Some notes on the terminology of brassinosteroids

Marco António Teixeira Zullo1,*, Ladislav Kohout2 and Mariangela de Burgos Martins deAzevedo1

1Phytochemistry Laboratory, Instituto Agronômico (IAC), P.O. Box 28, CEP 13001-970 Campinas, SP, Brazil;2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovonám. 2, CZ - 166 10 Praha 6, Dejvice, Czech Republic; *Author for correspondence (e-mail:mzullo@cec.iac.br; phone: +55 19 32415188-312; fax: +55 19 32415188-420)

Received 3 April 2002; accepted in revised form 29 July 2002

Key words: Brassin, Brassinolide activity, Brassinolide, Brassinosteroid analogues, Brassins, Natural brassinos-teroids

Abstract

In this paper the definitions of brassinolide, brassinolide activity and brassins are reviewed, and definitions forthe terms brassin, natural brassinosteroids and brassinosteroid analogues, based on biosynthetic reasoning andstructure similarity are proposed.

Introduction

After the discovery of brassinolide (Grove et al.1979), its synthesis (Ishiguro et al. 1980; Fung andSiddall 1980), and the preparation (Thompson et al.1979) or detection of similar compounds (Abe et al.1982), many terms were used to designate this familyof plant growth regulators, such as brassino steroids(Thompson et al. 1979), brassinolide-like substances(Abe et al. 1982), and brassinosteroids (Thompson etal. 1982). The last term is now in common usage, butsometimes in a broader sense, leading to misunder-standing or misuse of the terms brassin, brassins,brassinolide, brassinolides, brassinosteroid, brassi-nosteroids. One example which occurs very often inthe literature is the term “brassinolides”, which is to-tally wrong, as will be seen below. This article indi-cates how such misuse can be avoided. Althoughthere have been some efforts to systematize nomen-clature in the brassinosteroid field (Mandava 1988;Khripach et al. 1999), the question remains on whatis a brassinosteroid and what is its characteristic bio-logical activity. We propose and review some defini-tions, based on structural and biosynthetic reasons,for recognizing these compounds.

Natural brassinosteroids

To date, more than 40 brassinosteroids have been iso-lated or detected (Khripach et al. 1999; Adam et al.1999; Fujioka 1999; Soeno et al. 2000) (Figure 1).They present a 5�-cholestane, 5�-ergostane or 5�-si-tostane steroidal skeleton, mono- to trioxygenation inring A, and 22�,23�-dihydroxylation in the sidechain. Ring B can be fully saturated or include a ke-tone or lactone function at carbon 6.

An inspection of the structures shown in Figure 1reveals that all the compounds contain: a) a normalor a B-homo cholestane, ergostane or sitostane steroi-dal skeleton; b) an oxygen function at carbon 3 (� or� hydroxyl, ester, ether or ketone); c) trans-trans-trans fusion, i.e., all-trans fusion, between rings A/B,B/C and C/D; d) 20R (or 20�) configuration; e) �-cis(i.e. R,R) vicinal hydroxyls at carbons 22 and 23, andf) only carbon, hydrogen and oxygen in their mole-cules. It is noteworthy that none of these compoundslack an oxygen at carbon 3, none include A/B cis ringfusion or a carbon-carbon double bond between ringsA and B, and none have less than 27 carbon atoms.

Some partially characterised compounds were alsoclassified as brassinosteroids, such as those shown inFigure 2, by structure similarity. Two compoundsshown in Figure 2, �-epi-23-dehydrobrassinolide

1Plant Growth Regulation 39: 1–11, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Figure 1. Natural brassinosteroids (all-trans ring junctions unless otherwise noted).

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(Watanabe et al. 2000) and �-epi-23-dehydrocastast-erone (Adam et al. 1999), do not contain the vicinalhydroxyls in the side chain, but an R(�)-hydroxyke-tone, and are considered to be metabolites of knownbrassinosteroids (Fujioka 1999). The four partiallycharacterised oxo, carboxo or carboxy derivatives ofbrassinolide or dolicholide and the oxa and carboxo

derivatives of castasterone, also shown in Figure 2,found in the extract of immature seeds of Phaseolusvulgaris L. (Kim 1991), deviate also from the char-acteristics listed for the natural brassinosteroids ofknown structure.

Figure 1. Continued.

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Natural products of related structure

A number of natural steroids, of animal or plant ori-gin, contains one or more of the fragments seen in thestructures of brassinosteroids. Very few examples ofsteroidal B ring lactones are found in nature, one ofthem being asterasterol A, isolated from an Antarcticstarfish (De Marino et al. 1997) (Figure 3), but thelactone function is regioisomeric with that found inbrassinosteroids. While it is somewhat common tofind natural steroids oxygenated at carbons 20 and/or22, it is unusual to find 22,23-dioxygenation, a fewexamples of them being the water mould sex stimu-

lating steroid antheridiol (Arsenault et al. 1968),found in Achlya bisexualis, some steroidal sapo-genins, like isoplexigenin C (Freire et al. 1970), andthe withanolide ixocarpalactone A (Kirson et al.1979). In the case of steroidal sapogenins and witha-nolides, 22,23-dioxygenation is accompanied by atleast 16-oxygenation, and in the case of antheridiol,oxygen in C-23 is part of a 5-membered ring lactone.Ecdysteroids were soon recognised (Grove et al.1979) as having some structural similarity withbrassinosteroids, owing to cis-2,3-dihydroxy func-tion, a 6-ketone function (usually conjugated with7,8-unsaturation) and polyhydroxylation in a choles-

Figure 2. Partially characterized brassinosteroids (all-trans ring junctions unless otherwise noted).

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tane side chain, like in the Arthropod moulting hor-mone ecdysone (Butenandt and Karlson 1954), and inthe phytoecdysteroid muristerone (Canonica et al.1972). A number of polyhydroxylated sterol conju-gates were isolated from marine organisms (Aiello etal. 1999), some of them showing polyfunctionalisa-tion in rings A and B. The epimeric pair of tamoster-one sulphates (Fu et al. 1999), isolated from a spongeof the family Oceanapiidae, is a surprising exampleof a 22,23-dioxygenated animal steroid, in which theside chain is identical with that of brassinolide. Thereare at least two differences between tamosterones andthe up to now known brassinosteroids: ring A is 2,3,4-trioxygenated in tamosterones, while in brassinoster-oids 2,3-dioxygenation, and only very rarely 1,2,3-trioxygenation were found, and ring B is 6,7-dihydroxylated in tamosterones, while in

brassinosteroids 7-oxygenation occurs only when partof a 6-oxo-7-oxalactone function. In 14-epitamoster-one sulphate C/D ring junction is cis, while in brassi-nosteroids all ring junctions are trans.

Comparing the structure of compounds in Figures1 and 2 with that of other natural steroids resemblingbrassinosteroids, like those shown in Figure 3, onecan depict the general structural formula for naturalbrassinosteroids shown in Figure 4, accounting for allstructural variations found in fully characterised com-pounds of this class known up to now. It is seen thatthe only distinctive structural fragment is the(20S,22R,23R)-cholestane-22,23-diol side chain withan oxygen at carbon 3.

Figure 3. Some oxygenated natural steroids resembling brassinosteroids (all-trans ring junctions unless otherwise noted).

Figure 4. General formula of natural brassinosteroids (common features of natural brassinosteroids in bold face).

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Brassinolide biosynthesis

The present knowledge on brassinolide biosynthesis(Sakurai 1999; Asami and Yoshida 1999; Schrick etal. 2000; Kushiro et al. 2001), obtained by studyingdifferent plant species, is summarized in Figure 5.The biological activity of a compound in this biosyn-thetic route is more pronounced as it is closer tobrassinolide and the oxidation state of carbon 6 ishigher. Although some brassinolide precursors havenot been isolated from or detected in plant material,such as 6�-hydroxycampestanol (Suzuki et al. 1995)and 6�-hydroxycastasterone (Fujioka et al. 2000), itwas possible to prove their occurrence as endogenoussteroids in biosynthetic studies by using isotopicallylabelled precursors. In this way, 6�-hydroxy functionshould be considered also as a characteristic fragmentof brassinosteroids.

A number of brassinosteroid biosynthetic mutants,identified mainly in Arabidopsis thaliana, have beendiscovered in the few past years (Clouse and Sasse1998; Clouse and Feldmann 1999), some of them de-fective in steps prior to the synthesis of campestanol,the branching point in the early and late C-6 oxida-tion pathways in brassinolide biosynthesis. This mayhave provoked the wrong idea that brassinosteroidprecursors could also be considered as brassinoster-oids themselves.

Definitions

Based on the above information we suggest the useof the following definitions.

Brassinolide (Grove et al. 1979) is(20S,22R,23R,24S)-2�,3�,22,23-tetrahydroxy-24-methyl-B-homo-7-oxa-5�-cholestan-6-one, the firstisolated compound of this family of plant growth reg-ulators.

Natural brassinosteroids are considered only the3-oxygenated (22R,23R)-5�-cholestane-22,23-diols,of plant origin, bearing alkyl or oxy substituents, con-jugated or not to sugars or fatty acids, presenting thecharacteristic brassinolide activity. Applying this defi-nition to the biosynthetic route of brassinolide shownin Figure 5, one would consider as brassinosteroidsonly those compounds originated after the 22�,23�-dihydroxylation (i.e., those between teasterone or6-deoxoteasterone and brassinolide), and hence asbrassinosteroid precursors those before dihydroxyla-tion occurs (i.e., those compounds up to cathasterone

and 6-deoxocathasterone). Using this definition, asummary of the variations in the structure of fullycharacterised natural brassinosteroids is presented inFigure 6.

