Plant Physiol. Biochem.

, 1999, 37 (5), 363372

Physiology and molecular mode of action of brassinosteroids

Carsten Mssig*, Thomas Altmann
Max Planck Institut fr Molekulare Pflanzenphysiologie, Karl-Liebknecht-Str. 25, 14476 Golm, Germany
* Author to whom correspondence should be addressed (fax +49 3319772301; e-mail muessig@mpimp-golm.mpg.de)

(Received November 4, 1998; accepted January 4, 1999)

Abstract Brassinosteroids (BRs) comprise a group of polyhydroxysteroids, which show close structural similarity to steroid
hormones from arthropods and mammals. BRs are now accepted as a new class of phytohormones due to their ubiquitous
occurrence in plants, their highly effective elicitation of various responses and the identification of mutants defective in
BR-biosynthesis or -response. Important steps of BR-biosynthesis were elucidated with precursor-feeding experiments and by
the analysis of BR-biosynthesis-deficient mutants. The altered phenotypes of these mutants, particularly in Arabidopsis, revealed
the essential nature of BRs for normal growth and development. A major role of BRs is the positive regulation of cell expansion.
Furthermore, BRs modulate plant responses to biotic and abiotic stresses and to other phytohormones, and influence
differentiation processes of cells and tissues. BR-insensitive mutants such as bri1 hold the potential for uncovering BR-signalling
pathway(s) at the molecular level. The identification of BR-regulated genes demonstrates a genetic basis for BR mode of action
with reference to their multiple effects. This review focuses on the relevance of BRs to the control of various physiological
processes, BR-signalling and underlying molecular mechanisms by considering known mutants. Elsevier, Paris
Brassinosteroids / growth / cell elongation / signal transduction / interaction / gene regulation / light
BR, brassinosteroid / CK, cytokinin / GA, gibberellin / XET, xyloglucan endotransglycosylase

1. INTRODUCTION
Since the 1930s, the growth promoting effect of
pollen extracts has been known [44], but brassinolide
was first identified in 1979 as the effective compound [29] of a hydrophobic extract called brassin
[52]. Brassinolide and all other BRs are derivatives of
cholestane, thus resembling animal steroid hormones.
BRs contain a steroid scaffold consisting of four rings,
termed A through D, and a carbon side-chain at
position C17. Knowledge on the BR-biosynthetic
pathway was gained from studies on seedlings and cell
cultures of various plants (reviewed in [23, 57]). Feeding of labelled intermediates followed by analysis of
metabolites using GC/MS confirmed a number of
reactions through which campesterol is converted to
brassinolide. The so-called early and late C6-oxidation
pathways are synchronically active at least in Arabidopsis thaliana [24] and pea [54], as some intermediates of both pathways are found simultanously in
planta. Brassinolide is considered to be the endproduct of BR-biosynthesis as it shows the highest
biological activity among BRs [25]. In general, BRs
with a 6,7-lactone functionality, as exhibited by brassinolide, possess higher biological activity than 6-keto
steroids, and BRs lacking a B-ring oxygen function
Plant Physiol. Biochem., 0981-9428/99/5/ Elsevier, Paris

show minor activity. BRs are found throughout the

plant kingdom ([58] and references therein) and occur
at the highest levels in pollen and seeds and at a
nanomolar range in young leaves, shoots and flowerbuds [2, 36, 44].
Since their discovery, BRs have been shown to elicit
strong growth responses in a variety of test systems, a
subset of which was used later on as bioassay. Grove
et al. [29] tested the stimulation of cell elongation and
cell division in the bean second-internode bioassay. In
the Raphanus sativus bioassay, BRs stimulated growth
and H+-secretion of cotyledons [19]. In a number of
other systems, the growth promoting effect on hypocotyls, epicotyls and further tissues was shown
(e.g. [70, 78]). In contrast to the observed effects of
BRs on shoot growth, the responses of roots are less
consistent. Root development can be inhibited even at
moderate BR-concentrations. On the other hand, promotion of root growth was described in various
systems [56]. In addition to the growth promoting
effect, BRs stimulate a variety of physiological responses including changes in enzymatic activities,
membrane potential, DNA-, RNA-, and proteinsynthesis, photosynthetic activity and changes in the
balance of other endogenous phytohormones (reviewed in [44, 62]). Particular interest in BRs was

