EP0813510A4 - Methods and compositions for enhancing formyltetrahydropteroylpolyglutamate in plants - Google Patents

Abstract

Methods and compositions for enhancing plant growth provide for foliar application of a substance which enhances the accumulation of formyltetrahydropteroylpolyglutamate (C1-THF) in a treated plant. Treatment with substances that contribute to the structure of C1-THF increases the rate and quantity of carbon fixation by the plant. Thereafter, plant growth is further improved either by exposure of the plant to elevated oxygen, illumination and heat or by foliar input of single carbon fragment sources. Optimal results are obtained by combined treatment with a substance that can serve as a sink for C1 fragments produced in the leaf.

Description

METHODS AND COMPOSITIONS FOR ENHANCING FORMYLTETRAHYDROPTEROYLPOLYGLUTAMATE IN PLANTS

BACKGROUND OF THE INVENTION This application is a continuation-in-part of U.S. patent application Serial No. 08/399,399, filed March 6, 1995 which was a continuation-in part of U.S. patent application Serial No. 08/351,348, filed on December 9, 1994, which was filed as a PCT International Application PCT/US93/05676, on June 14, 1993, which was a continuation-in-part of application Serial No. 07/901,366, filed on June 19, 1992. The full disclosures of each of these patent applications are incorporated herein by reference.

1. Field of the Invention

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. The conversion of CO₂ into plant matter is generally referred to as carbon fixation and occurs by the C₃ cycle in most plants. Plants in which the C₃ cycle occurs are referred to hereinafter as "Cj plants". The C₃ 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. An important aspect of the C₃ cycle is that the RuBP pool remains charged during carbon uptake. Therefore, for every six carboxylation events, which yields twelve PGA's, two PGA's can be converted to hexose, while ten molecules of PGA are recycled to replace the six RuBP's initially carboxylated. A simplified illustration of the C₃ cycle is shown in Fig. 1.

Another event in the C₃ cycle shown in Fig. 1, is photorespiration, during which oxygen (O outcompetes CO₂ and is added to RuBP. As a result of oxidation, phosphoglycolate is formed. The phosphoglycolate is dephosphorylated to glycolate which is oxidized to glyoxylate. Glycine is made by attachment of ammonia (NH₃). The glycine is deaminated releasing NH₃ and further decarboxylated to CO₂ plus a single carbon (C-) fragment. This 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 NH₃ 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 C₃ cycle. Because of their imbalances, application of conventional fertilizers has never achieved optimal productivity during photorespiration.

For these reasons, it would be desirable to provide improved methods and formulations for promoting plant growth. It would be particularly desirable if such methods and compositions were able to maintain flows of C* fragments which enhance growth without toxicity. 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. SUMMARY OF THE INVENTION According to the present invention, methods and compositions are provided for stimulating carbon fixation and increasing the growth of plants by enhancing the availability of C--THF in the leaves of a plant. As shown in Fig. 6, 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. Foliar application of substances that increase the amount of C--THF, either by contributing any one of these components to the structure of C,-THF or otherwise promoting the formation of C.-THF, increases carbon fixation by the C, pathway and enhances plant growth. Such substances which increase the amount of C.-THF in the leaf of a plant are referred to herein as "enhancers" or "enhancer substances".

As provided herein, 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_rTHF. By adding to the C--THF content of a leaf, catalysis of C, fragments is enhanced, i.e. , the plant's capacity to metabolize C|-fragments by increasing the flow of carbon through the -THF pool and thereby fix carbon into cellular constituents is increased proportionately. If the level of C, fragments is then increased, this then results in increased plant growth according to this newly afforded capacity to metabolize what otherwise would have remained an underutilized carbon source. 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. When addition of a fertilizer having the components disclosed herein enhances the catalyst, 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_rinput substances".

Organic compounds can find passage through C--THF as a consequence of the metabolism of much larger molecules from which C, fragments arise. For example, the incorporation of formimino-amino acids is illustrated in Figs. 4 and 5, wherein, a C* fragment is transported as formiminotetrahydrofolate. In fact, 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. Within a leaf, 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 CO₂ 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. (1976) "A novel pathway for the synthesis of solanidine in the isolated chloroplast from greening potatoes," Eur J Biochem 67:275-282— and may provide an additional source of C, units. Therefore, CO₂ released from glyoxylate could be refixed via this direct pathway which is much more energy efficient. As can be deduced from Fig. 3, an absolute requirement for circumventing the GOGAT cycle is a source of glycine. In both cases shown in Figs. 1 and 3, the flow of carbon during photorespiration is dependent on C.-THF and glycine. Furthermore, when fixed carbon, such as, glutamine or glycolate, is added to a leaf, the carbons of serine no longer need to be recycled back to RuBP because the added carbon compensates for those normally lost to glycolate. In fact, even the sole addition of nitrogen in the form of nitrate or NH₃ could influence carbon partitioning since energy for nitrogen cycling and serine recycling would be reduced. Thus, both carbon and nitrogen metabolism are interdependent with C,-THF central to their flow. It follows, that when C.-THF is enhanced, the flow of carbon into growth of the plant can also be accelerated. Following enhancement of C,-THF, input of compounds that release C_t fragments or exposure to conditions that produces a flow of glycolate within leaves (thus providing a source of formate for the C_t-THF pool as shown in Figure 3) leads to improved efficiencies of carbon fixation. As a consequence, plant growth improves.

