BRing it on: new insights into the mechanism of brassinosteroid action

Journal of Experimental Botany, Jan 2004

Several recent breakthroughs have filled in key details of the brassinosteroid (BR) response. Identification of BAK1, a BRI1 interacting protein, the negative regulator BIN2, as well as direct targets of BIN2, BZR1 and BES1, provide a link between BR perception at the cell surface and regulation of gene expression in the nucleus. Global expression studies further defined the downstream events in this pathway, confirming the role of several factors acting in negative feedback regulation on BR levels. New links to the plant hormone, auxin, were also uncovered.

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BRing it on: new insights into the mechanism of brassinosteroid action

Journal of Experimental Botany BRing it on: new insights into the mechanism of brassinosteroid action Jennifer L. Nemhauser 1 Joanne Chory 0 1 0 Howard Hughes Medical Institute, The Salk Institute for Biological Studies , 10010 North Torrey Pines Road, La Jolla, CA 92037 , USA 1 Plant Biology Laboratory, The Salk Institute for Biological Studies , 10010 North Torrey Pines Road, La Jolla, CA 92037 , USA Several recent breakthroughs have ®lled in key details of the brassinosteroid (BR) response. Identi®cation of BAK1, a BRI1 interacting protein, the negative regulator BIN2, as well as direct targets of BIN2, BZR1 and BES1, provide a link between BR perception at the cell surface and regulation of gene expression in the nucleus. Global expression studies further de®ned the downstream events in this pathway, con®rming the role of several factors acting in negative feedback regulation on BR levels. New links to the plant hormone, auxin, were also uncovered. Arabidopsis; auxin; brassinosteroids; light Introduction Hormones are at the heart of all plant growth and development, yet the mechanism by which their effects are harnessed into speci®c morphological outcomes are largely unknown. Hormone activity has been implicated in responses to a wide array of biotic, abiotic, and developmental stimuli. Brassinosteroids (BRs), among the newest hormones to be identi®ed, play roles throughout the plant life cycle, including germination, root and stem elongation, seedling photomorphogenesis, vascular development, ¯oral organ elongation, and senescence. Brassinolide (BL), the most biologically active BR, was initially isolated in an ambitious experiment using over 200 kg of Brassica napus pollen as the starting material (Grove et al., 1979) . Subsequently, BRs have been identi®ed in all plant species examined to date. Puri®ed BL was shown to promote cell elongation in a number of bioassays encompassing diverse species and tissue types (Mandava, 1988) . A breakthrough in elucidating the crucial role of endogenous BRs came from the isolation of mutants defective in BR biosynthesis and perception (Clouse, 2002) . DET2 and CPD were the ®rst genes in the biosynthesis pathway to be cloned, and have been shown to encode a steroid 5a-reductase and a C23-steroid hydroxylase, respectively (Li et al., 1996; Szekeres et al., 1996) . Both mutants were originally identi®ed for exhibiting strikingly light-grown morphology, even when grown in the dark, and so were named constitutive photomorphogenesis and de-etiolation (cpd) and de-etiolated 2 (det2). These, and subsequently identi®ed mutants with reduced BR levels, also show growth defects when grown in the light (Clouse, 2002) . They are dark-green dwarfs with reduced internode, petiole, and leaf elongation, giving them a cabbage-like appearance. The mutants have reduced apical dominance, in¯orescence stems are reduced in length, and ¯owers are small with reduced fertility. Roots are also shorter than those of wild-type plants. In several different genetic screens, one loss-of-function BR-insensitive mutant, named bri1, was identi®ed (Clouse et al., 1996; Kauschmann et al., 1996; Li and Chory, 1997) . BRI1 is a leucine-rich repeat (LRR) receptor serine/ threonine kinase expressed throughout the plant (Friedrichsen et al., 2000; Li and Chory, 1997; Oh et al., 2000) . Several lines of evidence suggest that it is the BR receptor. A chimeric protein was constructed containing the N-terminus of BRI1, from extracellular through juxtamembrane domains, fused to the kinase domain of the rice gene Xa21 involved in pathogen response (He et al., 2000) . Application of BL to rice cell cultures Fig. 1. In low BL conditions, BIN2, a GSK3-like kinase, phosphorylates BES1 and BZR1. This