AU708417B2 - Methods and compositions for enhancing formyltetrahydropteroylpolyglutamate in plants - Google Patents

Description

WO 96/27572 PCT/US96/02444 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 (CO2) and water. The conversion of CO 2 into plant matter is generally referred to as carbon fixation and occurs by the C 3 cycle in most plants. Plants in which the C 3 cycle occurs are referred to hereinafter as "C 3 plants". The C 3 cycle involves the carboxylation of ribulose-1,5-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 C 3 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 C 3 cycle is shown in Fig. 1.

Another event in the C 3 cycle shown in Fig. 1, is photorespiration, during which oxygen (02) outcompetes

CO

2 and is added to RuBP. As a result of oxidation, phosphoglycolate is formed. The phosphoglycolate is dephosphorylated to glycolate NEENEd WO 96/27572 PCTIUS96/02444 2 which is oxidized to glyoxylate. Glycine is made by attachment of ammonia (NH 3 The glycine is deaminated releasing NH 3 and further decarboxylated to CO 2 plus a single carbon (C 1 fragment. This C 1 fragment from glycine is passed on to a FORMYLTETRAHYDROPTEROYLPOLYGLUTAMATE (CI-THF) pool, whereby, it is catalytically transferred in the form of 5,10-methylenetetrahydrofolate. Serine hydroxymethyltransferase (SHMT), an abundant enzyme of the Ci-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 amination of glyoxylate and deamination of glycine during photorespiration occurs through the GOGAT (glutamine: 2 -Qxo-glutarate amino transferase) cycle. The GOGAT cycle is the path by which NH 3 is assimilated by plants and follows the depiction given in Fig. 2, wherein, glutamine: 2 -oxo-glutarate amino transferase catalyzes the combining of glutamine with 2 -oxo-glutarate to form two molecules of glutamate.

A fertilizer that provides carbon, enhances uptake of carbon, or increases the efficiency of carbon metabolism would increase growth. Conventional fertilizers do not directly provide carbon as a nutrient nor do they improve carbon fixation even though carbon accounts for 80% or more of plant growth under the conventional

C

3 cycle. Because of their imbalances, application of conventional fertilizers has never achieved optimal productivity during photorespiration.

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.

WO 96/27572 PCTfUS96/02444 3 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 1 -THF in the leaves of a plant. As shown in Fig. 6, the C 1

-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 (Glu) chain. The formylpteroyl glutamide can be further subdivided into a Cl-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 Cj-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

1 -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 1 -THF. By adding to the C 1 -THF content of a leaf, catalysis of C, fragments is enhanced, the plant's capacity to metabolize Cl-fragments by increasing the flow of carbon through the Cj-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

1 -THF, 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 "Cl-input substances".

Organic compounds can find passage through C 1 -THF as a consequence of the metabolism of much larger molecules from which C, fragments arise. For example, WO 96/27572 PCT/US96/02444 4 the incorporation of formimino-amino acids is illustrated in Figs. 4 and 5, wherein, a C 1 fragment is transported as formiminotetrahydrofolate. In fact, C 1 -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 CI-THF, 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

1 fragment, formate, is incorporated through the intermediacy of the C-THF pool by its addition to glycine yielding serine which is ultimately converted to cellular constituents. Direct fixation of CO 2 to formate is known--e.g., S.S. Kent (1972) "Photosynthesis in the higher plant Viciafaba. II. The non-Calvin cycle origin of acetate and its metabolic relationship to the photosynthetic origin of formate," J. Biol. Chem.

247:7293-7302; or Ramaswamy et al. (1976) "A novel pathway for the synthesis of solanidine in the isolated chloroplast from greening potatoes," Eur J Biochem 6 7 2 7 5-282--and may provide an additional source of C, units. Therefore,

CO

2 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 Cj-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 NH 3 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 1 -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 CI-THF, input of compounds that release C, fragments or exposure to conditions that produces a flow of glycolate within leaves (thus providing a source of WO 96/27572 PCT/US96/02444 formate for the Cj-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 C 1 fragments, C, 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 "Cl-acceptor compounds", 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 1 -acceptor compounds are sources of polyglutamate.

C

1 -acceptor compounds may also be glycine or substances that may be metabolized to glycine.

Glycine serves as a sink for C 1 fragments by being converted to serine which is ultimately converted to sugars and other cellular constituents.

C

1 -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

1 -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, 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 1 -THF or readily metabolized precursors thereto, 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 (yM) concentrations. In contrast, mere application of single carbon sources such as methanol and formate require molar concentrations as much as a million times greater than folinate to show activity.

Folinate has the components of the C 1 -THF 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 1 -THF also promote plant WO 96/27572 PCT/US96/02444 6 growth. For example, pteridines formulated with aminobenzoates and Glu, sources would provide the components for the plant to assemble an entire CI-THF.

Alternatively, p-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 Cj-THF and thereby promote plant growth. As used herein, the term "C 1 -THF" refers to tetrahydrofolates carrying a one-carbon unit. The one-carbon unit can be carried in various oxidation states. In the most reduced form, it is carried as a methyl group. In more oxidized forms, it is carried as a formyl, formimino, or methenyl group.

Tetrahydrofolate is also called tetrahydropteroylglutamate

(H

4 PteGlu), see "Biochemistry" by Lubert Stryer, pp. 719-721 H. Freeman, 4th ed. (1995)). The terms "Cl-THF", "C,-THF pool(s)" and "tetrahydrofolate pool(s)" as used herein also includes such species that carry more than one glutamate residue, Cj-H 4 PteGlu,. We refer to compounds capable of contributing components to the molecular structure of the formylpteroylglutamate portion

C,-H

4 PteGlu 1 of the Cj-THF molecule as "enhancers".

Increasing the level of C-THF 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 C,-THF as C, fragments or by exposure of the plants to environmental conditions which increase the flow of C, fragments into and through the C 1 -THF pool. Such substances utilizable by C,-THF as C, fragments may be C, fragments themselves, formate, or may be substances which are metabolized to a C, fragment. All such substances are collectively referred to as "Cr-input substances". Environmental conditions which promote the flow of C, fragments into the C 1 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 0 2 -Uptake conditions. Exposure of plants to high levels of Cl-input substances and/or 0 2 -Uptake levels alone can be toxic because of buildup of intermediates to toxic levels WO 96/27572 PCTIUS96/02444 7 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 Cl-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 preferably from about 0.0001 to 0.1%.

Suitable pteridine compounds contribute to the structure of C,-THF and are represented by the formula below wherein: OH ROI

N^NR

H

2 N N N

R

o 0 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),, 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 CI-THF and are represented by the formula below, wherein: WO 96/27572 PCTIUS96/02444 8 R is H, hydrocarbyl, halogen; -OH; -SH, NH 2 N-linked amino acid, N-linked polypeptide,

-OR

3

-SR

3

NHR

3 wherein R 3 is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR 4

R

5 wherein

R

4 and R 5 which may be the same or different are independently selected from the group consisting of H, optionally substituted hydrocarbyl, alkyl, aryl, acyl, C-terminal linked amino acids, C-terminal linked polypeptide chains, or R 4 and R together with the nitrogen atoms to which they are linked form a heterocyclic ring;

R

1 and R 2 are independently selected from the group consisting of: optionally substituted hydrocarbyl groups, alkyl, aryl, acyl, aroyl, halo, cyano, thio, hydroxy, alkoxy, aryloxy, amino, alkylamino, aminoalkyl, arylamino, aminoaryl, acylamino, ureido, alkylureido, arylureido, hydrazino, hydroxamino, alkoxycarbonylamino, aryloxycarbonylamino, nitro, nitroso, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, carboxamido, monoalkylaminocarbonyl, dialkylaminocarbonyl, formyl, sulfo, sulfamoyl, sulfoamino, alkylsulfonyl, arylsulfonyl, 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 CI-THF 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 1 -input substance for foliar application is usually selected from the group consisting of components that contribute C, fragments to the tetrahydrofolate pool.

