The Plant Journal (1996) 10(1), 1-8
MINI-REVIEW
Molecular genetic studies confirm the role of
brassinosteroids in plant growth and development
Steven D. Clouse*
Department of Horticultural Science, 228 Kilgore Hall,
North Carolina State University, Raleigh, NC 27695-7609,
USA
Introduction
Brassinosteroids (BRs) are plant growth-promoting natural
products with structural similarities to animal steroid hor-
mones. When BRs are applied exogenously at nanomoiar
to micromolar levels in a number of test systems, they
have a marked effect on cell elongation and/or proliferation
that is distinct from auxins, cytokinins and gibbereliins;
although they interact with these plant hormones and also
environmental signals, such as light and temperature, in
complex ways (Adam and Marquardt, 1986; Mandava,
1988). Besides promoting cell elongation and division, BRs
have been shown to enhance tracheary element differenti-
ation, substitute for cytokinins in growth of cell cultures,
stimulate membrane hyperpolarization and ATPase activity,
promote ethylene biosynthesis via ACC synthase, control
microtubule orientation, alter mechanical properties of cell
walls, and modulate environmental stress signals (Arteca,
1995; Clouse and Zurek, 1991; Iwasaki and Shibaoka, 1991;
Mandava, 1988; Mayumi and Shibaoka, 1995; Wang et al.,
1993; Wilen et al., 1995; Zurek et al., 1994). BRs are
found throughout the plant kingdom at endogenous levels
sufficient to promote, in planta, the physiological effects
observed in bioassay systems (Sasse etal., 1992; Takatsuto,
1994). Together, these observations suggest that BRs may
serve as one of the critical signals controlling plant growth
and development (Sasse, 1991a).
Those few laboratories that have consistently worked
on BRs over the past decade have proposed that these
compounds deserve recognition as a sixth class of plant
hormones, equal in importance to auxins, cytokinins, gib-
berellins, abscisic acid and ethylene. However, the plant
community at large has been very slow to recognize BRs
as important growth regulators. This may have resulted
from a lack of unequivocal proof that BRs are essential for
plant growth and development, despite the large body of
physiological data gathered since the first publication of a
BR structure (Grove et al., 1979). Furthermore, the molecu-
Received2 April 1996;revised29 April 1996;accepted30 April 1996.
*Forcorrespondence(fax + 19195152505;e-mailsteve clouse@ncsu.edu).
lar mechanism of BR action is uncertain, although one
might argue from structural considerations that they act
by a mechanism similar to that of animal steroid hormones.
In general, such hormones act via a soluble receptor/
ligand complex that binds to nuclear sites to regulate the
expression of specific genes (Evans, 1988; Mangelsdorf
et al., 1995).
An impressive body of information on plant hormone
action has accumulated over the last decade by employing
transgenic plants and other molecular techniques to study
hormone-regulated gene expression. Mutants of Arabi-
dopsis thaliana that are deficient in or insensitive to plant
hormones have also been used extensively in the study of
abscisic acid, auxin, ethylene and gibberellin action (Ecker
and Theologis, 1994; Estelle and Klee, 1994; Finkelstein
and Zeevart, 1994). Hormone-deficient mutants usually
result from lesions in genes encoding biosynthetic enzymes
and are rescued to wild-type phenotype by treatment with
the hormone. Hormone-insensitive mutants may show the
same phenotype as deficient mutants but are not rescued
by hormone treatment. Insensitive mutants usually result
from lesions in genes encoding the hormone receptor or
elements of the signal transduction pathway (Ecker, 1995).
Within the past 3 years, the molecular genetic analysis of
BR action has begun. Genes regulated by BR have now
been cloned and mutants that are deficient in or insensitive
to BR have been identified. This short review will focus on
these very recent results which provide strong genetic
evidence that BRs are essential for normal plant growth
and development.
Brassinosteroids regulate gene expression
Both plant and animal hormones rapidly alter gene expres-
sion before the onset of cellular and physiological changes.
If BR is a true plant hormone, one would expect some effect
on gene expression in target tissues. Two-dimensional gel
analysis of in vitro translated mRNA first showed that
BRs regulate the expression of specific genes in soybean
hypocotyls and epicotyls (Clouse and Zurek, 1991; Clouse
et al., 1992), and peduncles and whole plants of Arabidopsis
(Clouse et al., 1993). Brassinosteroids were also shown to
alter the levels of specific polypeptides in wheat leaves
(Kulaeva etal., 1991) and pea stems (Sasse, 1991b). Further-
more, Arteca (1995) found that both BR and IAA increased
levels of ACC synthase transcripts in mung bean hypo-
cowls.
2 Steven D. Clouse
The first cloning of a gene regulated specifically by BRs
was accomplished in elongating soybean stem segments
(Zurek and Clouse, 1994), a system used nearly a decade
earlier to clone the first auxin-regulated genes (reviewed in
Hagen, 1995). BR-promoted elongation of young vegetative
stem tissue has been widely observed (Sasse, 1991b). It is
well known that auxin also promotes stem elongation
(Taiz, 1984) and comparison of BR and auxin-promoted
elongation shows that the kinetics of BR-promoted elonga-