Exposure of the plant to enhancers and conditions which increase the level of Cj fragments, e.g. , Ci sources, such as glycolate or carbon inputs leading to formate and formaldehyde, can lead to toxicity and leaf burn as a result of the build up of excess formate. Therefore, substances which serve as a sink for such C, fragments must normally be provided. Such substances which serve as a sink for C- fragments in the plant are referred to herein as "C,-acceptor compounds", i.e. , they serve as acceptors for C, fragments which would otherwise be converted to toxic metabolites. 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. Alternatively, 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. For example, when folinate is applied to foliage as an active component formulated with a Glu., source, the folinate is active at minute micromolar (μM) concentrations. In contrast, 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--TΗF 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. For example, the formulation may comprise two compounds such as pABA and potassium glutamate. In this case, the plant would not only be left to produce and attach a pteridine, but it would have to attach the pABA to the glutamate. Thus, there are several components and combinations thereof which may be formulated together for application to foliage to enhance C.-THF and thereby promote plant growth. As used herein, the term "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 (H₄PteGlu), see "Biochemistry" by Lubert Stryer, pp. 719-721 (W. H. Freeman, 4th ed. (1995)). The terms "C.-THF" , "C THF pool(s)" and "tetrahydrofolate pool(s)" as used herein also includes such species that carry more than one glutamate residue, e.g. , C,-H₄PteGlu_n. We refer to compounds capable of contributing components to the molecular structure of the formylpteroylglutamate portion (i.e. , CrH,PteGlu,) of the C,-THF molecule as "enhancers".

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. Such 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 O₂-Uptake conditions. Exposure of plants to high levels of Cj-input substances and/or O₂-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. Typically, 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:

R⁰¹ 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; and

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, NH₂, N-linked amino acid, N-linked polypeptide, -OR³, -SR³, NHR³, wherein R³ is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR R⁵ wherein R⁴ and R⁵ 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 R⁴ and R³ together with the nitrogen atoms to which they are linked form a heterocyclic ring; R¹ and R² 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, sulfeno, sulfino, alkylsulfino, arylsulfino; and salts thereof.

In specific cases, acidic enhancers may have very low solubility in cool water (25 °C) and, therefore, require solubilization. For example, pteroic acid, pteroyl(Glu)_n and substituted benzoates, do not dissolve at sufficiently high concentrations in water to promote plant growth. However, they are sufficiently soluble in 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. Some of the enhancers are activated by dissolving high concentrations in small volumes of alkali followed by adjusting to pH 7 prior to formulation into aqueous solutions. Solubilizers which aid dissolution of the enhancer and facilitate its penetration into the leaf are referred to as "Activators". Preferred 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.

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. Generally, 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. As mentioned earlier, C, fragment flow in the leaf can also be provided by exposure to O₂-Uptake conditions. O₂-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, O₂-Uptake occurs within the leaf, i.e. , the oxygenase activity of Rubisco outcompetes its carboxylase activity. As mentioned earlier, foliar formulations for use under O₂-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 O₂-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. 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. For example, 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. In this case, the formamidine portion serves as the polyglutamate source and the glycolate serves as the Crfragment. Similarly, 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.

As an example of the use of a Crinput, elevated concentrations of CO₂ can be applied to plants after application of an enhancer and a C_racceptor compound. The plant is exposed to the elevated CO₂ 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 O₂-Uptake conditions and a Cj-acceptor compound. Usually, the foliar application of C,-input substance(s) or the exposure to an O₂-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_rTHF 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. Alternatively, the first aqueous solution can contain a C_racceptor 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 O₂-Uptake conditions and application of the C,-acceptor compound.

In summary, 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 O₂-Uptake conditions.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a simplified depiction of the C₃ photosynthetic pathway in plants. Fig. 2 is an enlarged depiction of the relation between the GOGAT

(glutamine: 2-oxoglutarate amino transferase) cycle and oxygen uptake in the C₃ photosynthetic pathway in plants.

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 O₂-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 CO₂ gas exchange. Soy foliage was sprayed with 17 μM folinate, 40 mM glycine, 0.1 M dimethylsulfoxide and 0.05% Hamposyl™C (▼); or 5 M methanol, 40 mM glycine and 0.1 % Triton^® X-100 (V). Each point is the mean of 3 to 8 measurements. Fig. 8 is a graphic depiction of results of experimentation showing the long-term effects of Uvinul^® P-25 or formiminoglycine on CO₂ gas exchange. Leaves of sugar beets were sprayed with 0.5 mM Uvinul^® P-25, 13 mM glycine and 0.05% Hamposyl™C (o); or 2 mM formiminoglycine, 13 mM glycine and 0.05% Hamposyl™C(v). Each point is the mean of 3 to 6 measurements of foliar CO₂ gas exchange.

Fig. 9 is a graphic depiction of results of experimentation showing the long-term effects of formamidine acetate or potassium glycolate on CO₂ Assimilation (A). Soy foliage was sprayed with 30 mM formamidine acetate (FAM) plus 0.05% Hamposyl™C (left frame) or 30 mM potassium glycolate plus 0.05% Hamposyl™C (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% Hamposyl™C (^•), 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.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS The present invention provides methods and compositions for promoting the growth of green higher plants, that is, all plants which are actively photosynthetic. The phrase "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. The phrase "C₃ plants" refers to all plants capable of fixing carbon via the C₃ 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, hibiscus, gardenia, jasmine, camellia, marigold, daisy, stock, vinca, gerbera, carnation, cyclamen, peony, shooting star, bird-of-paradise, forget-me-not, petunia, lily, tulip, crocus, daffodil, lisianthus, borage, and the like; leafy plants, such as philodendron, ficus, and the like; fruit trees, such as apple, plum, peach, cherry, citrus, pistachio, almond, walnut, mango, papaya, guava, cocoa, banana, and the like; forest trees, such as maple, dogwood, oak, yew, fir, pine, redwood, cypress, juniper, elm, birch, palm, mahogany, teak, Christmas trees, and the like; grasses; ferns; and kelps. This list is intended to be exemplary and is not intended to be exclusive.

The methods and compositions of the present invention may be used to promote growth in photosynthetic parts of either juvenile or mature plants. Generally, however, it is desirable that 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. It will be desirable to minimize the amount of aqueous solution which is lost in the soil, with typically at least 75% of the aqueous solution being directed at the leaves, preferably at least 90% by weight, and more preferably substantially all of the solution being directed at the leaves. Such 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 (TeeJet™).