WO 96/27572 PCTUS96/02444 9 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 Ci-THF in the C, pathway can serve as a Cl-input substance.

C

1 -input substances include formimino-, methyl-, methenyl-, 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 formimino-amino acids, purines, histidines, thymidylates and S-adenosylmethionine all of which are metabolized to As mentioned earlier, C, fragment flow in the leaf can also be provided by exposure to 0 2 -Uptake conditions. 0 2 -Uptake conditions generally include specific stressful environmental conditions such as high light intensity, high temperature, elevated oxygen, and water deprivation, either alone or in any combination. Under conditions such as these, O 2 -Uptake occurs within the leaf, the oxygenase activity of Rubisco outcompetes its carboxylase activity. As mentioned earlier, foliar formulations for use under 0 2 -Uptake environments preferably require a C 1 -acceptor compound. An oxidized substance such as formamidine nitrate that can be reduced by electron carriers from photosynthesis is a preferred exemplary

C

1 -acceptor compound to ensure that metabolism of the C-fragments by the C, pathway under 0 2 -Uptake environments is non-toxic and beneficial to the growth of the plant.

C,-acceptor compounds provide Glu, sources and can be formulated with enhancers or C,-inputs or provided separately. When C 1 -acceptor compounds are formulated with enhancers or C,-inputs, they may be present as independent substances or they may be part of the same substance as the enhancer or the C 1 -input substance.

For example, a single compound that can act as both Cl-acceptor compound and C,-input (salts we denote as C 1 -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 C 1 -fragment. Similarly, an enhancer such as phthaloylglutamate contains, within its structure, a glutamate as Cl-acceptor compound. Other preferred single component enhancer substance formulations in which the C 1 -acceptor compound is inherent in the molecular make up of the enhancer include, but are not limited to, folinate, pteroyl(Glu),, phthaloyl(Glu)n, aminobenzoyl(Glu)., nitrobenzoyl(Glu)., and the like. Cl-acceptor compound substances include compounds such as but not limited to glycine, glutamate, nitrates and formamidines.

As an example of the use of a Cl-input, elevated concentrations of CO2 can be applied to plants after application of an enhancer and a Cl-acceptor compound. The plant is exposed to the elevated CO2 during daylight hours with continuous exposure to the Cl-acceptor 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 1 -THF pool. After passage of a time sufficient to allow for C 1 -THF accumulation, the plant is exposed to conditions which increase the flow of carbon, such as by foliar application of Cl-inputs and C 1 -acceptor compound or exposure to 0 2 -Uptake conditions and a Cl-acceptor compound. Usually, the foliar application of Cl-input substance(s) or the exposure to an 0 2 -Uptake condition will be done at least twice following each application of the enhancer substance. Often it will be more than twice over a period from 1 day to days following each enhancer substance application. It will be recognised that even though it is 15 preferable to apply the enhancer substance before the Cl-input substance, the order of application may be reversed.

;Thus, according to one embodiment of the invention, there is provided a method of promoting growth of a plant, said method comprising: applying to leaves of the plant an amount of an enhancer substance which increases the amount of Cl-tetrahydrofolate (C1-THF) in the leaves; and applying to the leaves a Cl-input substance which increases the metabolism of C 1 fragments through C 1 -THF and a Cl-acceptor compound which serves as a sink for Ci fragments in .OD* the leaves.

In another embodiment of the invention, there is provided a method of promoting growth of a plant, said method comprising: applying to leaves of the plant an amount of an enhancer substance which increases the amount of Cl-tetrahydrofolate (C 1 -THF) in the leaves and a Cl-acceptor compound which serves as a sink for C 1 fragments in the leaves; and exposing the leaves to conditions which promote oxidative metabolism of 30 photorespiration.

Plant growth promoting compositions according to the present invention may also be formulated to one-step application. Such one-step compositions will comprise an aqueous solution of enhancer(s) and/or Cl-input substances in combination with a Cl-acceptor compound. The enhancer compounds will be present in amounts sufficient to increase the amount of C 1 -THF available in foliage when applied to the plant. The Cl-acceptor compound will be selected and be present in amounts sufficient to act as a sink for the C 1 fragments generated by metabolism through C 1

-THF

by increasing Glun). Preferred enhancers for such a one-step application include, but are not limited to, folinate, nitrobenzoates and phthalates. Preferred Cl-inputs for such a one-step Sapplication include, but are not limited to, formamidine glycolate and formamidine formate.

[N:\LIBA]00558:SAK Preferred Cl-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

S

[N:\LIBA]00558:SAK increase Ci-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 Cl-input Ci-acceptor compound substance selected to increase the flow of carbon in a leaf. Preferred enhancer substances include but are not limited to folinate, pteroic acid, p-nitrobenzoic acid and substituted benzoates, such as terephthalic acid. Preferred Ci-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 Ci-acceptor compounds include but are not limited to glutamate, glutamine, glycine and formamidines. Alternatively, the first aqueous solution can contain a Cl-acceptor compound substance and the second aqueous solution can contain an enhancer and/or a Ci-input.

Another plant growth promoting composition for two-sep application according to the present invention is a first aqueous solution comprising an enhancer and a second aqueous solution comprising a Ci-acceptor compound. Application of the enhancer is followed by exposure to 02- Uptake conditions and application of the Ci-acceptor compound.

Thus, in another embodiment of the invention, there is provided a system comprising: C. a first aqueous solution containing, 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 1 -tetrahydrofolate (C 1 -THF) in leaves of the plant when applied to the leaves of the plant; and a second aqueous solution containing, or a nonaqueous material which when combined 20 with an aqueous carrier contains, an amount of a Cl-acceptor compound effective to serve as a sink for C 1 fragments in leaves of a plant when applied to the leaves of the plant and optionally containing a Cl-input substance which increases the metabolism of C 1 fragments through C 1 -THF when applied to the leaves of the plant; when used for promoting growth of the plant by applying both said solutions to the leaves of the plant.

In yet a further embodiment of the invention, there is provided a system comprising: a first aqueous solution containing, or a nonaqueous material which when combined with an aqueous carrier contains, an amount of a C 1 -acceptor compound effective to serve as a sink for

C

1 fragments in leaves of a plant when applied to the leaves of the plant; and a second aqueous solution containing, 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 1 -tetrahydrofolate (Cl-THF) in leaves of the plant when applied to the leaves of the plant and optionally containing a Cl-input substance which increases the metabolism of C 1 fragments through C 1 -THF when applied to the leaves of the plant; when used for promoting growth of the plant by applying both said solutions to the leaves of the plant.