tion are slower than those of auxin, but that BR is more
effective than auxin in promoting elongation after long
incubations. Moreover, in contrast to auxin, BR does not
rapidly induce members of the GH, SAUR or JCW auxin-
inducible gene families prior to the onset of elongation
(Clouse et al., 1992). This apparent independence of auxin
and BR-promoted elongation was further supported by the
findings that BR exerts the same physiological effects on
auxin-insensitive mutants of tomato and A. thaliana as it
does on the corresponding wild-type plants (Clouse et al.,
1993; Zurek et al., 1994). After this initial analysis of BR
vs. auxin-regulated gene expression, differential colony
hybridization was used to identify a gene in elongating
soybean epicotyls, named BRU1 (brassinosteroid _up-
regulated), whose transcript levels were increased within
2 h by BR but not auxin, cytokinin, gibberellin, or abscisic
acid treatment (Zurek and Clouse, 1994). Studies of endo-
genous transcript levels showed that BRU1 expression is
highest in young stem tissue, while in situ hybridization
analysis revealed detectable BRU1 message in epidermal,
cortical, vascular and pith tissue, with the most intense
expression localized in xylem parenchyma cells sur-
rounding tracheary elements (Oh and Clouse, unpub-
lished data).
With respect to the molecular mechanisms of BR-pro-
moted elongation, it is of interest whether BR-induced
growth, like auxin-induced cell elongation, is ultimately
mediated by cell wall loosening. The biochemical basis of
wall relaxation was predicted nearly 20 years ago to involve
enzymes that cleave and rejoin cell wall polymers, particu-
larly xyloglucans (Albersheim, 1976). Xyloglucan endo-
transglycosylase (XET) is an enzyme that specifically
cleaves a xyloglucan chain and transfers a fragment of
that chain to an acceptor xyloglucan. Several groups
(Fanutti etal., 1993; Fry etal., 1992; Nishitani and Tominaga,
1992) have proposed a wall-loosening function for this
enzyme during cell elongation, or it may have other import-
ant roles in elongation such as xyloglucan biosynthesis
or integration of new xyloglucan polymers into the wall
(McQueen-Mason et al., 1993). The BRU1 sequence has
extensive homology (48-74% identity) to numerous XET
genes from nasturtium, mung bean, Arabidopsis, soybean
and tomato (Arrowsmith and de Silva, 1995; Okazawa etal.,
1993; de Silva et al., 1993). BRU1 transcript levels are
correlated with the extent of BR-promoted stem elongation
and the increase in BRU1 message levels coincide with
BR-mediated increases in plastic extensibility of the cell
wall (Zurek etat., 1994). We recently found that recombinant
BRU1 protein has XET activity and that extractable XET
activity in soybean epicotyls is increased by BR treatment
(Romanow et al., unpublished data). Therefore, XETs may
play a role in BR-modulated stem elongation in soybean.
This is currently being tested in our laboratory with trans-
genic soybean plants expressing sense or antisense BRU1
transcripts.
Recent studies in Arabidopsis provide further evidence
that XETs are involved in BR-promoted elongation. In
Arabidopsis there are at least seven XET or XET-related
sequences (Xu et al., 1996). The TCH4 gene is a member
of this family that is regulated by touch, darkness, heat
shock, cold shock and auxins, and has been shown to
encode an active XET (Xu et al., 1995). Moreover, applica-
tion of 1.0 ~uM BR results in an increase in TCH4 transcripts
within 30 min, with a maximum at 2 h (Xu et al., 1995).
TCH4 is apparently the only member of the Arabidopsis
XET family that is regulated by BRs (Xu et al., 1996). A
TCH4 promoter:GUS fusion consisting of 958 bp of 5'
flanking region and 48 bp of the 5' untranslated leader
transformed into Arabidopsis, resulted in BR-regulated
GUS expression in elongating tissue (Xu et aL, 1995).
Subsequent experiments in Dr Braam's lab (Wu and Braam,
personal communication) have shown that a TCH4 pro-
moter fragment between -958 and -1 conferred the same
pattern of BR regulation of GUS expression in transgenic
plants as occurred with BR regulation of endogenous TCH4
expression, indicating that BR controls TCH4 expression
transcriptionally.
The availability of clones for BR-regulated genes allows
for the first time direct examination of the mechanism of
gene regulation by a plant steroid. We are collaborating
with Dr Braam's group to identify the cis-acting BR
response elements in the TCH4 promoter. The identification
of such sequences could lead to the isolation of steroid-
dependent DNA-binding proteins and help determine
whether plants and animals share common mechanism of
steroid-regulated gene expression. Surprisingly, results
from run-on transcription experiments in isolated epicotyl
nuclei suggest that BR regulates BRU1 expression at a
post-transcriptional rather than a transcriptional level. Post-
transcriptional regulation by steroid hormones, while not
as common as transcriptional regulation, has been found
in several cases (Nielson and Shapiro, 1990; Santos et al.,
1988). Brock and Shapiro (1983) showed that estrogen
stabilizes vitellogenin mRNA against cytoplasmic degrada-
tion and several other examples of post-transcriptional
regulation by plant growth regulators have also been
reported (reviewed by Gallie, 1993). Thus, analysis of the
regulatory regions of the TCH4 and BRU1 genes will
 Brassinosteroids are required for plant growth 3

