Classifications

• • 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
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/10—Aromatic or araliphatic carboxylic acids, or thio analogues thereof; Derivatives thereof
• • 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
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
  • A01N37/48—Nitro-carboxylic acids; Derivatives thereof
• • 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
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
• • 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
• • C—CHEMISTRY; METALLURGY
  • C05—FERTILISERS; MANUFACTURE THEREOF
  • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
  • C05F11/00—Other organic fertilisers
  • C05F11/10—Fertilisers containing plant vitamins or hormones
• • C—CHEMISTRY; METALLURGY
  • C05—FERTILISERS; MANUFACTURE THEREOF
  • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
  • C05G5/00—Fertilisers characterised by their form
  • C05G5/20—Liquid fertilisers
  • C05G5/23—Solutions
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
  • Y02W30/00—Technologies for solid waste management
  • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Definitions

• the present invention relates generally to methods and compositions for stimulating carbon nutrient uptake that yields enhanced growth in plants with improved water use efficiency.
• Photosynthesis is the process by which all photosynthetic plants utilize solar energy to build carbohydrates and other organic molecules from carbon dioxide (CO*) and water.
• CO 2 carbon dioxide
• the conversion of CO 2 into plant matter is generally referred to as carbon fixation and occurs by the C 3 cycle in most plants.
• Plants in which the C 3 cycle occurs are referred to hereinafter as "Cj plants”.
• the C 3 cycle involves the carboxylation of ribulose-l,5-bisphosphate (RuBP) to produce two molecules of the 3-carbon compound, 3-phosphoglyceric acid (PGA), the carbon skeleton for hexoses and other organic molecules.
• RuBP ribulose-l,5-bisphosphate
• PGA 3-phosphoglyceric acid
• An important aspect of the C 3 cycle is that the RuBP pool remains charged during carbon uptake.
• NH 3 ammonia
• Ci fragment from glycine is passed on to a FORMYLTETRAHYDROPTEROYLPOLYGLUTAMATE (C.-THF) pool, whereby, it is catalytically transferred in the form of 5,10-methylenetetrahydrofolate.
• Serine hydroxy methyltransferase (SHMT) an abundant enzyme of the C,-THF pool, reversibly catalyzes the attachment of a second molecule of glycine with the C* fragment to make serine.
• Photorespiration is a source of glyoxylate which is ultimately cleaved into C, fragments.
• the animation of glyoxylate and deamination of glycine during photorespiration occurs through the GOGAT (glutamine: 2-oxo-glutarate amino transferase) cycle.
• the GOGAT cycle is the path by which NH 3 is assimilated by plants and follows the depiction given in Fig. 2, wherein, glutamine: 2-oxo-glutarate amino transferase catalyzes the combining of glutamine with 2-oxo-glutarate to form two molecules of glutamate.
• a fertilizer that provides carbon, enhances uptake of carbon, or increases the efficiency of carbon metabolism would increase growth.
• Conventional fertilizers do not directly provide carbon as a nutrient nor do they improve carbon fixation even though carbon accounts for 80% or more of plant growth under the conventional C 3 cycle. Because of their imbalances, application of conventional fertilizers has never achieved optimal productivity during photorespiration.
• the present invention should further provide convenient methods resulting in increased photosynthesis for applying the compositions to photosynthetic plant surfaces. Additionally, it would be desirable if the methods and compositions of the present invention could promote rapid growth and maturing of the treated plant, increase sugar content and, otherwise, increase the quality of the plant, all the while, adjusting transpiration to reduce the watering requirement of the plant and enhance environmental tolerance.
• the C--THF molecule can be segmented into distinct components including a formylpteroyl glutamide (the example shown in Fig. 6 is folinate carrying multiple glutamates) and a polyglutamate (Gli chain.
• the formylpteroyl glutamide can be further subdivided into a Cj-fragment, a pteridine and an aminobenzoylglutamic acid.
• an enhancement of the carbon pathway focuses on modulating the flow of carbon through C,-THF in a manner that enhances fixation of C, fragments in plants.
• C--THF is a catalyst for C* metabolism, meaning that C, metabolism is dependent upon C r THF.
• catalysis of C, fragments is enhanced, i.e. , the plant's capacity to metabolize C
• C. pathway This flow, which we refer to as the C. pathway, is illustrated in Figures 4 and 5.
• the C, pathway is characterized by passage of C* fragments to C--THF, as described by Cossins (1980) and (1987), infra.
• C--THF i.e. , by increasing the formyltetrahydropteroylpolyglutamate pool, improved carbon fixation, water use efficiency and plant growth results.
• the level of C* fragments may be increased by exposure to appropriate environmental conditions or by the addition of substances which are capable of being metabolized to C* fragments. All such substances are referred to as "C r input substances".
• Organic compounds can find passage through C--THF as a consequence of the metabolism of much larger molecules from which C, fragments arise.
• C--THF is involved in manners illustrated in Figs. 4 and 5, representing modifications from Besson et al (1993) and Cossins (1980) and (1987), infra.
• a supramolecular complex in this scheme is thought to be a storage product, serine hydroxymethyltransferase • glycine • folyl-polyglutamate.
• such ternary complexes may be the natural selection for storage because of stability. Modulation of release permits control of the flow of CrTHF, glycine and SHMT. Based on the storage of the ternary complex, treatments are directed towards stimulating its conversion to maintain a high flow of carbon.
• the energy consumptive GOGAT cycle can be circumvented if, for instance, glyoxylate were to undergo decarboxylation in a manner depicted in Fig. 3.
• the remaining C, fragment, formate is incorporated through the intermediacy of the C--THF pool by its addition to glycine yielding serine which is ultimately converted to cellular constituents.
• Direct fixation of CO 2 to formate is known-- e.g. , S.S. Kent (1972) "Photosynthesis in the higher plant Viciafaba. II.
• the non-Calvin cycle origin of acetate and its metabolic relationship to the photosynthetic origin of formate J. Biol. Chem. 247:7293-7302; or Ramaswamy et al.