[N:\LIBA)OO558:SAK In summary, plant growth is promoted by enhancement of C 1 -THF in the leaf and increased flow of C 1 fragments in the Ci pathway by foliar application of Ci-acceptor compounds with enhancers, Ci-inputs, and/or with exposure to 02-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 C 3 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 C1 pathway, further illustrating paths for enhancing, C 1

-THF

and the flow of C 1 fragments.

Fig. 5 is a simplified depiction of the C 1 pathway, further illustrating paths for enhancers, Cjinputs, Ci-acceptor compounds and 02-Uptake.

*0

*S

*0

C

S

[N:\LIBA]OO558:SAK WO 96/27572 PCT/US96/02444 12 Fig. 6 is a structure of Ci-THF with bars corresponding to the various components that can be applied to foliage for enhancement. Attachment of the single carbon fragment at the 5-position of the pteridine structure is shown with regard to availability of the 10- and 5,10-positions, also.

Fig. 7 is a graphic depiction showing the effects of folinate or methanol on long-term CO 2 gas exchange. Soy foliage was sprayed with 17 /M folinate, 40 mM glycine, 0.1 M dimethylsulfoxide and 0.05% Hamposyl'C or 5 M methanol, mM glycine and 0.1% Triton® X-100 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 CO 2 gas exchange. Leaves of sugar beets were sprayed with 0.5 mM Uvinul* P-25, 13 mM glycine and 0.05% Hamposyl"C 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 CO 2 gas exchange.

Fig. 9 is a graphic depiction of results of experimentation showing the long-term effects of formamidine acetate or potassium glycolate on CO 2 Assimilation Soy foliage was sprayed with 30 mM formamidine acetate (FAM) plus 0.05% HamposylmC (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 ratio.

Cabbages were sprayed with 30 mM formamidine acetate and 0.05% Hamposyl'C but controls were not treated 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 WO 96/27572 PCT/US96/02444 13 fix carbon dioxide. "Higher" plants include all plant species having true stems, roots, and leaves, thus excluding lower plants, yeasts and molds. The phrase "C 3 plants" refers to all plants capable of fixing carbon via the C 3 photosynthetic pathway. Suitable plants which may benefit from C 1 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 the "seed leaves") or other substantial light-gathering surfaces, including, of course, the true leaves. Improved growth occurs as a result of enhancement of C 1 -THF as in Fig. 6 or via the C 1 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 WO 96/27572 PCTIUS96/02444 14 minimize the amount of aqueous solution which is lost in the soil, with typically at least 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 CI-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, C,-inputs, and Ci-acceptor compounds and exposure to 0 2 -Uptake conditions. Suitable light and temperature conditions may be achieved by prolonged exposure of the plant to direct sunlight or other suitable high light intensity illumination sources while maintaining optimal to hot temperatures, usually above 200 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, for carbon fixation, a CI-acceptor compound source is supplied with application of enhancer or Cl-input. Enhancer compounds include those which increase Ci-THF. Preferred contributors to Ci-THF include its fragments such as folinate, pteridines, and substituted benzoates. CI-input substances include those which increase the flow of C, fragments. Preferred C 1 -input substances include organic compounds such as formamidine *glycolate and formamidine. formate that yield C, fragments from CI-acceptor compound C-input salts.

Preferred Ci-acceptor compound compounds include those such as formamidine nitrate, glycine and glutamate which add to the Glu chain. Activator compounds are those in which enhancers will dissolve prior to mixture with an aqueous solution. Preferred Activators also benefit plant growth by contributing nutrients and include methanol, potassium carbonate, potassium hydroxide, calcium hydroxide and acetic acid.

WO 96/27572 PCT/US96/02444 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 250 to 35 0 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, above about 0.03%, typically being above about 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 Cj-THF by application of a Cl-acceptor 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, C-input and Cl-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 C-THF molecule. Enhancer, Cz-input and C-acceptor 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 Cl-acceptor compound substance.

Suitable enhancer substances include those compounds which are whole molecules, fragments or precursors of Cj-THF in the pathway, as well as all salts, hydrates, aldehydes, esters, amines, and other biologically or chemically equivalent compounds and substances which can be metabolized in the leaf to contribute a component to C,-THF. Preferred enhancer substances include Cj-THF compounds, pteridine compounds, and substituted benzoate compounds.

Suitable Ci-THF compounds include folinates and compounds which may be converted to N-formyltetrahydropteroyl(Glu), when applied to the treated plant.

Exemplary Cj-THF compounds include folinic acid; anhydroleucovorin; WO 96/27572 PCTUS96/02444 16 (depicted in Fig. 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 Ci-THF compounds will be applied to the plant as an aqueous solution having a concentration in the range from about 0.0001 by weight to 0.5% by weight, preferably to about 0.1% by weight.

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: OH ROI N RO

H

2 N N N

R

0 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)., 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 WO 96/27572 PCT/US96/02444 17 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, 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 alkylsulfates), nonionic 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 p-aminobenzoic acid and the corresponding polyethoxylated p-aminobenzoic acid (Uvinul® Suitable non-limiting examples of R 0 1 include lower-alkyl, alkyl, hydroxymethyl, hydroxyalkyl, 1-hydroxyalkyl, hydroxylower-alkyl, 1-hydroxylower-alkyl, alkoxyalkyl, 1-alkoxyalkyl, alkoxylower-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

C

1 -THF and are represented by the formula below, wherein: WO 96/27572 PCT/US96/02444 18 Ri

RCO-R

R is H, hydrocarbyl, halogen; -OH; -SH, NH2, N-linked amino acid, N-linked polypeptide,

-OR

3

-SR

3

NHR

3 wherein R 3 is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR 4

R

5 wherein R 4 and R 5 which may be the same or different and are independently selected from the group consisting of H, optionally substituted hydrocarbyl, alkyl, aryl, acyl, C-terminal linked amino acids, C-terminal linked polypeptide chains, or R 4 and R 5 together with the nitrogen atoms to which they are linked form a heterocyclic ring; R' and R 2 are independently selected from the group consisting of: optionally substituted hydrocarbyl groups, alkyl, aryl, acyl, aroyl, halo, cyano, thio, hydroxy, alkoxy, aryloxy, amino, alkylamino, aminoalkyl, arylamino, aminoaryl, acylamino, ureido, alkylureido, arylureido, hydrazino, hydroxamino, alkoxycarbonylamino, aryloxycarbonylamino, nitro, nitroso, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, carboxamido, monoalkylaminocarbonyl, dialkylaminocarbonyl, formyl, sulfo, sulfamoyl, sulfoamino, alkylsulfonyl, arylsulfonyl, sulfeno, sulfino, alkylsulfino, arylsulfino; and salts thereof.

Preferably,

R

1 and 2 will be at the 2, 3, or 4 position of the benzoate ring.

Cationic salts of the benzoates include cations selected from the group consisting of cations of alkali metals, alkaline earth metals, ammonium, organic ammonium (amine), quaternary ammonium, and mixtures of such salts.

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 m-nitrobenzoic acid, WO 96/27572 PCTIUS96/02444 19 p-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 Ci-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 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 1 -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 Cl-carbon required for rapid growth is preferably accomplished by application of C,-input with C-acceptor compound to the plant being treated. These Ci-input with C-acceptor compound compositions can be applied as distinctly separate formulations or in combination with enhancers. Ci-inputs with Cl-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 Cz-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

C

i -inputs with Cl-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 WO 96/27572 PCT/US96/02444 spaced apart by a period in the range from 7 days to 20 days, with aqueous solutions comprising the Ci-input such as formate with a Ci-acceptor compound such as glutamine being applied from 1 time to 50 times between such successive applications.