det2 cpd (cbb3)

I MUTATIONIN I MUTATIONIN
STEROID STEROID
REDUCTASE HYDROXYLASE

OH

.o :o
Campesterol Campestanol Cathasterone Teasterone

OH OH OH OH
~..~,]~/ BR SIGNALPERCEPTION
~u | SIGNALTRANSDUCTION
HO~ ~ 1 ~ . ~ v,, GENEEXPRESSION(TCH4)
Wild
Type
3-Dehydroteasterone Typhasterol Castasterone Brassinolide
T
/
"

BR B I O S Y N T H E S I S
I MUTATIONIN
BR RECEPTOR?
SIGNALINGMOLECULE?
REGULATORYPROTEIN?

DEVELOPMENTAL AND brfl

ENVIRONMENTAL SIGNALS (cbb2)

Figure 1. Model for the role of brassinosteroid biosynthesis and response mutants in A. thaliana.
The proposed biosynthetic pathway to brassinolide from campesterol is shown (Fujioka et al., 1995). In this model, normal plant development requires BR
activity which alters gene expression and leads to cell elongation. The TCH4 gene encodes a BR-regulated xyloglucan endotransgiycosylase that may act on
cell walls during BR-promoted growth (Xu et al., 1995; Zurek and Clouse, 1994), If BR levels are reduced by mutations in genes encoding BR biosynthetic
enzymes (det2 or cpd) a dwarf phenotype results. A similar dwarf phenotype occurs if BR perception and/or signal transduction genes are mutated (putative
role of bril and cbb2).

