Brassinosteroids

Brassinosteroids J Li, University of Michigan, Ann Arbor, MI, USA

© 2013 Elsevier Inc. All rights reserved.

This article is a revision of the previous edition article by J Chory, volume 1, pp 238–239, © 2001, Elsevier Inc.

Glossary 14-3-3 Proteins A large family of highly conserved ubiquitously expressed eukaryotic proteins capable of binding client proteins carrying phosphorylated serine or threonine residues. Apical dominance A plant growth phenomenon whereby the tip of the main stem inhibits the outgrowth of secondary or lateral stems. Brassinazole A highly specific brassinosteroid biosynthesis inhibitor. Lactone A cyclic ester resulted from a condensation reaction between a hydroxyl (–OH) group and a carboxylic acid group (–COOH) in the same molecule. Leucine-rich repeat A protein structural domain made up of repeating 20–30 amino acid motifs rich in the hydrophobic amino acid leucine. Each motif adopts a β-strand-turn-α-helix structure, while the assembled domain of many repeating motifs has a curved horseshoe

Introduction Brassinosteroids are a unique class of plant polyhydroxyster­ oids that are structurally similar to cholesterol-derived animal steroid hormones and are essential for normal plant growth and development. While the importance of animal steroids was known for over 80 years, the plant steroids acquired their hormonal status only in the mid-1990s. The discovery of plant steroid hormones was the result of over 30 years of research that was initiated in the 1960s by searching for novel growth-promoting substances in the pollen of many plant species. In 1970, a unique growth-stimulating activity was dis­ covered from the organic solvent extract of pollen from rapeseed (Brassica napus L.) and its active compounds were named brassins. Nine years later, the chemical nature of the most active brassin was determined by X-ray crystallography to be (22R,23R,24S)-2α,3α,22,23-tetrahydroxy-24-methyl-6,7­ s-5α-cholestano-6,7-lactone (named brassinolide) after purify­ ing 4 mg of the compound from over 500 pounds of bee-collected rapeseed pollen (Figure 1).

Natural Occurrence and Chemical Diversity Up to now, nearly 70 brassinolide analogs (collectively known as brassinosteroids) were identified by a combination of bio­ logical assays with gas chromatography and mass spectrometry from 53 flowering plants, 6 nonflowering seed-bearing plants, 1 nonseed vascular plant, 1 nonvascular plant, and 3 green algae. These naturally occurring brassinosteroids, all known to be 5α-cholestane derivatives, differ mainly in (1) the presence and configurations of hydroxyl groups at carbon 2 (C2) and C3 Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 1

structure with a convex α-helical array, a concave parallel β-sheet, and an internal hydrophobic core. Petiole The stalk that attaches a leaf to the plant stem. Photomorphogenesis A series of physiological and morphological changes of a dark-grown seedling in response to light. This developmental change is also known as de-etiolation or simply greening. Rapeseed A bright yellow flowering member of the Brassicaceae (mustard or cabbage) family. Receptor-like cytoplasmic kinase Plant proteins highly similar to receptor-like kinases in the kinase domain but lacking extracellular and transmembrane domains. The Baeyer–Villiger oxidation An oxidative cleavage reaction of a carbon–carbon bond adjacent to a carbonyl group, thus converting ketones to esters and cyclic ketones to lactones. Vascular plants Plant species having vascular tissues that transport water, mineral nutrients, sugars, and other biological molecules throughout the plant body.

positions in ring A; (2) the presence of an alcohol, ketone, or lactone function at C6 in ring B; (3) the number and stereo­ chemistry of hydroxyl groups at C22 and C23 in the side chain; (4) the presence or absence of a methyl (methylene) or ethyl (ethylidene) group at C24; and (5) conjugations of various hydroxyl groups with sugars, fatty acids, and sulfate (Figure 1). In flowering plants, brassinosteroids are found at extremely low levels in pollen, anthers, seeds, leaves, stems, roots, flowers, and young vegetative tissues, with pollen and immature seeds being the richest sources. Thus, brassinoster­ oids are ubiquitous plant steroids that likely evolved before the appearance of the first plant species on land.

Physiological Responses of Exogenously Applied Brassinosteroids Physiological studies via exogenous applications of synthetic, naturally occurring brassinosteroids and their analogs to var­ ious plant species revealed that the plant steroids could stimulate cell division and cell elongation, promote vascular differentiation, inhibit leaf and fruit abscission, regulate male fertility, enhance tolerance against biotic and abiotic stresses, and stimulate or inhibit gene expression. These biological assays also identified key chemical functions critical for the growth-promoting activity: (1) the cis-vicinal hydroxyls at C2 and C3 positions in ring A, (2) a ketone or a lactone function at C6 in ring B, (3) cis α-configured hydroxyl groups at C22 and C23 positions, and (4) an alkyl substitution at C24 (Figure 1). Thus, brassinolide and its immediate precursor castasterone, which has a ketone group at C6, are considered as the most active brassinosteroids. In addition, numerous field trials

doi:10.1016/B978-0-12-374984-0.00170-4

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(22R ,23R ,24S)-2α,3α,22,23­ Tetrahydroxy-24-methyl­ 6,7-s-5α-cholestano-6,7-lactone

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Figure 1 The structure and branched biosynthetic pathways of brassinolide. The four key functional groups and four biosynthetic segments (early or late C22/C6 oxidation) are indicated by colored background, while the key chemical reactions that convert campesterol to brassinolide are indicated by curly brackets. The structure of brassinolide is shown with the steroid carbon numbering system.

suggested that brassinosteroids could be used as ecologically safe, yield-enhancing plant growth stimulants. In spite of these dramatic physiological responses and potential agricultural applications, brassinosteroids were not accepted as plant growth hormones for over a quarter century because their physiological roles during normal plant growth and develop­ ment remained unknown.

Hormonal Functions of Endogenous Brassinosteroids It was the genetic studies in Arabidopsis thaliana, a favorite model organism for plant biology, in the mid-1990s that functionally demonstrated that brassinosteroids are true hormones that par­ ticipate with other plant hormones in regulating numerous aspects of plant growth and development. The first two known brassinosteroid-deficient mutants, de-etiolated2 (det2) and con­ stitutive photomorphogenesis and dwarfism (cpd), were independently identified in genetic screens for Arabidopsis mutants that exhibit the morphology of light-grown seedlings when they were actually grown in complete darkness. In addition to the dark-grown phenotype, the two Arabidopsis mutants share similar morphological and developmental abnormalities in the light, which include dwarfed stature, round and dark-green downward-curling leaves, short petioles, reduced cell size, delayed flowering and senescence, male sterility, reduced apical dominance, and abnormal vascular development. These pheno­ typic changes indicate that the protein products of DET2 and CPD genes and their associated processes are essential for the

normal growth and development of Arabidopsis. Molecular clon­ ing of the DET2 and CPD genes, biochemical studies of their protein products, and metabolic analysis of the corresponding mutants indicated that DET2 and CPD encode a steroid 5α-reductase and a steroid hydroxylase, respectively, that catalyze two crucial steps of the Arabidopsis brassinosteroid biosynthetic pathway. Interestingly, DET2 was able to catalyze the predicted Δ5 reduction with several animal steroid substrates in vitro and the two human steroid 5α-reductase genes were able to rescue the Arabidopsis det2 mutation, indicating evolutionary conservation of some steroid biosynthetic enzymes between the animal and plant kingdoms that were diverged at least 1 billion years ago. As expected for hormone-deficient mutants, exogenous application of active brassinosteroids and their biosynthetic intermediates downstream of the mutated steps could rescue the phenotypes of the two Arabidopsis mutants. Additional brassinosteroid-deficient dwarf mutants were discovered in Arabidopsis thaliana and other plant species, such as tomato (Solanum lycopersicum), garden pea (Pisum sativum), and rice (Oryza sativa). Subsequent studies of these mutants resulted in a wide acceptance of brassinosteroids being the sixth class of the plant hormones and helped to eluci­ date branched brassinosteroid biosynthetic pathways.

Biosynthesis and Metabolism Brassinosteroids are synthesized from three major phytosterols: campesterol, sitosterol, and stigmasterol, all of which are C24-alkylated cholesterols functioning mainly as common

Brassinosteroids constituents of cellular membranes. The conversion of these phytosterols to biologically active brassinosteroids involves the following key reactions: (1) reduction of the Δ5 double bond involving the DET2 steroid 5α-reductase, (2) addition of α-oriented vicinal hydroxyl groups at C22 and C23, (3) epimerization of a 3β-hydroxyl group to a 3α-hydroxyl group, (4) formation of the 2α-hydroxyl group, and (5) oxida­ tion of C6 and the subsequent Baeyer–Villiger-type oxidation

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to form an alcohol/ketone and a lactone function at C6, respec­ tively (Figure 2). Depending on when the C22 hydroxylation (before or after the Δ5 reduction) and C6 oxidation (right after the Δ5 reduction or at the third to last step) first occur, the brassinosteroid biosynthetic routes are termed the early and late C22 or C6 oxidation pathways that involve the same set of enzymes, most of which are members of the cytochrome P450 superfamily, acting on C22-non­

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Cytosol Figure 2 A current model of the brassinosteroid signal transduction pathway (see section Signaling Mechanism for details). P, phophorylated proteins.

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hydroxylated or hydroxylated or C6 deoxo or oxo brassinoster­ oid intermediates (Figure 1). The homeostasis of brassinosteroids is controlled largely by transcriptional regula­ tion of genes that encode rate-limiting enzymes of the brassinosteroid biosynthetic pathways, such as the C22 hydro­ xylase, and brassinosteroid-inactivating or brassinosteroid­ conjugating enzymes, including C25/C26 hydroxylases, 22-O­ sulfotransferases, and 23-O-glucosyltransferases.

Brassinosteroid Perception at the Cell Surface Genetic and biochemical studies using Arabidopsis mutants also led to the discovery of the first brassinosteroid receptor. A genetic screen for mutants exhibiting normal root elongation in the presence of high concentrations of active brassinoster­ oids known to inhibit the root growth of wild-type Arabidopsis seedlings isolated a single, brassinosteroid-insensitive (bri1) mutant, while genetic screens for det2-like Arabidopsis mutants that could not be rescued by exogenous brassinosteroid applica­ tion identified additional bri1 alleles, suggesting that BRI1 is a critical component of the steroid-sensing mechanism in Arabidopsis. Molecular cloning of the BRI1 gene and subsequent biochemical and structural studies demonstrated that BRI1, a member of the plant leucine-rich-repeat receptor-like serine/threonine kinase family, is a cell-surface-localized brassinosteroid receptor that uses a special ’island’ domain in its extracellular region to bind brassinosteroids and relies on its cytoplasmic kinase domain to transmit the steroid signal into plant cells (Figure 2). Such a cell-surface-initiated signaling pro­ cess is quite different than the classical signaling mechanism of animal steroid hormones. As lipophilic molecules, animal ster­ oids can easily move through the plasma membrane into the cytosol where the steroid hormones bind their intracellular receptors and the steroid-bound receptors can subsequently move into the nucleus to bind promoters of steroid-responsive genes, thus turning on or turning off their expression to bring about a defined set of cellular responses in animals.

Signaling Mechanism The extensive and intensive studies in the past decade have uncovered a linear signaling cascade that relies on protein phosphorylation to regulate the protein stability, subcellular localization, and DNA-binding activity of two highly similar, plant-specific transcription factors, known as bri1-EMS sup­ pressor 1 (BES1) and brassinazole-resistant 1 (BZR1), which directly bind to regulatory sequences of thousands of target genes to affect plant growth and development (Figure 2). Thus, in the absence of active brassinosteroids, BRI1 mainly exists as inactive homodimers on the plasma membrane although recent structural studies suggest that BRI1 unlikely forms a homodimer. A ligand-free BRI1 binds strongly with a small plasma membrane-associated protein called BRI1 kinase inhibitor 1 (BKI1), thus preventing the formation of an hetero­ tetrameric receptor complex between BRI1 and its co-receptor BRI1-associated receptor kinase 1 (BAK1), a much smaller leucine-rich-repeat receptor-like kinase. As a result, the consti­ tutively active brassinosteroid-insensitive 2 (BIN2), an Arabidopsis serine/threonine kinase highly similar to the animal glycogen synthase kinase 3 (GSK3), directly binds and phos­ phorylates BES1 and BZR1 to reduce their protein abundance,

to keep them in the cytosol via phosphorylation-dependent interaction with several 14-3-3 proteins, and/or to inhibit their DNA-binding activity, thus altering the expression of their target genes and inhibiting cell division and cell expan­ sion. Brassinosteroid binding to BRI1’s extracellular domain induces a rapid phosphorylation-dependent BKI1 dissociation from the plasma membrane and subsequent oligomeri­ zation and transphosphorylation of BRI1 and BAK1, causing a full activation of BRI1 that can phosphorylate serine, threonine, and tyrosine residues on itself and its substrates. The fully activated BRI1 phosphorylates several plasma membrane-associated receptor-like cytoplasmic kinases, including constitutive differential growth 1 (CDG1) and bras­ sinosteroid signaling kinase 1 (BSK1), which subsequently bind, phosphorylate, and activate BRI1 suppressor 1 (BSU1), a serine/threonine protein phosphatase. Intriguingly, this unique phosphatase, despite lacking the catalytic signature motif conserved in all known protein tyrosine phosphatases and dual-specificity phosphatases, is thought to dephosphory­ late an autophosphorylated tyrosine residue in the activation loop of BIN2, thus inactivating this GSK3-like kinase and relieving its inhibitory effect on BES1 and BZR1. Consequently, the newly synthesized BES1 and BZR1 remain unphosphorylated, while their phosphorylated counterparts are dephosphorylated by several members of the plant protein phosphatase 2A family. The non phosphorylated dephosphorylated BES1 and BZR1 accumulate in the cytosol and translocate into the nucleus to activate or inhibit the expression of their direct target genes. In addition to BES1 and BZR1, the brassinosteroid-induced changes in gene activ­ ities require other transcription factors, such as basic helix-loop-helix proteins and Myb-related proteins, and other nuclear proteins involved in transcription elongation and chro­ matin remodeling.

See also: Brassicaceae, Molecular Systematics and Evolution of; Arabidopsis Thaliana: The Premier Model Plant; Photomorphogenesis in Plants, Genetics of; Plant Hormones; Steroids.

Further Reading Clouse S (2011) Brassinosteroids. The Arabidopsis Book 9: e0151. doi:10.1199/tab.0151. Clouse SD, Langford M, and McMorris TC (1996) A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiology 111: 671–678. Clouse SD and Sasse JM (1998) Brassinosteroids: Essential regulators of plant growth and development. Annual Review of Plant Physiology and Plant Molecular Biology 49: 427–451. Grove MD, Spencer GF, Rohwedder WK, et al. (1979) Brassinolide, a plant growthpromoting steroid isolated from Brassica napus pollen. Nature 281: 216–217. Hayat S and Ahmad (eds.) (2011) Brassinosteroids: A Class of Plant Hormones. Dordrecht; Heidelberg; London; New York: Springer. Kim T-W and Wang Z-Y (2010) Brassinosteroid signal transduction from receptor kinases to transcription factors. Annual Review of Plant Biology 61: 681–704. Li J and Chory J (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90: 929–938. Li J, Nagpal P, Vitart V, McMorris TC, and Chory J (1996) A role for brassinosteroids in light-dependent development of Arabidopsis. Science 272: 398–401. Mitchell JW, Mandava NB, Worley JF, Plimmer JR, and Smith MV (1970) Brassin: A new family of plant hormone from rape pollen. Nature 225: 1065–1066. Szekeres M, Nemeth K, Koncz-Kalman Z, et al. (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85: 171–182.