Several hours before and/or for up to two weeks after application of a Cj-THF enhancing substance, the plants will be exposed to conditions which result in an increased activity for production of carbon flow according to the C, pathway of Figs. 4 and 5. Conditions include the foliar application of enhancers, Crinputs, and C,-acceptor compounds and exposure to O₂-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.

To be sure that there is sufficient Glu_D for carbon fixation, a ^-acceptor compound source is supplied with application of enhancer or -input. Enhancer compounds include those which increase C_rTHF. Preferred contributors to C,-THF include its fragments such as folinate, pteridines, and substituted benzoates. C_rinput 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_racceptor 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_rTHF 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;

10-formyl-THF; 5-methyl-THF; 5, 10-methenyl-THF; 5,10-methylene-THF;

5,6,7,8-THF; 5-formimino-THF; and ethoxylates, salts and hydrates thereof. Such

C_rTHF 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.

As a class, 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:

R⁰¹ 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; and

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.

For the purposes of this invention the term "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.

The term "optionally substituted 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.

The term "lower" as used herein in connection with organic 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. Such groups and radicals may be straight chain or branched. The term "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. The term "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). Suitable non-limiting examples of R⁰¹ 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, NH₂, N-linked amino acid, N-linked polypeptide, -OR³, -SR³, NHR³, wherein R³ is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR⁴R⁵ wherein R⁴ and R⁵ 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 R⁴ and R⁵ together with the nitrogen atoms to which they are linked form a heterocyclic ring;

R¹ and R² 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, sulfeno, sulfino, alkylsulfino, arylsulfino; and salts thereof.

Preferably, R¹ 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.

Among the suitable compounds are aminobenzoic acids, such as m-aminobenzoic acid and p-aminobenzoic acid, and 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 as, terephthalic acids, phthalic acids, phthaloyl amino acids, and phthalaldehydic acids; formylbenzoic acids; and esters, amides, hydrates and salts thereof. 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%.

In the process of formulating some enhancer compositions, the substance must first be activated by completely dissolving it in a compatible Activator solution prior to mixture into an aqueous medium. Suitable Activators 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. Other 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_rinput with Cj-acceptor compound to the plant being treated. These 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. Often, 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. It would be possible in some cases to apply the Crinputs with C acceptor compounds continuously between successive applications of another enhancer substance. For example, 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_rinput 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 acetamide; carbon dioxide; l,3,5-triazin-2-one or hexamethylenetetramine; and salts and hydrates thereof, and other sources of C, fragments including salts, esters, amines, alcohols, aromatics, aldehydes, carbamates, and the like. 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_rinputs. Thereby, application of high concentrations of Cj-inputs results in carbon-based growth rather than retarding or killing the plant. The more closely the C_racceptor compound resembles Glu„, the lower the energy requirement for metabolism; therefore, the simpler compounds of conventional fertilizers generally do not contribute to CrTHF as efficiently as do the preferred C,-acceptor compounds. The C,-acceptor compound may come as an inherent part of the enhancer and -input molecules to more fully enhance Cj-THF rather than being utilized strictly for nitrogen. Suitable Cracceptor compounds may also be linked to surfactants to enhance penetration into the leaf, such as, for example, with PEG glutamate or with Hamposyl™C.

Particularly preferred formulations are p-NBA and urea dissolved in methanol and a surfactant such as Tween^® 80. Another preferred formulation comprises phthalic anhydride and hexamethylenetetramine dissolved in methanol and Tween^® 80. Compositions of Crinpu with C_racceptor compound will typically be applied at a concentration ranging from about 0.001 % by weight to 5% by weight. Preferred C_rinput 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. Ornamentals and other tender nursery plants meant for indoor horticulture will frequently require lower concentrations and perhaps more frequent application than outdoor agricultural crops. 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.

While the 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. For example, 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, LED3A™, Teepol™, Tween^®, Triton^®, Latron™, Dawn™ dish detergent, and the like. Alternatively, or additionally, 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.

In addition to the above enhancers, Cj-inputs and Cracceptor compounds of the present invention, 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.

FIRST EXEMPLARY TWO-PART

COMPOSITION

ENHANCER IN ACTIVATOR WITH ^-ACCEPTOR COMPOUND

Component Concentration Broad Range Narrow Range -Nitrobenzoic acid 1-100 ppm 5-20 ppm Activator (Methanol) 0.1 % to 99% 1 % to 75% Urea 0.01 % to 1 % 0.1 % to 0.4% Surfactant (Triton^® X-100) 0.01 % to 0.5 % 0.03% to 0.1 % C_rACCEPTOR COMPOUND C_rINPUT SOLUTION

Component Concentration Broad Range Narrow Range

Formiminoglycine 0.001% to 1% 0.05% to 0.3% Surfactant (Triton^® CF10) 0.01% to 0.5% 0.1% to 0.2%

SECONDARY EXEMPLARY TWO-PART COMPOSITION

ENHANCER SOLUTION

Component Concentration Broad Range Narrow Range Calcium folinate 1 ppm to 500 ppm 5 ppm to 50 ppm

Surfactant (Tween^® 80) 0.01% to 0.5% 0.05% to 0.1%

DMSO 0.5% to 3% 0.8% to 1%

Glycine 0.01% to 1% 0.1% to 0.3%

CrACCEPTOR COMPOUND C,-INPUT SOLUTION

Component Concentration Broad Range Narrow Range

Formamidine • glycolate 0.01% to 5% 0.1% to 1% Surfactant (TWEEN^® 80) 0.01% to 0.5% 0.05% to 0.1% THIRD EXEMPLARY TWO-PART COMPOSITION