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C. Mssig, T. Altmann

elicited by observations of enhanced growth for seedlings and diminished phenotypic variance, increased
biomass production, increased crop yield (e.g. [31])
and enhanced stress tolerance of BR treated plants
(e.g. [33, 72]).
Despite the available information on the presence of
BRs at low levels in all plant species analysed and on
the elicitation of a broad range of physiological
responses of intact plants or explants, the importance
of BRs for normal growth and development was not
broadly acknowledged, since appropriate pharmacological, surgical or genetic tools were not available
until recently. The identification of mutants and the
isolation of the corresponding genes in A. thaliana,
Pisum sativum and Lycopersicon esculentum defective
in either BR-biosynthesis or -perception demonstrated
the essential role of BRs as plant growth regulators
and provided novel tools to study their precise function and their mode of action in plants.

2. BR-BIOSYNTHESIS DEFICIENT MUTANTS

The analysis, characterisation and verification of
BR-biosynthesis deficient mutants affected at different
steps of the biosynthetic pathway, which are discussed
in the following section, largely relied on the availability of information on the biosynthetic pathway and
of a large set of chemically synthesised pure intermediates. The observed phenotypic alterations in the
mutants corroborated various previous findings on BR
effects on cell growth and differentiation and uncovered additional functions, e.g. in the control of photomorphogenesis. While the overall phenotypic changes
are consistent among all known mutants, more subtle
differences between them require further detailed
analysis and may uncover various levels of genetic
redundancy (with respect to genes encoding biosynthetic factors) and/or yet unknown functions of BRs
hitherto mainly regarded as biosynthetic intermediates.

2.1. dwf1/dim
Like all other BR-biosynthesis deficient mutants,
the phenotypic defects of the Arabidopsis mutants
dwarf1/diminuto (dwf1/dim) [22, 35, 39, 64] can be
rescued specifically by application of exogenous BRs.
The dwf1-6 (cbb1) mutant was tested for its response
to known phytohormones and their inhibitors, including GA, indole-3-acetic acid, kinetin, jasmonic acid,
pCIP (an antiauxin), ethrel (an ethylene releasing
compound), a blocker of ethylene biosynthesis (AIB)
and a blocker of ethylene perception (AgNO3). dwf1-6
Plant Physiol. Biochem.

seedlings reacted to treatments with these substances

similarly to wild-type plants [35], but none of these
substances restored a wild-type phenotype or caused a
mutant phenocopy of the wild-type. In contrast, treatment of the dwf1-6 mutant with different brassinosteroids led to normalisation to an almost wild-type
phenotype. dim seedlings can be rescued especially by
addition of both castasterone and brassinolide [39],
while earlier precursors to brassinolide are less efficient. The BR-biosynthesis deficient mutants were
clearly distinguished from dwarf mutants affected in
GA-biosynthesis or perception by treatments of the
ga1-1, ga2-1, ga3-1, ga4-1, ga5-1, and the gai-1
mutants with 24-epibrassinolide. Hypocotyl length
was increased in all GA-mutants, but no phenotypic
normalisation of these mutants could be achieved [35].
The dwf1/dim mutants are affected in phytosterol
synthesis prior to the specific conversions involved in
BR-synthesis, whose primer is campesterol [39]. The
DWF1/DIM gene encodes for an oxidase, which possibly contains a domain involved in FAD-binding and
catalyses the reactions from 24-methylene-cholesterol
to campesterol via the intermediate 24-methyldesmosterol [39]. The levels of castasterone, typhasterol, and 6-deoxoteasterone are below the detection
limit in dim plants, which confirms the role of DIM in
BR-biosynthesis. The enzyme is a transmembrane
protein, consistent with the expected site of biosynthesis of hydrophobic sterols. Transiently expressed GFPDIM fusion protein seemed to be localised in the
endoplasmic reticulum in pollen tubes and BY2 cells.
Transgenic Arabidopsis plants overexpressing
DWF1/DIM do not show any obvious phenotypic
changes, indicating that DWF1/DIM does not catalyse
a rate-limiting step in BR-biosynthesis [39].
The dwf1/dim mutants are dwarfed with a stunted
axis and dark green epinastic leaves arranged in a
compact rosette and they develop a reduced root
system [35, 64]. Furthermore, dwf1/dim mutants develop a shortened hypocotyl and opened cotyledons
upon germination in darkness, thus displaying features
of de-etiolated/constitutive photomorphogenic mutants (reviewed in [68]). In comparison to mutants
affected in reactions specific to BR-biosynthesis, the
dwf1/dim mutants have a rather mild phenotype. Unlike, e.g., cpd/cbb3 and dwf4 plants (see below), the
dwf1/dim mutant plants develop inflorescences and
produce fertile seeds [22, 35, 64]. The reduced size of
the plants is due to a reduction of cell size rather than
a reduction of cell number, which also holds true for
other BR-biosynthesis deficient mutants [35, 64].
Thus, BR-deficiency is accompanied by reduced cell

