Advances in the Use of Brassinosteroids - ACS Symposium Series

Chapter 7

Advances in the Use of Brassinosteroids Horace G. Cutler

Downloaded by UNIV LAVAL on October 7, 2015 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch007

Richard B. Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 5677, Athens, GA 30613

The brassinosteroids are a unique class of plant growth regulators that have the potential to increase yields in economic and horticultural crops. Their original discovery occurred almost simultaneously in Japan and America, respectively in Distylium racemosum,"Isunoki,"an evergreen tree and in canola (Brassica napus) pollen. Extracts from 227 kg canola pollen, gathered by bees, gave America the lead in the field and the structure of brassinolide was determined. There followed an intense search for sources of brassinosteroids in plants and by chemical synthesis, especially in Japan and Europe. The discovery that 24epibrassinolide was active in field tests and was more readily available from synthesis led to the compound becoming the focus of attention in laboratory andfieldexperiments. Consequently, 24epibrassinolide and brassinolide were examined as fungal hormones, to producefruitingbodies, as antiecdysis compounds in insects and especially as yield enhancers infieldand horticultural crops. By 1994 it is projected that Japan will have treated more than 23,000 hectares of wheat in China where yields, so far, have been increased 8-15%. History has a unique way of fashioning events, especially in science, and sometimes significant discoveries are made independently at almost precisely the same time. But the odd feature is that these events occur, even in the age of rapid communication, without any connections having been made. Perhaps one of the best examples of this is the almost simultaneous discovery of the differential calculus by Sir Isaac Newton and Gottfried Leibniz in the latter part of the 17th century. While they worked independently, one in England and the other in Germany, the presentation of their work was so close that a dispute went on for years as to whose discovery camefirst.It is now conceded that Newton was a few

This chapter not subject to U.S. copyright Published 1994 American Chemical Society In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


86

NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS

months ahead of Leibniz though, of course, their synthesis was based on the work of all the mathematicians who had gone before them. An almost identical situation occurred with the discovery of the brassinosteroids and the logic which brought about their discovery had only one common link, the curiosity to find compounds that regulated plant growth with a view to increasing agricultural yields. Following World War Π, there had been a burst of energy in the agricultural chemical industry and this had been brought about in part by the fact that the world was in the process of rebuilding and that a growing population would need a food supply that was both plentiful and inexpensive. The catalyst that gave rise to this industry was the small, but important, handful of natural products that had been discovered in the 1930's and 1940*s. These included indole-3-acetic acid (Figure 1) upon which the synthetic homolog indole-3-butyric acid (1) was based (it is still used today for rooting plant cuttings) and in the early 1950's the production of gibberellic acid (GA ) (Figure 2) which was discovered by the Japanese in the late 1920's but hidden in the Japanese literature because few translations, if any, were made until after World War Π. Again, GA increased yields in certain crops and is used today in at least twenty-seven different horticultural and agronomic situations (i). One example is Thompson seedless grape where vines sprayed at 7-10 days after flowering with 20-40 mg/liter increase yields by ~ 150%. Prior to the use of GA vines had been girdled by cutting a ring around the base of the trunks at approximately a week following flowering and even the most careful operator occasionally killed plants by slicing through the vascular cambium. By 1955 the arsenal of natural products consisted of indole-3-acetic acid, GA , cytokinin and ethylene. The latter was produced by ripening fruits and found practical in use in shipping green bananas in the sealed holds of banana boats from Central and South America, and the West Indies. By the time the bananas reached their destination, they were well on the way to being a nice yellow color for immediate market use. But all these materials had the following significant properties. They were active at exceedingly low concentrations, and in many cases treatments at ΙΟ" M induced the desired response; they were non-toxic at the concentrations used; and they left no apparent residue in the treated product. It was believed that nature contained many such compounds and that it was only necessary to look in therightplaces to discover them. Consequently, in the early 1950's a number of bioassays were developed to evaluate plant extracts for plant growth regulatory activity in the hope of finding new biologically active natural products. 3

3

3

3

5

Brassinosteroid Research in the US and Japan. It seemed obvious to certain researchers, including J. W. Mitchell, who worked for the Agricultural Research Service, that plant organs that grew rapidly should be sources for growth stimulating substances, and among these were seeds and pollen. So Mitchell actively pursued bioassay systems and made pollen extracts in his search for new plant growth regulators. Unfortunately, there was a lag time between his initial verbal report to an evening meeting of the Potomac Division of the Plant Physiology Society at the National Arboretum in the winter of early 1963,

In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


7.