Suitable CI-input compounds that pass C 1 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; 1,3,5-triazin-2-one or hexamethylenetetramine; and salts and hydrates thereof, and other sources of Ci 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,.

Suitable Ci-acceptor compounds act as Ci sinks and/or enhance Glu. as a consequence of C,-THF metabolism. Formamidines, such as, formamidine nitrate, are exemplary Ci-acceptor compounds. Other suitable Ci-acceptor compounds include, but are not limited to, glycine, glutamine, glutamate, serine, sarcosine, homocysteine, cystathionine, methionine, hexamethylenetetramines, and formamide. Suitable Ci-acceptor compounds are also available as conventional nitrogen fertilizers. They include nitrates, ureas, and the like. Nitrate and urea Ci-acceptor compounds are utilized differently from conventional fertilizers in the present invention because they are used in combination with enhancers and Ci-inputs to enhance carbon fixation. They directly target increases of the polyglutamate component of C,-THF in the leaf. This has a safening effect that eliminates toxicity of CI-inputs. Thereby, application of high concentrations of Ci-inputs results in carbon-based growth rather than retarding or killing the plant. The more closely the C,-acceptor compound resembles Glu,, the lower the energy requirement for metabolism; therefore, the simpler compounds of conventional WO 96/27572 PCT/US96/02444 21 fertilizers generally do not contribute to CI-THF as efficiently as do the preferred CI-acceptor compounds. The C -acceptor compound may come as an inherent part of the enhancer and CI-input molecules to more fully enhance C 1 -THF rather than being utilized strictly for nitrogen. Suitable Ci-acceptor compounds may also be linked to surfactants to enhance penetration into the leaf, such as, for example, with PEG glutamate or with Hamposyl7C.

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® Compositions of C,-input with C,-acceptor compound will typically be applied at a concentration ranging from about 0.001% by weight to 5% by weight.

Preferred CI-input with CI-acceptor 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 C -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 Ci-acceptor compound substances with enhancers and Ct-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 WO 96/27572 PCT/US96/02444 22 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, Ci-inputs and Cl-acceptor 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 CI-ACCEPTOR

COMPOUND

Component Concentration Broad Range Narrow Range p-Nitrobenzoic acid 1-100 ppm 5-20 ppm Activator (Methanol) 0.1% to 99% 1% to Urea 0.01% to 1% 0.1% to 0.4% Surfactant (Triton® X-100) 0.01% to 0.5% 0.03% to 0.1% WO 96/27572 WO 9627572PCTfUS96/02444 23 1 -ACCEPTOR COMPOUND C 1 -INPUT SOLUTION Component Formiminoglycine Surfactant (Triton@ CF 10) Concentration Broad Range 0.001% to 1% 0.01% to 0.5% Narrow-Range 0.05% to 0.3% 0.1% to 0.2% SECONDARY EXEMPLARY TWO-PART

COMPOSITON

ENHANCER SOLUTION Component Calcium folinate Surfactant (TweenO 80) DMS0 Glycine Concentration Broad Ranp-e 1 ppm to 500 ppm 0.01 to 0.5% 0.5% to 3% 0.01% to 1% Narrow Rane 5 ppm to 50 ppm 0.05% to 0.1% 0.8% to 1% 0.1% to 0.3% CI- ACCEPTOR COMPOUND C,-INPUT SOLUTION Component Formamidine.- glycolate Sur-factant (TWEENO 80) Concentration Broad Range 0.01 to 5 0.01 to 0.5% Narrow Ranye 0.1% to 1% 0.05% to 0.1% WO 96/27572 PCTIUS96/02444 24 THIRD EXEMPLARY TWO-PART COMPOSITION ENHANCER IN ACTIVATOR WITH C 1 -ACCEPTOR COMPOUND SOLUTION Component p-Aminobenzoylglutamic acid Activator (KOH) Surfactant (TWEENO 80) FeHEEDTA Potassium glutamate Phosphate buffer Concentration Broad Range 10 ppm to 1000 ppm 0.01% to 1% 0.1% to 1% 0.1 ppm to 5 ppm 0.01% to 1% pH 5 to pH 7 Narrow Range 50 ppm to 100 ppm 0.1% to 0.3% 0.2% to 1 ppm to 3 ppm 0.1% to pH 5.5 to pH C-ACCEPTOR COMPOUND C,-INPUT SOLUTION Component Formamidine* glycolate Surfactant (TWEEN® 80) Concentration Broad Range 0.01% to 5% 0.2% to 1% Narrow Range 0.1% to 0.2% 0.2% to For use on plants subjected to 0 2 -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.

ELARYN-PART OMPOSITION FOR USE ON PLANTS WITH O,-UPTAKE Composition Concentration Broad Range 0.01% to 1% 0.01% to 0.3% Uvinul® P-25 Potassium nitrate Narrow Range 0.1% to 0.1% to 0.2% WO 96/27572 PCT/US96/02444 SECOND EXEMPLARY ONE-PART COMPOSITION

FOR

USE ON PLANTS WITH O,-UPTAKE Composition Formiminoglutamate Potassium glycolate Calcium nitrate Surfactant (HAMPOSYL™C) Concentration Broad Range 0.01% to 1% 0.01% to 1% 0.2% 0.01% to 1% 0.01% to 0.1% Narrow Range to 1% 0.1% to 3% 0.1% to 0.3% 0.02% to 0.05% THIRD EXEMPLARY ONE-PART COMPOSITION WITH ACTIVATOR FOR USE ON PLANTS WITHO 2

-UPTAKE

VA

Composition Terephthalic acid Activator (DMSO) Potassium glycolate Formamidine nitrate Surfactant (HAMPOSYLITC) Concentration Broad Range 1 ppm to 100 ppm 0.01% to 3% 0.001% to 0.3% 0.01% to 1% 0.03% to 1% Narrow Range 15 ppm to 50 ppm 0.5% to 1% 0.1% to 0.2% 0.1% to 0.3% 0.05% to 0.2% FOURTH EXEMPLARY ONE-PART

COMPOSITION

WITH ACTIVATOR FOR USE ON TURF Composition Phthaloylglutamate Activator (Methanol) Iron EDTA Surfactant (Latron B-1956") Concentration Broad Range 1 ppm to 100 ppm 0.1% to 99% 0.1 ppm to 3 ppm 0.01% to 0.5 Narrow Range 15 ppm to 50 ppm 3%to 0.5 ppm to 1 ppm 0.02% to 0.1% The following examples are offered by way of illustration, not by way of limitation.

WO 96/27572 PCT/US96/02444 26 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), m-aminobenzoic acid (MABA),p-aminobenzoic acid (pABA),p-aminobenzoic acid ethyl ester (Benzocainem), (p-aminobenzoyl)-L-glutamic acid (pABG),p-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 (MgC1 2 ),Tween® 80 and Triton® X-100,Sigma;p-nitrobenzoic acid (pNBA), Dupont; dimethylsulfoxide

(DMSO),

Gaylord; ethoxylated p-aminobenzoic acid (Uvinul* P-25), BASF; 14

CO

2

ICN;

2 4 -dinitrobenzoic acid (DNBA), 2 -chloro-4-nitrobenzoic acid (CNBA), 4 -chloro-2-nitrobenzoic acid (NCBA), 2 4 -dichlorobenzoic acid (2CIBA), Aldrich.