Physiology and molecular mode of action of brassinosteroids

elongation confirming the requirement of BRs for

normal growth and an essential role of BRs in the
control of cell (wall) expansion. Hints of the potential
mode of action of BRs in cell wall loosening, the
prerequisite of cell elongation [18], were obtained by
studying the expression of genes encoding for cell wall
proteins probably involved in cell wall weakening.
Thus, xyloglucan endotransglycosylases (XET) genes,
such as TCH4 whose expression can be stimulated by
BRs and auxins [73], and meri5 [51], were shown to
be expressed at low levels in the dwf1-6 mutant and to
be inducible to wild-type levels upon BR feeding [35].
These results corroborated previous observations on
the XET encoding gene BRU1 that is BR-induced in
soybean epicotyls and those induction coincides with
an increase in wall extensibility [77, 78]. Recent observations, however, suggest that EXPANSINS that act
through reversible disruption of non-covalent bonds
rather than hydrolytic enzymes are key mediators of
cell wall loosening [18, 50]. The level of EXPANSIN
geneexpressionispositivelycontrolledbyBRs(Kauschmann, Cosgrove and Altmann, unpubl. data), and
auxins [59] and cytokinins [20] are also known to
stimulate EXPANSIN expression. Furthermore, directional cell growth requires ordered deposition of cellulose microfibrils most probably controlled by cytoskeleton components. These processes may be
influenced by BRs, which were shown to be involved
in the control of cortical microtubule organisation [46].
Frequently, the tissues sensitive to BRs also exhibit
strong growth responses to auxins [76] and distinction
of auxin- and BR-effects is complicated. Differences in
provoked gene expression patterns indicated that auxin
induced growth may be based on a mechanism different from that elicited by BRs [78]. At least one
auxin-independent BR-signalling pathway exists, as
BRs promote hypocotyl elongation of auxininsensitive dgt tomato plants [78] and the two phytohormones differ in their growth induction kinetics. On
the other hand, there is evidence of mutual interaction
and dependency of BRs and auxins. In Cucurbita
maxima hypocotyls, application of BRs led to an
increased indole-3-acetic acid content [21]. Accordingly, a decreased IAA-content was also found in
BR-biosynthesis-deficient pea lkb mutants [54], which
show reduced internode elongation. Thus, BRs may
promote auxin synthesis implying that BR-effects
could at least partly be caused by changes in auxin
levels. In addition, the synergistic interaction of BRs
and auxins in some systems are possibly due to

365

BR-induced increases of the sensitivity of tissues to

auxin [34] or vice versa [37].
The pea homologue to DWF1/DIM probably is
LKB. The lkb mutant displays dwarfism and is rescued
by brassinolide and its biosynthetic precursors. lkb
plants are deficient for the same two reactions between
24-methylene-cholesterol and campesterol as the
dwf1/dim mutant [53, 54, 75]. Interestingly, cell wall
characteristics related to wall expansion are altered in
the lkb mutant [7]. The increased wall-yield threshold
observed for cells in the epicotyl growing zone, which
is indicative of reduced loosening of the cell walls, is
consistent with a role of BRs in the regulation of
expression and/or activity of factors involved in cell
wall modification leading to enhanced wall yielding.