• Cj fragments e.g. , Ci sources
• C,-acceptor compounds substances which serve as acceptors for C, fragments which would otherwise be converted to toxic metabolites.
• C,-acceptor compounds These substances can be provided either as part of the enhancer substance or as a separate substance. Frequently, these C*-acceptor compounds are sources of polyglutamate.
• C--acceptor compounds may also be glycine or substances that may be metabolized to glycine. Glycine serves as a sink for C, fragments by being converted to serine which is ultimately converted to sugars and other cellular constituents. C*-acceptor compounds, therefore, provide a means of feeding carbon into the leaves at much higher concentrations than would be possible in their absence.
• C,-acceptor compound sources may include exposure of the plant to environmental conditions which increase polyglutamate levels using existing plant constituents, but this reliance on natural sources lends itself to inconsistency. Most direct Glu n chain-lengthening sources, such as glycine, will decrease plant growth when applied alone; but when applied in combination with enhancer substance(s), they increase plant growth.
• Highest potency is achieved by foliar application of formulations which provide all the components of C.-THF or readily metabolized precursors thereto, i.e. , a C- fragment, a pteridine, an aminobenzoic acid and a glutamate, or metabolic precursors thereto.
• formulations which provide all the components of C.-THF or readily metabolized precursors thereto, i.e. , a C- fragment, a pteridine, an aminobenzoic acid and a glutamate, or metabolic precursors thereto.
• ⁇ M micromolar
• mere application of single carbon sources such as methanol and formate require molar (M) concentrations as much as a million times greater than folinate to show activity.
• Folinate has the components of the C--T ⁇ F molecule "preassembled" as a formylpteroyl glutamide. It is not necessary that the compositions disclosed herein be preassembled in this fashion. Combinations of substances that contribute to one or more of the previously identified components of the structure of C--THF also promote plant growth. For example, pteridines formulated with aminobenzoates and Glu,, sources would provide the components for the plant to assemble an entire C--THF. Alternatively, j?-aminobenzoylglutamate may be applied as a single active component, leaving the plant to produce and attach a pteridine. The C.-THF structure may be segmented even further into additional components.
• the formulation may comprise two compounds such as pABA and potassium glutamate.
• the plant would not only be left to produce and attach a pteridine, but it would have to attach the pABA to the glutamate.
• C THF refers to tetrahydrofolates carrying a one-carbon unit.
• the one-carbon unit can be carried in various oxidation states. In the most reduced form, it is carried as a methyl group. In more oxidized forms, it is carried as a formyl, formimino, or methenyl group.
• Tetrahydrofolate is also called tetrahydropteroylglutamate (H 4 PteGlu), see “Biochemistry” by Lubert Stryer, pp. 719-721 (W. H. Freeman, 4th ed. (1995)).
• C.-THF C THF pool(s)
• tetrahydrofolate pool(s) also includes such species that carry more than one glutamate residue, e.g. , C,-H 4 PteGlu n .
• CrTHF CrTHF Increasing the level of CrTHF as described above gives the plant a greater capacity to fix carbon by the C. pathway. This capacity is then exploited by treatment of plants with substances which can be utilized by CrTHF as C, fragments or by exposure of the plants to environmental conditions which increase the flow of C, fragments into and through the C j -THF pool.
• substances utilizable by CrTHF as C. fragments may be C, fragments themselves, e.g. , formate, or may be substances which are metabolized to a fragment. All such substances are collectively referred to as "Crinput substances".
• Environmental conditions which promote the flow of C, fragments into the C, pathway are generally those which promote oxidative metabolism of photorespiration, i.e, the photorespiratory cycle initiated by the oxygenase activity of RuBP carboxylase oxygenase (Rubisco). These conditions are generally referred to as O 2 -Uptake conditions. Exposure of plants to high levels of Cj-input substances and/or O 2 -Uptake levels alone can be toxic because of buildup of intermediates to toxic levels and because of consumption of products of photosynthesis. Therefore, as described earlier, enhancers and C,-acceptor compounds are supplied to route consumption of photosynthetic products back into synthesis of sugar.
• Enhancer substances are generally CI-THF, pteridines, pterins, pteroic acids, pteroyl derivatives, folinates, substituted benzoic acids, substituted benzoates and derivatives thereof; and salts, hydrates and surfactant-linked derivatives thereof.
• the enhancer is applied as an aqueous solution at a concentration in the range from about 0.0001 % to 0.5%, preferably from about 0.0001 to 0.1 %.
• Suitable pteridine compounds contribute to the structure of CrTHF and are represented by the formula below wherein:
• R 01 is hydrogen or is a hydrocarbyl group capable of being metabolized to a one carbon substituent having the oxidation state of a methyl, hydroxymethyl, formyl or formic acid residue;
• R° is independently selected from the group consisting of: methylene-aminobenzoate, optionally substituted on the benzoate ring; methylene-arninobenzoyl(Glu) n , wherein n is an integer from 0 to 10, optionally substituted on the benzoyl ring; and its corresponding dihydro- and tetrahydro-reduction products at positions 5, 6, 7, and/or 8 of the pteridine rings; and salts, hydrates and surfactant-linked derivatives thereof.
• Suitable substituted benzoates contribute to the structure of C,-THF and are represented by the formula below, wherein:
• R is H, hydrocarbyl, halogen; -OH; -SH, NH 2 , N-linked amino acid, N-linked polypeptide, -OR 3 , -SR 3 , NHR 3 , wherein R 3 is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR R 5 wherein R 4 and R 5 which may be the same or different are independently selected from the group consisting of H, optionally substituted hydrocarbyl, alkyl, aryl, acyl, C-terminal linked amino acids, C-terminal linked polypeptide chains, or R 4 and R 3 together with the nitrogen atoms to which they are linked form a heterocyclic ring; R 1 and R 2 are independently selected from the group consisting of: optionally substituted hydrocarbyl groups, alkyl, aryl, acyl, aroyl, halo, cyano, thio, hydroxy, alkoxy, aryloxy
• acidic enhancers may have very low solubility in cool water (25 °C) and, therefore, require solubilization.
