Microbes and Microbial Products as Herbicides - American Chemical

Chapter 4

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 8, 2016 | http://pubs.acs.org Publication Date: September 25, 1990 | doi: 10.1021/bk-1990-0439.ch004

Satellite Metabolites and Synthetic Derivatives of Abscisic Acid as Potential Microbial Product Herbicides Horace G. Cutler Richard B. Russell Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 5677, Athens, GA 30613

The recent isolation of abscisic acid (ABA) from the fungi Cercospora cruenta and blue light-irradiated Botrytis cinerea has led to renewed interest in the development of ABA and its derivatives as practical plant growth regula tors. The discovery of relatively large quantities of ABA has led to two developments: first, the synthesis of ABA derivatives that have plant regulatory activity and second, the isolation and identification of satellite metabolites. A review of the work and possible synthetic suggestions are presented and include the synthetic cy clization of ABA to produce bicyclics with distinct bio logical activities and the isolation of metabolites, in cluding (2Z,4E)-(+)-4'-hydroxy-gamma-ionylideneacetic acid, from C. cruenta and Stemphylium sp. The potential exists, with the bicyclic derivatives, to produce herbi cides. In the late sunmer of 1957, I.D.J. R i i l l i p s joined our labora tory at the Boyce Thcnpson Institute, Yohkers, New York, as a pre-îoctoral student. He was an exchange student from the labora tory of S i r P.F. Wareing, who had recently moved from the University of Manchester t o Aberystwyth, Wales, and P h i l l i p s was seconded t o Professor A.J. V l i t o s , for two years. IXiring that period I had the exceptional honor, and luck, of working with P h i l l i p s during which time he constantly referred t o the β-inhibitar that was present i n the dormant buds of Acer rrairtrolatanus. He explained, i n de t a i l , that the secondary metabolite was present i n the buds when they were l a i d down i n the sunmer and that the amount of inhibiting substance, on a gram weight basis of collected buds, remained high through the winter months, then proceeded t o drop i n December and January. By March when Spring started i n the B r i t i s h Isles (Phillips assured me that there i s a spring i n England), the t i t r e of ^-inhibitor had diu|jped t o exceedingly low levels. At a point when the inhibitor was no longer present, buds broke dormancy and This chapter not subject to U.S. copyright Published 1990 American Chemical Society

Hoagland; Microbes and Microbial Products as Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


4. CUTLER

SatMuMHakoluâMdSynthetkDtm

73

stem and leaf growth appeared. The experiments designed by P h i l l i p s ware elegant and included the separation, frxxn crude extracts, of the inhibitor i n zones on paper ciirxxnatograms which, at the time, had recently been introduced into the laboratory. In a typical separation, the crude material, usually obtained from a methanol extraction of buds (and, later, leaves), was spotted onto paper chromatxxjrams, developed i n a suitable solvent (isopropanol:aiaicnia:water), the chrxxnatograms were then dried, cut into ten equal parts (Rf 0.0-1.0) and the cut pieces were introduced into test tubes with approximately 2 ml of water. To these were added ten etiolated wheat coleoptiles, under suhdueri green safelight and, following treatment, the extension of the coleoptiles was recorded. The ^-inhibitor present i n sycamore buds was extremely active against etiolated wheat coleoptiles and these findings were published (1,2). Further, F h i l l i p s stated that the growth of leaf disks of Acer pseudcolatanus could be inhibited by the dconancy factor that had been eluted, i n water, from paper chromatograms. Discussions centered around the practical use of the inhibitor t o control the emergence of weed seeds by pre^lanting application t o t i l l e d fields and other "herbicidal applications. There was no doubt that Wareing i n the United Kingdom and Addicott i n the United States, who had discovered an identical compound i n cotton (Gosgypim fiirsutum L. ) bolls, were hot on the t r a i l of an inportant secondary metabolite. The B r i t i s h work implied that dormancy i n tree buds could be chemically explained, while that of the Americans c l a r i f i e d , i n cotton, the acceleration of abscission i n leaves and bolls. Inportantly, the methods by which t h i s chemically-unknown substance had been detected involved the exogenous application of the material t o either etiolated wheat (Triticum) coleoptiles i n the case of Acer nseudoplatanus buds, or Avena coleoptiles, i n the case of the cotton metabolite. In both bioassay species indole-3-acetic acid (IAA) caused curvature when applied to the etiolated t i p s of the seedlings but, IAA i n ocmbination with the inhibitor induced far less bending. The inhibitor also accelerated abscission when applied to cotton f r u i t or pedicels frxxn which the f r u i t had been removed. In a l l these cases, the effects were those observed on hJTher Plants following application, and not îniczoorganisms or vertebrates. The structure of the inhibitor was elucidated by Ohkuma et a l . , i n 1965 (3) and given the t r i v i a l name abscisin I I , later, t h i s was changed to abscisic acid (Figure 1). In 1965, Cornforth synthesized (±) -abscisic acid (ABA) (4), and relatively large samples of t h i s isomeric mixture became available for further work, as opposed t o the very limited milligram quantities of (+)-ABA that had been obtained from plant sources. The biologically active species i s the 2-cis(+) form; the 2-trans(-) isomer i s relatively inactive. This means that any evaluations wherein the synthetic form (±) i s used must be clearly stated as such. Over the intervening 25 years, there have been no major developments with respect to the use of abscisic acid, either i n the native or derivatized form, i n agriculture. The early promise that the compound showed for controlling the breaking of dormancy i n stored products such as I r i s h and sweet potatoes, onions, carrots, and other cxxnnodities, has not materialized. Neither has a use been 11