ENHANCER IN ACTIVATOR WITH ^-ACCEPTOR COMPOUND SOLUTION

Component Concentration

Broad Range Narrow Range p-Aminobenzoylglutamic acid 10 ppm to 1000 ppm 50 ppm to 100 ppm Activator (KOH) 0.01% to 1% 0.1 % to 0.3% Surfactant (TWEEN^® 80) 0.1 % to 1 % 0.2% to 0.5% FeHEEDTA 0.1 ppm to 5 ppm 1 ppm to 3 ppm Potassium glutamate 0.01 % to 1 % 0.1 % to 0.5% Phosphate buffer pH 5 to pH 7 pH 5.5 to pH 6.5

CrACCEPTOR COMPOUND C.-INPUT SOLUTION

Component Concentration

Broad Range Narrow Range

Formamidine • glycolate 0.01 % to 5% 0.1 % to 0.2%

Surfactant (TWEEN^® 80) 0.2% to 1 % 0.2% to 0.5 %

For use on plants subjected to O₂-Uptake, 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.

EXEMPLARY ONE-PART COMPOSITION FOR USE ON PLANTS WITH O-.-UPTAKE

Composition Concentration Broad Range Narrow Range

Uvinul^® P-25 0.01 % to 1 % 0.1 % to 0.5% Potassium nitrate 0.01 % to 0.3% 0.1 % to 0.2% SECOND EXEMPLARY ONE-PART COMPOSITION FOR USE ON PLANTS WITH O_rUPTAKE

Composition Concentration

Broad Range Narrow Range

Formiminoglutamate 0.01 % to 1 % 0.1 % to 1 % Potassium glycolate 0.01 % to 1 % 0.2% to 3% Calcium nitrate 0.01 % to 1 % 0.1 % to 0.3% Surfactant (HAMPOSYL^C) 0.01 % to 0.1 % 0.02% to 0.05 %

THIRD EXEMPLARY ONE-PART COMPOSITION WITH ACTIVATOR FOR USE ON PLANTS WITHO.-UPTAKE

Composition Concentration Broad Range Narrow Range

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 (HAMPOSYL™C) 0.03% to 1% 0.05% to 0.2%

FOURTH EXEMPLARY ONE-PART COMPOSITION WITH ACTIVATOR FOR USE ON TURF

Composition Concentration Broad Range Narrow Range

Phthaloylglutamate 1 ppm to 100 ppm 15 ppm to 50 ppm Activator (Methanol) 0.1 % to 99% 3% to 90% Iron EDTA 0.1 ppm to 3 ppm 0.5 ppm to 1 ppm Surfactant (Latron B-1956™) 0.01 % to 0.5% 0.02% to 0.1 %

The following examples are offered by way of illustration, not by way of limitation. Experimental Materials and Methods

Chemicals (abbreviations) and sources: sodium glutamate, Ajinomoto; formamidine formate (FAF), glycine (Gly), HAMPOSYL™C, 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 (Benzocaine™), (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

(FIGlu),p-formylbenzoic acid (pFBA), methanol (MeOH), methyl-2-anthranilate (MeoABA), nicotinamide adenine dinucleotide phosphate (NADP), terephthalic acid (CBA), N-phthalolyl-L-glutamate (CBG), potassium maleic acid (Malate), potassium chloride (KC1), pteroic acid (Pteroic), 5,6,7,8-tetrahydrofolate (THF), n-[2-hydroxyethyl]piperazine-N'-[ethanesulfonic acid], aprotinin, 2-mercaptoethanol, triethanolamine, ammonium formate, magnesium chloride (MgCl_), Tween^® 80 and Triton^® X-100,Sigma; -nitrobenzoic acid (pNBA), Dupont; dimethylsulfoxide (DMSO), Gaylord; ethoxylated -aminobenzoic acid (Uvinul^® P-25), BASF;¹⁴CO₂ , ICN; 2,4-dinitrobenzoic acid (DNBA), 2-chloro-4-nitrobenzoic acid (CNBA), 4-chloro-2-nitrobenzoic acid (NCBA), 2,4-dichlorobenzoic acid (2C1BA), Aldrich.

Radioisotopic ^,4CO₂ 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 μM folinate, 0.2% glycine, 1 % DMSO, and 0.1 % Triton^® X-100; 2) 40% MeOH, 0.2% glycine, and 0.05 % Hamposyl™C; 3) 0.5 mM pABG, 0.2% glycine, and 0.05% Hamposyl™C. At 24 h to 48 h, 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 O₂ during the acclimation period. A leaf plug, 3.67 cm², was then removed, and placed in a hermetically sealed Plexiglass™ leaf chamber containing pure O₂ being pumped at a rate of 2-3 L min^'1. The chamber was illuminated with 1,000 μmol photosynthetically active quanta nvV¹ directed through a fiber optic cable connected to a quartz halogen light similar to the one used for preillumination. After 1 min, 5 mL CO₂ containing 0.8 μCi Na^l4CO₂ (specific activity of 5 Ci mol^*1) was injected with a syringe to a final concentration of about 700 ppm CO**. The leaf plugs were allowed to incorporate ¹⁴CO₂ for 15, 60 or 180 s, and then fixation was immediately stopped. In other experiments the leaf plugs were pulsed for 15 s, then chased for 1 min or 3 min. The chase was carried out under ambient air. Fixation was stopped by placing the leaf disc in boiling EtOH containing formic acid. Stable fixed ¹⁴CO₂ containing products were separated by paper chromatography as previously described (R. D. Gates, O. Hoegh-Guldberg, M. J. McFall-Ngai, K. Y. Bil, and L. Muscatine (1995) "Free amino acids exhibit anthozoan "host factor" activity:They induce the release of photosynthate from symbiotic dinoflagellates in vitro," Proc. Natl. Acad. Sci. USA 92:7430-7434).

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).

Protein content of samples was determined by a modified Lowry Procedure, (J.N. Nishio, S.E. Taylor, N. Terry (1985) "Changes in Thylakoid Galactolipids and Proteins during Iron Nutrition-Mediated Chloroplast Development," Plant Physiol. 77:705-711).

