|Title of Invention||
"A COMPOSITION FOR ENHANCING GROWTH OF PLANT"
|Abstract||A composition for enhancing growth of plant, said composition comprising: (a) an aqueous solution containing an oxidant which induces NADPH: cytochrome P450 reductase in said plant, wherein said oxidant is selected from the group consisting of flavins; salts of flavins; hydrates of flavins; surfactant-linked derivatives of flavins; and combinations thereof; and (b) an aqueous solution containing a reductant which induces cytochrome P450 monooxygenase in said plant; wherein oxidant: reductant molar ratio is in the range of from 10 : 1 to 1 : 2.|
|Full Text||The present invention relates to a composition for enhancing growth of plant.
BACKGROUND OF THE INVENTION The present invention relates generally to methods and compositions for treating plants and for plant growth enhancement
Photosynthesis is the process by which all photosynthetic plants utilize solar energy to build carbohydrates and other organic molecules from carbon dioxide (CO,) and water. In general, photosynthesis is a complex sequence of electron and proton-transfer reactions. Optimally, photosynthesis involves serial electron-proton transfers leading to stable reduced metabolites; but when electrons or radicals accumulate along this chain, the resulting imbalance interferes with one after another system until growth decreases. The chemistry of living systems dictates that an electron acceptor is usually balanced by the presence of an electron donor, however, application of chemicals for biological response has generally been one-sided in the sense that xenobiotics applied to plants to elicit specific responses are generally formulated without regard for providing a balance of electron acceptors and donors. This historical one-sided approach often stresses the biological system when either oxidants or reductants abound. Our tests suggest that when a balance of electron couples is established by application of formulations selected for appropriate pairing, stress
components may be neutralized. The E0 values of the prospective couples are defined
within a range that is compatible to biological systems. In sunlight, a nontoxic balance is
especially important for minimizing damage by oxidants. Imbalances of electron couples
may be corrected by induction of Cytochromes P450 (CYP) and NADPH:Cytochrome P450
reductase (CPR) pathways that result in the utilization of reducing power.
Cytochromes P450 are a superfamily of hemoproteins that catalyze the
singular insertions of oxygen, i.e. monooxygenation, of endogenous and xenobiotic
hydrophobic substrates, wherein, the general reaction for hydroxylation by the Cytochromes
P450 system is,
RH + NADPH + H+ + O, - ROM + NADP* + Water,
and R represents a substrate compound. The CYP and flavin monooxygenase families are
noted for their broad substrate specificities and utilization of oxygen without being linked to
phosphorylation of adenosine diphosphate (ADP) and can mediate hydroxylations at nitrogen
and sulfur heteroatoms, epoxidations, dehalogenations, deaminations and dealkylations. In
general, the monooxygenations require one or two additional proteins to transfer electrons
from NADPH to the heme iron and these systems are placed in two groups: Class I, which
use an iron-sulfur protein to shuttle electrons from FAD-containing reductase to CYP in
mitochondria and bacteria; and Class II, in which NADPHiCytochrome P450 reductase
transfers electrons from NADPH to a CYP in microsomes. In plants, CYP comprises a wide
range of hydroxylases, epoxidases, peroxidases and oxygenases which are largely based upon
Class II monooxygenations.
Neither the direct connection of CYP and CPR to regulate photosynthesis nor
the formulations of cytochromes P450 and inducer substrates have been made previously.
We introduce novel methods for formulating compositions comprised of CYP and CPR
substrates and enzymes selectedjbr cofnpTetm^The"ne^Hs^ry^le^tfoncoupIes and inducing
For these reasons, it would be desirable to provide-sovel methods-and
formulations for activating cytochromes P450 enzymes. It would be particularly desirable if
such methods and compositions were able to regulate plant growth. Additionally, it would
be desirable if the compositions reduce toxicity of otherwise one-sided treatments. The
present invention should further provide convenient methods resulting in increased activities
of CYP and CPR electron couples for applying the novel compositions to plants. It is
desirable that the methods and compositions of the present invention promote rapid growth
and maturing of the treated plant, increase sugar content, improve blossoms and enhance the
quality and quantity of plants. Furthermore, it is generally desirable to provide methods for
enhancement of CYP and CPR related enzymes in all biological systems.
The structures and functions of CPR and CYP are reviewed with focus on
animal CYP, some of which metabolize more than fifty structurally diverse compounds. See,
e.g., H. W. Strobel, et al, "NADPH Cytochrome P450 Reductase and Its Structural and
Functional Domains," and C. von Wachenfeldt, et al., "Structures of Eukaryotic Cytochrome
P450 Enzymes," P. R. Ortiz de Montellano, ed. (1995) CYTOCHROME P450: STRUCTURE,
MECHANISM, AND BIOCHEMISTRY (Second Edition), Plenum Press, New York. Inducers of
cytochromes P450 in animal systems include aromatic hydrocarbons, proteins, phenobarbital,
peroxisome proliferators, steroids, aminopyrine and ethanol (see, J. P. Whitlock et al.,
"Induction of Cytochrome P450 Enzymes That Metabolize Xenobiotics" in P. R. Ortiz de
Montellano, ed. (1995) CYTOCHROME P450: STRUCTURE, MECHANISM, AND BIOCHEMISTRY
(Second Edition), Plenum Press, New York, pp 367-390); but no inducers of cytochromes
P450 for growth have been identified in green plant systems.
In avocado tissue, alcohols, aniline, /7-chloro-N-methylaniline, N, Ndimethylaniline,
cinnamic acid, dimethyl formamide, aryl hydrocarbons and fatty acids
showed binding to cytochromes P450. See, S. Cottrell, et al., "Studies on the cytochrome P-
450 of avocado (Persa americana) mesocarp microsomal fraction" Xenobiotica 20:711-726
(1990). In recent reviews of molecular cloning, plant pathways included cytochromes P450
catalysis of oxygen insertion for fatty acids, phenylpropanoids, flavonoids, terpenoids,
alkaloids, dyes, pesticides (see, e.g., G. P. Bolwell, et al., "Review Article Number 96.
Plant Cytochrome P450" Phytochemistry 37:1491-1506 (1994)); lignins, coumarins,
pigments, alkaloids, jasmonates and plant growth regulators (see, M. A. Schuler "Plant
Cytochrome P450 Monooxygenases" Critical Reviews in Plant Sciences 15(3):235-284
(1996)). Metolachlor is a herbicide that is detoxified by cytochromes P450 (see, D. E.
Moreland, et al., "Metabolism of Metolachlor by a Microsomal Fraction Isolated from Grain
Sorghum (Sorghum bicolor) Shoots" Z. Naturforsch 45c:558 (1990)). Beneficial effects of
flower inducement implicate binding of carbamates to cytochromes P450 (see, M.
Kusukawa, et al., ";V-(3,4-Methylenedioxyphenyl)carbamates as Potent Flower-Inducing
Compounds in Asparagus Seedlings as Well as Probes for Binding to Cytochrome P-450" Z
Naturforsch 50c:373 (1995)), where known inhibitors of cytochromes P450 including
piperonyl butoxide and frwiy-cinnamic acid 4-hydroxylase stopped the effect. The hormonal
action of the ecdysone-like brassinosteroids may encode CYP90 genes that regulate various
aspects of plant development (see, M. Szekeres, et al., "Brassinosteroids rescue the
deficiency of CYP90, a cytochrome P450 controlling cell elongation and de-etiolation in
Arabidopsis" Cell (Cambridge) 85:171 (1996)). Salicylate and aspirin caused elevation of rat
liver ethanol inducible cytochromes P450 (see, B. Damme, et al., "Induction of hepatic
cytochrome P4502E1 in rats by acetylsalicylic acid or sodium salicylate" Toxicology 106:99-
103 (1996)) and, although salicylates in plants are associated with systemic acquired
resistance, their relationships to plant cytochromes P450 has not been demonstrated (see,
e.g.. S. A. Bowling, et al., "A Mutation in Arabidopsis That Leads to Constitutive Expression
of Systemic Acquired Resistance" The Plant Cell 6:1845-1857 (1994)). Phenobarbital has
been shown to enhance the activity of CYPCC in non-photosynthetic plant tissue cultures. See,
Palazon, et al., "Effects of auxin and phenobarbital on morphogenesis and production of
digitoxin in Digitalis callus" Plant and Cell Physiology 36:247 (1995).
As a supplement to tissue culture, tyrosine has been found in specific natural
products during fermentation. See, Y. Hara, et al., "Effect of gibberellic acid on berberine
and tyrosine accumulation in Coptis japonica" Phytochemistry 36:643-646 (1994)).
Tyrosine is essential for flavin mononucleotide binding to cytochromes P450, (see, M. L.
Klein, et al., "Critical Residues Involved in FMN Binding and Catalytic Activity in
Cytochrome P450BM.3" The Journal of Biochemistry 268:7553-7561 (1993)) and plays a key
role in facilitating electron transfer between flavin mononucleotide and heme groups of other
cytochromes (see, C. S. Miles, et al., "Tyr-143 facilitates interdomain electron transfer in
flavocytochrome b2" Journal of Biochemistry 285:187-192 (1992)). Tyrosine is a substrate
for CYP56 and CYP79 in plants. See, B. M. Koch, et al., "The primary sequence of
cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of Lryrosine
ofp-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic
glucoside dhurrin in Sorghum bicolor (L.) Moench." Archives of Biochemistry and
Biophysics 323:177-186 (1995).
Among the early cytochromes P450 functional markers, para-nitrobenzoate
(pNBA) was used to screen the activity of liver microsomal cytochromes P450 substrates by
following the reduction to the primary amine. Thus, cytochrome P450 substrates were
defined by type I spectra characterized by a trough at 420 nm and a peak at 385 nm or type II
spectra characterized by a trough at 390 nm and a peak at 430 nm. See, H. A. Sasame, et al.,
"Studies on the Relationship between the Effects of Various Substances on Absorption
Spectrum of Cytochrome P-450 and the Reduction of/7-Nitrobenzoate by Mouse Liver
Microsomes" Mol. Pharmacol. 5:123 (1969); and J. R. Gillette "Reductive Enzymes"
Handbuch der experimentellen Pharmakologie 28/2:349 (1971). In addition to pNBA, other
oxidants have been identified including menadione, Mitomycin C, Adriamycin,
anthraquinone sulfonate, dinitrobenzene, and quinones, their association with cytochromes
P450 dependent upon E0 values residing within a range of -400 mV to -165 mV. See, J.
Butler, et al., "The one-electron reduction potential of several substrates can be related to
their reduction rates by cytochrome P-450 reductase" Biochimica et Biophysica Acta 1161:73
(1993). A review of chemical potentials for electron couples detailing one-electron
processes for reduction of oxidants (reduction of electron acceptor) and oxidation of
reductants (oxidation of an electron donor) gives values for approximately 700 compounds
(see, P. Wardman "Reduction Potentials of One-Electron Couples Involving Free Radicals in
Aqueous Solution" J. Phys. Chem. Ref. Data 18(4): 1637-1755 (1989) including flavin,
bipyridinium, nitroaryl, phenol, terpenoid, imidazole, amine, peroxide and Lndole
compounds. lodosobenzene and N-oxide of p-cyano-W.vV-dimethylalanine have been used
for oxidation reactions with CYP in chemical models (see, W. Nee, et al., "Use of//-oxide of
p-Cyano-jV.A^-dimethylalanine as an "Oxygen" Donor in a Cytochrome P-450 Model System"
J. Am. Chem. Soc. 104:6123 (1982)), but they have not been applied to plants or other
U.S. Patent No. 5,532,204 proposes foliar applied methanol at the R5 seed
growth stage of legumes. U.S. Patent No. 5,300,540 proposes preservation of freeze-dried
plant cells with barrier compositions containing, polyethylene glycol,/?-aminobenzoic acid,
acerylsalicylic acid, cinnamic acid, benzoic acid, blended alcohol and other organics. U.S.