74

MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES

found for abscisic acid i n prolonging dormancy i n f r u i t trees, especially i n the transition zones of the world where bud break brought on by unseasonably warm weather followed by a freeze leads to ccnplete y i e l d or crop destruction. Even the effects of (±)-ABA on seed germination appears to have gone unexploited as a possible selective pre-emergenoe herbicide. This despite the fact that (±)-ABA seems relatively non-toxic figures are not given i n the Merck Index). Paradoxically, 109 ABA analogs and metabo l i t e s have been isolated, synthesized, and their biological a c t i v i t i e s i n various assay systems reported without much advancement i n practical application (5). As i f to complicate the cxxundrum, abs c i s i c acid has been implicated i n controlling abscission, bud dor> mancy, seed dormancy, tuberization, flowering, stomatal closure, drought resistance, root growth, f r u i t ripening, senescence, and membrane transport (6) · Practical use of ABA seems to stick at one point: abscisic acid i s not readily transported across leaf c u t i cles. Earlier work demonstrated that ABA i s so poorly transported across leaf barriers that insuffcient material penetrates the c e l l to induce a response. E>qperiments with astomatous cuticles isolated from tomato f r u i t , and the upper epidermis of apricot leaves, pear, and orange, showed that penetration was linear with time, was greater through dewaxed than non-deuaxed cuticle and was more f a c i l e as the undissociated ion. Inportantly, naphthalene acetic acid and 2,4-&chlcrophenGKyaoetic acid were 3 - 6 times more effective that (±)-ABA i n cuticular penetration (7). More s i g n i f i cant was the finding that ABA was not metabolized to another chemical species during transport across the cuticle. I f the cause of response to ABA i n reaching i t s target site(s) when applied to higher plants i s due to leaf penetration, we are faced with an apparent paradox. ABA has recently been discovered i n the brains of pigs, rats (8), rodents and ruminants (9), and there are only two possible origins for t h i s material. F i r s t , i t may be obtained from dietary sources, i n which case the ABA must survive passage through the digestive system, cross into the blood stream, be carried to the proximity of the brain and pass through the blood brain barrier into the brain where i t i s , presumably, slowly metabolized. I t i s d i f f i c u l t to transfer chemical cxmpounds across the blood brain barrier, a constant nemesis for the medicinal chemist who must synthesize compounds with precise l i p o p h i l i c and hydrop h i l i c properties to reach the receptor sites i n the brain. I f t h i s i s the case for ABA reaching the brain i n the mammalian system, then i t i s something of a contradiction that ABA can be transferred across a number of membranes and barriers, a l l possessing differing properties, yet not penetrate leaf and stem tissure i n plants i n sufficient quantity to induce a significant response. However, i t i s also possible that ABA i n brain t i f i s w i s synthesized de novo, implying that the genetic code for manufacturing ABA exists i n microorganisms, plants and animals and has not been deleted during the evolutionary process. Two points have governed the possible development of ABA as an agpdcultural chemical. F i r s t , the relatively low yields of ABA from higher plants , as opposed to ndCTOorgarrisms, has precluded the isolation of biosynthetically dependent s a t e l l i t e metabolites which occur i n less quantity which may also have potent biological activ-


4. CUTLER

Satellite Metabolites and Synthetic Derivatives of Abscisic Acid

i t y . Second, there has been a preoccupation, especially i n Western science, with the node of action of ABA; and some of the earlier questions cxncerning the practical use of ABA have been held i n abeyance. Ihis may now be changing.