• pteroic acid, pteroyl(Glu) n and substituted benzoates do not dissolve at sufficiently high concentrations in water to promote plant growth.
• acids such as formic acid and acetic acid, alcohols such as methanol, alkali metal and alkaline earth metal bases such as potassium hydroxide and calcium hydroxide and carbonates to be formulated in aqueous solution with these agents.
• Such formulations permit dissolution of the enhancer and facilitate penetration of the enhancer into the leaf of the plant. This then allows enhancement of Cr HF levels in the leaf and promotion of plant growth.
• Activators are potassium hydroxide for aminobenzoic acids; normal potassium bicarbonate for folates and phthalic anhydrides; hexamethylenetetramine for pteroic acid; methanol for nitrobenzoic acids; and dimethylsulfoxide (DMSO) for terephthalates.
• Activators potassium hydroxide for aminobenzoic acids
• normal potassium bicarbonate for folates and phthalic anhydrides
• hexamethylenetetramine for pteroic acid
• methanol for nitrobenzoic acids
• DMSO dimethylsulfoxide
• the C j -input substance for foliar application is usually selected from the group consisting of components that contribute C, fragments to the tetrahydrofolate pool.
• any carbon-containing substance which can be metabolized by any of the metabolic pathways in the leaf to generate a C* fragment which can be utilized by C,-THF in the C, pathway can serve as a Crinput substance.
• C,-input substances include formimino-, methyl-, ethenyl-, methylene- and formyl-fragment sources.
• Examples include, but are not limited to formamidine salts of carboxylic acids, N-formyl amino acids, formimino amino acids, carboxylic acids, aldehydes, trialkyl orthoesters, N-formylated organic compounds and carbon dioxide.
• Formimino-fragments can originate from for imino-amino acids, purines, histidines, thymidylates and S-adenosylmethionine all of which are metabolized to 5-formimino-THF.
• C fragment flow in the leaf can also be provided by exposure to O 2 -Uptake conditions.
• O 2 -Uptake conditions generally include specific stressful environmental conditions such as high light intensity, high temperature, elevated oxygen, and water deprivation, either alone or in any combination. Under conditions such as these, O 2 -Uptake occurs within the leaf, i.e. , the oxygenase activity of Rubisco outcompetes its carboxylase activity.
• foliar formulations for use under O 2 -Uptake environments preferably require a ⁇ -acceptor compound.
• An oxidized substance such as formamidine nitrate that can be reduced by electron carriers from photosynthesis is a preferred exemplary ⁇ -acceptor compound to ensure that metabolism of the C,-fragments by the C, pathway under O 2 -Uptake environments is non-toxic and beneficial to the growth of the plant.
• C,-acceptor compounds provide Glu n sources and can be formulated with enhancers or C,-inputs or provided separately.
• Cracceptor compounds When Cracceptor compounds are formulated with enhancers or Crinputs, they may be present as independent substances or they may be part of the same substance as the enhancer or the C,-input substance.
• a single compound that can act as both C,-acceptor compound and C,-input (salts we denote as C,-acceptor compound • C,-input) would be formamidine • glycolate.
• the formamidine portion serves as the polyglutamate source and the glycolate serves as the Crfragment.
• an enhancer such as phthaloylglutamate contains, within its structure, a glutamate as ⁇ -acceptor compound.
• Other preferred single component enhancer substance formulations in which the C, -acceptor compound is inherent in the molecular make up of the enhancer include, but are not limited to, folinate, pteroyl(Glu) n , phthaloyl(Glu) n , aminobenzoyl(Glu) n , nitrobenzoyl(Glu) n , and the 10 like.
• Cj-acceptor compound substances include compounds such as but not limited to glycine, glutamate, nitrates and formamidines.
• elevated concentrations of CO 2 can be applied to plants after application of an enhancer and a C r acceptor compound.
• the plant is exposed to the elevated CO 2 during daylight hours with continuous exposure to the Cracceptor compound substance and high light intensity.
• the plant is moved back to air at night.
• Methods of the present invention may comprise two steps, where a formulation of enhancer(s) is applied to the leaves of the plant to initiate enhancement of the C,-THF pool. After passage of a time sufficient to allow for C,-THF accumulation, the plant is exposed to conditions which increase the flow of carbon, such as by foliar application of Crinputs and ⁇ -acceptor compound or exposure to O 2 -Uptake conditions and a Cj-acceptor compound.
• the foliar application of C,-input substance(s) or the exposure to an O 2 -Uptake condition will be done at least twice following each application of the enhancer substance. Often it will be more than twice over a period from 1 day to 15 days following each enhancer substance application. It will be recognized that even though it is preferable to apply the enhancer substance before the C, -input substance, the order of application may be reversed.
• Plant growth promoting compositions according to the present invention may also be formulated for one-step application.
• Such one-step compositions will comprise an aqueous solution of enhancer(s) and/or Crinput substances in combination with a Cj-acceptor compound.
• the enhancer compounds will be present in amounts sufficient to increase the amount of C r THF available in foliage when applied to the plant.
• the C,-acceptor compound will be selected and be present in amounts sufficient to act as a sink for the Cj fragments generated by metabolism through Cj-THF (e.g. , by increasing GluJ.
• Preferred enhancers for such a one-step application include, but are not limited to, folinate, nitrobenzoates and phthalates.
• Preferred Crinputs for such a one-step application include, but are not limited to, formamidine • glycolate and formamidine • formate.
• Preferred C,-acceptor compounds for such a one-step application include, but are not limited to, glycine, glutamate, glutamine, and formamidine nitrate.
• compositions according to the present invention include plant growth promoting systems comprised of a first aqueous solution and a second aqueous solution.
• the first aqueous solution contains an amount of an enhancer substance selected to increase C,-THF when applied to a plant.
• the second aqueous solution applied to the plant after application of the first aqueous solution, contains an amount of C,-input + Cracceptor compound substance selected to increase the flow of carbon in a leaf.