For gas exchange experiments, 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 CO₂ 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% HAMPOSYL™C 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. In the preparation of 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. 11, D. D. Davies, Ed., Academic Press, NY, Pp.317-353). 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.

In an experiment designed to test tolerance to drought stress, three soy plants were treated with 2 μM. folinate with Triton^® X-100 surfactant. The plants were watered daily to establish a baseline. Subsequently, water was withheld until the soil was extremely dry. The plants were rewatered after extensive prolonged wilt was observed in the controls. The assay relied on death as an endpoint for controls since water stress can be controlled to lethal dose. Treatments that prevented death provided a simple visible determination of remedy. Field tests were undertaken on plants first treated with folinate and followed by post-treatment with glycolate and glycine under conditions of limited and high-rate photorespiration.

In experiments to measure growth yields, plants were cultured in greenhouses or Percival™ 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. Shoot 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. Growth was inhibited in some detergent-treated controls as compared to controls that were not treated with detergent, therefore, depending on the plant type or intent of the experiment, controls were sprayed with water or with the diluent solution minus the active ingredient or controls went without any treatment as a means of comparing against situations that did not decrease growth, enzyme activity or photosynthesis. Based on tests of a gradient of concentrations,0.05% HAMPOSYL™C was found to yield 100% growth in investigations.

Results

As shown in Table 1, rate of initial uptake of⁴C0₂ after the 15 second pulse and 1 minute chase in treated plants was higher than in untreated controls. Fixation of ¹⁴C into the insoluble fraction is greater in treated plants than in controls at 3 minutes.

Table l.PULSMHASE SHOWS RAPID^wC0_ϊFIXATION

Compound 15 sec ¹⁴C0₂ 180 sec ¹²C0₂

Control 6.6 ± 0.5 pABG 3.3 ± 0.2

Folinate 4.2 + 0.2

In Table 2, below, our results show ¹⁴C0₂ uptake in light saturated pulse-chase experiments serine decreased in proportion to the contribution of applied substances to C_rTHF stnicture.The glycine: serine ratio increased mainly due to serine depletion, folinate treated leaves showing most extensive depletion of serine.

Table 2.¹⁴C0\FATE: HIGH GLYCINE:SERINE RATIO

In Table 3, below, ¹⁴C0₂light saturated photorespiratory pulse-chase results showed increases in treated plants over controls that were not treated for glycine + serine and 3-phosphoglycerate+phosphoric ethers of sugars at the initial pulse of ¹⁴C0₂. Thereafter, at the 60 second chase, the glycine + serine products decreased when the sugars increased in treated plants as compared to controls.

Table 3.^I4C0,PULSE^-CHASE: RAPID METABOLISM TO SUGAR

^'PGA: phosphoglyceric acid. PES: phosphoric ethers of sugars

As shown in Table 4, below, folinate, folate.pABA, pABG, Uvinul^®,

Benzocaine™, ABA, pNBA, pNBA 4- glycine, pFBA + glycine, CBA, pAH, FIGly and MeOH activated C,-THF enzymes, but glycine, Hamposyl™C, FAM and øABA did not. Folinate increased enzyme activity in com. The addition of glycine to pNBA did not depress the overall stimulation of C,-THF enzymes as compared against similar glycine levels that, alone, depressed enzyme activity. This result is indicative of the synergistic involvement of the metabolism of enhancers with C acceptor compounds. Table 4. C,-THF Enzyme Analysis

As shown in Table 5, below, 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, Benzocaine™ and MeOH. Gas exchange was significantly higher after Uvinul^® P-25 treatments as compared to pABA treatments. To a small extent, pAH stimulated gas exchange in soy. Neither øABA nor MeoABA showed increases in gas exchange.

Table 5. Effect of enhancers, ^-acceptor compounds and Crinputs on CO₂ Gas Exchange

*n - 3 for 24 h; *n = 2 for 24 h; '72 h after 3rd spray; '24 h after 3rd spray. Assimilation/Transpiration data yielded the following results given in Table 6, below, wherein reduced transpiration with increased CO₂ assimilation was recorded or treatment of foliage with folinate, pAH and MeOH.

Table 6. Effect of enhancers, Cracceptor compounds and Crinputs on A/T

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 μM folinate to 5 M MeOH and showing increased CO₂ 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. Each data point in this graph is the mean of samples such that two different leaves were measured on individual plants; for folinate, n = 16 except on days 9, 15, 16 and 21 when n=8; for MeOH, n=8 except on days 9, 15, 16 and 21 when n=6; and for the control, n = 12 on days 9, 15, 16 and 21 when n = 16.

Figure 8 shows that 0.5 mM Uvinul^® or 2 mM FIGly applications to sugar beet increased CO₂ 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 CO₂ assimilation maintained with reduced transpiration for the duration of the experiment. 36

Generally, 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.

The effects of treatments on the quantity of the C,-THF pool of leaves are given in Table 7, below. Treatments with MeOH, FIGly and folinate significantly increased the total C,-THF pool and the Glu„ chains over controls. Short-chained pteroyls increased significantly over controls when treated by 0.1 mM folinate, but not by MeOH or FIGly. Overall, folinate was a more effective enhancer of the Cj-THF pool than MeOH or FIGly. Treatments with 10 mM FIGly and 14.8 M MeOH showed equivalent increases in the total C,-THF pool; this result underscoring the increased quantities of MeOH required to achieve the same responses as those observed for treatments with small quantities of FIGly or even more minute quantities of folinate.

These increases in the CrTHF pool coπesponded closely to increased photosynthetic gas exchange and enzymes measured at the time of sampling.