Patent No. 3,897,241 proposes application of ethanolamine formulations with carboxylic
acids of less than 8 carbons, such as, oxalic acid, formic acid, acetic acid, phthalic acid and
glutaric acid to fruit-bearing plants. U. S. Patent No. 4,799,953, proposes polymeric
condensates of the sulfur-polymers of thiolactic and thioglycolic acids, increasing the rate of
growth and production of chlorophyll specific to tissue and hydroponic culture ofLemna
minor. European Patent 465 907 A1 proposes compositions for stimulating the growth and
ripening of plants comprised of at least one adduct of menadione bisulfite and a compound
chosen from a group including pAB A, nicotinamide, nicotinic acid, thiamine, tryptophan,
histidine, or adenine. U.K. Patent Application 2 004 856 proposes plant growth stimulating
compositions consisting of cysteine as the active component in formulations that also include
sulfosalicylic acid, folic acid, an aldehyde, a magnesium salt, and a buffer. European Patent
FR 2 689 905 Al proposes a method for cloning DNA sequences coding for an NADPH
Cytochrome P450 reductase implicated by survival of a deficient mutant of Saccharomyces
PCT W094/00009 is the published text of parent application
PCT/US93/05673 (published on 6 January 1994) and U. S. Patent No. 5,597,400 issued on
28 January 1997. South African patent 93/4341, which is also the equivalent of
PCT/US93/C5673, issued on 30 March 1994. South African patent 96/1637, which is also
the equivalent United States application Serial No. 08/610,928, filed on 5 March 1996,
which was filed as a PCT International Application PCT/US96/02444, on 20 February 1996.
SUMMARY OF THE INVENTION
As a first aspect, the present invention provides methods for treating plants.
The methods include (a) applying to the plant a first compound selected from the group
consisting of (/') NADPH:cytochrome P450 reductase enzyme and (/'/) oxidants that induce
NADPH:cytochrome P450 reductase in plants; and (b) applying to the plant a second
compound selected from the group consisting of (/') cytochrome P450 monooxygenase
enzyme and (»') reductants that induce cytochrome P450 monooxygenase.
As a second aspect, the present invention provides a second method for
treating a plant. The method comprises (a) applying to the plant an oxidant selected from the
group consisting of flavins, salts of flavins, hydrates of flavins, surfactant-linked derivatives
of flavins, and combinations thereof, and (b) applying to the plant a reductant that induces
cytochrome P450 monooxygenase.
As a third aspect, the present invention provides a method for increasing the
amount of cytochrome P450 in a photosynthetic plant. The method comprises (a) applying
to the plant an oxidant that induces NADPH:cytochrome P450 reductase in plants, and (b)
applying to the plant a reductant that induces cytochrome P450 monooxygenase. The
oxidant is selected from the group consisting of flavins, salts of flavins, hydrates of flavins,
surfactant-linked derivatives of flavins, and combinations thereof.
As a fourth aspect, the present invention provides a method for enhancing
growth of a plant. The method comprises (a) applying to the plant an amount of an oxidant
which induces NADPH:cytochrome P450 reductase in the plant, and (b) applying to the plant
an amount of a reductant which induces cytochrome P450 monooxygenase in the plant. The
oxidant is selected from the group consisting of flavins salts of flavins, hydrates of flavins,
surfactant-linked derivatives of flavins, and combinations thereof.
As a fifth aspect, the present invention provides a plant growth enhancing
system. The system comprises (a) an aqueous solution containing an amount of an oxidant
which induces NADPHxytochrome P450 reductase in the plant, and (b) an aqueous solution
containing an amount of a reductant which induces cytochrome P450 monooxygenase in the
plant. The oxidant is selected from the group consisting of flavins, salts of flavins, hydrates
of flavins, surfactant-linked derivatives of flavins, and combinations thereof.
As a sixth aspect, the present invention provides a composition for enhancing
growth of a plant. The composition comprises (a) an aqueous solution containing an amount
of an oxidant which induces NADPHicytochrome P450 reductase in the plant, and (b) an
aqueous solution containing an amount of a reductant which induces cytochrome P450
monooxygenase in the plant. The oxidant is selected from the group consisting of flavins,
salts of flavins, hydrates of flavins, surfactant-linked derivatives of flavins, and combinations
thereof.As a seventh aspect, the present invention provides a second composition for
enhancing growth of a plant. The composition comprises (a) a first compound selected from
the group consisting of (/) NADPHxytochrome P450 reductase enzyme and (//') oxidants that
induce NADPH:cytochrome P450 reductase in plants; and (6) a second compound selected
from the group consisting of tyrosine, tyrosine ester, and salts thereof.
As an eighth aspect, the present invention provides another method for
enhancing growth of a plant The method comprises applying to the foliage of a plant, a
composition comprising (z) a reductant and (if) an agronomically suitable surfactant. The
reductant is selected from the group consisting of tyrosine, tyrosine ester, tyrosine
methylester, and tyrosine methylester hydrochloride
As a ninth aspect, the present invention provides yet another method for
enhancing growth of a plant. The method comprises applying to the foliage of the plant, a
composition comprising (i) a flavin and (if) an agronomically suitable surfactant. The flavin
is selected from the group consisting of flavin mononucleotide, flavin adenine dinucleotide,
riboflavin, deazaflavin, salts thereof, hydrates thereof, surfactant-linked derivatives thereof,
and combinations thereof.
These and other aspects of the present invention are described further in the
detailed description and examples of the invention which follow.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a simplified and general schematic depiction of the electron
couple pairing to the catalytic cycle of cytochromes P450. Reductants are given in the left
vertical column, which would function through CYP. Products of the oxygen insertion are
given in the right vertical column. At the top center of the figure are examples of oxidants
which function through flavin reductases such as CPR. Products are given at the bottom
center of the figure. Improvement of plant growth is achieved by formulating CPR
substrates with CYP inducers.
DESCRIPTION OF THE PREFERRED EMBODIMENT
According to the present invention, methods, compositions, and systems are
provided for coinducing cytochromes P450 monooxygenases (CYP) and
NADPH:Cytochrome P450 reductases (CPR). Methods are provided for treating plants,
particularly photosynthetic plants with the compositions of the present invention.
Unless otherwise defined, all technical and scientific terms employed herein
have their conventional meaning in the art. As used herein, the following terms have the
meanings ascribed to them.
"Oxidant" refers to electron acceptors or reductase substrates that induce
CPR. Reductase substrates which induce CPR accelerate the metabolism of reductants by
"Reductant" refers to electron donors or oxidase substrates that induce CYP.
Oxidase substrates which induce CYP accelerate the metabolism of oxidants by CPR.
"Enhance(s) growth" or "enhancing growth" rejers to promoting, increasing or
improving the rate of growth of the plant or increasing or promoting an increase in.the.size of
the plant. Without wishing to be bound by any particular theory regarding the mechanism by
which the compositions of the present invention enhance the growth of a plant, it is believed
that when CYP and CPR enzymes are induced exogenously, they are enhanced beyond the
natural content of a plant and, thereby lead to the enhanced growth of the plant.
Enhancement of CYP and CPR increases the capacity of an organism to insert oxygen into
metabolites and xenobiotics.
"Plants" refers to virtually all live species with active light-gathering surfaces
capable of receiving treatments, particularly higher plants that fix carbon dioxide.
"Surfactant" refers to surface-active agents, i.e., which modify the nature of
surfaces, often by reducing the surface tension of water. They act as wetting agents,
spreaders, dispersants, or penetrants. Typical classes include cationic, anionic (e.g.,
alkylsulfates), nonionic (e.g., polyethylene oxides) and ampholytic. Soaps, alcohols, and
fatty acids are other examples.
"Surfactant-linked derivative" refers to a derivative of the parent compound,
the derivative having a surfactant covalently attached to the parent compound. A
representative example of a parent compound and a surfactant-linked derivative thereof is paminobenzoic
acid and its surfactant-linked derivative polyethoxylatedp-aminobenzoic acid
"Percent" or"%" is percent by weight unless otherwise indicated.
"ppm" refers to parts per million by weight.
"Alky!" refers to linear, branched or cyclic; saturated or unsaturated C,-CS
hydrocarbons. Examples of alkyl groups include methyl, ethyl, ethenyl, propyl, propenyl,
isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, cyclohexyl, octyl, and the like.
"Aqueous" with reference to solutions or solvents refers to solutions or
solvents which consist primarily of water, normally greater than 90 weight percent water,
and can be essentially pure water in certain circumstances. For example, an aqueous solutioi
or solvent can be distilled water, tap water, or the like. However, an aqueous solution or
solvent can include water having substances such as pH buffers, pH adjusters, organic and
inorganic salts, alcohols (e.g., ethanol), sugars, amino acids, or surfactants incorporated
therein. The aqueous solution or solvent may also be a mixture of water and minor amounts
of one or more cosolvents, including agronomically suitable organic cosolvents, which are
miscible therewith, or may form an emulsion therewith. Agronomically suitable organic
solvents include, for example, acetone, methanol, nitromethane, limonene, paraffin oils,
siloxanes, esters, ethers, and emulsifiers.
The compositions and methods of the present invention may be applied to
virtually any variety of plants. In particular, the compositions and methods of the present
invention may be advantageously applied to "higher plants." Higher plants include, but are
not limited to all species having true stems, roots, and leaves, thus excluding "lower plants"
such as bacteria, yeasts and molds. Plants which may benefit according to the present
invention include but are not limited to all crop plants, such as, alfalfa, anise, bach ciao,
barley, basil, blueberry, breadfruit, broccoli, brussels sprouts, cabbage, cassava, cauliflower,
celery, cereals, cilantro, coffee, com, cotton, cranberry, cucumber, dill, eggplant, fennel,
grape, grain, garlic, kale, leek, legume, lettuce, melon, mint, mustard, melon, oat, onion,
parsley, peanut, pepper, potato, saffron, squash, legume, lettuce, millet, parsnip, parsley, pea,
pepper, peppermint, pumpkin, radish, rice, sesame, sorghum, soy, spinach, squash, stevia,
strawberry, sunflower, sweet potato, sugar beet, sugar cane, tea, tobacco, tomato, turnip,
wheat, yam, zucchini and the like; pomes and other fruit-bearing plants, such as, apple,
avocado, banana, breadfruit, cherry, citrus, cocoa, fig, guava, macadamia, mango,
mangosteen, nut, olive, papaya, passion fruit, pear, pepper, plum, peach and the like; floral
plants, such as achillea, ageratum, alyssum, anemone, aquilegia, aster, azalea, begonia,
bird-of-paradise, bleeding heart, borage, bromeliad, bougainvillea, buddlea, cactus,
calendula, camellia, campanula, carex, carnation, celosia, chrysanthemum, clematis, cleome,
coleus, cosmos, crocus, croton, cyclamen, dahlia, daffodil, daisy, day lily, delphinium,
dianthus, digitalis, dusty miller, euonymus, forget-me-not, fremontia, fuchsia, gardenia,
gazania, geranium, gerbera, gesneriad, ginkgo, gladiolus, hibiscus, hydrangea, impatrens,
jasmine, lily, lilac, lisianthus, lobelia, marigold, mesembryanthemum, mimulus, myosotis,
New Guinea Impatiens, nymphaea, oenothera, oleander, orchid, oxalis, pansy, penstemon,
peony, petunia, poinsettia, polemonium, polygonum, poppy, portulaca, primula, ranunculus,
rhododendron, rose, salvia, senecio, shooting star, snapdragon, solanum, solidago, stock, ti,
torenia, tulip, verbena, vinca, viola, violet, zinnia, and the like; leafy plants, such as ficus,
fern, hosta, philodendron, and the like; trees, such as Abies, birch, cedar, Cornus, cypress,
elm, ficus, fir, juniper, magnolia, mahogany, maple, oak, palm, Picea, Finns, Pittosporum,
Plantago, poplar, redwood, Salix, sycamore, Taxus, teak, willow, yew, Christmas tree and
the like; grasses, such as Kentucky blue grass, bent grass, turf, festuca, pennisetum, phalaris,
calamogrostis, elymus, helictotnchon, imperata, molina, carex, miscanthus, panicum, and the
like; and thalloid plants such as algae. This list is intended to be exemplary and is not
intended to be exclusive. Other plants which may benefit by application of the compositions
and methods of the present invention will be readily determined by those skilled in the art.