• Preferred enhancer substances include but are not limited to folinate, pteroic acid, />-nitrobenzoic acid and substituted benzoates, such as terephthalic acid.
• Preferred Cj-inputs include but are not limited to carbon dioxide, glycolate, formamidine • glycolate; formamidine • formate; formiminoamino acids; and other formimino-, methyl-, methenyl-, methylene- and formyl-sources.
• Preferred C,-acceptor compounds include but are not limited to glutamate, glutamine, glycine and formamidines.
• the first aqueous solution can contain a C r acceptor compound substance and the second aqueous solution can contain an enhancer and/or a C,-input.
• Another plant growth promoting composition for two-step application according to the present invention is a first aqueous solution comprising an enhancer and a second aqueous solution comprising a C,-acceptor compound.
• Application of the enhancer is followed by exposure to O 2 -Uptake conditions and application of the C,-acceptor compound.
• plant growth is promoted by enhancement of CrTHF in the leaf and increased flow of C* fragments in the C, pathway by foliar application of C,-acceptor compounds with enhancers, C,-inputs, and/or with exposure to O 2 -Uptake conditions.
• Fig. 1 is a simplified depiction of the C 3 photosynthetic pathway in plants.
• Fig. 2 is an enlarged depiction of the relation between the GOGAT
• glucose 2-oxoglutarate amino transferase
• Fig. 3 is a simplified depiction of an alternate pathway for glyoxylate, bypassing the GOGAT cycle.
• Fig. 4 is a detailed depiction of the C. pathway, further illustrating paths for enhancing CrTHF and the flow of C, fragments.
• Fig. 5 is a simplified depiction of a C* pathway, further illustrating paths for enhancers, C,-inputs, Cracceptor compounds and O 2 -Uptake.
• Fig. 6 is a structure of CrTHF with bars corresponding to the various components that can be applied to foliage for enhancement. Attachment of the single carbon fragment at the 5-position of the pteridine structure is shown with regard to availability of the 10- and 5,10-positions, also.
• Fig. 7 is a graphic depiction showing the effects of folinate or methanol on long-term CO 2 gas exchange.
• Leaves of sugar beets were sprayed with 0.5 mM Uvinul ® P-25, 13 mM glycine and 0.05% HamposylTMC (o); or 2 mM formiminoglycine, 13 mM glycine and 0.05% HamposylTMC(v). Each point is the mean of 3 to 6 measurements of foliar CO 2 gas exchange.
• Fig. 9 is a graphic depiction of results of experimentation showing the long-term effects of formamidine acetate or potassium glycolate on CO 2 Assimilation (A). Soy foliage was sprayed with 30 mM formamidine acetate (FAM) plus 0.05% HamposylTMC (left frame) or 30 mM potassium glycolate plus 0.05% HamposylTMC (right frame). Arrows indicate when plants were treated. Each point is the mean of 3 separate measurements + SD. No SD shown is less than symbol size.
• Fig. 10 is a graphic depiction of results of experimentation showing the long-term effects of formamidine acetate on the Assimilation/Transpiration (A/T) ratio.
• Cabbages were sprayed with 30 mM formamidine acetate and 0.05% HamposylTMC ( • ), but controls were not treated (v). Each data point is the mean of 3 separate measurements.
• the line representing treated plants is a linear regression and the line representing controls is a bionomial regression.
• the present invention provides methods and compositions for promoting the growth of green higher plants, that is, all plants which are actively photosynthetic.
• green higher plants is intended to include virtually all species with active light-gathering surfaces capable of receiving foliar sprays, particularly higher plants that fix carbon dioxide.
• Higher plants include all plant species having true stems, roots, and leaves, thus excluding lower plants, yeasts and molds.
• C 3 plants refers to all plants capable of fixing carbon via the C 3 photosynthetic pathway.
• Suitable plants which may benefit from pathway carbon fertilization according to the present invention include crop plants, such as cranberry, cotton, tea, onions, garlic, leek, bach ciao, coffee, cassava, mustard, melon, rice, peanut, barley, broccoli, cauliflower, mint, grape, potato, eggplant, zucchini, squash, cucumber, legume, lettuce, kale, sugar beet, radish, kale, tobacco, alfalfa, oat, soy, turnip, parsnip, spinach, parsley, corn, sugar cane, Stevia, sorghum and the like; flowering plants, such as New Guinea Impatiens, geranium, passion fruit, breadfruit, poinsettia, Dusty Miller, mimulus, snapdragon, pansy, fuchsia, lobelia, carnation, impatiens, rose, coleus, chrysanthemum, poppy, gesneriads, bromeliads, bougainvillea, oleander,
• the methods and compositions of the present invention may be used to promote growth in photosynthetic parts of either juvenile or mature plants.
• the plants include at least the sprouted cotyledon (i.e. , the "seed leaves") or other substantial light-gathering surfaces, including, of course, the true leaves.
• Improved growth occurs as a result of enhancement of CrTHF as in Fig. 6 or via the pathway of Figs. 4 and 5.
• High foliar content of C,-THF maintains high rates of carbon fixation even under detrimental conditions and plant growth is improved.
• the aqueous solution of the C,-THF enhancer substance will be applied to the leaves of the plant, usually as a foliar spray, but also including dipping, brushing, wicking, misting, and the like of liquids, foams, gels and other formulations.
• Foliar sprays will comprise atomized or other dispersed droplets of the aqueous solution which are directed at the plant leaves in such a way as to substantially wet the surface of the leaf with the minimum amount of the aqueous solution being lost on the soil.
• foliar sprays can be applied to the leaves of the plant using commercially available spray systems, such as those intended for the application of foliar fertilizers, pesticides, and the like, and available from commercial vendors such as FMC Corporation, John Deere, Valmont and Spraying Systems (TeeJetTM).
• Conditions include the foliar application of enhancers, Crinputs, and C,-acceptor compounds and exposure to O 2 -Uptake conditions.
• Suitable light and temperature conditions may be achieved by prolonged exposure of the plant to direct sunlight or other suitable high light intensity illumination sources while maintaining optimal to hot temperatures, usually above 20° to 35°C.