Table 7. Effect of Treatments on the C,-THF Pool

In our test of water stress, after plants were rewatered, we observed that the plants treated with folinate survived the drought, whereas the shoots of all controls withered and died. Pretreatment of plants with folinate followed by posttreatment with glycolate and glycine improved turgidity of the plants in shade. Treatments with 40 mM glycolate or 40 mM glycine alone did not improve turgidity in the shade or in sunlight. Plants that were treated with 40 mM glycolate and exposed to direct sunlight were dead within a week. Plants under direct sunlight that were treated with 40 mM glycolate immediately followed by applications of 40 mM glycine showed healthy leaves with no phytotoxicity observed within a month. Folinate followed by 40 mM glycolate treatments in the sun resulted in wilt, whereas, following folinate with glycine in the sun resulted in turgid foliage. Post-folinate additions of glycolate with glycine were repeatable daily or as infrequently as once in three weeks. The practical nontoxic concentrations of glycolate and glycine are limited by penetration prior to evaporation of the water. Following folinate treatments, when used in the field under direct sunlight, only glycine supplementation was required because the plants were continuously producing glycolate from photorespiration within the leaf.

Foliar treatments with solutions containing folinate, Uvinul^® P-25, pABG, pNBA, DNBA, CNBA, NCBA, 2C1BA, CBA, CBG, pteroic acid, FIGly, pAH, pFBA, FAF or glycolate enhanced growth as compared to controls, shown in Table 8. No growth improvements were observed for Benzocaine™ and MeøABA. Co showed growth improvement by treatment with nitrobenzoates and folinate.

Table 8. Effect of Treatments on Plant Growth

"Active components were formulated in the following control solutions^*. (A) 0.05% HAMPOSYL™C surfactant, (B) 0.2% glycine + 0.05% (C) 200 ppm potassium formate + 0.2% glycine + 0.05 % HAMPOSYX™C, and (D) water. Folinate was followed by GO^lx once at 2 d or GO²" twice at 2d and 4d.

"Bean yield was measured at University of Wyoming. The treatments given in Table 8 were selected from a broad range of concentrations of each formulation that was applied and represent optimal doses for growth improvement in each case. The most effective concentration is directly related to the volume of solution applied, therefore, the actual concentration must be adjusted according to the quantity absorbed into foliage.

Although the foregoing invention has been described in detail for purposes of clarity of understanding, it will be obvious that certain modifications may be practiced within the scope of the appended claims.

All patents, patent applications and publications cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application or publication were so individually denoted.

Claims

WHAT IS CLAIMED IS:

1. A method of promoting growth of a plant, said method comprising: (a) applying to leaves of the plant an amount of an enhancer substance which increases the amount of Crtetrahydrofolate (CrTHF) in the leaves; and (b) applying to the leaves a Crinput substance which increases the metabolism of C, fragments through CrTHF and a C,-acceptor compound which serves a sink for C, fragments in the leaves.

2. The method of Claim 1, wherein the C,-acceptor compound increases the level of polyglutamate in the leaves.

3. The method of Claim 2, wherein the C,-acceptor compound is part of the molecular structure of the enhancer substance.

4. The method of Claim 1, wherein the enhancer is a substance which can be metabolized by the plant to CrTHF.

5. The method of Claim 1, wherein the enhancer substance promotes carbon fixation by the C, pathway.

6. The method of Claim 1, wherein the enhancer is selected from the group consisting of CrTHF compounds, pteridines, pterins, pteroic acids, pteroyl compounds, folinates, substituted benzoic acids, substituted benzoates and derivatives thereof; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from about 0.0001% by weight to 0.5% by weight. 7. The method of Claim 6, wherein the enhancer is a pteridine of the formula below, wherein:

R⁰¹ 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; and 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; and its corresponding dihydro- and tetrahydro-reduction products at positions 5, 6, 7, and/or 8 of the pteridine ring; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from about 0.0001 % by weight to 0.5% by weight.

8. The method of Claim 1, wherein the enhancer is in a solution comprising an activator substance selected from the group consisting of alcohols, organic nitrogenous bases, alkali metal hydroxides, alkaline earth metal hydroxides, hydrocarbyl acids and surfactants.

9. The method of Claim 6, wherein the enhancer is a Cr HF compound selected from the group consisting of folinic acid, anhydroleucoverin, 5-foιmyltetrahydropteroyl polyglutamate, 10-formyltetrahydropteroyl polyglutamate, 5-formyltetrahydrofolate, 10-formyl tetrahydrofolate, 5-methyltetrahydrofolate, 10-methyl tetrahydrofolate, 5,10-methenyltetrahydrofolate and 5,10-methylenetetrahydrofolate; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight. 10. The method of Claim 6, wherein the enhancer is a substituted benzoate derivative of the formula below, wherein:

R is H, hydrocarbyl, halogen, -OH, -SH, NH₂, N-linked amino acid, N-linked polypeptide, -OR³, -SR³, NHR³, wherein R³ is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR⁴R⁵ wherein R⁴ and R⁵ 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 and C-terminal linked polypeptide chains, or R⁴ and R⁵ together with the nitrogen atoms to which they are linked form a heterocyclic ring; R¹ and R² 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, sulfeno, sulfino, alkylsulfino and arylsulfino; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

11. The method of Claim 10, wherein the substituted benzoate derivative is selected from the group consisting of m-aminobenzoic acid, p-aminobenzoic acid, N-benzoyl amino acids, N-acyl-aminobenzoic acids, aliphatic aminobenzoate esters, aliphatic N-acyl-aminobenzoate esters, N-acylaminobenzoyl amino acids, N-formylaminobenzoic acids, 2-chloro-4-aminobenzoic acid, ethoxylated p-aminobenzoic acids, nitrobenzoic acids, m-nitrobenzoic acid, p-nitrobenzoic acid, N-nitrobenzoyl amino acids, polyethyleneglycol nitrobenzoate, 4-chloro-2-nitrobenzoic acid, 2-chloro-4-nitrobenzoic acid, terephthalic acids, phthalic acids, isophthalic acids, phthalic anhydrides, N-phthaloyl amino acids, phthalaldehydic acids and formylbenzoic acids; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

12. The method of Claim 10, wherein the substituted benzoate is derivatized with a surfactant comprising a polyoxyethylene chain.

