Conjugated and Unconjugated Brassinosteroids - ACS Symposium

Chapter 8 Conjugated and Unconjugated Brassinosteroids Hiroshi Abe, Kazuo Soeno, Naoko-N Koseki, and Masahiro Natsume

Downloaded by COLUMBIA UNIV on September 17, 2012 | http://pubs.acs.org Publication Date: December 28, 2000 | doi: 10.1021/bk-2001-0774.ch008

Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan

Research on brassinosteroids(BRs) started with the discovery of Distylium factors. Brassin technology is growing rapidly and advancing in the knowledge of biosynthesis and metabolism, and in molecular genetic analysis using BR-deficient mutants. B R is an essential hormone for regulation of plant growth and development. B R can be regarded as the most important discovery in the field of plant growth regulation. B R will be made avilable for increasing crop production and crop protection as a new type of plant growth regulator in the near future.

A m o n g new class o f plant hormones, brassinosteroid(BR) is a steroidal compound with a unique chemical structure and specific biological activity. More than 40 kinds of B R have been identified in plants, ferns and algae but it has not yet been found as a microbial metabolite. Natural B R occurrs in either an uncojugated or conjugated form, and the former is both in abundance and ubiquitous in plants. T w o biosynthetic routes for brassinolide(BL), a typical and the most active B R in nature, have been discovered. A variety of modified BRs have been also detected in metabolic studies using plant seedlings or plant cell cultures, including epimerization, hydroxylation, fetty acid esterification, and glycosylation. B R elicits

© 2001 American Chemical Society

In Agrochemical Discovery; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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growth promotion, stress tolerance enhancement and crop yield increase, indicating that it will be available as a new type of plant growth regulator for increasing crop production and crop protection. In this paper we describe the occurrence of conjugated and unconjugated biosynthetic and metabolic pathways

BRs in nature,

the

of BRs, and the prospect and

problems for agricultural application.


Unconjugated Brassinosteroids Natural BRs were purified from plant tissue through monitoring elongation or splitting activity in the bean first or second internode test and promotion activity in the rice-lamina inclination test. General purification procedure in our laboratory consisted of 5 steps; solvent extraction, acetonitrile-«-hexane partition, S i 0 or A 1 0 adsorption chromatography, reversed-phase Sep-Pak cartridge column chromatography, and finally C i reversed-phase H P L C . The purified active fraction was subjected to G C / M S analysis affer conversion into methaneboronate or methaneboronate-TMS, or subjected to L C / F A B M S analysis without derivatization. Accordingly, more than fcrty compounds have been isolated and identified. However, many compounds remain unidentified, so that the number of identified BRs is likely to increase. 2

2

3

8

The structure-activity relationship of naturally occurring BRs and related synthetic compounds were studied by means of the lamina inclination test. The results indicated that B R activity required two pairs cf vicinal diols at (2 a,3 a) and (22R,23R), B-ring of 7-oxalactone, one alkyl substituent at the C24 position, and the A/B-trans ring junction. The results of structural variation and their biological activity relationship proposed two hypothetical biosynthetic pathways for B L from campesterol. Metabolic studies in culture cells of Catharanthus roseus and its transformed cells, and biosynthetic mutants of Arabidopsis and Pisum sativum revealed that B L is synthesized from campesterol by either early C6-oxidation pathway or late C6-oxidation pathway as shown in Figure 1(7). However, 2-epiCastasterone(2-epiCS)(l), 3-epiCS(2), 2,3diepiCS(3) and 2 β,3 β -epoxyCS (4, secasterone) were not involved in the two pathways, suggesting that another biosynthesis pathway for these compounds may be involved in plants.



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Figure 1 Biosynthetic pathways ofbrassinolidefromcampesterol In Agrochemical Discovery; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


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(3)

Conjugated Brassinosterids Conjugation of BRs has been investigated intensively during the past 5 years and good progress was made concerning their chemistry and physiological significance. Conjugated BRs have been found as endogenous B R and metabolites converted from exogenously applied B R s . 25-Methyl dolichosterone-23 β -D-glucoside(5) and its 2 β isomer(6)fromPhaseolus vulgaris seeds(2), and teasterone-3 β -D-glucoside(TE-3 β -D-glucosideX7) (J), TE-3-laurate (18) and TE-3-myristate (19)(4) from Lilium longiflorum pollen have been identified as endogenous BRs (Figures 2, 3). In addition to the endogenous conjugates, a variety of conjugated B R s have been found in metabolic studies including glycosylation, esterification and acylglycosylation. Glucosyl conjugation has been observed in metabolic studies (Figure 2). 23 β -D-glucoside (8)(5) was found in B L metabolism using mung bean expiant. 25 β -D-Glucoside (9) and 26 β -Dglucoside (10)(d) were major metabolites of 24-epiBL in tomato culture cells. This metabolism indicated that hydroxylation at C25 and C26 was performed prior to glucosylation. The same conjugation pathway has been observed in the metabolism of 24-epiCS, using tomato culture cells, which



(5),

(ρ)

(η

(61 (Ρ)

(14), (m)

(Ρ, m)

R=-0-D-glc(15) R=- j3-D-glc-(l->6)-j8 -D-glc (16) R=- β -D-glc-(l->4)-j8 -D-gal (17)

Figure 2

Structures of glycosylated brass inosteroid conjugates detected endogenously(p) and in metabolism(m).



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(24) (m)

(25) (m)

Figure 3 Structures of esterified brass inosteroid conjugates detec endogenously(ρ) and in metabolism(m).



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was converted into 25 β -D-glucoside(ll) and 26 β -D-glucoside(12)(6). However, the metabolism of 24-epiCS by tomato culture cells also produced 2 β -(13) and 3 β -glucosides(14)(6) in addition to the side chain glucosides. The results indicated that a to β epimerization of the corresponding 3-hydroxy 1 was important prior to the formation of the glucosyl conjugation. Requirement of epimerization prior to the conjugation has been observed at the C2- or C3 -hydroxy 1 in the metabolism of BL and its 24-epimer by cucumber seedlings or in culture cells(7), although the structure elucidation remains to be determined. Glucosylation was observed in the metabolism of TE. TE-glucoside(7) was found in metabolites of TE afforded by meristem cell cultures of Lilium longiflorum(3). 24-epiTE-3 β -D-glucoside(15), its (1-6)- and (l-4)-3 β-Όglucosides(16,17) have been obtained from exogenously applied 24-epiTE in cell cultures of Lycopersicon esculentum L.(8). On the other hand, esterification has been observed in the metabolism ofBR(Figure 3). 3 β -Lauryl(20), 3 j3-myristy 1(21) and 3 β -palmityl(22) derivatives were obtained as metabolites of 24-epiBL by Ornithopus sativus culture cells(6). The formation of the ester conjugation (23,24,25)(

Conjugated and Unconjugated Brassinosteroids - ACS Symposium

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