CUTLER

Advances in the Use of Brassinosteroids

87

when he exhibited time-lapse movies showing the effects of rape pollen extract (Brassica napus L.) on bean second node extension, and his formal publication in 1970 in Nature (2). And the lag time is essential in understanding the historical development and division of opinion as to whether the primacy of discovery of the brassinosteroidsfirstoccurred in the United States, or Japan. Events in Japan had followed a slightly different course. For years scientists worldwide had observed that insect induced galls in plants grew rapidly. Therefore, it was surmised that the spurt in growth was, most probably, under the influence of a plant growth regulatory substance produced either by the insect, or by the plant, that was extremely potent. If the substance could be isolated it could have important agronomic and commercial implications. And while many projects were mounted, the literature surrounding specific natural products that controlled galling was sparse until the research carried out in Japan on an evergreen tree, Distylium racemosum Sieb. et Zucc, commonly called "Isunold," came into play. It transpired that the very young leaves of the tree, when attacked by the aphid Neothoracaphis yanonis in the spring, rapidly produced 1-2 mm galls which, in two to three months, grew to - 1 cm. Doubtless this observation had been made for years but, at some point in the 1960's, work began in earnest to isolate the "Distylium factor." Another vital point in this work was the selection and development of a bioassay system and in 1965 the circumstances surrounding the rice-lamina inclination assay were published (5). Surprisingly, this assay and the second internode bean assay used by Mitchell were specific for detecting the brassinosteroids and without either the discovery of this class of compounds would not have been possible at the time because other assays were insufficient to detect their activity. Early work showed that the active material occurred not only in galls but also in healthy Distylium leaves. Incredibly, 430 kg of fresh leaves were harvested in 1966 (4), extracted with methanol to give a neutral fraction which was soluble in ether, and active in therice-laminaassay. On chromatography this fraction gave 751 μ% of Distylium factor A 50 /*g of factor A and 236 μ% of B: all had biological activity. But, obviously, there was not enough material for chemical work, so the biological and isolation results were published in 1968 (5). u

2

Bees as pollen harvesters. American research approached the problem of collecting enough rape (canola) pollen in an ingenious way to obtain enough material for extraction. At the time, in the 1960's, the health food industry was starting to collect pollen from bee hives and some of the pollen was also used in baked products. It was also known that a healthy bee colony can collect about 34 kg of pollen during a season (6). In addition, the maximum distance that a bee can fly for harvesting is 2 miles but, like all other creatures, they prefer to forage close to home. If you put all these ingredients together and place the hives in the middle of afloweringcanolafieldin Canada, pure canola pollen can be gathered. Special traps are placed at the hive entrance to collect the pollen so that the pure pollen pellets fall onto a wire screen and these can be suitably harvested. Approximately 227 kg were harvested in this manner. Finally, after solvent extraction, several chromatographic separations (both open column and high pressure liquid chromatography) - 4 mg of brassinolide (Figure 3) crystals were obtained from a




CH2COOH

Figure 1. Indole-3-acetic acid (Rhizopus suinis).

Figure 3. Brassinolide.


7. CUTLER


89

methanol fraction though this represented only approximately one-sixth of the pollen sample. That is, 23 mg of crystals would have been found in 227 kg of pollen (7). Again, two pieces of serendipity occurred. First, the material was suitably crystalline for X-ray analysis and, second, the necessity for synthesizing heavy atom derivatives for X-ray detennination had only been superseded in ~1973.