13. The method of Claim 10, wherein the substituted benzoate is in a solution comprising a surfactant or an activator substance selected from the group consisting of alcohols, organic nitrogenous bases, alkali metal hydroxides, alkaline earth metal hydroxides, hydrocarbyl acids and surfactants.

14. The method of Claim 1, wherein the C,-input substance is selected from the group consisting of formamidine salts of carboxylic acids, N-formyl amino acids, formimino amino acids, carboxylic acids, aldehydes, trialkyl orthoesters, N-formylated organic compounds, acetamides and carbon dioxide; and salts and hydrates thereof, applied as an aqueous solution at a concentration in the range from 0.001 % by weight to 5 % by weight.

15. The method of Claim 14, wherein the C,-input substance is a formamidine salt 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 acetamide; l,3,5-triazin-2-one or hexamethylenetetramine; and salts and hydrates thereof, applied as an aqueous solution at a concentration in the range from 0.001 % by weight to 5% by weight.

16. The method of Claim 1, wherein the C,-acceptor compound is selected from the group consisting of formamidine amino acids, formamidine carboxylates, formamidine salts of mineral acids, amino acids, hexamethylenetetramines, and formamides.

17. The method of Claim 16, wherein the C,-acceptor compound is an amino acid selected from the group consisting of glycine, serine, glutamate, methionine, cystathionine, homocysteine and sarcosine.

18. The method of Claim 16, wherein the C_racceptor compound is formamidine glycolate.

19. The method of Claim 1, wherein the enhancer is p-nitrobenzoic acid, the Crinput is potassium glycolate, and the C,-acceptor compound is a urea.

20. The method of Claim 1, wherein the enhancer is terephthalic acid, the Crinput is formic acid, and the C,-acceptor compound is glycine.

21. The method of Claim 1, wherein the enhancer is calcium folinate, the Crinput is glycolic acid, and the C,-acceptor compound is hexamethylenetetramine.

22. The method of Claim 1, wherein the enhancer is m-nitrobenzoic acid, the C,-input is methanol, and the C,-acceptor compound is acylglycine.

23. The method of Claim 1, wherein the enhancer is PEG-p-nitrobenzoate, and the Cj-acceptor compound and Crinput is formamidine glycolate. 24. A method of promoting growth of a plant, said method comprising: (a) applying to leaves of the plant an amount of an enhancer substance which increases the amount of C,-tetrahydrofolate (CrTHF) in the leaves and a C,-acceptor compound which serves as a sink for C, fragments in the leaves; and (b) exposing the leaves to conditions which promote oxidative metabolism of photorespiration.

25. The method of Claim 24, wherein the C,-acceptor compound increases the level of polyglutamate in the leaves.

26. The method of Claim 24, wherein said conditions are selected from the group consisting of high light intensity, elevated oxygen levels, water deprivation, high temperatures and low humidity.

27. The method of Claim 24, wherein the enhancer is selected from the group consisting of CrTHF compounds, pteridines, pterins, pteroic acids, pteroyl compounds, folinates, substituted benzoic acids, substituted benzoates and derivatives thereof; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

28. The method of Claim 27, wherein the enhancer is a pteridine of the formula below, wherein:

R⁰¹ 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; and 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, 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, applied as an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

29. The method of Claim 24, wherein the enhancer is in a solution comprising an activator substance selected from the group consisting of alcohols, organic nitrogenous bases, alkali metal hydroxides, alkaline earth metal hydroxides, hydrocarbyl acids and surfactants.

30. The method of Claim 27, wherein enhancer is a C,-THF compound is selected from the group consisting of folinic acid, anhydroleucoverin, 5-formyltetrahydropteroyl polyglutamate, 10-formyltetrahydropteroyl polyglutamate, 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, 5-methyltetrahydrofolate, 10-methyltetrahydrofolate, 5, 10-methenyltetrahydrofolate and 5, 10-methylenetetrahydrofolate; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

31. The method of Claim 27, wherein the enhancer is a substituted benzoate is of the formula below, wherein:

R is H, hydrocarbyl, halogen, -OH, -SH, NH₂, N-linked amino acid, N-linked polypeptide, -OR³, -SR³, NHR³, wherein R³ is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR⁴R⁵ wherein R⁴ and R³ 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 and C-terminal linked polypeptide chains, or R⁴ and R⁵ together with the nitrogen atoms to which they are linked form a heterocyclic ring; wherein R¹ and R² 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, sulfeno, sulfino, alkylsulfino and arylsulfino; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

32. The method of Claim 31, wherein the substituted benzoate is selected from the group consisting of rø-aminobenzoic acid, p-aminobenzoic acid, N-benzoyl amino acids, N-acyl-aminobenzoic acid, aliphatic aminobenzoate esters, aliphatic N-acyl-aminobenzoate esters, N-acylaminobenzoyl amino acids, N-formylaminobenzoic acids, 2-chloro-4-aminobenzoic acid, ethoxylated p-aminobenzoic acids, nitrobenzoic acids, /Ti-nitrobenzoic acid, p-nitrobenzoic acid, N-nitrobenzoyl amino acids, polyethyleneglycol nitrobenzoate, 4-chloro-2-nitrobenzoic acid, 2-chloro-4-nitrobenzoic acid, terephthalic acids, phthalic acids, N-phthaloyl amino acids, phthalaldehydic acids and formylbenzoic acids; and salts, hydrates and surfactant-linked derivatives thereof, applied as an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

33. The method of Claim 31, wherein the substituted benzoate is derivatized with a surfactant comprising a polyoxyethylene chain.

