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Abscisic acid analogs: probes for basic research in plant science and practical applications in agriculture.

Abscisic Acid Analogs: Probes for Basic Research in Plant Science and Practical Applications in Agriculture

Abscisic Acid (ABA, 1) is a highly functionalized monocyclic sesquiterpene that is ubiquitous in higher plants and regulates many stages of plant growth and development. It is implicated in germination inhibition, transpiration, induction and maintenance of dormancy, and senescence. For example, under drought conditions, ABA levels in leaves increase, and exogenous application of ABA induces closure of stomates, the pores in leaves through which gases and water pass. ABA is considered to act as an endogenous antitranspirant. Plant scientists have demonstrated abscisic acid is involved in plant resistance to stresses such as cold, heat, salinity and drought.

Recently, the bio-organic chemistry group at the Plant Biotechnology Institute has been working on aspects of ABA chemistry. We are collaborating with biologists in diverse disciplines on studies of the mode of action of the hormone, and on applications of compounds related to abscisic acid for plant growth regulation in agriculture and forestry.

Through this collaborative effort, we have identified synthetic compounds with ABA-like activity, and discovered compounds with the opposite, or anti-ABA, effects. These chemical switches have the potential to aid in uncovering basic principles of plant hormone action. As well, analogs are plant growth regulators potentially useful in improving plant performance and crop yields.

Abscisic acid, the natural hormone, is rapidly degraded under field conditions. Light photoequilibrates the cis double bond of the sidechain to the trans form, resulting in loss of activity. Bacteria on the surface of plants degrade the hormone to carbon dioxide and water, and the plant's natural enzymes rapidly metabolize ABA to inactive products. Because of the liability of ABA, coupled with the high cost of producing synthetic compounds, abscisic acid is not a practical growth regulator.

Over the past 25 years, chemists have synthesized many analogs of ABA mainly to establish structure-activity relationships. Employing standard ABA assays, researchers have tested the analogs for biological activity like germination inhibition, growth inhibition and stomatal closure. Analogs are generally much less active than the natural hormone and are active in some ABA assays but not in others. Compounds having deviations from the carbon skeleton of ABA show reduced activity, while their oxidation levels are relatively less important. Compounds containing triple bonds in place of the trans double bond of the side chain have good biological activity. Analogs with trans substituting for the cis double bond are only weakly active.

Some unnatural analogs show high activity because they are converted by plants into ABA. Millborrow demonstrated that epoxy-beta-ionylideneacetic acid (2) is converted into ABA. The epoxide is structurally related to xanthoxin, a putative biosynthetic precursor of ABA. The epoxide is inactive in the stomatal closure assay, but is four times more active than ABA as a growth inhibitor. This unnatural substrate is considered to act as a slow release form of ABA. The hormone level inside the plant can be higher and more long-lived than that achieved by spraying a more concentrated solution of ABA on the surface of the plant. It is thought that esters of ABA owe their activity to a similar slow release phenomenon, and that hydrolysis of inactive esters affords high internal concentrations of the hormone.

ABA, used in plant tissue culture studies to suppress germination of embryos, enhances embryogenic callus formation from explants. Research at PBI has shown that two analogs of ABA biotransformed into the hormone compare well with ABA for their effects on wheat embryos in culture (Qureshi et al 1989). In this study, Qureshi determined the number of plants that could be regenerated from 100 embryos of each of ten different cultivars of wheat with the different treatments. In some cultivars ABA was most effective, with 400 plants being regenerated per 100 embryos. In other cultivars, using led to twice as many plants being regenerated, while in another group, methyl abscisate was more effective than the other two compounds. This study is the first successful example of ABA analogs being used as tools in plant tissue culture and could lead to manipulating plant cells as desired.

ABA is implicated in induction and maintenance of dormancy in plants and in acclimation of those species capable of cold hardening. Spraying ABA on plants generally does not substitute for endogenous ABA synthesized in response to environmental and other cues and is not effective in inducing dormancy or resistance to stress. ABA analogs have the potential to act as agents to induce or maintain dormancy. Cold acclimated plants lose their hardiness rapidly when temperatures rise at the end of winter.