Brassinosteroid Production. As soon as the structure for brassinolide had been proved, an immediate search followed to find systems that would give greater quantities for practical applications. This followed two routes: examination of organisms that produced not only brassinolide but, hopefully, other brassinosteroids possessing higher specific activity and, concomitantly, chemical synthesis. Fermentation and Plant Sources. It was hoped that fermentation of a suitable microorganism would yield sufficient quantities of brassinolide for industrial use but although a project was apparently mounted the yields were extremely poor (personal communications), and none of the information was published. Also, while there have been rumors of gene splicing to produce brassinosteroids in bacteria no official publications support this endeavor. The reasons for taking the fermentation approach are logical because all the major plant growth regulators, indole-3-acetic acid, abscisic acid, cytokinins (Figure 4), gibberellic acid and ethylene have been found in microorganisms and higher plants (S). Indole-3-acetic acid and gibberellic acid were originally discovered in microorganisms and the yields were relatively large. Their yields from plant sources were minuscule. Conversely, abscisic acid and cytokinins were discovered in higher plants (kinetin was initially found in stale fish sperm) in low quantities and later in microorganisms in much higher amounts. For example, 93 mg/L of abscisic acid have been isolated from cultures of Botrytis cinerea irradiated at 366 nm (5). Thus, the genetic mechanism for producing these plant growth regulators seems to have been preserved during the course of evolutionfrommicroorganisms to the higher plants. Furthermore, the isoprene precursors for the production of the brassinosteroids are, presumably, operative in microorganisms. At the present time, over sixty types of brassinosteroids have been detected in plants but of these only thirty-one have been characterized (10) (Tables I, Π, and ΠΙ). Table I. Brassinosteroid Sources of Green Algae and Gymnosperms Green Algae 24-epicastasterone Hydrodyction retriculatum 24-ethylbrassinone Gymnosperms Pinus thunbergii pollen 2-deoxycastasterone Picea sitchensis shoots Castasterone Typhasterol Pinus sylvestris cambium Castasterone Brassinolide


90


Table Π. Brassinosteroid Sources of Monocotyledonous Plants and Dicotyledonous Plants Monocotyledonous Plants Oryza sauva shoots Zea mays pollen


Typha latifolia pollen

Castasterone Dolichosterone Castasterone Typhasterol Teasterone 2-Deoxycastasterone

Dicotyledonous Plants Alnus glutinosa pollen

Brassinolide Castasterone Castanea crenata insect gallsBrassinolide 6-Deoxodihydrocastasterone Brassinone Castasterone Castanea crenata stems, leaves 6-Deoxodihydrocastasterone flowers Castasterone Pharbitis purpurea fruit Brassinone Brassinolide Thea sinensis leaves Castasterone Typhasterol Teasterone Vicia faba pollen Brassinolide Castasterone 24-Epibrassinolide

Generally, they are found in such small amounts that processing plant material to isolate sufficient quantities is not yet practical. Among the highest yielding sources are seeds. With few exceptions, all these isolations have been carried out in Japanese laboratories and the obvious academic and industrial support for brassinosteroid research and development in Japan will play a very decisive role in crop production, as we shall discuss later. One system that may present a commercial source of brassinosteroids is tissue culture. Crown gall cells from Catharanthus roseus D. Don (Vinca rosea L.) have yielded brassinolide and castasterone at ~ 30-40 /xg/kg fresh weight of tissue and while this does not compare to the yield for brassinolide from rape pollen ( - 100 /ig/kg) the material is easier to obtain and handle. The crown gall cells were generated from C. roseus through transformation with a strain of Agrobacterium tumefaciens (A208) carrying the Tiplasmid, pTi-T37, to produce C. roseus (V208) and this cell line is characterized by the biosynthesis of the amino acid nopaline as opposed to octopine in other cell lines. The titre of brassinosteroids was increased by the addition of plant growth



Brassinolide Castasterone 24-Epibrassinolide 3,24-Diepicastasterone 1/S-Hydroxycastasterone 3-Epi-1 a-hydroxycastasterone 25-Methyldolichosterone 2-Epi-25-methyldolichosterone 2,3-Diepi-25-methyldolichosterone 2-Deoxy-25-methyldolichosterone 3-Epi-2-deoxy-25-methyldolichosterone 6-Deoxodihydro-25-methyldolichosterone 23-Q-j8-D-gluœpyranosyl-25-methyldolichosterone 23-û-j8-D-glucopyranosyl-2-epi-25-methyldohchosterone

Dolicholide - 200 j*g/kg: equivalent to brassinolide in rape pollen) Dolichosterone Homodolicholide Homodolichosterone 6-deoxodihydrocastasterone 6-deoxodihydrodolichosterone Brassinolide Castasterone

Table ΠΙ. Brassinosteroid Sources of Immature Seed Plants

Phaseolus vulgaris Brassinolide Castasterone Dolicholide Dolichosterone 6-Deoxodihydrocastasterone 6-Deoxodihydrodolichosterone 6-Deoxodihydrohomodolichosterone Typhasterol Teasterone 2-Epicastasterone 3-Epicastasterone 2,3-Diepicastasterone

Vicia faba

Immature Seed Dolichos lablab


92



regulators, including indole-3-acetic acid, or naphthalene acetic acid, or 2,4dichlorophenoxyacetic acid to the cell cultures (ii). While increasing yieldsfromplant or microbial sources have not yet been fully successful, the discovery of new brassinosteroids is significant because of the questions raised. Are any of these congeners more or less active than either brassinolide or 24-epibrassinolide? Are they specific for select active sites in the plant? Do they have a synergistic effect with each other, or other endogenous plant growth regulators? At leastfromthe chemical perspective they offer new templates for synthesis with, of course, synthesis of analogs not presently found in nature, for other exploratory work. Synthesis of brassinosteroids. One of the limiting factors to the use of brassinosteroids in the field has been the limited availability of sufficient quantities of chemical. Apartfromproving the structure and therefrom the biological activity, the first synthesis of brassinolide isomers was reported in 1979 (12). The starting material was ergosterol tosylate and the final products, which contained the steroid nuclei of brassinolide, had the opposite configuration at C22, C23, and C24 in one case, but in the other only the orientation of the methyl group at C24 was different. Later, brassinolide was synthesized from sterols and their degraded products including brassicasterol, bisnor dinorcholenic acid, pregnenolone, and stigmasterol (13). Brassinolide, castasterone, teasterone, and typhasterol have also been synthesized relatively easilyfromthe intermediate (22R,23R,24S)-3a,5-cyclo22,23-diacetoxy-5a-ergostan-6-one (14). It transpired, during the course of evaluating the biological properties of various brassinosteroids, that 24epibrassinolide (Figure 5) elicited responses much like brassinolide in bioassays (15). Coincidentally, brassicasterol was detennined to be present at levels of 1020% in the sterolfractionof rapeseed oil and using solvent extraction, followed by recrystallization, the compound could be obtained in high yield (16). A straightforward series of reactions consisted of treating brassicasterol 3-Q-mesylate with sodium carbonate to give the isoform which was oxidized with chromic acid to yield the 3,5-cyclo-6-ketone. Acid isomerization of this compound gave the 2,22-dien-6-one which, when treated with catalytic amounts of the oxidant, osmium tetroxide and Ν^βΛν^οφηοΙίηβ-Ν-οχίαβ, yielded a 3:5 ratio of 2a,3a,22R,23Rtetrol and 2a,2a,22S,23S-tetrol, respectively. The mixture was separated and each compound was subjected to Baeyer Villiger oxidation giving 24-epibrassinolide and the 22S,23S-epimer (i7), respectively. A recent chemical scheme uses a chinchona alkaloid derivative as the starting material to produce 2a,3a,22R,23R epibrassinolide, as a major product (17). Although 24-epibrassinolide is only about 10% as active in certain bioassays, including radish and tomato, its activity in field trials is approximately equal to brassinolide and it is an attractive candidate for industrial development because of the relative ease with which it can be synthesized. Another synthetic route leadsfromstigmasterol in thefirststep to produce 3j8-acetoxy-bisnor-cholenic acid followed by steps to the next critical intermediate, 2a,3a-isopropyHdenedioxy-6-ethylenedioxy-bisnor-5a-cholanal(18). Presumably, this will be a relatively efficient reaction.




CUTLER


94


Brassinolide is now available in a highly pure form in milligram quantities and 24-epibrassinolide and homobrassinolide in gram quantities from Beak Consultants, Brampton, Ontario, Canada and it is probably only a questions of time before other brassinosteroids are on the market, at which point other research avenues will be opened. But the commercial availability of large quantities also means that synthetic derivatives can now be made for other disciplines, including medicine.


Applications for the Brassinosteroids. We now turn our attention to those areas for which some of the brassinosteroids arefindingapplication. These include their potential as fungal growth regulators, insecticides, andfieldapplication on selected economic and horticultural crops.

Fungal Growth Regulators. The last published report concerning the effects of brassinosteroids on higher fungi was that of Adam, et al. showing results which held great practical promise for the future (20). Higher fungi have two distinct periods in their life cycle, insofar as their culture is concerned. One is the mycelial, orfilamentousstage and the other is the fruiting stage. It is the fruiting stage upon which the lucrative mushroom industry depends to supply soup manufacturers, and processed food markets with Agaricus campestris bisporus. The mycelial stage, relative to the basidiocarp, is not readily visible but remains just below the soil surface. At some point during the life cycle of the fungus, the mycelium begins to aggregate and thefruitingbody is formed (19). It is thought, but not yet proved, that the formation of the basidiocarp in the Basidiomycetes is in response to a specific endogenous hormone. However, in the case of the common mushroom, A. campestris bisporus, production has been streamlined and is carried out underrigorousconditions, for example, in limestone caves in Pennsylvania. The more exotic fungi, like the morels which belong to the Discomycetes, produce afruitingbody called an apothecia, and this is commonly referred to as a sponge mushroom, while the truffles which are also Discomycetes produce an ascocarp which grows underground (19). But to reiterate, all these fruiting bodies have their genesis in their respective mycelia. In addition there are numerous other gastronomic fungi awaiting market development of which some seventeen Boletus sp. are included. In each case, the mycelial stage is relatively easy to grow in the laboratory but the difficulty has been to induce the mycelium to produce fruiting bodies. Obviously, thefirstgroup to accomplish and patent the process will have a thriving business. Three brassinosteroids have been used to speed up the life cycle of fungi in vitro. These are brassinolide, 24-epibrassinolide, and 22S,23S-homobrassinolide all of which increased mycelial growth by a factor of 2-3 and induced earlier sporocarp formation in Psilocybe cubensis and Gymnopilus purpuratus (20). P cubensis forms part of the group known as the sacred mushrooms employed satisfactorily in religious ceremonies by Mexican Indians. One of the biologically active compounds contained in Psilocybe is psilocybin which has been isolated, synthesized and used in the study of schizophrenia (19). The effects with 22S,23S-


7. CUTLER


95

homobrassinolide included a one-third increase in biomass, a one-quarter decrease in the number of weeks to produce fruiting bodies and a quadrupling in the number of fruiting bodies, compared to controls, with lfr ppm treatment. The exact role of the brassinosteroids in this process has not been determined. Whether the effect is to stimulate the mycelial growth and compress the life cycle or to induce mycelial aggregation has yet to be elucidated. Unfortunately, this work has been terminated because of a lack of funds and was the victim of financial restructuring of Germany following the collapse of the German Democratic Republic (correspondence with Dr. G. Adam, 1992). But the rewards for practically producing luxury fungi are too much of a temptation for the work to lie fallow for long (Table IV). Downloaded by UNIV LAVAL on October 7, 2015 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch007

2

Table TV. Fungi Awaiting Commercial Development Discomycetes Morchella conica "Morels" M. hybrida Ai. vulgaris "Truffles" Tuber aestivium T. rufitm Basidiomycetes Boletus appendicular B. erythropus B. auranticus B. granulatus B. badius B. luridus B. bovus B. luteus B. castaneus B. chrysenteron B. queletu B. scaber B. cynescens B. subtomentosus B. edulis B. viscidus B. elegans Insect Growth Regulators. The structural connections between the brassinosteroids and the ecdysteroids are apparent and have led to some detailed examinations of the antiecdysteroid effects of brassinosteroids. The fact that both groups of compounds are exceedingly active in their respective domains makes them interesting models for testing in reciprocal systems and for structural modifications to pursue structure activity relationships. Again, only those brassinosteroids that are available in relatively abundant amounts have been evaluated and these include brassinolide, 22S,23S-homobrassinolide and 22S,23Shomocastasterone (Figure 6). These are similar in structure to ecdysone, 20hydroxyecdysone and ponasterone A (Figure 7). Examination of the brassinosteroids and ecdysteroids used in insect experiments shows that the major differences are as follows: 1) The juncture between the A and Β rings is cis with the ecdysteroids and trans with the brassinosteroids. 2) The Β ring in many, but not all of the brassinosteroids, is