Radioisotopic 1 4

CO

2 was applied to plants to determine the fate of active substances and changes in the path of carbon fixation. Cabbage plants were sprayed with one of the following three solutions: 1) 90 /M folinate, 0.2% glycine, 1 DMSO, and 0.1% Triton® X-100; 2) 40% MeOH, 0.2% glycine, and 0.05% Hamposyl"C; 3) 0.5 mMpABG, 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 02 during the acclimation period. A leaf plug, 3.67 cm 2 was then removed, and placed in a hermetically sealed PlexiglassM leaf chamber containing pure 02 being pumped at a rate of 2-3 L min- 1 The chamber was illuminated with 1,000 imol photosynthetically active quanta m-2 s- directed through a fiber optic cable connected to a quartz halogen light similar to the one used for WO 96/27572 PCT/US96/02444 27 preillumination. After 1 min, 5 mL CO 2 containing 0.8 pCi Na 1 4

CO

2 (specific activity of Ci mol- 1 was injected with a syringe to a final concentration of about 700 ppm CO 2 The leaf plugs were allowed to incorporate

"CO

2 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 14CO 2 containing products were separated by paper chromatography as previously described D. Gates, 0. 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 Ci-THF-synthase, (EC 5,10-methenyl-THF-cyclohydrolase

(EC

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, 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

CO

2 Gas Exchange was determined by taking measures with with a portable gas exchange system.

The quantity of the CI-THF pool was determined in two steps. Sugar beets ((Beta vulgaris 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% HAMPOSYLC and 0.3% glycine. After treatment with FIGly (20 h) and folinate 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 C,-THF synthases showed approximately 20% stimulation over the WO 96/27572 PCT/US96/02444 28 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 1 -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 supemrnatant 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 CI-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 C 1 -THF 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 IM 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 M 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 WO 96/27572 PCT/US96/02444 29 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

h C was found to yield 100% growth in investigations.

WO 96/27572 PCT/US96/02444 Results As shown in Table 1, rate of initial uptake ofV 4 C0 2 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 1.PULSE-CHASE SHOWS RAPID"CO 2

FIXATION

Compound Treatment 15 sec "CO 2 15 sec "14CO 2 Concentration 60 sec 12C0 2 180 sec 12CO2 Control 0 4.8 0.2 6.6 pABG 1 mM 8.2 0.5 3.3 0.2 Folinate 0.05 mM 8.5 0.8 4.2 0.2 In Table 2, below, our results show 1 4 C02 uptake in light saturated pulse-chase experiments serine decreased in proportion to the contribution of applied substances to Ci-THF structure.The glycine:serine ratio increased mainly due to serine depletion, folinate treated leaves showing most extensive depletion of serine.

Table 2."CO 2 FATE: HIGH GLYCINE:SERINE

RATIO

Compound Treatment Serine Glycine: Serine Concentration 3 min Control 0 13.51 0.76 MeOH 5000 mM 8.81 0.84 pABG 1 mM 7.58 2.13 Folinate 0.05 mM 3.06 4.38 In Table 3, below, 14CO2light 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.

WO 96/27572 PCT/US96/02444 31 Thereafter, at the 60 second chase, the glycine+serine products decreased when the sugars increased in treated plants as compared to controls.

Table 3.

1 COPULSE-CHASE: RAPID METABOLISM TO SUGAR Compound 14

CO

2 2 C0 2 PGA+PES* Sugars Glycine+Serine Control 15 s 1.98 0.16 1.79 15 s 60 s 1.64 1.64 3.36 pABG 15 s 5.23 0.39 1.73 s 60 s 1.74 2.94 1.27 Folinate 15 s 4.92 0.26 2.09 s 60 s 1.43 3.46 1.29 'PGA: phosphoglyceric acid. PES: phosphoric ethers of sugars As shown in Table 4, below, folinate, folate,pABA, pABG, Uvinul®, Benzocaine", mABA, pNBA, pNBA glycine, pFBA glycine, CBA, pAH, FIGly and MeOH activated Cj-THF enzymes, but glycine, Hamposyl"C, FAM and oABA did not. Folinate increased enzyme activity in corn. The addition of glycine to pNBA did not depress the overall stimulation of Cj-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.

WO 96/27572 WO 9627572PCTIUS96/02444 Table 4. CI-THF Enzyme Analysis Treatment Species {Concentration HAOSYL-C Sugar beet 12 mM Cyclohydrolase iiyIItheaI se! Dehydrogenase Treatment Activity/Control Activity

I

Sample HAMPOSYL-C Cabbage 2 mIM 0.51 0.86 0.86 3 HAMPOSYL'C Soy 2 mM 0.96 0.78 0.98 3 Folinate Sugar beet 0.1 mnlv 2.58 2.23 1 2.66 2 Folinate Corn 0.2 nm 0.97 1.45 T1.26 3 Folate Cabbage 0.11 MM 1.12 1.10 0.83 2 pABA Sugar beet 0.5 mm 4.1 1.48 2.05 2 pABA Cabbage 0.5 nm 1.7 1.17 1.84 2 pA.3G Soy 0.5 mM 1.87 1.43 1 1.26 2 Uvinul* Sugar beet 0.5 mM 1.97 1.14 1.93 6 Benzocaine- Sugar beet 0.06 mM 2.5 1.82 4.03 3 Benzocaine- Cabbage 0.06 mM 1.1 1.07 1.16 3 mABA Sugar bect 5 nm 1.73 1.24 2.04 6 oA.BA Sugar beet 0.5 mM 0.61 0.68 0.51 3 oA.BA Cabbage 0.5 mM 0.69 0.76 0.41 3 pNBA Sugar beet 1 mM 1.37 1.49 1.56 6 pNBA Cabbage 1 mM 1.24 0.95 1.09 pNBA+ Glycine Cabbage 1 mM+30 mM 1.38 1.05 1.21 3 CBA Sugar beet 0. 15 mM 1.58 1.47 1.52 3 Glycine Cabbage 71 mm 0.93 0.69 0.97 2 FAM Cabbage 30 mM 0.43 0.25 0.77 3 FAM Sugar beet 30 mM 0.65 0.77 0.7 6 pA.H Sugar beet 0.5 mM 0.87 0.85 1.19 3 pAH Cabbage 0.5 mM 1.91 0.5 0.93 2 FIGly Sugar beet 2 mM 2.86 1.46 2.09 7 FIGly Sugar beet 10 mM 1.27 1.1 1.4 pFBA+ Glycine Cabbage 0.5 mM 1.54 1.50 1.56 3 MeOti Sugar beet 14,800 mM 1.4 1.4 A. J.

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'8 P-25, FIGly, FAM, FAM with GO, Benzocaine7 m and MeOH. Gas exchange was significantly higher after Uvinul@8 P-25 treatments as WO 96/27572 PCTfUS96/02444 compared to pABA treatments. To a small extent, pAH stimulated gas exchange in soy.

Neither oABA nor MeoABA showed increases in gas exchange.