The methods and compositions of the present invention may be used to
enhance growth in juvenile and mature plants, as well as cuttings and seeds. Generally,
however, it is desirable that the plants include at least the sprouted cotyledon (i.e., the "seed
leaves") or other substantial light-gathering surfaces including the true leaves.
As provided herein, enhancement of CYP and CPR focuses on modulating
electron and oxygen transfer through CYP and CPR in a manner that shifts the flow of
electrons in plants. Figure 1 is a schematic depiction of the electron couple pairing to the
catalytic cycle of cytochromes P450. By inducing or adding to the CYP and CPR of a leaf,
the reductive capacity for growth is enhanced. An enhanced pool of CYP allows increased
leaf capacity for electron transfer via CPR and vice versa. Accordingly, the compositions
and methods of the present invention include, in general, an oxidant component and a
II. Methods and Compositions
The present invention provides methods for treating plants, for increasing the amount
of cytochrome P450 in a photosynthetic plant, and for enhancing the growth of a plant.
These methods typically involve the application of an oxidant component and the
application of a reductant component to the plant.
As noted above, oxidants are compounds which induce NADPH:cytochrome
P450 reductase. Any compound capable of inducing such reductase will be useful as the
oxidant component in the methods, compositions, and systems of the present invention.
Accordingly, reductases, particularly CPR, may be utilized as the oxidant component of the
methods, compositions, and systems of the present invention. In addition, a number of other
suitable oxidants will be readily determinable by those skilled in the art.
Preferred oxidant compounds exhibit a one electron reduction potential (E0)
between about -400 mV and about -165 mV inclusive, more preferably between about -396
mV and about -240mV. Some multiple electron reductions are also biologically important
with CYP and oxygen. Examples of suitable reductants include but are not limited to
ferredoxin-NADP* reductases, including the reductases listed hereinabove as well as, flavins,
nitrobenzoate compounds, nicotinic acids, nitrobenzoic acid compounds, haloaryl
compounds, amine oxides, formamidines, glycolates and glycolic metabolites, cytochrome
reductases, azo compounds, quinone compounds, bipyridinium compounds, and all salts,
hydrates, aldehydes, esters, amines, surfactant-linked derivativates, and other biologically or
chemically equivalent derivatives thereof and combinations thereof.
Specific examples of flavins which are useful as oxidants in the methods and
compositions of the present invention include but are not limited to flavin mononucleotide
(FMN), flavin adenine dinucleotide (FAD), deazaflavin, riboflavin, lumichrome, lumizine,
alloxazine, salts of any of the foregoing flavins, hydrates of any of the foregoing flavins,
surfactant-linked derivatives of any of the foregoing flavins, and combinations thereof.
Specific examples of nitrobenzoate compounds include but are not limited to
p-nitrobenzoate, polyethylene glycol nitrobenzoate, and combinations thereof.
Specific examples of nitrobenzoic acid compounds include but are not limited
to m-nitrobenzoic acid, p-nitrobenzoic acid (pNBA), 4-chloro-2-nitrobenzoic acid, 2-chloro-
4-nitrobenzoic acid, p-nitrophenol, nitrophenolates, salts thereof, hydrates thereof, and
Specific examples of haloaryl compounds include but are not limited to
iodobenzoic acid, iodosobenzene, and combinations thereof.
Specific examples of amine oxides include but are not limited to tertiary
amine-N-oxide, N,N-dimethylhexadecylamine N-oxide, N,N-dimethylisooctadecaneamine
N-oxide, N,N-dimethyloctadecylamine N-oxide, N,N-dimethyloctylamine N-oxide, N,Ndimethyltetradecylamine
N-oxide, cocoamide N-(3-dimethylamino) propyl N-oxide, C6-C,4
alkyl dimethylamine N-oxide, bis (2-hydroxyethyl)-3-(decycloxy)propylamine N-oxide,
cocoalkyldimethylamino N-oxide, and combinations thereof.
Specific examples of formamidines include but are not limited to
formamidine acetate, formamidine hydrochloride, formamidine glycolate, formamidine
formate, formamidine sulfuric acid, formiminoglutamate, formiminoglycine, and
Specific examples of glycolates and glycolic metabolites include but are not
limited to glycolate, potassium glycolate, glycolic acid, formate, oxalate, C,-tetrahydrofolate,
salts thereof, hydrates thereof, and combinations thereof.
Specific examples of cytochrome reductases include but are not limited to
cytochrome/ cytochrome c, cytochrome b5, flavocytochrome P450, nitric oxide synthase,
and combinations thereof.
Specific examples of azo compounds include but are not limited to azo dyes,
azodicarboxamide, diazolidinylurea, and combinations thereof.
Specific examples of nicotinic acids include but are not limited to niacin,
NAD, NADP, and combinations thereof.
Specific examples of quinone compounds include but are not limited to
anthraquinone sulfonate, 1,4-bis[(2-ethylhexyl)amino]anthraquinone, tert-butyl
hydroquinone, and combinations thereof.
and combinations thereof.
Compounds selected from the aforementioned classes based solely upon
optimal E0 values would, for example, include anthraquinone sulfonate (-390mV),
bis(dimethylaminocarbonyl)propylbipyridinium (-399 mV),
ethylpropenylmethoxyethylbipyridinium (-396 mV), the oxygen radical (-330), all of which
are operational in the present invention, but may be impractical due to cost. Examples of
preferred oxidants whose selection is based on E0 values and beneficial metabolism include
p-nitrobenzoic acid (-396 mV), glycolic acid (-290 mV), riboflavin (-292 mV), FMN (-313
mV), FAD (-241 mV) and salts, hydrates and surfactant-linked derivatives of any of the
Currently preferred oxidants for use in the methods and compositions of the
present invention include but are not limited to FAD, FMN, pNBA, p-nitrophenolate,
glycolate, and salts, hydrates and surfactant-linked derivatives thereof. FMN is a
particularly preferred oxidant in the compositions, methods and systems of the present
invention, primarily because it is cost effective.
As noted above, the oxidant employed in the present invention may comprise
any two or more of the foregoing oxidants in combination. For example, in one preferred
embodiment, the oxidant comprises a combination of FMN and FAD. In the embodiment of
the invention wherein two or more oxidants are combined, the two or more oxidants are
typically provided in equimolar quantities to provide the oxidant component of the
compositions and methods of the present invention.
Reductants are compounds which induce cytochrome P450 monooxygenase.
Any compound capable of inducing such enzyme will be useful as the reductant component
in the present invention. The reductant is usually selected from the group consisting of
components that are capable of accepting activated oxygen from metalloporphyrins.
Reductants can be hydroxylated, dealkylated, oxidatively deaminated, sulfoxidized, oxidized,
peroxidized, epoxidized and oxidatively dehalogenated by CYP.
a reduction potential (E0) between about 1 and about 2000 mV, and more preferably between
about 600 mV and about 900 mV. Examples of suitable reductants include but are not
limited to cytochromes, peroxisome proliferators, amines, cinnamates, retinoids, fatty acids,
carbamates, manganese, pteridines, terpenoids, alcohols, ketones, pyridines, indoles,
brassinolides, barbiturates, flavones, salts of any of the foregoing, esters of any of the
foregoing, phosphates of any of the foregoing, hydrates of any of the foregoing, surfactantlinked
derivatives of the foregoing, and combinations thereof. Plant metabolites are also
Specific examples of cytochromes which may be employed as reductants in
the methods of the present invention include but are not limited to, hemoglobin, human CYP,
insect CYP, animal CYP, fungal CYP, plant CYP, bacterial cytochromes, viral cytochromes,
microsomes, salts, hydrates, and surfactant-linked derivatives thereof, and combinations
Specific examples of peroxisome proliferators which may be employed as
reductants in the methods of the present invention include but are not limited to
dihydroxytetraeicosatrienoic acid, thiazolidinedinone-4-carboxylic acid, and pimelic acid.
Specific examples of amines which may be employed as reductants in the
methods of the present invention include but are not limited to tyrosine, tyrosine ester, Nacetyltyrosine,
tyrosine methylester, tyrosine methylester hydrochloride, tyramine,
alanyltyrosine, levodopa, aminopyrine, phosphonomethylglycine, and combinations thereof.
Specific examples of terpenoids which may be employed as reductants
include but are not limited to cinnamates such as /ra/w-cinnamic acid; orcinols such as
resorcinol; and hydroxybenzoates such as salicylates and aspirin; and combinations thereof.
A specific example of a retinoid which may be employed as a reductant is
Specific examples of fatty acids which may be employed as reductants
include but are not limited to lauric acid, palmitic acid, arachidonic acids, linoleic acid, and
Specific examples of alcohols which may be employed as reductants include
but are not limited to alkanols such as methanol, ethanol, phenol, alcohol amines such as
triethanolamine, and combinations thereof.
A specific ketone which may be employed as a reductant is acetone.
Specific examples of pyridines which may be employed as reductants include
pyridine, and alkyl substituted pyridines.
Specific examples of pteridines which may be employed as reductants include
but are not limited to aminobenzoic acids such as m-aminobenzoic acid.p-aminobenzoic
acid, PEG-25 p-aminobenzoic acid; tetrahydrofolates such as tetrahydrobiopterin; and
Specific examples of carbamates which may be employed as reductants
include but are not limited to N-(3,4-methylenedioxyphenyl)carbamates, 3-iodo-2-
propynylbutylcarbamate, ammonium carbamate, o-chlorophenyl N-methylcarbamate, and
Specific examples of indoles which may be employed as reductants include
indole-3-glycerol phosphate, indole-3-acetic acid, indole-3-butyric acid, and combinations
Specific examples of barbiturates which may be employed as reductants
include phenobarbital and hexobarbital.
A specific flavone which may be employed as the reductant is isoflavone.
Preferred reductants include various forms of tyrosine such as tyrosine (640
mV), .V-acetyltyrosinamide (650 mV), alanyltyrosine (850 mV), tyrosine methylester (870
mV); tyrosine methylester hydrochloride; amines, particularly aminopyrine; pteridines,
particularly p-aminobenzoic acid, and PEG-25 p-aminobenzoic acid; and hydroxybenzoic
acids (>500 mV).