modi®cation leads to the destabilization of BES1 and BZR1 and their degradation by the 26S proteosome. Once BL is perceived by BRI1 at the cell surface, BIN2 is inactivated by an unknown mechanism leading to the accumulation of BES1 and BZR1 in the nucleus. This activates the expression of a number of genes, including those involved in cell wall modi®cation and growth. It also leads to the rapid repression of several BR biosynthetic genes. BAK1, like BRI1, is a transmembrane LRR kinase. Although it has been shown to interact with BRI1, its exact role in the BR response has not been shown. expressing this chimeric protein mimicked the cell death response observed when cells expressing Xa21 were challenged with pathogen. In addition, BL-binding capacity observed in microsomal fractions of wild-type plants is lost when the extracellular domain of BRI1 is disrupted and increased in plants overexpressing BRI1 (Wang et al., 2001) . In the last year, several new proteins have been described, shedding light on how the BR signal is transmitted from BRI1 at the extracellular surface into the cytoplasm and the nucleus. BR signalling: from a lone player to a crowded ®eld In 2002, a number of exciting new ®ndings were described, greatly advancing understanding of BR signalling pathways. The gene responsible for the hypermorphic BRinsensitive phenotype of bin2 was cloned and found to encode a previously identi®ed member of the Glycogen Synthase 3/SHAGGY family of kinases, called GSKh (Li and Nam, 2002) . Recapitulation lines transformed with the BIN2 gene carrying the bin2-1 lesion showed a range of phenotypic severity that was correlated with transgene expression. Several lines containing the wild-type BIN2 gene under the viral 35S promoter showed a reduction in levels of the endogenous gene and could partially suppress a weak bri1 mutant. However, as BIN2 is part of a closely related family of proteins, it is possible that this effect is the result of reduced expression of another GSK3 or a combined effect of several. As GSK3-type kinases are known to be negatively regulated by phosphorylation in other systems, it is tempting to speculate that BRI1 might act as a direct regulator in this pathway. Although several approaches were taken to ®nd such a connection between the two proteins, no evidence to support such a link was found. At the same time, ultracurvata1 (UCU1), which, when mutated, caused a severe dwarf phenotype, was cloned and found to encode the same gene as BIN2 (Perez-Perez et al., 2002) . Perez-Perez and colleagues showed that the dwar®sm observed in ucu1/bin2 mutants results from a severe defect in cell expansion, which is particularly severe on the abaxial (ventral) surface of leaves. They also showed that leaves of ucu1/bin2 plants contain additional internal layers of cells contributing to the increased thickness of the organs. Physiological analysis of ucu1 roots revealed an increased sensitivity to the synthetic auxin, 2,4-D and insensitivity to 24-epibrassinolide. The close relationship of brassinosteroids and auxin was also observed in the synergistic interaction of ucu1 with semidominant auxin resistant mutants, axr2 and shy2. A major breakthrough came with the cloning and characterization of BES1 and BZR1, which provides a connection between the cytoplasmic BR response and the nucleus (Wang et al., 2002; Yin et al., 2002) . bes1 and bzr1, were identi®ed as suppressors of bri1 phenotypes, as well as being resistant to brassinozole, a BR biosynthesis inhibitor. BES1 and BZR1 encode closely related novel proteins that accumulate in the nucleus following BR treatment. Identical dominant mutations identi®ed in both genes stabilize the respective proteins and increase their nuclear accumulation. By tracking the expression of a BZR1 translational fusion with cyan ¯uorescent protein, it was possible to correlate nuclear accumulation with elongating regions of etiolated seedlings (Wang et al., 2002) . Perhaps most importantly, BES1 and BZR1 can be phosphorylated by the negative regulator BIN2, resulting in their turnover (He et al., 2002; Yin et al., 2002) . Accumulation of unphosphorylated proteins is greatly reduced in bin2/BIN2 heterozygotes, consistent with their strong growth defects. These ®ndings provided the scaffold for a new model of BR signalling (Fig. 1). In mid-2002, a pair of papers was published describing an LRR II receptor-like protein kinase called BAK1 (Li et al., 2002; Nam and Li, 2002) . BAK1 was uncovered in two laboratories using different approaches. In one, BAK1 was found to interact with the BRI1 cytoplasmic kinase domain in yeast two-hybrid analysis (Nam and Li, 2002) . Moreover, phosphorylated products of the expected size were only observed when full-length clones of both proteins were co-expressed in yeast, suggesting transphosphorylation may be required for kinase activation of both BRI1 and BAK1. In another approach, BAK1 was identi®ed as an activation-tagged suppressor of a weak allele of bri1 (Li et al., 2002) . Interestingly, unlike bzr1 and bes1 mutants, overexpression of BAK1 is not able to suppress a biosynthetic mutant or a strong bri1 allele. Both groups showed that loss-of-function bak1 alleles are less sensitive to exogenous BL, although the phenotype is quite subtle. This may result from some degree of redundancy among the several BAK1 homologues found in Arabidopsis, homologous with the SERK family in Daucus carota. Loss-of-function bak1 alleles enhance a weak bri1 phenotype, while strong bri1 alleles are epistatic to loss of bak1 function. When BAK1 is overexpressed from its own promoter it shows a similar phenotype to BRI1OX and an increased sensitivity to exogenous BL in roots. Interestingly, when BAK1 is expressed from the constitutive viral 35S promoter, the phenotype is less marked, and no increased sensitivity to BL is observed. Transgenic plants containing high levels of a kinase-dead bak1 protein show a strong bri1-like phenotype, suggesting that this mutation may create a dominant negative effect. This ®nding combined with data showing BRI1 and BAK1 interacting in vivo, led to an intriguing model where BAK1 acts near the point of BR perception, perhaps as a coreceptor with BRI1. Further analysis of BAK1's role in BL binding, the effects of BL binding on BRI1-BAK1 interactions, as well as detailed characterization of the transphosphorylation events between the two proteins should prove quite informative about the mechanism of BR perception. Shedding light on the function of endogenous BRs BRs have also been closely linked to the process of deetiolation. Mutations causing decreased BR levels or decreased BR response, as well as treatment with BR biosynthesis inhibitors, cause dark-grown plants to deetiolate (Asami and Yoshida, 1999; Li et al., 1996) . BAS1, a steroid 26-hydroxylase involved in the regulated inactivation of BRs, provides one possible mechanistic link between brassinosteroid biosynthesis and light (Neff et al., 1999). Increased expression of BAS1 results in severely reduced production of BL and is able to suppress both intermediate and null alleles of phyB fully in red light. Antisense lines of bas1 are hyper-responsive to BL, and show a decreased response to white, blue, and far-red light, but no change in their red light response. Recently, brassinosteroids have been implicated in repressing some PHYA-mediated responses (Luccioni et al., 2002) . Luccioni and colleagues performed a mutant screen, looking for plants with enhanced very low ¯uence responses (VLFR) by screening in hourly far-red light pulses and looking for shorter hypocotyls and opened cotyledons. One such mutant, called eve1, was found to be allelic to dwf1/dim, a mutant in a gene involved in BR biosynthesis. In addition to its seedling phenotypes, eve1 also shows enhanced VLFR and reduced high irradiance responses when chlorophyll and anthocyanin accumulation are measured in either hourly pulses or continuous far-red light. This same relationship was observed in det2 seedlings. Interestingly, when eve1/dwf1 plants were germinated in sunlight, there was little difference in hypocotyl length, but when germinated in canopy shadelight, BR de®ciency resulted in signi®cantly shorter hypocotyls, suggesting a role for BRs in optimizing growth in different light environments. Another recent paper suggests a mechanism for communication between the light receptors and BR biosynthesis (Kang et al., 2001) . Pra2, a dark-inducible, phytochrome-repressed small G protein from pea was used as bait in a yeast two-hybrid screen. A cytochrome P450 hydroxylase, which they named DDWF1, was identi®ed and shown to have an overlapping expression pattern with Pra2 in the elongating region of the etiolated pea epicotyl. In light, expression was detected only in the root. Pra2 and DDWF1 were shown to interact in vitro and to co-localize on the ER membrane of onion epidermal cells. Etiolated tobacco seedlings overexpressing pea Pra2 had short hypocotyls and are probably cosuppressed for the tobacco Pra2 homologue. Recombinant DDWF1 was shown to