34. The method of Claim 31, wherein the substituted benzoate is in a solution comprising an activator substance selected from the group consisting of alcohols, organic nitrogenous bases, alkali metal hydroxides, alkaline earth metal hydroxides, hydrocarbyl acids and surfactants.

35. The method of Claim 24, wherein the C,-acceptor compound is selected from the group consisting of formamidine amino acids, formamidine carboxylates, formamidine salts of mineral acids, carboxylic acids, amino acids, hexamethylenetetramines and formamides.

36. The method of Claim 35, wherein the C,-acceptor compound is an amino acid selected from the group consisting of glycine, serine, glutamate, methionine, cystathionine, homocysteine and sarcosine.

37. The method of Claim 35, wherein the C_racceptor compound is formamidine glycolate.

38. The method of Claim 24, wherein the enhancer is a surfactant-linked substituted benzoic acid and the C,-acceptor compound is a formamidine.

39. The method of Claim 24, wherein the enhancer is p-nitrobenzoic acid and the Cracceptor compound is hexamethylenetetramine.

40. The method of Claim 24, wherein the enhancer is a phthalate and the Cj-acceptor compound is a formamidine.

41. The method of Claim 24, wherein the enhancer is a folinate and the C₍-acceptor compound is a formimino amino acid.

42. A plant growth promoting system comprising: a first aqueous solution or a nonaqueous material which when combined with an aqueous carrier contains an amount of a C,-acceptor compound effective to serve as a sink for fragments in leaves of a plant when applied to leaves of the plant; and a second aqueous solution or a nonaqueous material which when combined with an aqueous carrier contains an amount of an enhancer substance effective to increase the amount of C tetrahydrofolate (CrTHF) in leaves of the plant when applied to the leaves of the plant and optionally containing a -input substance which increases the metabolism of C* fragments through CrTHF when applied to the leaves of the plant.

43. The system of Claim 42, wherein the Cracceptor compound is selected from the group consisting of formamidine amino acids, formamidine carboxylates, formamidine salts of mineral acids, carboxylic acids, amino acids, hexamethylenetetramines and formamides.

44. The system of Claim 43, wherein the C, -acceptor compound is an amino acid selected from the group consisting of glycine, serine, glutamate, methionine, cystathionine, homocysteine and sarcosine.

45. The system of Claim 43, wherein the C,-acceptor compound is formamidine glycolate.

46. The system of Claim 42, wherein the enhancer is selected from the group consisting of C,-THF compounds, pteridines, pterins, pteroic acids, pteroyl compounds, folinates, substituted benzoic acids, substituted benzoates and derivatives thereof; and salts, hydrates and surfactant-linked derivatives thereof.

47. The system of Claim 46, wherein the enhancer is a pteridine of the formula below, wherein:

R⁰¹ 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; and 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, and its conesponding 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.

48. The system of Claim 46, wherein the enhancer is a C,-THF compound is selected from the group consisting of folinic acid, anhydroleucoverin, 5-formyltetrahydropteroyl polyglutamate, 10-formyltetrahydropteroyl polyglutamate, 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, 5-methyltetrahydrofolate, 10-methyltetrahydrofolate, 5,10-methenyltetrahydrofolate and 5,10-methylenetetrahydrofolate; and salts, hydrates and surfactant-linked derivatives thereof.

49. The system of Claim 46, wherein the enhancer is a substituted benzoate is of the formula below, wherein:

R is H, hydrocarbyl, halogen, -OH, -SH, NH₂, N-linked amino acid, N-linked polypeptide, -OR³, -SR³, NHR³, wherein R³ is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR⁴R⁵ wherein R⁴ and R⁵ 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 and C-terminal linked polypeptide chains, or R⁴ and R⁵ together with the nitrogen atoms to which they are linked form a heterocyclic ring; R¹ and R² 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, sulfeno, sulfino, alkylsulfino and arylsulfino; and salts, hydrates and surfactant-linked derivatives thereof, in an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

50. The system of Claim 49, wherein the substituted benzoate is selected from the group consisting of /7j-aminobenzoic acid, p-aminobenzoic acid, N-benzoyl amino acids, N-acyl-aminobenzoic acids, aliphatic aminobenzoate esters, aliphatic N-acyl-aminobenzoate esters, N-acylaminobenzoyl amino acids, N-formylaminobenzoic acids, 2-chloro-4-aminobenzoic acid, ethoxylated p-aminobenzoic acids, nitrobenzoic acids, m-nitrobenzoic acid, p-nitrobenzoic acid, N-nitrobenzoyl amino acids, polyethyleneglycol nitrobenzoate, 4-chloro-2-nitrobenzoic acid, 2-chloro-4-nitrobenzoic acid, terephthalic acids, phthalic acids, N-phthaloyl amino acids, phthalaldehydic acids and formylbenzoic acids; and salts, hydrates and surfactant-linked derivatives thereof, in an aqueous solution at a concentration in the range from 0.0001 % by weight to 0.5% by weight.

51. The system of Claim 49, wherein the substituted benzoate is in a solution comprising an activator substance selected from the group consisting of alcohols, organic nitrogenous bases, alkali metal hydroxides, alkaline earth metal hydroxides, hydrocarbyl acids and surfactants.

52. A mixture for promoting growth of a plant comprising an aqueous solution of p-nitrobenzoic acid, a formamidine salt of a carboxylic acid and agronomically suitable additives. 53. The mixture of Claim 52, wherein the formamidine salt is formamidine glycolate.

54. The mixture of Claim 52, wherein the formamidine salt is formamidine formate.

55. A mixture for promoting growth of a plant comprising an aqueous solution of folinic acid, a formamidine salt of a carboxylic acid and agronomically suitable additives.

56. The mixture of Claim 55, wherein the formamidine salt is formamidine glycolate.

57. The mixture of Claim 55, wherein the formamidine salt is formamidine formate.

58. A mixture for promoting growth of a plant comprising an aqueous solution of formamidine glycolate and agronomically suitable additives.