Compounds that inhibit cytochromes P450 are not generally useful in the
compositions, methods and systems of the present invention. These compounds include
compounds that accelerate degradation or that bind to the heme iron atom or to the
prosthetic heme group. Unsuitable compounds include in general carbon monoxide, carbon
tetrachloride, cyanide, cimetidine, allylisopropylacetamide, piperonyl butoxide, l-[4-(3-
hydrogen peroxide, cumene hydroperoxide, phenylimidazole, aminoglutethimide,
terconazole, fluconazole, saperconazole, miconazole, metyrapone, ketoconazole, parathion,
carbon disulfide, thiourea, tienilic acid, diethyldithiocarbamate, isothiocyanate,
mercaptosteroid, chloramphenicol, dichloroacetamides, undecynoic acid, ethynylpyrene,
ethynylprogesterone, ethynylnaphthalene, secobarbital, dihydropyridine, dihydroquinoline,
1,1-disubstituted hydrazine, acyl hydrazine, alkyl hydrazine, aryl hydrazine, phenelzine,
aminobenzotriazole; syndones, 2,3-bis(carbethoxy)-2,3-diazabicyclo[2.2.0]hex-5-ene, and
Certain of the oxidants and reductants are, by themselves useful in methods of
treating plants and in methods of enhancing the growth of plants. For example, the flavins,
by themselves, or together with an agronomically suitable additive may be useful in the
methods of the present invention without the additional application of a reductant. As
another example, tyrosine and tyrosine esters such as tyrosine methylester and tyrosine
methylester hydrochloride are useful by themselves or together with an agronomically
suitable additive in the methods of the present invention without the additional application of
Typically, however, the oxidant component and the reductant component are
co-applied to achieve beneficial results in methods of treating plants, enhancing growth, and
increasing cytochrome P450 in photosynthetic plants. The co-application of an oxidant and a
reductant does not require the simultaneous application of these components. The methods
of the present invention include the simultaneous application of the oxidant and reductant
from separate sources, the separate application of the oxidant and reductant wherein the
oxidant is applied first followed by the application of the reductant, and the separate
application of the oxidant and the reductant wherein the reductant is applied first followed by
the application of the oxidant. When the oxidant and reductant are separately applied, they
are typically applied at or near the same time, and generally one is applied within a 24 hour
period of the other, preferably within a 12 hour period, more preferably within a 3 hour
period and most preferably within a 1 hour period. In addition, the oxidant and the reductant
may be formulated into a single composition and thereby simultaneously applied to the plant.
Although the oxidant and reductant components may be applied in a solid
form, it is often advantageous to provide the oxidant and the reductant in liquid form, such as
by solubilizing the component in an aqueous or agronomically suitable organic solvent or
carrier to produce aqueous or organic solutions of the oxidant and/or reductant for
application to the plant. The amount of oxidant which is solubilized in the carrier will
depend upon the particular oxidant selected and the method of application. The oxidant may
be solubilized in the carrier by adding the oxidant to the carrier and allowing the oxidant to
dissolve. In some instances, the application of stirring, agitation, or even heat may facilitate
the dissolution of the oxidant in the carrier.
Typically, the oxidant is applied as an aqueous solution having an oxidant
concentration in the range between about 0.0001% and about 1% by weight of the
composition inclusive, preferably between about 0.01% and about 0.5% inclusive. For
example, a flavin mixture of FAD:FMN at a ratio of 829:456 is preferred to match equimolar
ratios generally found in CPR and will range from 8 ppm:5 ppm to 829 ppm:456 ppm. For
glycolate, preferably from about 0.2% to 0.8% glycolate solutions are suitable for germlings;
and from about 0.5% to 5% glycolate solutions are suitable for mature crop plants, more
preferably from about 0.3% to 0.6% potassium glycolate solutions are used for open field
crops. For /?NBA, preferably from about 50 ppm to 300 ppmpNBA solutions are suitable
for seedlings and from about 150 ppm to 900 ppmpNBA solutions are suitable for mature
crop plants, more preferably from about 600 ppm to 800 ppm pNB A solutions are suitable
for open field crops of strawberries.
Similarly, the amount of reductant which is solubilized in the carrier will
depend upon the particular reductant selected and the method of application. The reductant
may be solubilized in the carrier by adding the reductant to the carrier and allowing the
reductant to dissolve. In some instances, the application of stirring, agitation, or even heat
may facilitate the dissolution of the reductant in the carrier. Typically, the reductant is
applied as an aqueous solution having a reductant concentration in the range between about
0.0001% and about 10% by weight of the composition inclusive, preferably between about
0.01% and about 0.3% inclusive. In one preferred embodiment, the reductant is provided at
or below a concentration of about 0.1 %, more preferably at about 0.05%. For example,
salicylates are typically provided in carrier solutions at a concentration of from about 50 ppm
to about 200 ppm by weight for seedlings and sensitive plants and more preferably about
300 ppm to 900 ppm for open field crops. Tyrosines are preferably provided in an aqueous
solution at a concentration from about 600 ppm to 2000 ppm for seedlings and sensitive
plants and more preferably from about 900 ppm to 9000 ppm for open field crops.
In the embodiment wherein the oxidant and the reductant are combined into a
single composition for use in the methods of the present invention, the composition includes
an aqueous or agronomically suitable organic solution having solubilized, dispersed, or
otherwise contained therein, an amount of the oxidant that induces NADPH:cytochrome
P450 reductase in the plant, and an aqueous solution having solubilized dispersed or
otherwise contained therein, an amount of the reductant that induces cytochrome P450
monooxygenase in the plant. The solution containing the oxidant and reductant may be
prepared using the general techniques set forth above for solubilizing oxidant or reductant
Compositions containing both the oxidant and the reductant are advantageous
in that they permit the one-step application of both components to the plant. The one-step
compositions of the invention will comprise an aqueous solution or agronomically suitable
organic solvent emulsion of one or more reductants in combination with one or more
oxidants. Typically, the amount of reductant present is sufficient to balance the amount of
oxidant present, in terms of electron transfer potential, when both are applied to the plant.
The preferred oxidant:reductant molar ratio will be in the broad range of from about 10:1 to
about 1:2, preferably from about 3:1 to about 1:1.
Compositions containing both the oxidant and the reductant component in a
single solution may include any combination of oxidants and reductants selected from those
described hereinabove. Preferred oxidants for one-step compositions include, but are not
limited to glycolates, FAD, FMN andpNBA. Preferred reductants for one-step compositions
include, but are not limited to, alcohols, aminopyrine, aspirin, p-aminobenzoic acid, orcinol,
levodopa, trans-Klinoic acid, tyrosine, and tyrosine esters including tyrosine methylester and
tyrosine methylester hydrochloride. For example, one composition according to the present
invention includes glycolate and tyrosine methylester. Another composition according to the
present invention includes glycolate and alanyltyrosine. Another composition according to
the present invention includes riboflavin and aspirin. Another composition according to the
present invention includes pNBA and aminopyrine.
Compositions of oxidants with reductants will typically be applied at a
concentration ranging between about 0.0001% and about 10%. Preferred combined oxidant
and reductant compositions include: (l)pNBA and aminopyrine applied as an aqueous
solution, each at a concentration in the range of from about 0.001% to about 1%; (2)
FAD:FMN and retinoic acid applied as an aqueous solution each at a concentration in the
range of from about 0.001% to about 0.1%; (3) glycolate and tyrosine methyl ester
hydrochloride applied each at a concentration in the range from about 0.01% to about 5%;
and (4) potassium glycolate applied as an aqueous solution each at a concentration in the
range from about 1000 ppm to about 8000 ppm in combination with salicylate in the range
from about 50 ppm to about 900 ppm.
While the compositions of the present invention may consist essentially of the
aqueous solutions of oxidant and reductant, oil soluble compounds may be formulated in
agronomically suitable organic solvents. For example, 2-methyl-l,4-naphthalenedione and
naphthalic anhydride may be formulated as concentrates with paraffin oil as the carrier for
application in appropriate crop emulsions, hydrosols or organic films.
The compositions of the present invention may also include any of a wide
variety of agronomically suitable additives, adjuvants, or other ingredients and components
which improve or at least do not hinder the beneficial effects of the compositions of the
present invention (hereinafter "additives"). Generally accepted additives for agricultural
application are periodically listed by the United States Environmental Protection Agency.
For example, foliar compositions may contain a surfactant and a spreader present in an
amount sufficient to promote wetting, emulsiflcation, even distribution and penetration of the
active substances. Spreaders are typically organic alkanes, alkenes or polydimethylsiloxanes
which provide a sheeting action of the treatment across the phylloplane. Suitable spreaders
include paraffin oils and polyalkyleneoxide polydimethylsiloxanes. Suitable surfactants
include anionic, cationic, nonionic, and zwitterionic detergents, amine ethoxylates, alkyl
phenol ethoxylates, phosphate esters, PEG, polymerics, polyoxyethylene fatty acid esters,
polyoxyethylene fatty diglycerides, sorbitan fatty acid esters, alcohol ethoxylates, sorbitan
fatty acid ester ethoxylates, ethoxylated alkylamines, quaternary amines, sorbitan ethoxylate
esters, alkyl polysaccharides, block copolymers, random copolymers, trisiloxanes,
CHELACTANTS™ and blends. Surfactant preference is for polyalkylene oxides,
polyalkylene glycols, and alkoxylate-fatty acids. Blends are highly effective such as our
organosiloxane/nonionic surfactant SILWET® Y14242 (Y14242) blend which use is
demonstrated in our examples. Preferred commercial aqueous surfactants include
Hampshire LED3A; HAMPOSYL®; TEEPOL®; TWEEN®; TRITON®; LATRON™;
PLURONIC®; TETRONIC®; SURFONIC®; SYNPERONIC®; ADMOX®; DAWN®, and
the like. Commercial emulsifiers for combination with organic solvent formulations include
WITCANOL®, RHODASURF®, TERGITOL® and TWEEN®. Commercial spreaders
include TEGOPREN®, AGRIMAX™, DOW CORNING® 211, X-77®, SILWET® and the
like. Penetrants such as sodium dodecylsulfate, formamides and lower aliphatic alcohols,
may be used. Alkoxylation of an active component or otherwise chemically modifying the
active components by incorporating a penetrant substance is useful because formulation
without additional surfactant is achieved.
Macromolecules such as CPR and CYP pose problems related to cellular
penetration. Addition of diatomaceous earth, carborundum, fine sand or alumina may be
added to the compositions of the present invention to scratch the leaf surface and assist with
penetration of macromolecules. Small quantities (0.03-0.3%) of sterile diatomaceous earth
are preferred additions to the adjuvant formulation to enhance penetration of
macromolecules. In some cases such as cabbage, in which cells are tough, gentle movement
of the diatoms across the leaf surface by mechanical rubbing or high pressure treatments may
In addition to the foregoing additives, the compositions of the present
invention may also advantageously include one or more fertilizers. Suitable fertilizers for
inclusion in the compositions, methods and systems of the present invention will be readily
determinable by those skilled in the art and include conventional fertilizers containing
elements such as nitrogen, phosphorus, potassium, elevated carbon dioxide, hydrogen
peroxide and the like. Nitrogenous fertilizers (i.e., fertilizers containing nitrogen) are
currently preferred; particularly nitrogenous fertilizers containing not more than 1.5%
ammoniacal nitrogen (i.e., nitrogen in the form of ammonia or ammonium ion), preferably
not more than 1.2% ammoniacal nitrogen, more preferably less than 1% ammoniacal
nitrogen. Nitrate fertilizers (containing less than 1.5% ammoniacal nitrogen) are preferred
fertilizers for inclusion in the methods of the present invention. In particular, in cases
requiring foliar fertilizers, nitrate fertilizers are preferred. Low concentrations of ammonia
fertilizers may be fed to plants at least 2 days after treatment, preferably through the roots.
