. No. 5,833,861, describing chromatography methods and matrix geometry's which permit high resolution, high productivity separation of mixtures of solutes, particularly biological materials. For example, as described below in the Examples, a net photosynthesis enhancing agent or bacterial isolate of the invention can be in the form of crude extracts of bacterial cultures. In the Examples, microbial extracts are isolated from cultured Sinorhizobium meliloti as fractions using column chromatography and HPLC. The fractions are subsequently demonstrated to increase net photosynthesis in an alfalfa model plant system. Fractions which were able to increase net photosynthesis, as measured increased radioactive carbon-14 in the plant, were further analyzed by as variety of techniques, including mass spectrometry, proton nuclear magnetic resonance, and UV-visible absorbance analysis. Acetylphloroglucinols As Net Photosynthesis-Enhancing Agents The methods of the invention also use agents comprising triacetylphloroglucinol, diacetylphloroglucinol and monoacetylphloroglucinol in amounts effective for increasing net photosynthesis in a plant. These compounds were initially identified because they have general structural identity to the bacterial extract compounds D and Y of the invention (see below). Analysis of diacetylphloroglucinol (as described above for compounds D and Y, below), found that it increases net photosynthesis in plants by as much as 50% when it is applied to roots in nanomolar concentrations. The triacetylphloroglucinol, diacetylphloroglucinol (DAPG) and monoacetylphloroglucinol agents of the invention can be generated synthetically (i.e., in vitro organic synthesis), or, can be isolated from any one of a number of bacterial which generate (and, in many cases, secrete) these compounds as natural products. For example, a number of soil bacteria, including, e.g., Pseudomonas species, produce diacetylphloroglucinol. See, e.g., Shanahan (1992) J. of Chromatography 606:171-177, describing purification of 2,4-diacetylphloroglucinol from Pseudomonas fluorescens by HPLC. See also, e.g., Shanahan (1993) Analytica Chimica Acta 272:271-277, describing a preparative chromatographic isolation method involving thin-layer and liquid chromatography to isolate monoacetylphloroglucinol and DAPG from bacteria. For description of the synthesis of triacetylphloroglucinol, see, e.g., Gulati (1943) Org. Synth. Coll. Vol II, page 522; Broadbent (1976) Phytochemistry 15:1785. For the synthetic generation of the triacetylphloroglucinol, DAPG and monoacetylphloroglucinol agents of the invention; see also, e.g., Campbell (1951) J Am. Chem. Soc. 73:2708-2712; Cronin (1997) FEMS Microbiol. Ecology 23:95-106. See, e.g., Yamaki (1994) Phytotherapy Res. 8:112-114, for a description of several phloroglucinols isolated from Chinese herbal drugs; and, Arisawa (1990) Chem. Pharm. Bull. 38:1624-1626; Bowden (1965) J Pharm. Pharmacol. 17:239-242; Hisada (1972) Yakugaku Zasshi 92:1124-1128. Alternatively, bacteria or other microorganisms can be recombinantly manipulated to generate (or generate more) triacetylphloroglucinol, diacetylphloroglucinol and monoacetylphloroglucinol (or any of the other net photosynthesis-enhancing agents of the invention, as described herein). For example, see, e.g., Thomashow, et al., WO 97/01572, describing DNA sequences which function specifically in the synthesis of 2,4-diacetylphloroglucinol, and bacterial strains recombinantly manipulated to secrete this compound. See also, e.g., Barea (1998) Applied and Environmental Microbiol. 64:2304-2307; describing an genetically engineered Pseudomonas strain which is an overproducer of diacetylphloroglucinol; and, Naseby (1998) Molecular Ecology 7:617-625; Brimecombe (1998) Letters in Applied Microbiol. 26:155-160; Fenton (1992) Applied and Environmental Microbiol. 58:3873-3878. Applying and Enhancer of Net Photosynthesis to a Plant Selection of Plants The methods of the invention and the net photosynthesis enhancing agents or bacterial isolates of the invention can be used to enhance net photosynthesis in essentially any plant. Thus, the invention incorporates use of (application to) a broad range of plants, including, e.g., species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea, to name just a few. Application Methodologies The invention involves applying a net photosynthesis enhancing agent or a bacterial isolate to any part of a plant, including, e.g., roots, stems, leaves, seeds and shoots. For example, if the compound is to be applied to the root, it can be in a liquid applied to soil. Alternatively, it can be applied as organism per se, such as a soil-dwelling or root-colonizing microbe, and subsequently generated and secreted by to microorganism. Alternatively, the net photosynthesis enhancing agent or bacterial isolate can be sprayed on leaves, injected into a stalk, and the like. See also, e.g., Lloyd, et. al., U.S. Pat. No. 5,739,081, describing water dispersible granules suitable for agricultural application, where biologically active substances are loaded into preformed absorbent granules. Luthra, et. al., U.S. Pat. No. 5,652,196, describes means to apply water soluble agents to plants in a variable, controlled release manner. Behel, Jr., et. al., U.S. Pat. No. 5,632,799, describes a dried particulate, hydrophilic gel as micronutrient delivery system to plants in soil. Aoki, et. al., U.S. Pat. No. 5,676,726, describes a matrix for application as a plant culture medium which can be used as a microorganism-immobilizing support capable of delivering a large population of microorganisms with long-term viability and improved colonization and growth rates, to plants in soil. The net photosynthesis enhancing agent or bacterial isolate of the invention can also be applied to, e.g., seedlings or germinations incubated under hydroponic systems (see, e.g., Wang (1996) Biol Trace Elem Res 55:147-62. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |