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
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
. A breakthrough in elucidating the
crucial role of endogenous BRs came from the isolation
of mutants defective in BR biosynthesis and perception
. 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
(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
. 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
et al., 1996; Kauschmann et al., 1996; Li and Chory,
. 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.,
. 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
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.,
. 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
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
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.,
. 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
(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.,
. 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
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
(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
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
(Asami and Yoshida, 1999; Li et al., 1996)
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
(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
(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
(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
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
(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
. 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
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.,
. 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
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.,
. 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.
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. In particular, continued studies of how light and
other hormone response pathways are integrated with BRs
should provide many more interesting years ahead.
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