Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
1998-11-17
2002-04-02
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C047S05810R, C504S116100, C504S353000
Reexamination Certificate
active
06365406
ABSTRACT:
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
7
to about 10
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
Joseph Cecillia M.
Phillips Donald A.
Lankford , Jr. Leon B.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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