7. CUTLER


97


98


seven membered and contains an extra oxygen: this is a function of the lactonic Β ring. Both brassinolide and 22S,23S-homobrassinolide have a lactonic Β ring; 22S,23S-homocastasterone is an example of six membered Β ring and contains a ketonic function. 3) Ecdysone, 20-hydroxyecdysone, and ponasterone have an enone Βringand they more resemble 22S,23S-homocastasterone. 4) The A ring of these ecdysteroids have β hydroxyls at C2 and C3 while the A ring of the brassinosteroids have a hydroxyls. 5) Brassinosteroids have no hydroxyls on the D ring. 6) The number of carbons differs on the side chain as do the positions of hydroxyl and methyl groups. While a good deal of discussion has centered around the definition of true ecdysteroids relative to activity versus structure of those compounds that may be regarded as analogs, the reality is that certain brassinosteroids do possess antiecdysteroid activity. However, these effects appear to be species specific and caution should be exercised in accepting these compounds as general antiecdysteroids until more data are forthcoming. Early experiments were conducted using crude extracts of rape pollen, from which the brassinosteroids werefirstisolated, against the cockroach, Periplaneta americana. The ethanolic extract was fed to cockroaches on the 11th day of the last larval instar, on powdered rat food, and resulted in delay of moult by ~ 10 days. The length of instar was, therefore, increased approximately 33%. Subsequently, 22S,23S-homobrassinolide and 22S,23S-homocastasterone were fed to cockroaches and only 22S,23S-homobrassinolide delayed moult ~ 11 days, but only at the rate of 50 mg/4 grams food/10 larva (21). More recently, 22S,23S-homobrassinolide and 22S,23S-homocastasterone have shown a neurodepressing effect. In experiments with P. americana, it was noted that the efferent spike activity of the nervus corporis cardiaci Π was affected in isolated brains of larval last instars. While the effect was dose depended for the two brassinosteroids, and was equivalent to that of 20-hydroxyecdysone, a threefold concentration increase in 22S,23S-homocastasterone and a tenfold increase in 22S,23S-homobrassinolide decreased spike activity by 50% (22). Whether other brassinosteroids will be more or less active remains to be seen. Effects of brassinosteroids on other insects have given different results. For example, the data obtained in assays measuring the differentiation of imaginai disks in two flies, Phormia terraenova and Calliphora vicina were similar when 20hydroxecdysone was used and evagination occurred. But in C. vicina, neither of the brassinosteroids elicited activity. Conversely, both castasterone and homodolidolide caused a modest promotion or evagination of imaginai disks in Phormia (22). 22S,23S-Homocastasterone does not possess the seven numbered Β ring, as does 22S,23S-homobrassinolide, and is generally considered to be a more active antiecdysis agent because of this feature (vide infra, where a threefold increase in 22S,23S-homocastasterone relative to 20-hydroxyecdysone induced an effect as opposed to a tenfold increase in 22S,23-homobrassinolide).


7. CUTLER


99


Economic and Horticultural Crop Growth Regulators. The topics discussed up to this point have considered the brassinosteroids from their potential as fungal, or insect growth regulating agents. But there exists a large body of work which can no longer be placed in the category of purely experimental evaluation and that is the effects of the brassinosteroids on economic and horticultural crop production. Some of the information is sketchy and some is substantial. The American experience with the brassinosteroids, specifically the brassins which were a mixture, has been well documented and one is struck by the singular lack of success infieldtrials, with few exceptions. In 1974-75, soybean and barley seeds were treated with brassins (the brassinosteroids were not synthesized until 1979) both in North America and Brazil to see if yields from small seed were equal to those from large seed, but no differences were found. In 1975, soybean seed were treated prior to planting at 7 locations in the United states with brassins to determine whether an increase in yield would result. Again in 1975, barley seed was treated, sorted into 3 sizes and planted. There were no results of economic importance. When the synthetic materials became available in 1979,fieldtrials were conducted on beans, corn, lettuce, peppers, tomatoes, and radishes. These were treated at time of soil emergence with 0.01 ppm brassinosteroid. Lettuce heads were significantly increased by 25-32%, radishes ~ 20%. Peppers were increased by 9% and beans by 6%, but these increases in fruit weight in these two crops were not statistically different. There were no increases in corn or tomato yields. But by this time the American project was coming to a close and no further field work was planned. Sadly, another two years of experiments would have shown that the time of application was critical to increasing yields (7). For example, when seeds were soaked with solutions of brassins there was little effect on plant growth while in later experiments, increases in yields were noted in radishes, leafy vegetables and potatoes when young seedlings were treated, in greenhouse experiments (7). It was approximately at this point that the Japanese, spurred on by the American disclosure of the synthesis of the brassinosteroids, went through a logarithmic effort in both the evaluation and synthesis of these plant growth regulators. The first inkling that something big was afoot, insofar as field applications and results were concerned, came to light through personal communications in 1988 (Cutler, personal communications) when it appeared that wheat yields had been increased -15 % in both China and Russia. The latter results were only alluded to but werefinallyrevealed, albeit in a brief form, in the summer of 1990. In fact, the brassinosteroids, specifically 24-epibrassinolide and homobrassinolide, had been used to treat not wheat, but barley, lucerne, and some horticultural crops in Russia. Barley yields after treatment with 24-epibrassinolide were dramatically increased up to 25% with applications of 50 or 100 μg per hectare in 500 L of water. Increases in lucerne seed were also noted: 16% with homobrassinolide and up to 26% with epibrassinolide (25). Russia had obtained its brassinolides, if the sources are correct,fromJapan.