catalyse the conversion of typhasterol to cathasterone and feeding experiments of cosuppressed tobacco plants suggested that Pra2 was required for full DDWF1 activity. Although similar cosuppression experiments did not work in Arabidopsis for Pra2, overexpression of pea DDWF1 caused elongated hypocotyls. Phytochrome-mediated repression of Pra2 transcription has been studied in some detail and these ®ndings suggest one mechanism, operating at least in pea and tobacco, for communication between light and BR levels. However, other studies which quanti®ed BR levels in dark- and lightgrown seedlings fail to detect any change in hormone level with different light treatments (Symons and Reid, 2003) . Additional genetic and biochemical studies are clearly needed to determine the molecular mechanism underlying the interaction between light and BRs; such knowledge can then be applied to the broader question of whether these mechanisms are conserved across plants adapted to different light environments. BR effects in the nucleus In addition to effects on BR biosynthetic enzymes, another potential site of cross-talk between different factors affecting seedling development is at the level of gene regulation. Several recent studies have shed light on the nuclear end of the BR response. Three papers using Affymetrix Arabidopsis oligonucleotide microarrays have yielded the ®rst global glance at BR-mediated changes in gene expression (Goda et al., 2002; Mussig et al., 2002; Yin et al., 2002) . Each group used quite different starting materials for RNA isolation. Plants varied in age from 7± 50 d and encompassed several genetic backgrounds, including three biosynthetic mutants and bri1. In addition, the concentration and length of the BR treatment varied markedly from study to study. Not surprisingly, perhaps, the exact genes identi®ed were not well matched. Despite the discrepancies between the behavior of individual genes in each experimental condition, several important trends were detected in all studies (Table 1). Two classes of genes implicated by earlier studies on BR-regulated gene expression were con®rmed in these studies. In the ®rst case, several genes encoding cytochrome P450, most notably CPD and DWF4, were strongly repressed following BR application, re¯ecting the tight negative feedback regulation acting on the BR biosynthetic pathway. BAS1 was also up-regulated by BRs. Another major category of BR-regulated genes are those involved in cell-wall modi®cation and cellular metabolism, several of which have been detected previously, and re¯ect the dramatic effects on growth provoked by BRs (Friedrichsen and Chory, 2001) . Perhaps the most surprising outcome of these studies is the modest nature of the BR response. Overall, expression changes were 2±5-fold, quite different from the 10±100fold differences observed in the application of other phytohormones (Zhao et al., 2002) . Importantly, where tested, all BR responsive genes required functional BRI1. In bes1-D mutants which display a dramatic constitutive BR phenotype, all of the BR-induced genes examined show either higher basal levels of expression or hyperresponsivity to exogenous BL (Yin et al., 2002) . The small effect on gene expression observed in these studies may re¯ect the real strength of the BR response or, alternatively, these small changes may result from a previously unsuspected complexity in the localization of the BR response. For instance, if only a small subset of cells is fully competent to respond to increased endogenous or applied BRs then this could result in an overall dampening of the apparent changes in gene expression. A detailed analysis of distribution patterns of BR biosynthetic genes and signalling components is needed to distinguish between these possibilities. In another approach to dissecting the nuclear response to BRs, three early BR response genes were identi®ed and examined in greater detail (Friedrichsen et al., 2002) . BEE1, BEE2 and BEE3 encode proteins with conserved basic helix-loop-helix motifs. Although the BEE genes show only a 2-fold induction by BR, plants lacking all three gene products show reduced BR responses, con®rming that the small differences observed in the microarray experiments are probably relevant to BR signalling. Interestingly, these three BR early response genes are also regulated by other hormones. Most strikingly, they are all repressed by the application of abscisic acid (ABA), a known antagonist of BR signalling. Triple mutants lacking expression of all three genes show no ABA phenotype, but roots of plants overexpressing BEE1 show a reduced response to exogenous ABA. Taken together with the results of the microarray studies, the results with the BEE genes strongly suggest that small changes in gene expression are an important part of the BR response. BR links to auxin Another remarkable outcome from the global expression studies was the large proportion of auxin-regulated genes which also exhibited BR-responsivity. There are many examples of cross-talk between hormones in plant biology. For instance, recent elegant work has demonstrated that the growth-promotive effects of auxin in the root are largely mediated through the action of gibberellins (Fu and Harberd, 2003) . Auxin and BRs have been linked to many of the same growth processes, including vascular differentiation, ¯ower and fruit development, and root growth, in addition to their roles in seedling photomorphogenesis and shade avoidance (Mandava, 1988) . In addition, auxin and BRs have synergistic effects on cell elongation in a wide variety of bioassays, including soybean and cucumber hypocotyls, azuki bean and pea epicotyls, and rice lamina joints (Katsumi, 1985; Mandava, 1988; Yalovsky et al., 1990; Yopp et al., 1981) . Exogenous brassinolide has little effect on hypocotyl elongation in Arabidopsis mutants defective in biosynthesis or response to other hormones, with the notable exception of an auxin-response mutant axr2 which shows a 2±3-fold increase in hypocotyl elongation (Szekeres et al., 1996) . Also, while mutants defective in gibberellin and ethylene signalling show a normal growth response to increased auxin levels provoked by temperature increases, a mutant de®cient in BR synthesis, det2, shows signi®cantly reduced elongation (Gray et al., 1998) . Global analyses of gene expression in BR-treated plants reveal one possible mechanism for the interaction between these two hormones. Several previously identi®ed auxin early-response genes are also up-regulated by BRs (Yin et al., 2002) . Genes from all known classes of auxin earlyresponse genes, GH3, Aux/IAA, SAUR, were represented. In addition, expression changes in putative auxin ef¯ux carriers, auxin conjugating enzymes, and cytochrome P450 enzymes known to be involved in auxin synthesis were also detected in different studies. Regulation of these genes does not seem to follow a simple pattern. As described by Goda and colleagues, genes in the known auxin-responsive families fall into four classes on closer examination: those that are speci®cally induced by auxin, those that are induced by both BRs and auxin, those that are induced by BRs and not auxin, and those which are induced by auxin but repressed by BRs (Goda et al., 2002) . Promoters of most auxin-responsive genes identi®ed contain an auxin responsive element [T/A]GTCTC (Guilfoyle et al., 1998) . A synthetic construct containing ®ve repeats of this element, called DR5, has been shown to provide high sensitivity to auxin either in vitro or in vivo (Ulmasov et al., 1997) . To investigate the nature of the BR:auxin connection further, the response of transgenic seedlings carrying the DR5:GUS reporter was examined. Consistent with the microarray data, the GUS staining was greatly enhanced in plants exposed to exogenous BL (J Nemhauser and J Chory, unpublished data). These ®ndings suggest that BRs either act directly on auxin-responsive elements or are able to sensitize cells in some manner to auxin. Future studies aimed at dissecting the precise relationship between these two hormone pathways will undoubtedly shed light on the response to the individual hormones. Future prospects It has been a banner year for BRs. With an increasingly de®ned biosynthetic pathway and ever-expanding model of BR signalling, asking more sophisticated questions about BR effects are possible. It will now be possible to connect BR signalling to the cell mechanics of expansion and division, an area that remains poorly understood. A large number of putative transcription factors and proteins of unknown function are also BR-regulated and provide fertile ground for future investigations into the BR response. Moreover, it is clear that to understand the function of BRs fully, they must be placed in the context of the myriad other pathways acting throughout plant development. 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Jennifer L. Nemhauser, Joanne Chory. BRing it on: new insights into the mechanism of brassinosteroid action, Journal of Experimental Botany, 2004, 265-270, DOI: 10.1093/jxb/erh024