2.2. det2
Due to reduced cell size, light-grown de-etiolated2
(det2) mutants of A. thaliana are smaller than wildtype and even dwf1/dim plants [35, 43, 64]. The dark
green plants have reduced apical dominance, and male
fertility, flowering and senescence are delayed [43].
Initially, det2, like det1 [13], was selected for deetiolated growth in darkness [12] and det1 and det2
were postulated to connect CK- and lightsignalling [14]. CK-treated, dark-grown wild-type
Arabidopsis seedlings have similarities with light
grown seedlings [14, 61]. In addition, Arabidopsis
amp1 plants containing increased CK-levels display
photomorphogenesis in darkness [10]. However, det1
and det2 show no changes in CK-content compared
with wild-type plants [14]. Later on, upon isolation of
the DET2 gene, det2 was shown to be BRdeficient [43] and to be specifically blocked in BRbiosynthesis [27]. det2 is deficient for the conversion
of campesterol to campestanol. The contents of BRs in
the det2 mutant are drastically reduced and range in
the case of castasterone, 6-deoxocastasterone, typhasterol and 6-deoxotyphasterol at 10 % of wild-type
levels [27]. Residual amounts of BRs, present in det2
despite the total loss of function of DET2, may
implicate the presence of another DET2-like gene. The
deduced amino acid sequence of the DET2 gene shows
significant sequence identity with steroid 5reductases in mammals [43] and DET2 catalyses reduction of animal steroids such as testosterone and
progesterone. Additionally, a human steroid 5reductase gene was shown to complement det2 upon
introduction into the mutant via genetic transformation [42]. Thus, the structural and functional conservation between DET2 and mammalian steroid 5reductases and the BR-deficiency of det2 plants
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C. Mssig, T. Altmann

support the proposal that DET2 encodes for a 5reductase in the BR-biosynthetic pathway.
As mentioned above, det2 mutants differ from
wild-type plants that develop a long hypocotyl and
small, closed cotyledons when germinated and grown
in darkness. det2 plants (as well as other Arabidopsis
BR-mutants) display opened cotyledons, a short, thick
hypocotyl, emergence of primary leaves and accumulation of anthocyanins under these conditions [12, 43].
This is another feature distinguishing BR-related mutants from dwarfed GA-biosynthesis deficient or GAinsensitive mutants such as ga4-1, ga5-1 and gai-1,
which exhibit wild-type skotomorphogenesis [35].
The morphological changes of dark-grown det2 plants
have been shown to be accompanied by increased
expression of light-regulated genes. The accumulation
of anthocyanin in dark-grown det2 mutants is due to
the expression of genes such as chalcone synthase
(CHS) which normally are only expressed in illuminated plants [12]. Furthermore, the expression levels
of light-regulated nuclear RNAs as rbcS (coding for
the small subunit of ribulose 1,5-bisphosphate carboxylase) and CAB (chlorophyll a/b-binding protein)
and of several chloroplast RNAs, were 10- to 20-fold
higher than those in dark-grown wild-type seedlings [12]. Thus, det2 is a member of a class of at least
sixteen det (de-etiolated), cop (constitutive photomorphogenesis), and fus (fusca) mutants, which exhibit
aspects of photomorphogenesis when germinated and
grown in darkness, including the development of a
short hypocotyl, opened cotyledons, the accumulation
of anthocyanin, and the expression of light-regulated
genes (reviewed in [68, 71]). An additional feature of
a subset of these mutants is chloroplast development in
darkness, which however does not occur in the BRdeficient mutants dim [64] and det2 [12]. DET/COP/FUS genes are thought to encode light-inactivated
repressors of photomorphogenesis. Direct evidence for
such a function of COP1 was given by McNellis et
al. [49]. Transgenic Arabidopsis seedlings overexpressing COP1 displayed reduced light sensitivity and
partial inhibition of photomorphogenesis. At least nine
COP/DET/FUS genes (COP8COP10, DET1,
FUS4FUS6, FUS11 and FUS12) are required for the
nuclear accumulation of COP1 [69]. Loss of function
of any one of these genes leads to a pleiotropic
photomorphogenic phenotype. Nuclear localisation of
COP1 in dark-grown hypocotyl cells was found to
occur even in the absence of functional DET2, thus
separating DET2 function from that of other known
repressors of photomorphogenesis. Furthermore, different responses of det1 and det2 towards application
Plant Physiol. Biochem.

of BRs in darkness [43] and analysis of det1-det2

double mutants [12] exclude a function of DET2 and
BRs in the same regulatory pathway as DET1.
The pea mutant lk corresponds to the Arabidopsis
det2 mutant. lk displays dwarfism which could be
normalised by treatment with BRs. The endogenous
levels of BRs (especially of campestanol) in lk seedlings were drastically reduced [38] due to a block in
campestanol formation [53]. Thus, the LK gene from
pea appears to have the same function as the Arabidopsis DET2 gene.

2.3. dwf4
The further metabolism of campestanol to castasterone involves hydroxylation and oxidation of the
B-ring (at the C6 position), hydroxylation of the side
chain (at C22 and C23), epimerisation of the hydroxyl
group in the A-ring (at position C3) and hydroxylation
of the A-ring (at C2).
The hydroxylation at C22 is mediated by the DWF4
gene product of Arabidopsis, a cytochrome P450
termed CYP90B [5, 11]. This reaction is supposed to
be a rate-limiting step in BR-biosynthesis [26]. The
dwf4 phenotype is very similar to that of det2. Like
det2, the dwf4 mutant displays dwarfism, reduced
apical dominance, and retarded development and senescence. As in det2, the reduced growth of dwf4 is
due to reduced cell elongation. The dwf4 plants, like
det2, suffer from reduced fertility, which in the case of
dwf4 was shown to be caused by reduced growth of
stamen filaments [5]. This led to pollen deposition on
the ovary wall and thus prevented fertilisation. Hand
pollination of dwf4 with either wild-type or mutant
pollen resulted in normal seed set. The intensive dark
green colour of the leaves was caused by the decreased
cell volume containing approximately the same number of chloroplasts.
Similar to det2, dark-grown dwf4 plants display
short hypocotyls, opened cotyledons, and leaf primordia lacking developed chloroplasts [5]. In contrast to
det2, however, dwf4 showed no CAB gene expression
in darkness, indicating the absence of dark expression
of light-induced genes in dwf4 [5]. As an alternative to
a possible direct regulatory role of BRs in the control
of photomorphogenesis, a close vicinity of the apical
meristem to the growth medium brought about by
reduced cell elongation in the seedling was proposed
to be a potential cause for the de-etiolated growth of
dwf4. Even wild-type plants show open cotyledons,
leaf development, and flowering in darkness when
they are grown on vertically oriented plates [5] or in
liquid culture [4]. Thus, the de-etiolated phenotype of

Physiology and molecular mode of action of brassinosteroids

dwf4 could be a secondary effect and further work will

be required to determine the precise role of BRs in the
control of photomorphogenesis, be it direct or indirect.