The amount of fertilizer added to the compositions of the present invention will depend upon
the plants to be treated, and the nutrient content of the soil. Typically, the conventional
fertilizer is included in the amount of between about 1 ppm and about 1000 ppm, preferably
between about 10 ppm and about 400 ppm, and more preferably between about 25 ppm and
about 50 ppm by weight of the composition.
In addition to the conventional fertilizers, the compositions of the present
invention may also include the novel C,-C7 alkyl glucoside fertilizers which are the subject
of copending Application Serial No. , Attorney docket number 15190-000700
filed concurrently herewith in the name of the presently named inventors, the disclosure of
which is incorporated herein by reference in its entirety. Preferred C,-C7 alkyl glucosides
include methyl glucosides, particularly a-methyl glucoside and p-methyl glucoside; ethyl
glucoside, propyl glucoside, and combinations thereof. Currently, the preferred alkyl
glucosides for inclusion in the compositions, methods, and systems of the present invention
are the a-methyl glucoside, P-methyl glucoside, and combinations thereof. As with
conventional fertilizers, the amount of alkyl glucoside fertilizer included in the compositions
of the present invention will depend upon the plants to be treated, and the nutrient content of
the soil. Typically, the alkyl glucoside is included in the amount of between about 0.1% and
about 10.0%, preferably between about 0.5% and about 5.0%, and more preferably between
about 0.6% and about 2.0%.
The .compositions of the present invention may also include any of various
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, which are conventionally formulated in foliar fertilizers. Other
conventional fertilizer constituents which may be added to the compositions of the present
invention include pesticides, fungicides, antibiotics, plant growth regulators, gene therapies
and the like.
The compositions of the present invention may be applied to the plants using
conventional application techniques. Plants nearing or at maturity may be treated at any time
before and during seed development. Fruit bearing plants may be treated before and after
the onset of bud or fruit formation. Improved growth occurs as a result of inducing CYP
The compositions of the present invention may be applied to the plant at a
location including leaves, shoots, root, seed, and stem. The compositions may be applied to
the leaves, seed or stem by spraying the leaves with the composition. The composition may
be applied to the shoot or root by spraying the shoot or root, or dipping the shoot or root in a
bath of the composition, or drenching the soil in which the plant is being cultivated with the
composition, or spray-drenching the leaves and stem of the plant such that the soil in which
the plant is being cultivated becomes saturated with the composition.
Foliar application (i.e., application of the composition to one or more leaves
of the plant) of the compositions of the present invention are currently preferred. The
composition will normally be applied to the leaves of the plant using a spray. However,
other means of foliar application such as dipping, brushing, wicking, misting, electrostatic
dispersion and the like of liquids, foams, gels and other formulations may also be employed.
Side dressing is also applicable. 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®). If desired, oxidant
and reductant compounds may be applied to plants in rapid sequence from separate nozzles
in separate reservoirs. Chemically compatible combined mixtures may be preferred for many
applications to produce improved plant growth. High foliar content of CYP and CPR
maintains high rates of growth during day and night, with greatest response when plants are
exposed to water stress, warmth and high light intensity consistent with prolonged
photorespiration. High potency is achieved by foliar application of compositions containing
oxidant in combination with reductant or readily metabolized precursors, thereto. For
example, the oxidant pNB A is formulated with the reductant aminopyrine; or the oxidant 5'-
deazaflavin may be formulated with the reductant levadopa.
In the embodiment wherein the root and/or shoot is dipped in a bath of the
composition, it is preferred to pulse the application of the composition of the present
invention by dipping the shoot and/or root in the bath containing the composition for a
period of time and then removing the shoot and/or root from the composition. The dipping
period may be from 1 minute to 30 minutes, and is preferably from 10 to 15 minutes.
The compositions of the present invention may also be applied to plant
tissues, such as cell suspensions, callus tissue cultures, and micropropagation cultures. Such
plant tissues may be treated with the compositions of the present invention by adding the
composition to the culture medium in which the plant tissues are being cultivated.
In the methods of the present invention, the compositions are typically applied
in the amount of between about 3 gallons per acre and about 200 gallons per acre, depending
upon the application method. For horticulture applications, the compositions are preferably
applied in the amount of between about 75 gallons per acre and about 125 gallons per acre.
For ground rig row crop applications, the compositions are preferably applied in the amount
of between about 10 gallons per acre and about 40 gallons per acre. For aerial applications
by helicopter or airplane crop dusters, the compositions are preferably applied in the amount
of between about 1 gallon per acre and about 5 gallons per acre. The compositions may be
applied in a single application, or in multiple applications interrupted by a period of
photosynthetic activity. Ornamentals and other tender nursery plants meant for indoor
horticulture will frequently require lower concentrations and perhaps more frequent
application than outdoor agricultural crops.
In general agricultural practice, withholding fertilization of the crop for 2 days
prior to and following treatment with crop enhancers is recommended to prevent
interference. Suitable light and temperature conditions may be achieved by treating plants
within 4 hours of sunrise. Optimal to hot temperatures, usually above 15 ° C and preferably
above 30°C, are required after treatment. 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 three hours after treatments. Sufficient nutrients
should be present to support healthy growth.
Throughout the growing season after treatments, either sun or artificial
illumination should have an intensity and duration sufficient for prolonged high rates of
photosynthesis. A minimum suitable illumination intensity is 200/^mol photosynthetically
active quanta (400-700 nm) m'V, with direct sunlight normally providing much higher
illumination. Prior to treatment, leaf temperature should be sufficiently high for optimal
growth or hotter, usually above 10°C to35°C. After treatment, the leaf temperature will
normally drop as a consequence of improved photosynthetic efficiency. It is preferable that
the plant be exposed to at least a week of intense illumination preferably greater than
SOO^mol photosynthetically active quanta m'V following application of the compositions
of the present invention.
Compositions according to the present invention may be tailored for specific
uses, including enhanced performance or tolerance under environmental stress; enhanced
yield; elongation of growing seasons; aftermarket caretaking; flower retention; fruit
optimization; safening of xenobiotics; and in all areas of agriculture in which optimal growth
is beneficial. Compositions may also be formulated at very low concentrations without
surfactant or spreader for treatments of roots and liquid suspension culture media.
In addition to the methods and compositions described hereinabove,. the
present invention also includes a plant growth enhancing system. The system includes (a)
an aqueous solution containing an amount of an oxidant which induces NADPHxytochrome
P450 reductase in the plant, and (b) an aqueous solution containing an amount of a reductant
which induces cytochrome P450 monooxygenase in said plant. Typically, the oxidant is
selected from the group consisting of flavins, salts of flavins, hydrates of flavins, surfactantlinked
derivatives of flavins, and combinations thereof, although any of the oxidants
described hereinabove may be employed in the systems of the present invention. The
reductants employed in the systems of the present invention may also be selected from those
described hereinabove. Preferred reductants for use in the systems of the present invention
include but are not limited to hemoglobin, tyrosine, tyrosine ester, tyrosine methylester,
tyrosine methylester hydrochloride, N-acetyl tyrosine, tyramine, alanyltyrosine, levodopa,
aminopyrine, salicylates, orcinol, trans-retinoic acid, lauric acid, palmitic acid, maminobenzoic
acid, p-aminobenzoic acid, PEG-25 p-aminobenzoic acid, indole-3-glycerol
phosphate, indole-3-acetic acid, methanol, acetone, pyridine, manganese,
tetrahydrobiopterin, phenobarbital, and combinations thereof. The aqueous solutions
described hereinabove for compositions, using the same types of aqueous carriers. One
preferred system according to the present invention includes flavin mononucleotide as the
oxidant and/7-aminobenzoic acid as the reductant. Another preferred system according to
the present invention includes flavin mononucleotide as the oxidant and PEG-25
p-aminobenzoic acid as the reductant. Another preferred system according to the present
invention includes flavin mononucleotide as the oxidant and tyrosine methylester (e.g.,
tyrosine methylester hydrochloride) as the reductant.
The following examples are provided to further illustrate the present
invention, and should not be construed as limiting thereof. The present invention is defined
by the claims which follow. In these examples, glycine (gly), HAMPOSYL® C, 50%
potassium glycolate (GO), 45% potassium hydroxide (KOH), chelated manganese LED3A,
and purified water were obtained from Hampshire Chemical Corporation.
Human Cytochrome P450 (CYP 2E1) and Human NADPHiCytochrome P450
Reductase (Human CPR) were obtained from PanVera Corporation.
W-acetyltyrosinamide (NATA), alanyltyrosine (AlaTyr) aminopyrine (AP),
ascorbic acid, CELITE®, ethanol (Ethan), glycerol, levadopa, potassium chloride (KC1),
potassium cyanide (KCN), methanol (MeOH), polyvinylpolypyrrolidone (PVPP), potassium
phosphate, sodium bicarbonate, and L-tyrosine (Tyr) were obtained from Fisher Scientific.
jV-acetyltyrosine (NAT), adenosine triphosphate (ATP), amino-n-caproic acid, aprotinin,
benzamidine HCI, 7-benzyloxyresorufm (7B), bovine serum albumin (BSA), trans-cinnamic
acid (cinnamic), cytochrome c, dithiothreitol (DTT), ethylenediamine tetraacetic acid
(EDTA), ethylene glycol-bis(p-aminoethyl ether) (EOTA), flavin adenine dinucleotide
(FAD), formaldehyde, formamidine acetate (FAM), formic acid, HEPES, leucovorin,
leupeptin, magnesium chloride (MgCl2), nitromethane (NM), nicotinamide adenine
dinucleotide phosphate (NADP), orcinol monohydrate (OR), 4-aminobenzoic acid (PABA),
pepstatin, potassium maleic acid (malate), pteroic acid (pteroic), trans-relinoic acid (RET),
riboflavin (B,), salicylic acid (sali), triethanolamine, TRITON® X-100, tyrosine methyl ester
HCI (TyClMe), and valine (Val) were obtained from Sigma.
4-Nitrobenzoic acid (pNBA) was obtained from Nordic Synthesis.
Ethoxylated PABA (UVINUL® P-25) and PLURONIC® L-92 were obtained
14CO2 was obtained from ICN.
SYNPERONIC® and TWEEN® were obtained from ICI.
Flavin mononucleotide sodium salt(FMN) was obtained from Roche Vitamins
SILWET® Y14242 and 408, OSi; ADMOX® 10, 12,14, Albemarle;
iodosobenzene (IO) were obtained from TCI.
AGSOLEX® 1 was obtained from ISP.