WO 96/27572 PCT/US96/02444 34 Table 5. Effect of enhancers, CI-acceptor compounds and Ci-inputs on CO 2 Gas Exchange Assimilation (Treatment/Control) (h after treatment) n Treatment Species 24 48 Folinate 0.02 mM Soy 2.86 1.08 1.65 0.44 8 Folinate 0.09 mM Sunflower 1.34 0.20 3 Folate 0.11 mM Cabbage 2.49 1.1 1.31 0.12 2 pABA 0.50 mM Cabbage 1.16 0.17 1.53 0.13 6 pABA 0.07 mM Kale 1.01 0.11 1.14 0.11 3 pABA 0.15 mM Kale 0.94 0.11 1.20 0.01 3 pABA 0.29 mM Kale 1.09 0.04 1.17 0.11 3 pNBA 0.10 mM Sunflower 1.86 0.42 2 pFBA 0.10 mM Cabbage 1.46 0.56 6 Uvinul® 0.50 mM Cabbage 1.34 0.32 1.25 0.06 6* pAHL 0.50 mM Soy 1.08 0.29 0.99 0.07 8 Benzocaine" 0.06 mM Kale 1.04 0.23 1.05 0.06 3 Benzocaine" 0.12 mM Kale 1.03 0.01 3 Benzocaine" 0.24 mM Kale 1.65 0.31 1.02 0.11 3 FIGly 2 M Cabbage 1.29 0.9 1.20 0.23 6* FIGly 2 mM Soy 1.47 0.08 1.17 0.24 6* FIGly 2 mM Sugar beet 1.23 0.21 1.29 0.14 4§ FAM 30 mM Sunflower 1.22 0.23 3 FAM+GO 30 mM Sunflower 2.00 0.5 3 MeOH 5000 mM Soy 6 *n 3 for 24 h; 'n 2 for 24 h; *72 h after 3rd spray; '24 h after 3rd spray.

WO 96/27572 PCT/US96/02444 Assimilation/Transpiration data yielded the following results given in Table 6, below, wherein reduced transpiration with increased CO 2 assimilation was recorded for treatment of foliage with folinate, pAH and MeOH.

Table 6. Effect of enhancers, C,-acceptor compounds and Ci-inputs on A/T Assimilation (Treatment/Control) (h after treatment) n Treatment Species 24 48 Folinate 0.02 mM Soy 1.27 0.12 1.46 0.14 8 pAH 0.50 mM Soy 1.33 0.17 0.92 0.10 8 MeOH 5000 mM Soy 1.36 0.21 6 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 IM folinate to 5 M MeOH and showing increased CO 2 gas exchange over soy controls lasting three weeks. Plants were treated at the start and at 120 h. Peak activity was observed during the first and second weeks, the response to folinate far exceeding responses to MeOH. 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, 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 CO 2 gas exchange for ten days following treatments.

Figure 9 shows that 3 foliar sprays of 30 mM FAM or 30 mM GO formulations increased gas exchange over controls for approximately a week between treatments.

Figure 10 shows the relationship between assimilation and transpiration.

Measurements of the assimilation/transpiration ratio in cabbage leaves treated with a formulation containing 30 mM FAM showed CO 2 assimilation maintained with reduced transpiration for the duration of the experiment.

WO 96/27572 PCTIUS96/02444 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 CI-THF pool of leaves are given in Table 7, below. Treatments with MeOH, FIGly and folinate significantly increased the total Ci-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 C,-THF pool than MeOH or FIGly. Treatments with 10 mM FIGly and 14.8 M MeOH showed equivalent increases in the total CI-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 C 1 -THF pool corresponded closely to increased photosynthetic gas exchange and enzymes measured at the time of sampling.

Table 7. Effect of Treatments on the C-THF Pool C,-THF Pool (Treatment/Control) Standard deviation) n Treatment Species Short Glu, Total Chain C -THF Pterovls Pool Folinate 0.1 mM Sugar beet 1.27 1.46 0.14 8 0.12 pAH 10 mM Soy 1.33 0.92 0.10 8 0.17 MeOH 14,800 mM Soy 1.36 6 0.21 WO 96/27572 PCT/US96/02444 37 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 BenzocaineM and MeoABA. Corn showed growth improvement by treatment with nitrobenzoates and folinate.

WO 96/27572 PCT/US96/02444 38 Table 8. Effect of Treatments on Plant Growth Compound(*) ppm Plant Fresh Wt. Dry Wt.

plants) (m/Oplants) Control Folinate (C) Folinate+GOX(B)3 Folinate+GO2x(B) Pteroic (B) 50 7+2000 7+2000 15 All Plants Radish Wheat Wheat Radish Radish Wheat Rice Radish pABG (C) pAH (B) Uvinul® (D) pNBA (A)

(B)

DNBA (B) CNBA (B) NCBA (B) 2CIBA (B) CBA (B) CBG (B) pFBA (B) pFBA (B) MeoABA (B) FIGly

(B)

FAF (B) 1265 Radish Radish Corn Radish Radish Radish Radish Radish Radish Radish Soy Bean- Wheat Radish Wheat Radish 2.38 0.23 0.25 1.29 0.90 4.54 1.72 1.57 1.08 1.59 1.28 1.35 1.09 0.90 1.17 1.28 1.17 1.26 1.21 3.67 1.20 3.74 0.84 100 138 134 148 115 111 121 124 115 157 173 111 114 131 161 114 138 131 102 112 95 110 121 112 150- 31 33 70 60 38 26 90 60 110 890 90 60 60 62 76 70 70 211 340 70 350 51 100 131 141 150 110 119 123 136 113 150 179 119 113 141 177 119 135 134 114 119 99 114 130 106 GO (B) HAMPOSYLI (D) Glycine (D) 5000 Wheat 160 Wheat 2000 Wheat 4.34 1.15 1.06 "Active components were formulated in the following control solutions: 0.05% HAMPOSYL"C surfactant, 0.2% glycine 0.05% HAMPOSYL~"C, 200 ppm potassium formate 0.2% glycine 0.05% HAMPOSYL"C, and water. Folinate was followed by GO"x once at 2 d or GO' twice at 2d and 4d.

-Bean yield was measured at University of Wyoming.

WO 96/27572 PCT/US96/02444 39 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 (50)

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

2. A method according to claim 1, wherein the Cl-acceptor compound increases the level of polyglutamate in the leaves.

3. A method according to claim 1 or claim 2, wherein the Cl-acceptor compound is part of the molecular structure of the enhancer substance.

4. A method according to any one of claims 1 to 3, wherein the enhancer is a substance which can be metabolised by the plant to C 1 -THF. S, 5. A method according to any one of claims 1 to 4, wherein the enhancer substance promotes carbon fixation by the C 1 pathway.

6. A method according to any one of claims 1 to 5, wherein the enhancer is selected from the group consisting of C 1 -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, applied as an aqueous solution at a concentration in the range from about 0.0001% by weight to 0.5% by weight.

7. A method according to any one of claims 1 to 6, wherein the enhancer is a pteridine of the formula below, wherein: OH R 01 I I N N R H 2 N N N R 01 is hydrogen or is a hydrocarbyl group capable of being metabolised to a one carbon substituent having the oxidation state of a methyl, hydroxymethyl, formyl or formic acid residue; and SR 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. A method according to any one of claims 1 to 7, wherein the enhancer is in a solution A/ comprising an activator substance selected from the group consisting of alcohols, organic [N:\LIBA]OO558:SAK nitrogenous bases, alkali metal hydroxides, alkaline earth metal hydroxides, hydrocarbyl acids and surfactants.