2.4. cbb3/cpd
The cpd (constitutive photomorphogenesis and
dwarfism) mutant of Arabidopsis was identified by
screening for mutants with defects in hypocotyl and
root elongation during skotomorphogenesis [63]. The
allelic mutant cbb3 (cabbage3) was identified in a
screening for dwarf phenotypes and shown to be
rescued specifically by BR-feeding [35]. The CPD
gene encodes for a cytochrome P450 termed
CYP90A1 [63], which catalyses the hydroxylation of
cathasterone at the C23 position. As it was shown by
the introduction of a CPD promoter-uidA reporter
construct into Arabidopsis plants, expression of the
CPD gene initially occurs in the cotyledons and shifts
during development to the leaf primordia [45]. Subsequently, CPD expression is restricted to leaf buds and
young rosette leaves and was localised to the mesophyll and adaxial stomata. In the inflorescence, CPD
expression is restricted to cauline leaves and sepals.
The CPD gene is not active in mature roots and
siliques leading to the conclusion that BR-synthesis
may take place in a limited set of organs. A negative
feed-back regulation known from animal steroidogenic P450 genes was found for the CPD promoter [45]. The extent of CPD inhibition by BRs was
correlated with their biological activity and the inhibition itself was dependent on the synthesis of a
negative transcriptional regulator since cycloheximid
abolished the feed-back regulation. No inhibition of
CPD gene expression was obtained by application of
other signal molecules such as auxin, ethylene, GA,
CK, jasmonate, or salicylic acid.
Light-grown cbb3/cpd plants displayed a dwarf
phenotype reminiscent of that of det2 and dwf4, which
consisted of an extremely stunted stem, a compact
rosette with small, dark green leaves, and a reduced
root system. Histological analysis revealed an altered
adaxial leaf epidermis structure of the cpd mutant with
straightened cell walls and the occurrence of paired
stomata placed in direct contact to each other without
intervening epidermis cells. The cpd mutant displayed
male sterility caused by the inability of its pollen to
elongate upon germination, apparently in contrast to
the situation in dwf4. Loss of pollen growth upon BR
deficiency, however, is consistent with previous observations on the stimulation of pollen tube elongation by
BRs [30]. Stem cross sections of cpd mutant plants
revealed impaired cell differentiation leading to a

367

higher number of phloem cells at the expense of xylem

cells [63]. The latter observation is in agreement with
the promoting effect of BRs on xylem formation
shown earlier. BRs led to a 10-fold increase in xylem
differentiation in cultured explants of Helianthus
tuberosus [16] and were shown to be necessary for
tracheary-element differentiation in Zinnia elegans
mesophyll cells [32]. In the latter system, auxin and
cytokinin were shown to be necessary for the initiation
and for the progression of this differentiation process,
while BRs are essential for the transition at an intermediate step leading to the irreversible determination
towards tracheary-element formation [28].
Very similar to the situation in the dwf1-6 mutant,
the decreased cell elongation in cbb3 in the absence of
BRs is accompanied by decreased mRNA levels of the
XET genes meri5 and TCH4 and of EXPANSIN genes,
whose expression was strongly stimulated by BR
treatment ([35]; Kauschmann, Altmann and Cosgrove,
unpubl.). The expression levels of several stress
related genes such as chalcone synthase (CHS), alcohol dehydrogenase (ADH), lipoxygenase (LOX2),
S-adenosylmethionine synthase and HSP18.2 were
found to be increased in cpd mutants [63]. This observation may be interpreted as an increased stress
susceptibility in the mutant and relates to the aforementioned reports on enhanced stress tolerance of
BR-treated plants towards abiotic stress (e.g. [72]). In
line with enhanced resistance to pathogen infection of
plants achieved through BR-application (e.g. [33]), a
number of pathogenesis-related genes (PR1, PR2 and
PR5) are downregulated in the mutant and are significantly induced in CYP90A1 overexpressing plants.
The latter effect, however, could have been caused
either by enhanced BR levels resulting from the
overexpression of CYP90A1 or by the production of
oxygen radicals due to overaccumulation of the
CYP90A1 protein.
The D (DWARF) gene from tomato encodes a
cytochrome P450 related to CYP90A1 (termed
CYP85) [8], which is also involved in BRbiosynthesis as indicated by the castasterone deficiency of the corresponding dwarf (d) mutant
(Bishop). The D gene was isolated by using the maize
transposable element Ac introduced into transgenic
tomato plants [8]. In addition to an efficient way of
identifying the D gene at the molecular level, this
approach also provided a chance to study variegated
plants that harbour mutant (BR deficient) sectors and
wild-type (revertant) sectors covering entire leaves, or
parts of leaves and stems. The co-existence of mutant
and revertant sectors on individual leaves point tovol. 37 (5) 1999