• the plants should remain exposed to the sunlight or high intensity illumination for a period of time sufficient to allow for incorporation of treatments. Usually, the plants should remain exposed to sunlight or other illumination during daylight photoperiods for at least two hours before and for two weeks following fertilizer application. Sufficient nutrients must also be supplied to support healthy growth.
• Enhancer compounds include those which increase C r THF.
• Preferred contributors to C,-THF include its fragments such as folinate, pteridines, and substituted benzoates.
• C r input substances include those which increase the flow of C, fragments.
• Preferred C,-input substances include organic compounds such as formamidine • glycolate and formamidine • formate that yield C. fragments from -acceptor compound • C r input salts.
• Preferred C r acceptor compound compounds include those such as formamidine nitrate, glycine and glutamate which add to the Glu,. chain.
• Activator compounds are those in which enhancers will dissolve prior to mixture with an aqueous solution. Preferred Activators also benefit plant growth by contributing nutrients and include methanol, potassium carbonate, potassium hydroxide, calcium hydroxide and acetic acid.
• Plant illumination either sunlight or artificial, should have an intensity and duration sufficient to enhance photorespiration. A minimum suitable illumination intensity is 100 mmol photosynthetically active quanta (400-700 nm) m' 2 s' 1 , with direct sunlight normally providing much higher illumination.
• Leaf temperature should be sufficiently high for optimal growth or hotter, usually above 25° to 35 °C. It is preferable that the plant be exposed to at least two and preferably twelve hours of intense illumination following application of formulations.
• Elevated carbon dioxide levels will be above normal atmospheric levels, i.e. , above about 0.03%, typically being above about 1 %, and preferably being above about 10%.
• Such elevated carbon dioxide levels may be provided in controlled high light intensity environments, such as greenhouses, treatment chambers, protective crop and bedding covers, phytotrons, and other controlled-environment sealed enclosures for plant culture, and the like. Plants are initially treated with an enhancer to stimulate the rate of carbon dioxide fixation. Enhancement of the Glu,, portion of C,-THF by application of a Cracceptor compound according to the present invention is necessary with exposure of plants to elevated carbon dioxide levels which, in the absence of such, would be toxic to many or all treated plants.
• Enhancer, Cj-input and C,-acceptor compound substances suitable for use in the methods and compositions of the present invention may be selected with reference to Figs. 2, 3, 4, and 5 which are depictions of the C- pathway and Fig. 6 which depicts a representative CrTHF molecule.
• Enhancer, C,-input and Cracceptor compound formulations may be applied to plants in sequence or in combination for improved plant growth. For consistency of results and to prevent toxicity, formulations generally include a Cracceptor compound substance.
• Suitable enhancer substances include those compounds which are whole molecules, fragments or precursors of C r THF in the pathway, as well as all salts, hydrates, aldehydes, esters, amines, and other biologically or chemically equivalent compounds and substances which can be metabolized in the leaf to contribute a component to Cj-THF.
• Preferred enhancer substances include CrTHF compounds, pteridine compounds, and substituted benzoate compounds.
• Suitable CrTHF compounds include folinates and compounds which may be converted to N-formyltetrahydropteroyl(Glu) n when applied to the treated plant.
• Exemplary Cj-THF compounds include folinic acid; anhydroleucovorin; 5-formyltetrahydropteroyl(Glu) 0 (depicted in Fig. 5); 10-formyltetrahydropteroyl(Glu) n ;
• C r THF compounds will be applied to the plant as an aqueous solution having a concentration in the range from about 0.0001 % by weight to 0.5% by weight, preferably to about 0.1 % by weight.
• pteridines are expected to promote plant growth. These include, but are not limited to neopterin, biopterin, leucopterin and the like. Suitable pteridine compounds contribute to the structure of C,-THF and are represented by the formula below, wherein:
• R 01 is hydrogen or is a hydrocarbyl group capable of being metabolized to a one carbon substituent having the oxidation state of a methyl, hydroxy methyl, formyl or formic acid residue;
• R° is independently selected from the group consisting of: methylene-aminobenzoate , optionally substituted on the benzoate ring; methylene-aminobenzoyl(Glu) n , wherein n is an integer from 0 to 10, optionally substituted on the benzoyl ring; its corresponding dihydro- and tetrahydro-reduction products at positions 5, 6, 7, and/or 8 of the pteridine rings; and salts, hydrates and surfactant-linked derivatives thereof.
• hydrocarbyl shall refer to an organic radical comprised of carbon chains to which hydrogen and other elements are attached.
• the term includes alkyl, alkenyl, alkynyl and aryl groups, groups which have a mixture of saturated and unsaturated bonds, carbocyclic rings and includes combinations of such groups. It may refer to straight chain, branched-chain, cyclic structures or combinations thereof.
• hydrocarbyl refers to a hydrocarbyl group which can optionally be mono-, di-, or tri-substituted, independently, with hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl.
• radicals or compounds respectively defines such with up to and including 6, preferably up to and in including 4 and more preferably one or two carbon atoms.
• groups and radicals may be straight chain or branched.
• surfactant refers to surface-active agents, i.e. , which modify the nature of surfaces, often by reducing the surface tension of water. They act as wetting agents, dispersants or penetrants. Typical classes include cationic, anionic (e.g. , alkylsulfates), nonionic (e.g. , polyethylene oxides) and ampholytic. Soaps, alcohols and fatty acids are other examples.
• surfactant-linked derivative refers to a derivative of the parent compound, the derivative having a surfactant covalently attached to the parent compound.
• a representative example of a parent compound and a surfactant-linked derivative thereof is />-aminobenzoic acid and the corresponding polyethoxylated /j-aminobenzoic acid (Uvinul ® P-25).
• R 01 include lower-alkyl, alkyl, hydroxymethyl, hydroxyalkyl, 1-hydroxyalkyl, hydroxylower-alkyl, 1 -hydroxylower-alkyl, alkoxyalkyl, 1-alkoxyalkyl, alkoxy lower-alkyl, 1-alkoxylower-alkyl, haloalkyl, 1-haloalkyl, 1-halolower-alkyl, aminoalkyl, 1-aminoalkyl, 1 -aminolower-alkyl, thioalkyl, 1-thioalkyl and 1-thiolower-alkyl.