ABA and Associated Metabolites i n Rmai. The recent discovery of (+) -cis-ABA (Figure 1) i n relatively large abundance i n fungi, especially Oercospora rosicola (10), C. cruenta (11), and a blue light (360 nm) sensitive strain of Botrvtis cinerea. which produced 9.3 mg/100 mL of l i q u i d culture (12), may mark the turning point for the discovery of homologs and analogs of ABA for agricultural use. Because yields of ABA can be increased by subjecting producer microorganisms t o stress, the associated satell i t e metabolites may be oonoomitantly produced i n greater quantit i e s , isolated i n pure state and then u t i l i z e d or derivatized f o r specific biological use. Concurrently, the associated metabolites may be used to deduce, by suitable labelling, the biosynthetic pathways for ABA. Isolation of the s a t e l l i t e metabolites, followed by introduction of those metabolites t o other fungi which, presumably, more e f f i c i e n t l y convert them t o other ABA precursors, has furnished valuable information. For example, i n addition to the discovery of (+)-ABA i n Ç. rosicola a further compound, (+)-(2Ζ,4Ε)-4·-αχοôc-ionylideneacetic acid (13) (Figure 2), which occurred i n small quantities, was discovered. Later, i t was found that another fungal species, £. cruenta could produce large quantities of (+)-ABA and (+) - (2Z, 4E) -4 -axo-a-icnylideneaœtic acid (11). Biere followed the discovery that by feeding (+)-(2Z 4E)-o~ionylideneacetic acid (Figure 3) to £. cruenta the micxœrganism could convert the compound t o (+)-4·-1^ΰτΌ^-α-1οτν1άχΪΒηΒ3σβ^σ acid; (+)-(2Ζ,4Ε)-4·-οχο-or-ionylideneaoetic acid; and (+)-ABA (14) (Figure 3), thereby estab lishing some of the biosynthetic sequence for (+)-ABA. Two points of interest are that the 4' position of (+)-cîorylideneacetic acid has been p a r t i a l l y oxidized t o produce the OH derivative, or com pletely oxidized t o biosynthetically make the (+)-4'-oxo product. The metabolite (+) -ot-ionylidene acid has been shown t o e l i c i t the same plant growth inhibitory activity as (+)-ABA(14) ; and the (+)-4'-axo derivative does not appear t o have been tested. A fur ther development with respect to (+)-ABA metabolites was the i s o l a tion of (+)-(2Z,4E)-trarjs-l,4 -dihyto^ acid (Figure 4) from Ç. cruenta. The major difference between t h i s compound and those previously mentioned i s the l position which has been oxidized t o produce an hydroxyl function. This metabolite more closely resembles (+)-cis-ABA with the exception that the 4 -oxo i s now an hydroxyl, the 5'-6' position i s saturated (this corresponds to the 2'-3' unsaturation i n ABA, which i s numbered anticlockwise i n the cyclohexene ring), and there i s a methylene function at 6 (the corresponding position i s 2 i n ABA from which i s subtended a CH group). These changes are substantial enough t o a l t e r the biological activity so that (+) -trans-i ,4 -dihydroxy-y- ionylideneacetic acid i s only, approximately, one-tenth as active as (+)-ABA i n r i c e seedling assays (15). I t i s somewhat surprising that the minor addition of two protons at the 4 position and transition of the methyl to methylene reduces biological activity of the molecule by 90% compared with (+)-ABA. 1

/

,

1

,

1

1

3

1

1

1


75

MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES


76


CUTLER

SateOite MHaboUta amd SynthetkDenvatwesofAbsciskAcid

H C 3

V

XH

3

ÇK


COOH (+)-(2Z, 4 E W - i o n y l i d e n e a c e t i c acid (Cercospora cruenta)

(+H2Z, 4 E ) - 4 -oxo-of

ABA

ionylideneacetic acid

COOH

(+)-4'-hydroxy-*-ionylideneacetic acid

Figure 3.

(+)-(2Z,4E)-

Microbes and Microbial Products as Herbicides - American Chemical

Recommend Documents