An investigation is now under way in Larry Gusta's lab at the Crop Development Centre, University of Saskatchewan, on the application of ABA analogs to prevent dehardening of cold-acclimated cereal crops. The unnatural prohormone 2a was applied as a foliar spray to cold hardened wheat, rye and triticale and the plants, after receiving two-day warm treatment, were subjected to slow freezing. The treated plants survived temperatures of --17 [degrees] C, while the water-treated control plants all died. The analog treatment maintains the plant's hardiness through the warming period. Injury prevention due to early spring warming to fruit crops such as apples, peaches and grapes are other applications in Canadian agriculture.

ABA analogs have been tested in many studies for possible use as antitranspirants. An acetylenic analog 3 is one of the promising structures. Derivatives of 3 are currently being evaluated by BASF as a synthetic antitranspirant on crops such as barley and tomato. Although this compound is inactive in short term stomatal closure assays, it is effective in long term tests and BASF has reported irrigation can be reduced up to 30% without reducing crop yield.

Another major application in which ABA analogs have potential is in conditioning of conifer seedlings. About one billion seedlings are grown for reforestation projects in Canada per year. The mortality of the transplanted seedlings in the forest is high (greater than 40%) due to dessication in severe heat. Analogs could be used in watering nursery seedlings to make them more resistant to stress. Ideally, analogs would induce dormancy and increase viability of seedlings transplanted to the forest. The economic benefits to Canadian forestry would be considerable. Research in this field is being carried out jointly by the Plant Biotechnology Institute, Terry Blake's group at the University of Toronto, Faculty of Forestry, and Gary Hunt at Balco Canfor Reforestation Ltd. in Kamloops, BC.

In the course of our germination assays with structural variants of ABA, we have found a number of analogs promote rather than inhibit germination in a number of seeds including cress, corn, wheat, barley and canola. The germination of corn seeds was especially promoted at 10 [degrees] C, a low temperature at which corn normally will not germinate. Treated wheat, grown in pots in the greenhouse, emerged more rapidly and flowered two to three days earlier than the control plants.

In collaboration with Rob Hill at the University of Manitoba, we are studying the effects of promoting compounds on enzymatic processes in germinating barley seeds. In the normal germination process, gibberellic acid levels rise, and mediate the synthesis of alpha-amylase, a protein which hydrolyzes starch to provide energy for seedling growth. ABA inhibits germination by causing the production of a different protein that forms a one-to-one complex with alpha-amylase. Treating seeds with analog PBI-11 results in alpha-amylase levels comparable to those induced by GA. The analog is acting in a sense opposite to that observed for ABA. The mechanism by which ABA-related compounds promote germination is not at all understood. It is interesting to speculate that the analogs may be acting by interfering with ABA biosynthesis or catabolism, and may function as competitive inhibitors at ABA receptor sites.

The natural hormone has the S-(+) configuration, but oddly enough, both antipodes are active in ABA assays such inhibition of germination and growth. The biological activity of the R form has been ascribed to the near symmetry of the ABA molecule. In one important assay, in closing stomates, the pores through which transpiration takes place, the unnatural form R--(-)--ABA is not active. It is currently thought the putative receptor controlling stomatal closure has strict structural requirements. Biological studies of ABA action are commonly carried out using the synthetic racemic mixture because of its relatively lower cost and ready availability. It is known the two enantiomers are metabolized at different rates in plant systems, and it has recently been hypothesized, to different metabolites. Results from biological tests using the racemic mixture do not reflect accurately the effect of the natural hormone.

We have recently developed an NMR method to determine the enantiomeric composition of residual ABA in cell cultures fed the racemic mixture. The recovered ABA is converted to its salt, and proton NMR spectra of gammacyclodextrin complexes of the salt in deuterium oxide obtained. Diastereomeric complexes are formed between the chiral cyclodextrin and the two enantiomers of ABA. Integration of the signals of the two doublets can be used to determine the ratio of R to S ABA.