100

NATURAL A N D ENGINEERED PEST M A N A G E M E N T AGENTS

The extent of the Japanese work in China also became evident in the summer of 1990. By that time, Nobuo Ikekawa had synthesized sufficient quantities of 24-epibrassinolide to test large areas of wheat in China with his colleague Yu-Ju Zhao. Over a six year period, they had applied 24-epibrassinolide on 3,333 hectares of wheat using concentrations of 0.1 to 0.001 ppm (24). The results consistently showed an increase of up to 15% in wheat, a significant figure that could well alter future wheat markets throughout the world. Since 1990, further trials have been conducted on wheat in China and the total area now treated, as a result of the 1990-1991 season, has been increased to a total of 4,000 hectares. Again, yields have been increased 8-15% during this period. The upshot is that a grant has been made by the Chinese Academy of Sciences to Japan to apply 24-epibrassinolide to wheat and the total land use will be increased to over 23,000 hectares during the next 3 years (1992-1994). The amount of 24epibrassinolide needed for these experiments is 100 g. Horticultural crops have also been treated in China with 24-epibrassinolide and these include corn, tobacco, watermelon and cucumber. All exhibited increased yields in 1990 and 1991 though hardfiguresare not yet available. Grape, strawberry, orange, rape, eggplant, and sesame have also been treated at time of flowering, which appears to have been a critical time for application, and increased yields were due to increased fruit set, though in grape there was increased fruit weight, sugar content and maturity. Increased mushroom growth was also observed in plants treated with 24epibrassinolide and this serves to confirm the work mentioned earlier with P. cubensis and G. purpuratus (20). Sesame yields were increased 17% (25). Undoubtedly, thefiguresfrom these trials will be published later. It has been stated that the success of 24-epibrassinolide may be attributed to the lack of good cultural practice in China and that yields could easily be increased ~ 10% by proper use of fertilizer. While the point is well taken, it must be argued that if a few grams of a plant growth regulator can replace several tons of fertilizer then a more efficient agriculture exists. By analogy, it is far more economical to spray GA on grapevines to increase yields than to pay a girdler who may take days to do the job. 3

Conclusion. In reviewing the history of the brassinosteroids it is now obvious that these natural products, especially 24-epibrassinolide, have a future in agriculture for practical application. The fact that 24-epibrassinolide has very low toxicity, 1 g/kg (oral) for the mouse 2 g/kg (oral and dermal) for the rat, and was negative in the Ames test for mutagenicity, in addition to the very low amounts used per hectare, indicates that it has a bright future. It is also interesting to note that towards the end of the American experience with the brassinosteroids there were increases of ~ 20% in certain horticultural crops infieldtests, but it was precisely at this point that research ceased. The one lesson to be learned from the brassinosteroid experience is that a long term commitment has to be made in a research endeavor of this sort. In 1979, the Japanese had no hectares under test with the brassinosteroids. By 1994 they will have over 23,000 hectares treated with 24-


7. CUTLER


101


epibrassinolide in China alone with, one feels certain, operations going on elsewhere. And the environmental considerations augur well for the brassinosteroids for they are natural products that are target specific, apparently biodegradable, with very high specific chemical activity. If yields are consistently higher following brassinosteroid treatment then it means that reduced land mass can be used to produce the same amounts of food and this will entail the use of less fuel and pesticides. In any event, it is obvious that the world food supply will become, in this case, dependent upon the importation of Japanese agrochemicals for success. Acknowledgement. I am grateful to Dr. N. Ikekawa for the preliminary results of his trials with 24-epibrassinolide in China on various economic and horticultural crops. Literature Cited. 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13.