Following the above definition, synthetic brassi-nosteroids or brassinosteroid analogues would referto those compounds, of synthetic origin, obtained bychemical or biotechnological means, exhibiting anystructural similarity with natural brassinosteroidsand/or brassinolide activity, examples being presentedin Figure 7. Falling into this broad category are com-pounds presenting one or more of the fragments ob-served in natural brassinosteroids but with a structuraldiversity not found in nature in these types of com-pounds. Included are by-products in the synthesis ofnatural brassinosteroids [like 22,23,24-triepibrassino-lide (Thompson et al. 1979)], regioisomeric ketonesor lactones [such as the 7-keto-6-deoxo-2,24-diepi-castasterone (Anastasia et al. 1985) and 6-oxa-7-oxo-28-homobrassinolide (Takatsuto et al. 1987)], epimersof natural brassinosteroids [like 2,3-diepisecasterone(Voigt et al. 1995)], lactams or thialactones [like 28-homobrassinolide lactam (Kishi et al. 1986)], ethers[like 6-deoxo-28-homobrassinolide (Kishi et al.1986)], ketones [like 6a-homocastasterone (Baron etal. 1998)], norbrassinosteroids [such as 26,27-bisnor-brassinolide (Takatsuto et al. 1984) or the androstaneanalogue of brassinolide (Kohout et al. 1991)]. Com-pounds presenting unusual hydroxylation for the nat-ural brassinosteroids [such as 28-hydroxycastasterone(Kametani et al. 1988), 12�-hydroxy-24-epibrassino-lide (Voigt et al. 1993) or 5�-hydroxy-28-ho-mocastasterone (Brosa et al. 1998)] are also includedin this category. The 5�-isomers of natural brassinos-teroids [such as 5-epi-28-homocastasterone (Brosa etal. 1996)], ethers [like 22,23-dimethylbrassinolide(Luo et al. 1998)], epoxides [like TS-303 (Kamuroand Takatsuto 1999)], aromatic compounds [like thephenyl analogue of brassinolide (Hayashi et al.1989)], heterocycles [as the dioxonane (Lichtblau etal. 1999)], and halogen derivatives [like 5�-fluoro-homocastasterone (Ramirez et al. 2000)], as well assome compounds like the castasterone analogues ofthe spirostane (Marquardt et al. 1988) and solanidane(Quyen et al. 1994) skeletons, are also included in thiscategory. Although the above examples are com-pounds of steroidal nature, there are a few non-steroi-dal compounds that mimic brassinolide activity(Andersen et al. 2001).

We would like to point out that an overlap mightexist between natural brassinosteroids and synthetic

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Figure 5. Biosynthesis of brassinolide (all-trans ring junctions unless otherwise noted).

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brassinosteroids or brassinosteroid analogues, espe-cially if an analogue is firstly synthesised and onlythen it is described in or isolated from natural mate-rial [e.g. 24-epibrassinolide, detected in the Vicia fabapollen (Ikekawa et al. 1988) many years after its syn-thesis (Thompson et al. 1979)]. However, this is onlyminor overlap. A very important point is that it is notpossible to use the term “brassinolides”, as this wordmeans nothing. Brassinolide is one compound onlyand all other compounds with similar structure and/oractivity are brassinosteroids, either natural or syn-thetic analogues.

There are two more terms which should be men-tioned here - brassins and brassin.

Brassins (Mitchell et al. 1970) is the complex mix-ture of lipids purified from rape (Brassica napus L.)pollen which contain a very small amount of natural

brassinosteroid(s) (usually fraction of percent or less).Brassin is therefore an extract containing one naturalbrassinosteroid or a mixture of natural brassinoster-oids.

Brassinolide activity is the characteristic biologi-cal activity – elongation, swelling, curvature andsplitting of the treated internode – in the bean secondinternode bioassay (Grove et al. 1979). Several otherbioassays have been used to evaluate brassinolide ac-tivity (Yopp et al. 1981; Mandava et al. 1981; Adamand Marquardt 1986), among them the rice laminainclination bioassay (Wada et al. 1981) and the wheatleaf unrolling bioassay (Wada et al. 1985). Great caremust be taken in choosing a brassinolide activity bio-assay when looking for compounds useful for agri-cultural applications, since compounds which showhigh activity in one or more bioassays may show lit-

Figure 6. Structural variations in natural brassinosteroids.

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tle, if any, activity in field trials (Kamuro and Takat-suto 1999). This lack of correlation between the ac-tivity of a compound in brassinolide activitybioassays and in field trials was recognised even be-fore the isolation of brassinolide (Steffens 1991).Brassinosteroids, their analogues or complexes, maynot be active in the rice lamina inclination assay (De

Azevedo et al. 2001), depending upon the cultivarsused. Compounds active in the bean second internodebioassay but without or with marginal activity in ricelamina inclination assay, like the androstane analogueof brassinolide (2�,3�,17�-trihydroxy-5�-androstan-6-one), can be active in field trials (Kohout et al.2001).

Figure 7. Examples of brassinosteroid analogues (all-trans ring junctions unless otherwise noted).

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Acknowledgements

Support from Fapesp (grants 99/07907-2, 01/05711-5and 99/05119-7) and GA CR (No. 203/01/0083), andby research project Z4 055 905 is gratefully acknowl-edged.

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