One of the unanswered questions in ABA research is what is the active form of the hormone in the various physiological processes ABA is thought to regulate. That is, in cases where addition of abscisic acid produces a biological effect, is ABA itself the chemical signal that initiates the physiological process, or is ABA metabolized, and are the metabolites the actual hormones? While the biosynthetic pathway by which ABA is formed remains obscure, the degradation pathway has been clearly established. The first even that occurs is the hydroxylation of the methyl group on the same face of the ring as the hydroxyl group, perhaps through the intermediacy of a P-450 oxygenase. The molecule thus formed, 8'-hydroxyabscisic acid, has been isolated only once. It is exceedingly labile, and rearranges through Michael attack of the oxygen on the enone to a bicyclic compound, phaseic acid. It has been suggested this process is also controlled enzymatically, but closure to this ring system has been found to occur spontaneously in synthetic studies. In some plants, and plant cell cultures, phaseic acid accumulates. In others, the product of reduction of the ketone, dihydrophaseic acid, is the major metabolite. Phaseic acid and dihydrophaseic acid have only been available in minute quantities from isolation of extraction of plant material. These metabolites are somewhat light sensitive and are difficult to isolate in pure form. Quantities of pure, characterized compounds have not generally been available for proper biological testing.

In our laboratory at the NRC in collaboration with Gusta's group at the University of Saskatchewan's Crop Development Centre, we have two approaches to producing mg quantities of ABA metabolites. We are employing plant cell cultures to transform ABA to dihydrophaseic acid. The system is quite convenient in that the metabolites are excreted into the culture medium. When S-ABA alone is fed to the cells only one product, dihydrophaseic acid is observed. Using this approach milligram amounts of dihydrophaseic acid can be obtained. This is not a convenient method to obtain phaseic acid as the subsequent methylation, oxidation and saponification steps proceed with low recovery.

Phaseic acid is the metabolite of real interest, and a practical total synthesis of this compound has just been completed in our laboratories. The required functionalization of the methyl group is effected through a Barton reaction on the nitrite ester of 4-hydroxy-2,2,6-trimethylcyclohexanone. The key compound in the synthesis in a bicyclic intermediate accessible from isooxophorone in seven steps. Phaseic acid is produced from this compound by alkylation with an acetylide of a protected enynal, followed by standard functional group manipulations. Using this procedure batches of 10 to 15 mg of racemic phaseic acid have been produced for biological testing which is now underway in studies on cold acclimation of plants, and germination inhibition in cereals.

At the practical agricultural level, ABA analogs have many applications as plant growth regulators for improving crop productivity. Control of germination of seeds and greater water use efficiency by plants are two immediate examples.

Abscisic acid-related compounds have great potential as tools for determining how the plant hormone functions in plants. Using specifically designed compounds, it should be possible to identify proteins acting as receptors and metabolizing enzymes. No ABA receptors have been identified yet.

PHOTO : Suzanne Abrams, MCIC, at work in her laboratory at the Plant Biotechnology Institute.

PHOTO : Culture of wheat embryos collected 25 days after pollination left -- in the absence of

PHOTO : methyl ABA, note embryo germination and lack of embryogenic callus right -- in the

PHOTO : presence of methy ABA, note suppression of germination and induction of embryogenic

PHOTO : callus (callus capable of in vitro plant regneration).

PHOTO : Effects of germination promoters as compared to control corn. Acknowledgements The chemistry described in this article was done at the NRC by Garth Abrams, Lloyd Nelson and Angela Shaw. Bruce Evans and Martin Reaney carried out the biological assays in Larry Gusta's laboratory at the University of Saskatchewan. References D.C. Walton in Abscisic Acid. F.T. Addicott, Ed., Praeger Press, New York. 113 (1983). T. Oritani and K. Yamashita. Phytochem. 22 1909 (1983), and references contained therein. J.A. Qureshi, K.K. Kartha, S.R. Abrams, L. Steinhauer. Plant, Cell and Organ Tissue Culture in press. M.J.T. Reaney and L.V. Gusta. Plant Physiology 83, 423 (1987). W. Rademacher, R. Maisch, J. Liessegang, and J. Jung. In: Plant Growth Regulators for Agricultural and Amenity Uses A.F. Hawkins and A.D. Stead. BCPC Publications, Croydon, G.B. (1987). S.R. Abrams, M.J.T. Reaney, G.D. Abrams, T. Mazurek, A.C. Shaw and L.V. Gusta. Phytochemistry in press. SUZANNE R. ABRAMS, MCIC Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Sask.
COPYRIGHT 1989 Chemical Institute of Canada
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Copyright 1989 Gale, Cengage Learning. All rights reserved.

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Author:Abrams, Suzanne R.
Publication:Canadian Chemical News
Date:Jul 1, 1989
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