In these examples, "L" means liter; "ml" means milliliter; "cm" means
centimeter; "cm2" means centimeters squared; "nm" means nanometer; "g" means grams;
"mg" means milligrams; "M" means molar; "mM" means millimolar; "nM" means
nanomolar; "uM" means micromolar; "mol" means moles; "umol" means micromoles;
"mg/ml" means milligrams per milliliter; "ml/cm2" means milliliters per centimeter squared;
"ppm" means parts per million based on weight;"%" or "percent" means percent by weight
(of the composition); "kDa" means kiloDaltons; "L/min" means liters per minute; "h" means
hour(s); "min" means minute(s); "s" means second(s); "Xg" means multiple of centrifugal
gravitational force; "°C" means degrees Centrigrade (all temperatures are in °C, unless
Following are examples of specific compositions according to the present
invention which may advantageously be employed in the methods of the present invention to
treat plants, and to enhance growth in plants to increase cytochrome P450 in plants. The
following exemplary compositions are intended to provide further guidance to those skilled
in the art, and do not represent an exhaustive listing of compositions within the scope of the
Fifth Exemplary Composition: Foliar
Component Grams Range
FMN 17.34 0.5-2X
Uvinul®P-25 96.14 0.5-3X
Surfactant (dry powder) 100 0.5-3X
The slurry is warmed to 40 °C and stirred into 20 °C to 40 °C water to a final volume of 76
liters. The solution is adjusted within a range of pH 6 to pH 7 with suitable buffer.
Addition of a spreader is recommended prior to application. The solution is sprayed on
foliage or may be applied to any plant part.
Sixth Exemplary Composition: Dry Powder
Component Grams Range
Tyrosine methyl ester HC1 139 0.5-2X
Potassium glycolate 575 0.5-3X
Surfactant (dry powder) 100 0.5-3X
The homogenous dry powder is stirred into tap water at about room temperature to a final
volume of 100 liters. The solution is adjusted within a range of pH 5 to pH 7 as needed with
base or suitable buffer. Addition of a spreader is recommended prior to application. The
solution is sprayed on foliage or may be applied to any plant part.
The following example illustrates the application of numerous compositions
according to the present invention to many varieties of plants. The data demonstrate the
efficacy of the methods and compositions of the present invention in the treatment of plants.
Materials and Method
Plants tested under controlled and greenhouse conditions for growth response
included radish cv Cherry Bell, pepper cv Bell Boy, wheat cv Geneva, pansy cv Delta Pure
White, impatiens cv Super Elfin Violet and corn cv Butter Sugar. Gas exchange and
metabolic analyses were undertaken on soy (Glycine max cv Corsoy variety 9007 and 9008
Pioneer, Johnston, Iowa), sugar beet (Beta vulgaris L cv NBlxNB4 (United States
Agricultural Research Station, Salinas, California) or cv Monohikari (Seedex, Longmont,
Colorado), sunflower (Helianthus anuus), cabbage (Brassica oleracea var. Capitata) and red
beet. Radish cv Cherry Bell and Pepper cv Bell Boy were the preferred cultivars for our
standard screening assays. Radish was ready for treatment 7 d after planting and yielded
significant weight differences 7 d to 14 d after treatment. Radish showed changes in root
and shoot yields. Pepper was responsive within a week of treatment. In general,
determination of whether a formulation was phytobland or phytotoxic was visibly evident
within 5 days. The following plant varieties were treated in commercial greenhouses:
To compare the effects of treatments under tightly controlled conditions,
seeds were sown in individual 12 to 16 cm diameter plastic pots containing METRO-MIX®
350 growing medium (Grace Horticultural Products, W.R. Grace & Co., Cambridge, MA) or
PETER'S® Professional Potting Soil (Scotts-Sierra Horticultural Products Co., Marysville,
Ohio) containing complete nutrient pellets (Sierra 17-6-12 Plus Minors, Grace Sierra,
Milpitas, California) or fertilizers such as Hoagland nutrients were added regularly as
needed. Culture was in controlled environmental growth chambers (16 h light:8 h dark
photoperiod, 400-700 ^mol photosynthetically active quanta-m'V, 24-30°C and 30% RH) at
the University of Massachusetts or the University of Wyoming. Alternatively, plants were
cultured in greenhouses with the option of supplemental light provided by 1,000 watt metal
halide arc lights (16:& h photoperiod). In University of Wyoming greenhouses, physical
conditions were controlled and in these as well as other greenhouses, treatments and controls
being compared were made simultaneously and were subjected to identical conditions
consistent with good laboratory practices. Each survey pool held 20 or more replicates per
compound tested and these were matched with equal numbers of controls. Plants were
generally harvested and analyzed in the vegetative stage within two weeks after treatment.
Plants in individual pots received 1 ml to 5 ml of solution per treatment applied with a small
hand-held sprayer constructed of an atomizer head attached to a 5 ml syringe or with larger
commercial sprayers. Plants in trays received approximately 50 ml of solution per treatment
with even distribution and pressures as would be expected of commercial sprayers.
Generally, individual plants received approximately 0.1 ml/cm2 of solution to leaves. Plants
were watered daily with measured amounts of purified water.
The performance of compounds was surveyed by comparing yields against
untreated controls and 18 mM glycolate + 6 mM tyrosine positive controls. Yields were
optimized by bracketing around the following concentrations: 10 /*M retinoid, 500 ^M
flavin, 200 ^M aminopyrine, 5 mM substituted aryl, 10 mM glycolate and generally 6 mM
for others in aqueous solution. Separated active components were included as positive
controls to initial tests of mixtures.
Surfactants were compared and phytotoxicity was observed at effective
surfactancy levels of TWEEN® 80, HAMPOSYL® C and TRITON® X-100. SILWET®
Y14242 surfactant blend effectively wet foliage of plants at 400 ppm to 1200 ppm without
phytotoxicity, but concentrations above 2400 ppm reduced growth and caused foliar damage
in laboratory and greenhouse investigations. As a standard procedure, 800 ppm Y14242 was
added to formulations unless otherwise noted.
Some fertilizers were less effective than others. To test effects of ammonia as
compared to nitrate fertilizers, young begonia and radish plants were fed with Hoagland
solution nutrients modified to contain either nitrate or ammonia as nitrogen sources. In these
preliminary tests comparing nitrogen sources, treatments withpNBA were followed with
daily irrigations of 50 ppm nitrate or 50 ppm ammonia modified Hoagland solution
nutrients. Growth enhancement was observed with nitrate, but not with ammonia fertilizer;
therefore, ammonia fertilizers were eliminated or minimized to 1.5% of the nitrogen nutrient
composition or less during the course of the investigations.
For the majority of tests of productivity yield, plants were harvested within 1-
4 week(s) of treatment. The plants were removed from pots and the roots were rinsed clean.
Shoot and root lengths and fresh and dry weights were determined. Changes in shoot and
root growth were recorded in all cases and are variously presented to model growth of plants.
Where appropriate, harvested populations were subjected to analysis of variance and mean
separation by LSD test and showed significance within 95% confidence limits.
Trials were undertaken in commercial greenhouses to verify practical
application methods and beneficial outcome of various treatments. Automated plantings and
large populations in commercial settings provided uniformity of results. Plastic trays with up
to 512 cells were labeled, filled with media and sown by machine. Transplants to plastic 36
to 48 cell flats were undertaken after 5 to 8 weeks of culture depending on variety and
schedules. Media such as BERGER® and METRO® mixes appropriate to the plant types
were used to filled cells. Commercial foliar nutrient formulas were applied manually or by
automated overhead systems. Irrigation with water was supplied daily, but nutrients were
withheld 2 days before and after treatments. Plants in plug trays were generally treated at
emergence of the first true leaves. Treatments consisted of foliar sprays and control
solutions. Untreated controls were allocated in most cases. Baselines of 100% growth were
established for growth of controls as bases for comparisons against each active substance.
The percentage of change in growth caused by the tested substance is presented from which
the control data can be back-calculated. Mixtures of active materials contained adjuvants
because they did not show activity otherwise, therefore, laboratory controls included plants
that were treated with the adjuvants at equivalent dilutions. In commercial trials, controls
were left untreated. Diseased or aberrant plants were eliminated prior to test. Insects were
controlled by regular treatments with appropriate commercial pesticides.
One lead compound, nitrobenzoic acid, is an oxidant and a precursor to
pteridines. Initially, we characterized pNB A without pairing it with a reductant. Though
effective in laboratory trials, unpaired pNBA was not as consistent in commercial trials as
paired pNBA+Reductant formulations. Nitrobenzoates have known relation to CPR and,
consistent with our method of selection, pNB A showed high potency and consistent
enhancement of plant growth when formulated with reductants. Previous research on
nitrobenzoates had been derived from animal liver microsomes (see, for example, H. Sasame,
et al., A/o/. Pharmacol. 5:123 (1969)); therefore, given our observed plant responses to
pNBA, the possibility of plant response to human CPR was examined. Frozen human
NADPH:Cytochrome P450 reductase (hCPR) pellets from a recombinant DNA source were
diluted in chilled water adjusted to pH 7 to pH 7.5 with dilute KOH. Penetration into cells of
foliage by the macromolecular 76.5 kDa enzyme was achieved by addition of sterilized 0.1%
CELITE® (diatomaceous earth) kept in suspension with shaking as the mixture was applied
to foliage. Hand pumped sprayers were held within 2 to 3 cm of leaf surfaces to maximize
pressure and flow of the treatment solution across the leaf surface. Rubbing the solution into
the leaves to enhance the microincisive action of CELITE® was required for leaves with
thick cuticles such as cabbage, but the spray pressure allowed penetration into pepper and
radish leaf cells without additional mechanical intervention. Initially, a concentration
gradient was assayed on radish and 10 nM hCPR induced turgidity within an hour, enhancing
vegetative yield within two weeks of foliar treatments. Thereafter, 10 nM hCPR was
formulated in water adjusted to pH 7.5 with dilute 1 mM KOH, 0.1% CELITE® and 800
ppm Y14242. Formulations were sprayed on foliage within an hour and kept chilled on ice
to prevent degradation.
After establishing that foliar treatment with hCPR enhanced plant growth,
direct effects of substrates on hCPR and on sugar beet CPR was measured by preparation of
microsomes for quantification against CPR and cytochrome c. For the initial preparations,
ultracentrifuge-derived microsomal preparations of sugar beet CPR (sbCPR)were assayed
against combinations of GO, NAT and FMN as substrates. Preliminary preparation of sugar
beets involved germination in growth chambers followed by transplantation to greenhouses.
Substrate formulations were dissolved in water with 0.12% Y14242 surfactant blend and
sprayed onto the foliage of sugar beets. Controls included equal concentrations of each
individual substrate in surfactant and water and untreated plants under otherwise identical
conditions of culture. Enzyme assays were undertaken 2 days after treatments. The
procedure followed previously described methods (e.g., M. Markwell, et al., Methods of
Enzymology 72:296-303 (1981); C.A. Mihaliak, et al., Methods in plant biochemistry
19:261-279 (1993); R. Donaldson, et al.,Arch. Biochem. Biophys. 152:199-215 (1972); M.
Persans, et al., Plant Physiol. 109:1483-1490 (1995)) and includes a two phase partitioning
employing a 5.6% polymer concentrations with no K.C1 and the grinding buffer and 0.1 M
phosphate assay buffer contained protease inhibitors such as aprotinin (2 mg/mL),
amino-n-caproic acid (5 mM), benzamidine HCI (1 mM), leupeptin (2mg/mL), and pepstatin
A (2mg/mL). Leaves of 54 day-old sugar beet were collected and 24.5g was weighed and
chopped. All preparations were kept chilled at 0-4 °C . Chopped leaves were homogenized
by mortar and pestle in 100 mL of 50 mM HEPES (pH 7.6), 230 mM sorbitol, 1 mM DTT,
ImM EDTA, 3 mM EGTA, 0.5% BSA, 10 mM KC1, 5% glycerol, insoluble
polyvinylpolypyrrolidone (1.25 mg/mL), 20 mM sodium ascorbate and protease inhibitors.