Cutler, H. G.; Schneider, B. A. In Plant Growth Regulator Handbook of the Plant Growth Regulator Society of America 3rd Edition; Plant Growth Regulator Society of America. 1990. Mitchell, J. W.; Mandava, N.; Worley, J. F.; Plimmer, J. R.; Smith, M. V. Nature 1970, 255, 1065. Maeda, E. Physiol Plant. 1965, 78, 813. Abe, H.; Marumo, S. In Brassinosteroids; Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474; American Chemical Society; Washington, DC 1991; p. 19. Marumo, S.; Hattori, H.; Abe, H.; Nonoyama, Y.; Munakata, K. Agric. Biol Chem. 1968, 32, 528. Dadant, M.G. In The Hive and theHoneybee;Grout, R.A.; ed; Dadant and Sons, Hamilton, Illinois; 1949; pp 203-212. Steffens, G. L.InBrassinosteroids;Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474, American Chemical Society; Washington, DC 1991; p. 9. Cutler, H. G. CRC Critical Reviews in Plant Sciences. 1988, 6, 323. Marumo, S.; Konno, E.; Natsume, M.; Kanoh, K. Proc. 14th Ann. Meeting Plant Growth Regulator Soc. America; 1987, 146. Kim, S.-K.InBrassinosteroids; Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474, American Chemical Society; Washington, DC 1991; p. 26. Sakurai, Α.; Fujioka, S.; Saimoto, H. In "Brassinosteroids", Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474, American Chemical Society; Washington, DC 1991; p. 97. Thompson, M. J.; Mandava, Ν. B.; Flippen-Anderson, J. L.; Worley, J. F.; Dutky, S. R.; Robbins, W. E.; Lusby, W. J. Org. Chem. 1979, 44, 5002. Marumo, S. Proc. 14th Ann. Meeting Plant Growth Reg. Soc. America. 1987, 14, 174.


102 14. 15. 16. 17. 18.


19. 20.

21. 22. 23.

24. 25.


Aburatani, M.; Takeuchi, T.; Mori, K. Agric. Biol. Chem. 1987, 51, 1909. Takatsuto, S.; Ikekawa, Ν. Chem. Pharm. Bull. 1984, 32, 2001. Tsuda, K.; Sakai, K.; Ikekawa, N. Chem. Pharm. Bull. 1961, 9, 835. Ikekawa, N.; Zhao, Y.-J. In Brassinosteroids', Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474, American Chemical Society; Washington, DC 1991; p. 281. McMorris, T. C.; Donaubauer, J. R.; Silveira, M. H.; Molinski, T. F. In Brassinosteroids-, Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474; American Chemical Society; Washington, DC 1991; p. 41. Alexopoulos, C. J. In Introductory Mycology; J. Wiley and Sons, Inc., New York, 1962, pp. 613. Adam, G.; Marquardt, V.; Vorbrodt, H. M.; Hörhold, C.; Andreas, W.; Gartz, J. In Brassinosteroids; Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474; American Chemical Society, Washington, DC 1991; p. 83-84. Richter, K.; Koolman, J. In Brassinosteroids; Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474; American Chemical Society, Washington, DC 1991; p. 265-272. Richter, K. Naturwissenschaften. 1991, 78, 138. Khripach, V. Α.; Zhabinskii, V. N.; Litvinovskaya, R. P. In Brassinosteroids; Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474; American Chemical Society, Washington, DC 1991; p. 53. Ikekawa, N.; Zhao, Y.-J. In Brassinosteroids; Cutler, H. G.; Yokota, T.; Adam, G.; eds; ACS Symposium Series No. 474; American Chemical Society, Washington, DC 1991; p. 284-290. Ikekawa, N. Unpublished results 1992.

RECEIVED April 30, 1993


Advances in the Use of Brassinosteroids - ACS Symposium Series

Recommend Documents