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C. Mssig, T. Altmann

wards a very limited transport capability of the

CYP85-enzyme dependent intermediates of BRbiosynthesis. Thus far, reports on the occurrence of BR
transport are limited. The movement of tritiumlabelled BRs from the root to the shoot including the
metabolisation into water soluble intermediates was
demonstrated in rice [74]. Slow transport from the
shoot into the roots was detected as well. Furthermore,
feeding of BR-biosynthesis deficient mutants, such as
dwf1-6 and cbb3, through roots and leaves normalises
shoot development (e.g. [35]).
As pointed out in this section, BR biosynthesis
deficient Arabidopsis mutants in general show similar
phenotypes but several differences are evident from
the available descriptions. Clearly, further work will
be necessary to determine the causes, which in the
simplest scenarios would be due to differences in the
residual BR-levels in the different mutants (e.g. due to
various levels of genetic redundancy) or differences in
the growth conditions applied in the different studies.
Like other phytohormones, BRs undergo different
modifications leading to various derivatives and putative degradation products (reviewed in [3, 62]). BRs
isolated from cell culture systems have been described
to be hydroxylated, glycosylated, acylated, or to be
metabolised to pregnane compounds. These reactions
may serve as means of regulation of BR-levels
(through inactivation or degradation of BRs), the
storage of BRs, e.g. in seeds, or the increase of their
hydrophilicity in order to facilitate BR-transport. Mutants affected in genes encoding for factors responsible
for these modifications have not been isolated. Accordingly, no BR-overproducing mutants are currently
available that would provide additional information
about the action of BRs in the plant. Such plants may
soon be available, however, through application of
transgenic approaches involving overexpression of the
isolated biosynthetic genes.

3. BR SIGNAL TRANSDUCTION
As it was shown by the analysis of mutants impaired
in the response to other phytohormones like abscisic
acid (reviewed in [9]) or ethylene (reviewed in [48]),
hormone insensitive mutants may provide efficient
means of identifying and studying components of the
corresponding signal transduction pathways. In analogy, BR insensitive mutants may have defects in
components of the signal transduction pathway(s) such
as receptors, mediators, or transcription factors and
may allow insight into the mechanism(s) of BR-signal
transduction. BR-insensitive Arabidopsis mutants
Plant Physiol. Biochem.

have been selected on the basis of a specific loss of

root growth inhibition [17] or by habitual similarity to
BR-deficient mutants [35, 41]. The isolated mutants,
termed bri1 (brassinosteroid-insensitive1), cbb2 (cabbage2), and bin (brassinosteroid-insensitive) turned
out to be allelic [41]. Morphologically almost indistinguishable from BR-biosynthesis deficient Arabidopsis
mutants, bri1/cbb2 are dark green dwarfs and display
reduced apical dominance, male sterility and deetiolated growth in darkness. In contrast to det2,
cbb3/cpd and dwf4, the bri1/cbb2 mutants could not be
rescued by exogenous application of BRs, whereas
they retained sensitivity to other plant hormones [17,
35]. The insensitivity of cbb2 to BRs was also shown
at the gene expression level. Neither TCH4 nor meri5
gene expression could be induced by exogenous application of BRs while the meri5 gene retained normal
inducibility through GA treatment [35].
The BRI1 gene was shown to encode a putative
leucine-rich repeat (LRR) receptor-like kinase (RLK)
with highest similarity to ERECTA, CLV1 and Xa21
that probably are involved in the transmission of
signals responsible for the regulation of developmental
processes or plant pathogen interactions [15, 41, 60,
67]. The predicted BRI1 protein contains several
distinct domains. A large extracellular domain of the
protein consists mainly of 25 copies of a 24-amino
acid leucine-rich repeat (LRR) with a 70-amino acid
island between the 21st and 22nd LRR. LRR domains
have been identified in a variety of proteins and have
been implicated in protein-protein interactions [40]. A
N-terminally located leucine-zipper motif is present in
BRI1 that probably allows homo- or heterodimerisation of the protein possibly leading to interactions, e.g.
with other RLKs that could provide a basis for linking
BR-signalling to developmental processes and defence
responses. The intracellular domain exhibits all subdomains and conserved amino acids known from
eucaryotic serine/threonine protein kinases. The function of the BRI1 kinase domain has been demonstrated
in vitro (Li, pers. comm.; Clouse, pers. comm.) and its
importance was shown in vivo by analysis of several
mutant alleles carrying mutations in residues predicted
to be essential for kinase activity. The structural
features of BRI1 thus indicate a function as a receptor.
Taken together with the pleiotropic effects that result
from loss of BRI1 activity, which very strongly resemble BR deficiency, this points towards a role of
BRI1 as BR receptor that initiates a phosphorylation
cascade leading to the activation of BR responsive
genes and other potential non-genomic effects (see
figure 1). Thus, steroid hormone signalling may be