9. A method according to any one of claims 1 to 6, wherein the enhancer is a C 1 -THF compound selected from the group consisting of folinic acid, anhydroleucoverin, formyltetrahydropteroyl polyglutamate, 10-formyltetrahydropteroyl polyglutamate, formyltetrahydrofolate, 10-formyltetrahydrofolate, 5-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.

10. A method according to any one of claims 1 to 6, wherein the enhancer is a substituted benzoate derivative of the formula below, wherein: 0~ kg 9 *9 4 9 '4 4. S 0949 94 9 9 b R is H, hydrocarbyl, halogen, -OH, -SH, NH 2 N-linked amino acid, N-linked polypeptide, -OR 3 -SR 3 NHR 3 wherein R 3 is selected from the group consisting of optionally substituted hydrocarbyl, 15 alkyl, acyl, amino acids or polypeptide chains, -NR 4 R 5 wherein R 4 and R 5 which may be the same or different and are independently selected from the group consisting of H, optionally substituted hydrocarbyl, alkyl, aryl, acyl, C-terminal linked amino acids and C-terminal linked polypeptide chains, or R 4 and R 5 together with the nitrogen atoms to which they are linked form a heterocyclic ring; R 1 and R 2 are independently selected from the group consisting of: optionally substituted hydrocarbyl groups, alkyl, aryl, acyl, aroyl, halo, cyano, thio, hydroxy, alkoxy, aryloxy, amino, alkylamino, aminoalkyl, arylamino, aminoaryl, acylamino, ureido, alkylureido, arylureido, hydrazino, hydroxamino, alkoxycarbonylamino, aryloxycarbonylamino, nitro, nitroso, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, carboxyamino, 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. A method according to 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, polyethleneglycol 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 [N:\LIBA]00558:SAK 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. A method according to claim 10 or claim 11, wherein the substituted benzoate is derivatised with a surfactant comprising a polyoxyethylene chain.

13. A method according to any one of claims 10 to 12, 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. A method according to any one of claims 1 to 13, wherein the Cl-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. A method according to any one of claims 1 to 14, wherein the Cl-input substance is a o* 15 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; and acetamide selected from the group consisting of acetamide, methyl acetamide and dimethyl acetamide; 1,3,5-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 25 weight.

16. A method according to any one of claims 1 to 15, wherein the Cl-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. A method according to any one of claims 1 to 16, wherein the Cl-acceptor compound is 30 an amino acid selected from the group consisting of glycine, serine, glutamate, methionine, cystathionine, homocysteine and sarcosine.

18. A method according to any one of claims 1 to 16, wherein the Cl-acceptor compound is formamidine glycolate.

19. A method according to any one of claims 1 to 6, 10, 11 and 13 to 15, wherein the enhancer is p-nitrobenzoic acid, the Cl-input is potassium glycolate, and the Cl-acceptor compound is a urea. A method according to any one of claims 1 to 6, 10, 11 and 13 to 17, wherein the enhancer is terephthalic acid, the Cl-input is formic acid, and the Cl-acceptor compound is glycine. [N:\LIBA]00558:SAK

21. A method according to any one of claims 1 to 6, 9 and 14 to 16, wherein the enhancer is calcium folinate, the Cl-input is glycolic acid, and the Cl-acceptor compound is hexamethylenetetramine.

22. A method according to any one of claims 1 to 6, 10, 11 and 13 to 16, wherein the enhancer is m-nitrobenzoic acid, the Cl-input is methanol, and the Cl-acceptor compound is acylglycine.

23. A method according to any one of claims 1 to 6, 10 to 16 and 18, wherein the enhancer is PEG-p-nitrobenzoate, and the CI-acceptor compound and Cl-input is formamidine glycolate.

24. A method of promoting growth of a plant, said method comprising: applying to leaves of the plant an amount of an enhancer substance which increases the amount of Cl-tetrahydrofolate (CI-THF) in the leaves and a Cl-acceptor compound which serves as a sink for C 1 fragments in the leaves; and exposing the leaves to conditions which promote oxidative metabolism of photorespiration. 15 25. A method according to claim 24, wherein the Cl-acceptor compound increases the level of polyglutamate in the leaves.

26. A method according to claim 24 or claim 25, wherein said conditions are selected from the group consisting of high light intensity, elevated oxygen levels, water deprivation, high temperatures and low humidity.

27. A method according to any one of claims 24 to 26, wherein the enhancer is selected from the group consisting of C 1 -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, applied as an aqueous solution at a concentration in the range from 0.0001% by weight to 0.5% by weight. 25 28. A method according to any one of claims 24 to 27, wherein the enhancer is a pteridine of the formula below, wherein: OH R 0 1 IN N R H 2 N N N R 01 is hydrogen or is a hydrocarbyl group capable of being metabolised to a one carbon substituent having the oxidation state of a methyl, hydroxymethyl, formyl or formic acid residue; and RO 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 [N:\LIBA100558:SAK 44 as an aqueous solution at a concentration in the range from about 0.0001% by weight to 0.5% by weight.

29. A method according to any one of claims 24 to 28, 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, hydrobcarbyl acids and surfactants. A method according to any one of claims 24 to 27, wherein the enhancer is a C 1 -THF compound selected from the group consisting of folinic acid, anhydroleucoverin, formyltetrahydropteroyl polyglutamate, 10-formyltetrahydropteroyl polyglutamate, 1o formyltetrahydrofolate, 10-formyltetrahydrofolate, 5-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. A method according to any one of claims 24 to 27, wherein the enhancer is a substituted 15 benzoate of the formula below, wherein: 'CO-R R 2 R is H, hydrocarbyl, halogen, -OH, -SH, NH 2 N-linked amino acid, N-linked polypeptide, -OR 3 S" -SR 3 NHR 3 wherein R 3 is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR 4 R 5 wherein R 4 and R 5 which may be the same or different and are independently selected from the group consisting of H, optionally substituted hydrocarbyl, alkyl, aryl, acyl, C-terminal linked amino acids and C-terminal linked polypeptide chains, or R 4 and R 5 together with the nitrogen atoms to which they are linked form a heterocyclic ring; wherein R 1 and R 2 are independently selected from the group consisting of: optionally substituted hydrocarbyl groups, alkyl, aryl, acyl, aroyl, halo, cyano, thio, hydroxy, 25 alkoxy, aryloxy, amino, alkylamino, aminoalkyl, arylamino, aminoaryl, acylamino, ureido, alkylureido, arylureido, hydrazino, hydroxamino, alkoxycarbonylamino, aryloxycarbonylamino, nitro, nitroso, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, carboxyamino, 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. A method according to claim 31, wherein the substituted benzoate is selected from the group consisting of m-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, L ethoxylated p-aminobenzoic acids, nitrobenzoic acids, m-nitrobenozic acid, p-nitrobenzoic acid, N- [N:\LIBA)00558:SAK nitrobenzoyl amino acids, polyethyleneglycol nitrobenzoate, 4-chloro-2-nitrobenzoic acid, 2-chloro-4- nitrobenzoic acid, terephthalic acids, phthalic acids, N-phthaloyl amino acids, phthalaldehydic acid 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. A method according to claim 31 or claim 32, wherein the substituted benzoate is derivatised with a surfactant comprising a polyoxyethylene chain.