The homogenate was forced through six layers of cheese cloth. The crude microsome pellet
was prepared by differential centrifugation. The filtrate was centrifuged three times at
1700Xg for 5 min to remove debris. The supernatant was centrifuged at 27KXg for 30 min
to sediment mitochondria and chloroplasts. The pellet was resuspended in the assay buffer
(3 mL). The supernatant from the 27KXg spin was centrifuged at 75KXg for 1 hr. The
crude microsome pellets were saved in 1.2 mL assay buffer. The unused microsomal pellets
and 27KXg pellets were resuspended in 0.1 M potassium phosphate (pH 7.4). The pellets,
suspensions and the 75KXg supernatant were frozen at -20° C for later use. Protein
contents of microsomes, 75KXg supernatant and 27KXg pellets were determined by
modified Lowry method using BSA as standards. NADPH-dependent reduction of
cytochrome c was monitored for increase in absorbancy at 550 nm at room temperature. The
incubation medium contained 60 mM phosphate buffer (pH 7.4), NADPH (1 mM), 0.5 mM
KCN and microsomes in a 1 mL cuvette with a 1 cm path length. The reaction was initiated
by addition of horse heart cytochrome c to a 50 mM final concentration. The absorbancy at
550 nm was measured every 30 seconds for 5 min. One unit is defined as an absorbancy
change of 1.0 /min at 550 nm at 25 ° C in a 1 cm light path. This corresponds to reduction of
0.0476 ^mole of cytochrome c per minute per milliliter of reaction mixture. The rate was
determined by the difference between the samples with or without NADPH. This method of
assay was selected over Cytochrome P450 quantification by CO difference spectroscopy to
avoid interference with pigments and sample turbidity. The reaction cocktail contained the
following components (0.5 mM KCN was added to inhibit the cytochrome c oxidase
activity): water (200 ml), phosphate (600 ml), KCN (50ml), microsome (50 ml), NADPH
(50 ml), cytochrome c (50 ml).
Gas exchange, osmotic potential, enzyme and radioisotope assays were
undertaken in the laboratory of Professor John N. Nishio at the Department of Botany,
University of Wyoming, Laramie, WY. Photosynthetic CO2 gas exchange was measured
with a CIRAS-1 (PP Systems, Bedford, MA) portable gas exchange system. Foliage was
treated with compounds dissolved in standard aqueous solutions with surfactants. Gas
exchange was measured after treatments as a means of checking the health and
responsiveness of plants particularly when the tissues were sampled. Gas exchange was
nearly doubled in response to some treatments. For quantification of response of plants to
stress after treatment with oxidants and reductants, plants were adapted to 23 °C, 500
umol/nr/s light intensity and 80% to 90% humidity for more than 2 weeks prior to the start
of the experiment. Photosynthesis and respiration were measured polargraphically in a
Clark-type Rank oxygen electrode. Foliage was sprayed or compounds were added directly
into the oxygen chamber. Formulations and combinations included 100 mM glycolate, 50
mM salicylate, 50 mM tyrosine or 50 mM pNBA. Specific settings were 1.77 cm2 leaf
discs, 300 to 400 mm slices, 2 ml of 40 mM HEPES at pH 7,10 mM sodium bicarbonate in
HEPES at pH 7, registration speed 1 cm/min, light intensity 1350 nmol/nr/s, chamber
temperature 42°C, adaptation of slices to temperature given 5-8 min in room light, and time
of zero oxygen was 7 to 12 min. In general, plants treated with oxidant+reductant
formulations stood erect with turgidity when placed under environments where controls
wilted in midday, indicating that some treatments enhanced tolerance to water stress.
Therefore, osmolality was measured with a 51OOB vapor pressure osmometer (Wescor). The
osmometer was calibrated with a paper disc and standard plugs were placed in the tray for
Radioisotopic MC02 was applied to plants to determine the fate of active
substances and changes in the path of carbon fixation. Plant specimens were sprayed with
the formulations. 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 the laboratory conditions for 15-30 min. To test photorespiration, a leaf was
placed in an open chamber that was constantly flushed with pure O2 during the acclimation
period. Leaf plugs 3.67 cm2 were removed and placed in a hermetically sealed
PLEXIGLASS® leaf chamber containing pure O2 being pumped at a rate of 2-3 L/min. To
test ambient conditions, air was used instead of pure oxygen gas. The chamber was
illuminated with 1,000 /^mol photosynthetically active quanta m"2s' directed through a fiber
optic cable connected to a quartz halogen light similar to the one used for preillumination.
After 1 min, 5 mL CO, containing 0.8 jaCi Na'4CO2 (specific activity of 5 Ci mol'1) was
injected with a syringe to a final concentration of about 700 ppm C02. The leaf plugs were
allowed to incorporate I4CO2 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 ethanol containing formic acid. Stable fixed 14CO2 containing products were
separated by paper chromatography as previously described in R. D. Gates, et al., Proc. Nat I.
Acad. Sci. USA 92:7430-7434 (1995). Protein content of samples was determined by a
modified Lowry Procedure, previously described in J.N. Nishio, et al., Plant Physiol.
Nuclear magnetic resonance (NMR) 13C in vivo studies were undertaken in
the laboratory of Professor Roland Douce at the University of Grenoble, Laboratories of
Plant Cell Physiology, Centre d'Etudes Nucleaires de Grenoble, CEN-G, 85-X, F-38041
Grenoble Cedex, France utilizing a Bruker AMX400. Uniformly 13C labeled glycolate was
prepared by Professor A. A. Benson. Solutions of 10 mM 13C labeled glycolate+5 mM Ltyrosine+
800 ppm Y14242 were prepared and continuously supplied to sycamore cells.
Collections of 9 g treated cells were perfused with oxygen and placed in 25 mm diameter
tubes. The spectra at 900 scans/hour were taken under the following conditions: 30 [is
impulses at 60° and 4 s; decoupler Waltz sequence of 9 watts (0.38 s) with 0.5 watt (3.64 s)
period of acquisition. Fourier transform was performed at 16000 points acquired per 16000
zero filling points. Measurements and analyses were compared against reference standards
such as hexamethyldisiloxane (resonance peak 2.9 ppm). Other reference resonances
corresponded to intracellular compositions typical of 100 (iM/g cells.
As shown in Table 2 below, foliar solutions containing nM human CPR
(hCPR) or mM CPR substrates enhanced growth significantly. At harvest, plants treated
with hCPR and tyrosine were larger and stiffer with turgidity as compared to tyrosine treated
and untreated control plants. To the contrary, plants treated with hCPR plus glycolate did
not show significantly higher yields than controls, i.e., growth was not enhanced. Glycolate
was, therefore, placed in the oxidant category and tyrosine was appropriately placed in the
reductant category. Controls were formulated without the active component, but contained
equivalent Y14242 and CELITE®. Pepper plants tabulated in Table 2 were grown side-byside
In all cases examined and shown in Table 3, paired oxidants and reductants
showed greater growth enhancement than separate oxidant or reductant treatments. In
another related growth experiment, we found that the formulation of FMN+FAD+Tyrosine,
given in Table 3, was as effective at increasing pansy shoot dry weight yields (113%) within
10 d as it was at improving radish root yields over controls.
Surveys of different sets of paired oxidants with reductants resulted in
enhanced vegetative yields as shown in Table 4, below.
Table 4. Effect of Paired Oxidant+Reductant Treatments on Plant Growth
The oxidant+reductant formulation containing glycolic+salicylic, having been
proven effective as a plant growth enhancer (see Table 3), was assayed for effect on the
catalyst, CPR. The results of treatment of CPR with glycolic and salicylic acids are given in
Table 5 below, in which specific activities, expressed as nanomoles of reduced cytochrome
c per minute per mg of protein, are summarized. The pellet referred to in Table 5 is from
Table 5. hCPR Specific Activity
Reductase pellet (treatment)
Control 75K x g pellet
GO+Sali 75K x g pellet
75K x g supernatant control
75K x g supernatant GO+Sali
Control 27K x g pellet
GO+Sali 27K x g pellet
After treatment with GO+Sali, specific activity of the reductase was 20 times
higher than the control.
In Table 6, below, the specific activities of oxidants and reductants were
measured separately and combined against human CPR and sugar beet CPR. Individual
treatments of glycolate and salicylate each increased hCPR activity by 13%, whereas,
GO+Sali increased hCPR activity by 20 percent. Similarly, when the specific activities of
glycolate, N-acetyltyrosine and FMN were measured separately and combined against
microsomal sbCPR, combinations of substrates induced the enzymes more than individual
substrates. In fact, when treated with NAT alone, specific activity dropped, but formulations
of GO+NAT increased specific acitivity by nearly half-again. Notably, FMN showed
significantly higher induction than any other individual substrate tested. The combination of
Table 6. Reductants and Oxidants Synergistically Enhance
Human CPR and Sugar Beet CPR
In Table 7 below, assimilation rates and assimilation/transpiration (A/T) rates
of five soybean plants were measured in the morning 15, 16, and 17 days after treatment.
Plants were grown in the greenhouse at the University of Wyoming and measurements were
made 57 days from sow date. All 15 measurements per treatment were pooled to calculate
average rates (n=15). Probability calculated as a two-tailed Student's T-test is 0.000 for
FMN and 0.002 for FAD against controls for A/T supporting the observation that
photosynthesis increases for long durations after FMN and FAD treatments.
Table 7. FMN and FAD enhance photosvnthetic gas exchange for a lone duration.
Plants treated with 125 ppm FMN showed a higher degree of turgidity than
controls, especially when controls showed signs of water stress. Water potential of
FMN-treated sugar beets and controls was measured with an osmometer. FMN-treated sugar
beet showed improved water potential values, an average of 3 plant measurements at -3.83
milliPascals, as compared to controls which averaged -4.71 milliPascals. Probability
calculated as a two-tailed Student's T-test was 0.058.
In Table 8, below, pepper plants were treated with 10 mM GO+2.5 mM
Tyr+800 ppm Y14242. Gas exchange was measured under clear morning skies in a glass
greenhouse. Table 8 shows two runs of the experiment that were undertaken. Carbon
dioxide uptake occurred at significantly higher rates in plants treated with GO+Tyr as
compared with untreated controls.
Further characterization of glycolate+L-tyrosine treatments showed
quantifiably higher osmotic pressure corresponding to visually observed turgidity
enhancement over controls. Pepper plants that were treated with GO+Tyr showed an
improved osmotic pressure of-24.3 Bars as compared to controls without treatments that
showed an osmotic pressure of-21.1 Bars. The improved osmotic pressures measured for
GO+Tyr treatment corresponded to the visual observations of clearly higher turgidity in
treated plants in contrast to wilted untreated controls.
Plants that were treated with pairs of oxidant+reductant tolerated stress with
enhanced photosynthesis as compared with controls and as shown in Table 9, below.
Consistent with other plant responses, the data in Table 9 shows that treatments with
substrates of CYP and CPR enhance photosynthetic oxygen evolution (umol/m2/s). When
oxidase and reductase substrates are combined at appropriate ratios and concentrations,
enhancement is greater than when either substrate is added alone.
NMR spectra of glycolate and tyrosine treated sycamore cells elucidated
inhibitory action of the paired formulation. Without tyrosine, the 13C labeled glycolate was
passed on to other metabolites. Preabsorbed tyrosine inhibited metabolism of I3C labeled
glycolate in nonphotosynthetic sycamore cells.
Our results show that when CYP and CPR are induced, photosynthesis and
plant growth are enhanced. With few exceptions, when either of the CYP or CPR substrates
was supplied without the necessary electron couple or enzyme partner, treatments were
ineffective or inconsistent. The most potent treatment was foliar nanomolar CPR with
reductant substrates such as tyrosine. Of the CPR substrates which stimulated growth at uM
concentrations, FMN may be ranked as the most practical oxidant, being both safe and
effective. In all cases, CYP inducers did not enhance growth as much as when applied with
CPR or its substrates; however, induction of human CPR with oxidants and reductants
indicates a deeper tandem involvement of the reductase than had been known previously.
Results that showed paired treatments enhanced gas exchange are consistent with inhibition
of glycolate metabolism observed by NMR. Furthermore, physiological and biochemical
enhancement caused by treatments are descriptive of plant growth and yield enhancements in
the long term. Our studies provide conclusive evidence that induction of cytochromes P450
is key to plant growth.
Formulations that coupled pNB A with reductants generally showed high
potency and consistently higher yields than other pairs. The observed plant responses to
human CPR by coapplication of the enzyme with reductants in our experiments was
consistent with our hypothesis for the role ofpNBA. Selection of reductants and oxidants
based on one electron reduction of compounds (see, e.g., P. Wardman, J. Phys. Chem. Ref.
Data 18(4): 1637-1755 (1989)) within potentials associated with CPR reductase (see, e.g., J.
Butler, et al., Biochimica et Biophysica Acta 1161:73 (1993)) proved successful given the
substrates we discovered to improve yields. The combinations of cytochromes P450
reductases with monooxygenase substrates are numerous and underscore the potential of the
Photorespiration is a universal plant response to light, heat, O2 and CO2. N.
Tolbert, et al., Proc. Natl. Acad. Sci. USA 92:11230-11233 (1995). Radicals generated
during photorespiration damage the photosynthetic apparatus. By nature of its interference,
once photorespiration is controlled, enhanced productivity of all plants becomes possible. In
our studies, growth of plants was enhanced under conditions favoring photorespiration after
foliar treatments with formulations designed to enhance CPR and CYP. Photorespiration is a
major endogenous source of glycolate and this chemical imbalance sends signals to stop
related metabolic functions. A. Angerhofer, et al., Photochemistry and Photobiology 63:11-
38 (1996). From our nuclear magnetic resonance studies, inhibition of exogenous glycolate
metabolism was evident. Glycolate production inhibits CO, fixation. See, M. Badger, et al.,
Photosynthesis Research 37:177-191 (1993); A. Miller, et al., Plant Physiol. 91:1044-1049
(1989); T. Takabe, et al., Biochemistry 19:3985-3989 (1980); and C. Wendler, et al., J. Plant
Physiol. 139:666-671 (1992). In contrast, when we combined foliar applications of glycolate
with CYP reductants, we observed increased CO2 fixation and enhanced tolerance to the
photorespiratory stimuli. Initially, we selected glycolate as an electron donor, but to our
surprise, we observed that 5 mM to 30 mM glycolate concentrations synergistically enhanced
the activity of reductants, but not oxidants.
Work over the past decades has taken our knowledge of cytochromes P450
from identifying enzymes without function to highly characterized proteins with defined
catalytic electron transfer functions. See, C. von Wachenfeldt, et al., Structures of
Eukaryotic Cytochrome P450 Enzymes, P. R. Ortiz de Montellano, ed. (1995) CYTOCHROME
P450: STRUCTURE, MECHANISM, AND BIOCHEMISTRY (Second Ed.), Plenum Press, New
York, pp 183-223 and H. Strobel, et al., NADPH Cytochrome P450 Reductase and Its
Structural and Functional Domains, P. R. Ortiz de Montellano, ed. (1995) CYTOCHROME
P450: STRUCTURE, MECHANISM, AND BIOCHEMISTRY (Second Ed.) Plenum Press, New
York, pp 225-244. Exploitation of CYP has not previously been reduced to practice in
plants, but from investigations of biochemical pathways, it has been known that CYP
enzymes are involved in the metabolism of single carbon fragments, abscisic acid, ethylene,
gallic acid, cytokinin, lignin, furanocoumarin, anthocyanin, gibberellic acid, limonene,
geraniol, nerol, dhurrin, bisbenzylisoquinoline alkaloids, jasmonic acid,
phophonomethylglycine, sulfonylurea, phenylurea, aryloxyphenoxypropionate, metflurazon,
sethoxydim, bentazon and insecticides. See, M. Schuler, Critical Reviews in Plant Sciences
15(3):235-284 (1996). Some compounds which may be metabolized into phytotoxicity,
might enhance herbicidal action. For example, if activity of a reductant herbicide is
targeted, then formulating it with an oxidant such as N-3-nitrophenyl-N'-phenylurea may
speed its action. Furthermore, oxidants such aspNBA, l,4-bis[(2-
ethylhexyl)amino]anthraquinone or 1,4-bis(2-methylanilino)anthraquinone may be
compatible with a reductant herbicide such as phophonomethylglycine. Our methods are
also appropriate to stimulate enhancement of blooms. Interaction of N-phenylcarbamates
with CYP has been shown to induce flowering in asparagus seedlings. See, M. Kusukawa, et
al., Z. Naturforsch 50c:373-379 (1995). The results of our experiments with formamidines
support the relationship of CYP to flowering, combinations of oxidants with reductant
formamidines showing potential for floricultural product development.
Of the reductants that we surveyed for pairing, tyrosine is notable. Without
oxidant additions, the effects of tyrosine on plant growth were inconsistent. In our
experiments the derivative of tyrosine with the highest electron reduction potential, tyrosine
methyl ester (870 mV) showed the most consistent plant responses as compared against those
with lower electron potentials. Combinations of tyrosines withpNBA, glycolate, FMN, and
FAD yielded nontoxic and practical formulations.
The requirement that we have shown for electron couples to elicit plant
growth responses is consistent with monoxygenase and reductase necessary for metabolism
of typical human cytochromes P450 substrates in bacteria. Transformed Escherichia coli
metabolized monooxygenase substrates when CPR reductase was coexpressed with CYP
monoxygenase (A. Parikh, et al., Nature Biotechnology 15:784-788 (1997)), in this case,
accomplished with a bicistronic vector. Expression of intergeneric cloning of yeast CPR
reductase has been demonstrated. See, E. Kargel, et al., Yeast 12:333-348 (1996). Specific
CPR reductase sequences encoded for glycolate, once isolated, may find expression during
periods of light saturation. For example, tyrosines are closely associated with CYP (see,
e.g.,E. Halkier, et al., Plant Physiol. 96:10-17 (1991)), and in fact, all forms of tyrosine that
we tested showed consistent growth responses when paired with reductants. The reaction
center of the photosystem II oxygenic electron transport chain contains two redox-active
tyrosines, Tyrl60 Y sub D and Tyrl61 Y sub Z (see, e.g., G. MacDonald, et al., Proc. Natl.
Acad. Sci. USA 90:11024-11028 (1993)) and these tyrosines are involved as electron donors
to the water-oxidizing complex of photosynthesis in the cytochrome c mediated reduction of
photooxidized chlorophyll. See, J. Wachtveitl, et al., Biochemistry 32:10894-10904 (1993).
Given the fundamental relationship of tyrosine to photosynthesis in association with the
primary sequence of CYP P450tyr (see, B. Koch, et al., Archives of Biochemistry and
Biophysics 323:177-186 (1995)), plants might be genetically altered and bred for increased
levels of the enzymes associated with such CYP functions. For example, if expression of
CYP P450tyr is engineered to be triggered by photorespiration, the inhibition of glycolate we
observed by exogenous application of tyrosine may prove as beneficial to the enhancement
of plant growth as that which we observed in growth studies. Expression of an oxygen
transport complex foreign to plants, such as hemoglobin, has been demonstrated in tobacco
by fusion of coding sequences of globins to chloroplastidic transit peptide of the small
subunit of Rubisco from pea. See, W. Dieryck, et al., Nature 386:29-30 (1997). Transgenic
tobacco expressing haemoglobin exhibits enhanced growth and metabolites. See, N.
Holmberg, Nature Biotechnology 15:244-247 (1997). Similar techniques may be applied for
the insertion and amplified expression of coding sequences for CYP to give long-term results
similar to our foliar treatments.
Based on the results of our biochemical, physiological and growth studies,
we conclude that our treatments of plants to induce CYP and CPR cause increases in the rate
and quantity of carbon fixation. The ubiquity of CYP and CPR provides universal
applicability of these compositions and methods for selection of components which endows
plants with a means of resistance to environmental and chemical stresses while gaining ever
greater photosynthetic productivity for all plants.
The foregoing is illustrative of the present invention and is not to be construed as
limiting thereof. The invention is defined by the following claims, with equivalents of the
claims to be included therein.
1. A composition for enhancing growth of plant, said composition comprising:
(a) an aqueous solution containing an oxidant which induces NADPH: cytochrome P450 reductase in said plant, wherein said oxidant is selected from the group consisting of flavins; salts of flavins; hydrates of flavins; surfactant-linked derivatives of flavins; and combinations thereof; and
(b) an aqueous solution containing a reductant which induces cytochrome P450 monooxygenase in said plant;
wherein oxidant: reductant molar ratio is in the range of from 10 : 1 to 1 : 2.
2. The composition as claimed in claim 1, wherein said oxidant is selected from the group consisting of flavin mononucleotide, flavin adenine dinucleotide, riboflavin, deazaflavin, salts thereof, hydrates thereof, surfactant-linked derivatives thereof, and combinations thereof.
3. The composition as claimed in claim 1, wherein said reductant is selected from the group consisting of cytochromes, amines, cinnamates, retinoids, fatty acids, pteridines, terpenoids, alcohols, ketones, pyridines, indoles, brassinolides, barbiturates, and combinations thereof.
4. The composition as claimed in claim 1, wherein said reductant is selected from the group consisting of hemoglobin, tyrosine, tyrosine ester, tyrosine methylester, tyrosine methylester hydrochloride, tyramine, alanyltyrosine, aminopyrine, phosphonomethyl glycine, salicylates, trans-retinoic acid, carbamate, p-aminobenzoic acid, PEG-25 aminobenzoic acid, indole-3-glycerol phosphate, methanol, acetone, phenobarbital, and combinations thereof.
5. The composition as claimed in claim 1, wherein said oxidant is flavin mononucleotide and said reductant is tyrosine methylester hydrochloride.
6. The composition as claimed in claim 1, wherein said oxidant is flavin mononucleotide and said reductant is p-aminobenzoic acid.
7. The composition as claimed in claim 1, wherein said oxidant is flavin mononucleotide
and said reductant is PEG-25 p-aminobenzoic acid.
8. The composition as claimed in claim 1, wherein the said composition further comprising
C1-C7 alkyl glucoside.
9. The composition as claimed in claim 8, wherein the preferred C1-C7 alkyl glucoside
include methyl glucosides, particularly α-methyl glucoside and ß-methyl glucoside; ethyl
glucoside, propyl glucoside, and combinations thereof.
|Indian Patent Application Number||8/DEL/2003|
|PG Journal Number||33/2010|
|Date of Filing||01-Jan-2003|
|Name of Patentee||ARTHUR M. NONOMURA|
|Applicant Address||311 DEPORT ROAD, BOXBOROUGH, MASSACHUSETTS, 01719, USA|
|PCT International Classification Number||A01N 31/00|
|PCT International Application Number||N/A|
|PCT International Filing date|