• Exemplary pteridine compounds are pterin; pteroic acid; pteroyl(Glu) n such as folic acid, pteropterin and pteroylhexaglutamylglutamic acid (PHGA); dihydrofolate; Rhizopterin; xanthopterin, isoxanthopterin, leucopterin; and ethoxylates, salts and hydrates thereof.
• Such pteridine compounds will be applied to the plant as an aqueous solution in a concentration in the range of about 0.0001 % to 0.5% by weight, preferably in the range of about 0.0001 % to about 0.1 % .
• Suitable substituted benzoate compounds contribute to the structure of CrTHF and are represented by the formula below, wherein:
• R is H, hydrocarbyl, halogen; -OH; -SH, NH 2 , N-linked amino acid, N-linked polypeptide, -OR 3 , -SR 3 , NHR 3 , wherein R 3 is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR 4 R 5 wherein R 4 and R 5 which may be the same or different and are independently selected from the group consisting of H, optionally substituted hydrocarbyl, alkyl, aryl, acyl, C-terminal linked amino acids, C-terminal linked polypeptide chains, or R 4 and R 5 together with the nitrogen atoms to which they are linked form a heterocyclic ring;
• R 1 and R 2 are independently selected from the group consisting of: optionally substituted hydrocarbyl groups, alkyl, aryl, acyl, aroyl, halo, cyano, thio, hydroxy, alkoxy, aryloxy, amino, alkylamino, aminoalkyl, arylamino, aminoaryl, acylamino, ureido, alkylureido, arylureido, hydrazino, hydroxamino, alkoxycarbonylamino, aryloxycarbonylamino, nitro, nitroso, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, carboxamido, monoalkylaminocarbonyl, dialkylaminocarbonyl, formyl, sulfo, sulfamoyl, sulfoamino, alkylsulfonyl, arylsulfonyl, s
• R 1 and 2 will be at the 2, 3, or 4 position of the benzoate ring.
• Cationic salts of the benzoates include cations selected from the group consisting of cations of alkali metals, alkaline earth metals, ammonium, organic ammonium (amine), quaternary ammonium, and mixtures of such salts.
• aminobenzoic acids such as m-aminobenzoic acid and p-aminobenzoic acid
• aminobenzoic acids such as m-aminobenzoic acid and p-aminobenzoic acid
• derivatives such as N-benzoyl amino acids, N-acyl-aminobenzoic acids, aliphatic aminobenzoate esters, aliphatic N-acyl-aminobenzoate esters, N-acyl-N'-aminobenzoyl-amino acids, N-formylaminobenzoic acids, 2-chloro-4-aminobenzoic acid, and ethoxylates such as Uvinul* P-25; nitrobenzoic acids and derivatives such as -nitrobenzoic acid, ?-nitrobenzoic acid, nitrobenzyl amino acids, polyethyleneglycol nitrobenzoate, 4-chloro-2-nitrobenzoic acid, 2-chloro-4-nitrobenzoic acid; phthalates, such
• Such substituted benzoates that contribute to the structure of -THF will be applied to the plant as an aqueous solution in a concentration in the range from about 0.0001 % by weight to 1 % by weight, preferably about 0.0001 % to 0.5%.
• Activator solution dissolve with enhancer substances in water and include organic acids, particularly hydrocarbyl acids and aliphatic alkyl acids such as, for example, formic acid, acetic acid, propionic acid and the like.
• Activators include alkali and alkaline earth hydroxides (KOH, NaOH, ammonium hydroxide, Ca(OH) 2 and the like); alcohols such as methanol, isopropanol, and ethanol; alkali and alkaline earth carbonates and organic bases such as pyridine, diethylamine; surfactants; and penetrants such as organic solvents, particularly dipolar aprotic solvents such as DMSO.
• Preferred Activators which are also C,-inputs include methanol, trimethylorthoformate, hexamethylenetetramine and DMSO.
• Penetrants are typically organic solvent-based carriers which enable the applied substance to penetrate into the plant leaf.
• the sustained flow of Crcarbon required for rapid growth is preferably accomplished by application of C r input with Cj-acceptor compound to the plant being treated.
• Cj-input with C,-acceptor compound compositions can be applied as distinctly separate formulations or in combination with enhancers.
• Crinputs with ⁇ -acceptor compounds are preferably applied separately after enhancer substances, usually at least about 6 hours after enhancer substances had been applied, and preferably at least one day to one week after the enhancer substance had been applied.
• the Cj-inputs with C,-acceptor compounds will be applied at least twice between successive applications of an enhancer substance, frequently being applied from 2 to 10 times between such successive applications.
• the Crinputs with C acceptor compounds continuously between successive applications of another enhancer substance.
• the aqueous solution containing an enhancer substance such as folinate may be applied periodically with successive applications being spaced apart by a period in the range from 7 days to 20 days, with aqueous solutions comprising the Cj-input such as formate with a C,-acceptor compound such as glutamine being applied from 1 time to 50 times between such successive applications.
• Suitable C r input compounds that pass C, fragments include, but are not limited to, formamidine carboxylate salts selected from the group consisting of formamidine glycolate, formamidine acetate and formamidine formate; a formimino amino acid selected from the group consisting of formiminoglycine, formiminoglutamate, formiminoalanine, and formiminoaspartate; a carboxylic acid selected from the group consisting of glycolate, oxalate and formate; an aldehyde selected from the group consisting of formaldehyde and acetaldehyde; a trialkyl orthoester selected from the group consisting of trimethylorthoformate, triethylorthoformate; an N-formylated organic compound selected from the group consisting of diformylhydrazine, formamide, methyl formamide and dimethyl formamide; an acetamide selected from the group consisting of acetamide, methyl acetamide and dimethyl
• the formamidine carboxylates and formimino amino acids exemplified above can act simultaneously as C,-acceptors, since the formamidine and formimino portions can provide sources of Glu n .