34. A method according to any one of claims 31 to 33, 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. A method according to any one of claims 24 to 34, wherein the Cl-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. A method according to any one of claims 24 to 35, wherein the CI-acceptor compound is an amino acid selected from the group consisting of glycine, serine, glutamate, methionine, cystathionine, homocysteine and sarcosine.

37. A method according to any one of claims 24 to 35, wherein the Cl-acceptor compound is formamidine glycolate.

38. A method according to any one of claims 24 to 27, 31 to 35 and 37, wherein the enhancer is a surfactant-linked substituted benzoic acid and the CI-acceptor compound is a formamidine.

39. A method according to any one of claims 24 to 27, 31, 32, 34 and 35, wherein the enhancer is p-nitrobenzoic acid and the Ci-acceptor compound is hexamethylenetetramine.

40. A method according to any one of claims 24 to 27, 31, 32, 34, 35 and 37, wherein the enhancer is a phthalate and the Cl-acceptor compound is a formamidine.

41. A method according to any one of claims 24 to 27 and 30, wherein the enhancer is a folinate and the C 1 -acceptor compound is a formimino amino acid.

42. A system comprising: .*30 a first aqueous solution containing, or a nonaqueous material which when combined with an aqueous carrier contains, an amount of a Cl-acceptor compound effective to serve as a sink for C 1 fragments in leaves of a plant when applied to the leaves of the plant; and a second aqueous solution containing, or a nonaqueous material which when combined with an aqueous carrier contains, an amount of an enhancer substance effective to increase the 35 amount of C 1 -tetrahydrofolate (C 1 -THF) in leaves of the plant when applied to the leaves of the plant and optionally containing a Cl-input substance which increases the metabolism of C 1 fragments through C 1 -THF when applied to the leaves of the plant; when used for promoting growth of the plant by applying both said solutions to the leaves of the plant.

43. A system comprising: [N:\LIBA]00558:SAK a first aqueous solution containing, or a nonaqueous material which when combined with an aqueous carrier contains, an amount of an enhancer substance effective to increase the amount of Ci-tetrahydrofolate (C 1 -THF) in leaves of the plant when applied to the leaves of the plant; and a second aqueous solution containing, or a nonaqueous material which when combined with an aqueous carrier contains, an amount of a C 1 -acceptor compound effective to serve as a sink for Ci fragments in leaves of a plant when applied to the leaves of the plant and optionally containing a Cl-input substance which increases the metabolism of C 1 fragments through C 1 -THF when applied to the leaves of the plant; when used for promoting growth of the plant by applying both said solutions to the leaves of the plant.

44. A system according to claim 43, wherein the second aqueous solution contains both a C 1 -acceptor compound and a Cl-input substance. A system according to any one of claims 42 to 44, wherein the C 1 -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.

46. A system according to any one of claims 42 to 45, wherein the Cl-acceptor compound is an amino acid selected from the group consisting of glycine, serine, glutamate, methionine, cystathionine, homocysteine and sarcosine. 20 47. A system according to any one of claims 42 to 45, wherein the C 1 -acceptor compound is formamidine glycolate.

48. A system according to any one of claims 42 to 47, wherein the enhancer is selected from the group consisting of C 1 -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,

49. A system according to any one of claims 42 to 48, wherein the enhancer is a pteridine of the formula below, wherein: OH Ro 1 N Ro g* oS.) H 2 N N N R 01 is hydrogen or is a hydrocarbyl group capable of being metabolised to a one carbon S3o substituent having the oxidation state of a methyl, hydroxymethyl, formyl or formic acid residue; and *o o RO 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. [N:\LIBAOO0558:SAK A system according to any one of claims 42 to 48, wherein the enhancer is a C 1 -THF compound is selected from the group consisting of folinic acid, anhydroleucoverin, formyltetrahydropteroyl polyglutamate, 10-formyltetrahydropteroyl polyglutamate, formyltetrahydrofolate, 10-formyltetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methenyltetrahydrofolate and 5,10-methylenetetrahydrofolate; and salts, hydrates and surfactant-linked derivatives thereof.

51. A system according to any one of claims 42 to 48, wherein the enhancer is a substituted benzoate of the formula R 1 R2-CO- R wherein: R is H, hydrocarbyl, halogen, -OH, -SH, NH 2 N-linked amino acid, N-linked polypeptide, -OR 3 -SR3, NHR 3 wherein R 3 is selected from the group consisting of optionally substituted hydrocarbyl, alkyl, acyl, amino acids or polypeptide chains, -NR 4 R 5 wherein R 4 and R5 which may be the same or different and are independently selected from the group consisting of H, optionally substituted 15 hydrocarbyl, alkyl, aryl, acyl, C-terminal linked amino acids and C-terminal linked polypeptide chains, or R 4 and R 5 together with the nitrogen atoms to which they are linked form a heterocyclic ring; wherein R 1 and R 2 are independently selected from the group consisting of: optionally substituted hydrocarbyl groups, alkyl, aryl, acyl, aroyl, halo, cyano, thio, hydroxy, alkoxy, aryloxy, amino, alkylamino, aminoalkyl, arylamino, aminoaryl, acylamino, ureido, alkylureido, arylureido, hydrazino, hydroxamino, alkoxycarbonylamino, aryloxycarbonylamino, nitro, nitroso, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, carboxyamino, 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. 25 52. A system according to claim 51, wherein the substituted benzoate is selected from the group consisting of m-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, m-nitrobenozic acid, p-nitrobenzoic acid, N- so nitrobenzoyl amino acids, polyethyleneglycol nitrobenzoate, 4-chloro-2-nitrobenzoic acid, 2-chloro-4- nitrobenzoic acid, terephthalic acids, phthalic acids, N-phthaloyl amino acids, phthalaldehydic acid 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.

53. A system according to claim 51 or claim 52, wherein the substituted benzoate is in a solution comprising an activator substance selected from the group consisting of alcohols, organic [N:\LIBA]OO558:SAK nitrogenous bases, alkali metal hydroxides, alkaline earth metal hydroxides, hydrocarbyl acids and surfactants.

54. A system comprising: an aqueous solution containing, or a nonaqueous material which when combined with an aqueous carrier contains, an amount of a Cl-acceptor compound effective to serve as a sink for C 1 fragments in leaves of a plant when applied to the leaves of the plant; and another aqueous solution containing, 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 1 -tetrahydrofolate (C 1 -THF) in leaves of the plant when applied to the leaves of the plant; wherein one, but not both, of said solutions optionally contains a Cl-input substance which increases the metabolism of C 1 fragments through C 1 -THF when applied to the leaves of the plant; when used for promoting growth of the plant by applying both said solutions to the leaves of the plant, substantially as hereinbefore described, with reference to any one of the examples. 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,

56. A mixture according to claim 55, wherein the formamidine salt is formamidine glycolate.

57. A mixture according to claim 55, wherein the formamidine salt is formamidine formate.

58. 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.

59. A mixture according to claim 58, wherein the formamidine salt is formamidine glycolate. 0 60. A mixture according to claim 58, wherein the formamidine salt is formamidine formate.

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

62. A mixture for promoting growth of a plant, substantially as hereinbefore described with reference to any one of the examples. 0 Dated 11 May, 1999 *:30 Arthur M. Nonomura John N. Nishio Andrew A. Benson Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [N:\LIBA]00558:SAK