Physiology and molecular mode of action of brassinosteroids

369

Figure 1. Potential brassinosteroid (BR) signal transduction pathway(s). BRI1, a putative BR receptor, may bind BRs directly or via interaction
with another steroid binding protein at its extracellular leucine-rich repeat (LRR) domain. Activation of the intracellular BRI protein kinase domain
may lead to the phosphorylation of intracellular targets, e.g. transcription factors (TF) such as TCH4-BF1. Non-genomic targets (e.g. metabolic
enzymes or components of the cytoskeleton) may be phosphorylated causing metabolic and developmental changes in the cell. BRI1 may interact
with other receptor-like kinases (RLKs) thus integrating BR-signalling into a larger signalling network. The presence of additional BR-receptors
(e.g. nuclear receptor-like (NRL) factors) and the potential existence of BRI1 independent BR signal transduction pathways cannot be excluded.

different in plants and animals. In the latter, steroid

hormone action has been attributed mainly to interactions with members of the nuclear receptor superfamily that upon ligand-binding move into the nucleus and
regulate gene expression [6, 65, 66]. Nevertheless,
steroid action at the plasma membrane of animal cells
has been observed, which leads to non-genomic effects
mediated via as yet unknown receptors [47, 55].
BR-binding to BRI1, which remains to be demonstrated, may occur directly at the 70-aa island within
the LRR domain or via another BR-binding protein. A
putative substrate of the BRI1 kinase is TCH4-BF1
(Mssig, Kauschmann and Altmann, unpubl.), a protein containing a PHD-finger found in a variety of
(putative) transcription factors such as members of the

Drosophila Polycomb and trithorax group genes and

several transcription factor coactivators and corepressors (reviewed in [1]). TCH4-BF1 has been shown to
be phosphorylated by the BRI kinase domain in vitro
and to bind to a specific promoter fragment of the BR
inducible TCH4 gene (Oh and Clouse, unpubl. results)
and can therefore be regarded as a putative component
of a BR-signal transduction pathway. Beside transcription factors, phosphorylation targets could include
metabolic enzymes or components of the cytoskeleton.
In summary, only very limited information on the
mechanism of BR signal transduction is hitherto available. BRI1 is an essential component required for BR
signal perception and transduction potentially acting
as a BR-receptor. The possible existence of BRI1
vol. 37 (5) 1999

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C. Mssig, T. Altmann

independent BR signalling pathways, however, cannot

be excluded. An alternative role of BRI1 in the
establishment of the competence of cells to react to
BRs, rather than in immediate perception of BRs,
would also be consistent with the currently available
data and with the bri1 mutant phenotype. Characterisation of genes from other species, such as LKA from
pea [54], which upon mutation confer BRinsensitivity, may help to uncover further components
of BR-signalling pathway(s).

4. CONCLUSION
A number of genes involved in BR-biosynthesis has
been isolated. They provide insights into the molecular
basis of BR-biosynthesis and are highly valuable for
demonstrating the role of BRs in the control of
developmental and physiological processes. Through
knowledge on the regulation of BR-biosynthesis, and
on the rate-limiting steps and the corresponding genes,
specific and efficient modulation of BR-levels in plants
will be possible. Thus, antisense and overexpression
approaches will provide the means of characterising
BR-effects more precisely than through the use of the
currently available knock-out mutants and to differentiate spacially and temporally the sites of BR synthesis
and action. Further components of the signal transduction pathway, as well as the corresponding genomic
and non-genomic targets, need to be identified to
clarify the position of BRs in the whole plant signalling network and to gain insight into the potential
cross-talk between various signalling pathways.

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