• Suitable Cracceptor compounds act as C, sinks and/or enhance Glu,, as a consequence of C,-THF metabolism.
• Formamidines, such as, formamidine nitrate are exemplary ⁇ -acceptor compounds.
• Other suitable Cracceptor compounds include, but are not limited to, glycine, glutamine, glutamate, serine, sarcosine, homocysteine, cystathionine, methionine, hexamethylenetetramines, and formamide.
• Suitable Cj-acceptor compounds are also available as conventional nitrogen fertilizers. They include nitrates, ureas, and the like. Nitrate and urea C,-acceptor compounds are utilized differently from conventional fertilizers in the present invention because they are used in combination with enhancers and C,-inputs to enhance carbon fixation. They directly target increases of the polyglutamate component of CrTHF in the leaf. This has a safening effect that eliminates toxicity of C r inputs. Thereby, application of high concentrations of Cj-inputs results in carbon-based growth rather than retarding or killing the plant.
• compositions of Crinpu with C r acceptor compound will typically be applied at a concentration ranging from about 0.001 % by weight to 5% by weight.
• Preferred C r input with Cracceptor compound formulations include trimethylorthoformate with formamidine nitrate applied as an aqueous solution at a concentration in the range of 0.01 % by weight to 1 % by weight; formate and potassium glutamate applied as an aqueous solution at a concentration in the range from 0.001 % by weight to 5 % by weight; and glycolate and formamidine nitrate applied as an aqueous solution with glycolate at a concentration in the range from 0.01 % by weight to 0.5% by weight and formamidine nitrate in the range from 0.001 % to 1 % by weight.
• the preferred Cr input: C,-acceptor compound ratio will be in the broad range from 1,000: 1 to 1: 100 to a narrow range of 5:1 to 1: 1.
• compositions of the present invention may consist essentially of the aqueous solutions of Cracceptor compound substances with enhancers and C,-inputs, they will usually contain other ingredients and components which improve performance in various ways.
• compositions will usually contain a surfactant present in an amount sufficient to promote leaf wetting and penetration of the active substances, and optionally other components.
• Suitable surfactants include anionic, cationic, nonionic, and zwitterionic detergents, such as ethoxylated alkylamines, quaternary amines, LED3ATM, TeepolTM, Tween ® , Triton ® , LatronTM, DawnTM dish detergent, and the like.
• penetrants such as, dimethylsulfoxide (DMSO), sodium dodecylsulfate (SDS), formamides, and lower aliphatic alcohols, may be used. Ethoxylation of an active component or otherwise chemically modifying the active components by incorporating a penetrant substance is preferred because, as exemplified by Uvinul ® P-25, formulation without additional surfactant is achieved.
• formulations will often include one or more conventional fertilizer constituents such as nitrogen, phosphorus, potassium, and the like.
• Compositions may further comprise secondary nutrients, such as sources of sulfur, calcium, and magnesium, as well as micronutrients, such as chelated iron, boron, cobalt, copper, manganese, molybdenum, zinc, nickel and the like. Incorporation of such plant nutrients into foliar fertilizer formulations is well described in the patent and technical literature.
• Other conventional fertilizer constituents which may be added to the compositions of the present invention include pesticides, fungicides, antibiotics, plant growth regulators, and the like.
• compositions according to the present invention may be tailored for specific uses, including water use efficiency; enhanced performance under environmental stress, and in all areas of agriculture in which heightened carbon fixation is beneficial. Compositions may also be formulated at very low concentrations for liquid suspension culture media.
• Formiminoglycine 0.001% to 1% 0.05% to 0.3%
• Surfactant Triton ® CF10
• Triton ® CF10 0.01% to 0.5% 0.1% to 0.2%
• Formamidine • glycolate 0.01 % to 5% 0.1 % to 0.2%
• one-part compositions may be formulated.
• the one-part composition is typically composed oflow concentrations of substances in combination with a surfactant in a single solution.
• Exemplary one-part compositions follow.
• Terephthalic acid 1 ppm to 100 ppm 15 ppm to 50 ppm Activator (DMSO) 0.01 % to 3% 0.5% to 1 % Potassium glycolate 0.001 % to 0.3% 0.1 % to 0.2%
• Formamidine nitrate 0.01 % to 1 % 0.1 % to 0.3%
• Surfactant HAMPOSYLTMC 0.03% to 1% 0.05% to 0.2%
• Chemicals (abbreviations) and sources sodium glutamate, Ajinomoto; formamidine formate (FAF), glycine (Gly), HAMPOSYLTMC, potassium glycolate (GO), potassium phosphate (KOH), and purified water,Hampshire Chemical Corporation; adenosine triphosphate (ATP), -aminobenzoic acid (MABA),/?-aminobenzoic acid (pABA), -aminobenzoic acid ethyl ester (BenzocaineTM), (p-aminobenzoyl)-L-glutamic acid (pABG),/>-aminohippuric acid (pAH), anthranilate (oABA), ascorbic acid, ethanol (EtOH), folic acid (Folate), calcium folinate (Folinate), formaldehyde, formamidine acetate (FAM), formic acid, formiminoglycine (FIGly), formiminoglutamate
• FAF formam
• FIGlu p-formylbenzoic acid
• pFBA methanol
• MeoABA methyl-2-anthranilate
• NADP nicotinamide adenine dinucleotide phosphate
• CBA terephthalic acid
• CBG N-phthalolyl-L-glutamate
• K1 potassium maleic acid
• KC1 potassium chloride
• Pteroic pteroic acid
• THF 5,6,7,8-tetrahydrofolate
• THF 1,6,7,8-tetrahydrofolate
• aprotinin 2-mercaptoethanol
• triethanolamine ammonium formate
• magnesium chloride MgCl_
• -nitrobenzoic acid pNBA
• Dupont dimethylsulfoxide (DMSO)
• DMSO dimethylsulfoxide
• DMSO dimethylsulfoxide
• Radioisotopic ,4 CO 2 was applied to plants to determine the fate of active substances and changes in the path of carbon fixation.
• Cabbage plants were sprayed with one of the following three solutions: 1) 90 ⁇ M folinate, 0.2% glycine, 1 % DMSO, and 0.1 % Triton ® X-100; 2) 40% MeOH, 0.2% glycine, and 0.05 % HamposylTMC; 3) 0.5 mM pABG, 0.2% glycine, and 0.05% HamposylTMC.
• plants were removed from the glass house and placed under a quartz halogen light (type EKE, 21 V, 150 watt) at room temperature and allowed to acclimate to our laboratory conditions for 15-30 min.
• a leaf that was to be used for experiments was placed in an open chamber that was constantly flushed with pure O 2 during the acclimation period.
• a leaf plug, 3.67 cm 2 was then removed, and placed in a hermetically sealed PlexiglassTM leaf chamber containing pure O 2 being pumped at a rate of 2-3 L min '1 .
• the chamber was illuminated with 1,000 ⁇ mol photosynthetically active quanta nvV 1 directed through a fiber optic cable connected to a quartz halogen light similar to the one used for preillumination.
• Enzymes related to C,-THF were tested for enhancement following treatments.
• the three enzymes that comprise C,-THF-synthase, 10-formyl-THF-synthetase (EC 6.3.4.3), 5,10-methenyl-THF-cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF-dehydrogenase (EC 1.5.1.5) were assayed according to methods of Edwin A. Cossins (University of Alberta, Edmonton, Canada).
• plants were soil-cultured in controlled environmental growth chambers or plants were cultured in greenhouses with supplemental light. Plants were watered daily with measured amounts of reverse osmosis purified water. Photosynthetic CO 2 Gas Exchange was determined by taking measures with with a portable gas exchange system.
• the quantity of the C,-THF pool was determined in two steps.
• Sugar beets (Beta vulgaris L.) cv Monohikari (Seedex, Longmont, Colorado)) were grown under standard greenhouse conditions described above.
• Foliage was treated with 14.8 M MeOH.XX 10 mM FIGly or 0.1 mM folinate dissolved in standard aqueous solutions of 0.05% HAMPOSYLTMC and 0.3% glycine. After treatment with FIGly (20 h) and folinate (20 h), gas exchange was measured to insure that plants were healthy and responsive when the tissues were sampled at that time. Gas exchange was nearly doubled by these treatments and the Cr HF synthases showed approximately 20% stimulation over the controls.
• the MeOH sample was collected 72 h after treatment when gas exchange measured approximately 50% and enzymes were approximately 20% over the controls.
• tissue extracts for C,-THF pool quantification 0.5 g fresh live leaf tissue from different plants was placed in ascorbate buffer. The samples were then immediately placed in a boiling water bath and cooled. The tissues were homogenized and centrifuged. The supernatant was collected. Assays were based on microbial assay methods of D. W. Home and D. Paterson (1988) Clinical Chemistry 34: 2357-2359; and methods for determination of C,-THF pools in plants (Cossins, E. A. 1987. Folate Biochemistry and the Metabolism of One-Carbon Units. In The Biochemistry of Plants, v.
• Assays determined the content of short-chained pteroyls and total CrTHF pool.
• the Glu., value is the difference between the short-chained and total pool values. For each sample, 4 replicate measurements were made.
• plants were cultured in greenhouses or PercivalTM growth chambers. In greenhouses, no special control of physical conditions was attempted, but all comparable treatments were made simultaneously and were subjected to normal greenhouse conditions. Plants were generally harvested and analyzed in the vegetative stage 6 to 8 days after treatment. Plant and root lengths and fresh and dry weights were determined. Data on roots is given only for radish tests while data on shoot growth is given for other plants. All solutions were applied as aqueous foliar sprays.
• gas exchange was enhanced significantly in soy, sunflower, cabbage, kale and sugar beet by foliar treatments with folate, folinate, pABA, pNBA, pFBA, Uvinul ® P-25, FIGly, FAM, FAM with GO, BenzocaineTM and MeOH.
• Gas exchange was significantly higher after Uvinul ® P-25 treatments as compared to pABA treatments.
• pAH stimulated gas exchange in soy. Neither ⁇ ABA nor MeoABA showed increases in gas exchange.
• Figures 7, 8, 9 and 10 summarize results of the long-term effects of various treatments on gas exchange.
• Figure 7 compares the effects of foliar sprays of 17 ⁇ M folinate to 5 M MeOH and showing increased CO 2 gas exchange over soy controls lasting three weeks. Plants were treated at the start and at 120 h. Peak activity was observed during the first and second weeks, the response to folinate far exceeding responses to MeOH.
• Figure 8 shows that 0.5 mM Uvinul ® or 2 mM FIGly applications to sugar beet increased CO 2 gas exchange for ten days following treatments.
• Figure 9 shows that 3 foliar sprays of 30 mM FAM or 30 mM GO formulations increased gas exchange over controls for approximately a week between treatments.
• Figure 10 shows the relationship between assimilation and transpiration. Measurements of the assimilation/transpiration (A/T) ratio in cabbage leaves treated with a formulation containing 30 mM FAM showed CO 2 assimilation maintained with reduced transpiration for the duration of the experiment. 36
• gas exchange increases were observable several hours after treatment. For example, within the first 2 to 3 h of application, no significant difference in gas exchange was noted in soy foliage treated with folinate; but after 6 h, the same leaves showed elevated gas exchange rates as compared to controls that were not treated. After 6 h, the average overall increase in gas exchange on folinate treated plants was approximately 35% greater than in controls. Three weeks after treatment, increased gas exchange was still observed. When treatments were repeated once per week, increased gas exchange continued to the termination of the study at one month. Treatment with pABA required more time to show increases in gas exchange than most other compounds. Generally, sometime after 24 h and within 48 hours, pABA treatments showed an increase in gas exchange over controls.
• Active components were formulated in the following control solutions * .

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  • 1996-02-23 WO PCT/US1996/002444 patent/WO1996027572A1/en not_active Application Discontinuation
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