provide insights into multiple levels of BR control of gene
expression.
De-etiolated mutants take on a new identity as genetic
lesions in BR biosynthesis
Numerous structure-function analyses have defined the
specific requirements for BR activity (McMorris etal., 1994;
Takatsuto et al., 1983). These include a cis-vicinal glycol
at C-2 and C-3, a keto or lactone function at C-6 and
hydroxylation (with proper stereochemistry) of the C-17
side chain. In order to transform a commonly occurring
plant sterol such as campesterol into an active BR, the cell
must accomplish a series of reductions, oxidations and
epimerizations. The complete biosynthetic pathway of the
BR, brassinolide, via campesterol, has now been defined
by several groups in Japan (Fujioka et al., 1995). The
endogenous levels of BRs and related molecules were
established in Caranthus roseus cell cultures which were
then fed deuterium-labeled compounds whose fates were
monitored by gas chromatography-mass spectrometry.
The nine-step pathway shown in Figure 1 is the outcome
of several such studies.
With the BR biosynthetic pathway now established, it
should be possible to identify mutants that affect rate-
controlling steps in the pathway, resulting in reduced BR
levels in the mutant plants. The phenotype of such mutants
would provide a critical test of the importance of BR in
normal plant growth and development. For example, if BR
is required for cell elongation, BR-deficient mutants would
be expected to display a dwarf phenotype, similar to the
gal, ga2 and ga3 gibberellin-deficient mutants of Arabio
dopsis, which are dwarfs that can be restored to wild-
type height by spraying with gibberellins (Finkelstein and
Zeevart, 1994). The GA1 locus has been cloned by genomic
subtraction (Sun et al., 1992) and shown to encode ent-
kaurene synthetase A, a key enzyme in gibberellin bio-
synthesis (Sun and Kamiya, 1994). A direct search for BR
biosynthetic mutants has not been undertaken, but re-
examination of Arabidopsis dwarfs that have been selected
by mutant screens for physiological processes apparently
independent of BRs, has uncovered a series of very excit-
ing results.
When dicotyledonous plants, such as Arabidopsis, are
grown in darkness they show an etiolated phenotype which
includes an elongated hypocotyl, a pronounced apical
hook, small folded cotyledons, undeveloped chloroplasts
and repression of light-regulated gene expression. When
4 Steven D. Clouse
Figure 2. Phenotype of brassinosteroid mutants in A. thaliana.
In (a) - (c), each pair was grown for the same length of time under identical conditions for comparison of mutant (left) vs. wild-type (right) growth habit.
(a) det2, (b) cpd, (c) bril, (d) close-up of cpd, (e) close-up of bril. (a) Courtesy of Drs Li and Chory, Salk Institute; (b and d) courtesy of Csaba Koncz, Max
Planck Institute.
such plants are grown in the light, hypocotyl elongation is
inhibited, cotyledons open, primary leaves and mature
chloroplasts develop and transcript levels of genes such
as ribulose-bisphosphate carboxylase and chlorophyll A/B
binding protein increase dramatically. To study how light
controls developmental pathways, a number of Arabi-
dopsis mutants known as det (de-etiolated), cop (constitu-
tive photomorphogenesis) and fus (fusca), have been
identified that have characteristics of light-grown plants
even when grown in complete darkness (Chory and Susek,
1994). Loss-of-function mutations in the DET, COPand FUS
genes lead to pleitropic defects in development in both
the dark and the light and altered expression of light-
regulated genes. Figure 2(a) shows that one such mutant,
det2, when grown in the light results in an extremely
dwarfed phenotype (due to reduced cell size), dark green
leaves, reduced male fertility and apical dominance,
delayed flowering and a delay of leaf and chloroplast
senescence (Chory et al., 1991). Such a broad spectrum
of effects shows that DET2 plays an important role in
development throughout the Arabidopsis life cycle.
The DET2 gene has recently been cloned by chromosome
walking and sequence analysis revealed a surprising homo-
logy to mammalian steroid 5c{-reductases that catalyze the
NADPH-dependent conversion of testosterone to dihydro-
testosterone (Li et al., 1996). There is 38-42% identity
between the DET2 protein and the mammalian reductases
and the sequence similarity increases to 54-60% when
conservative substitutions are taken into account. Even
more convincing, 80% of the conserved residues in the
mammalian enzymes are found in the DET2 protein includ-
ing a glutamate that is absolutely required for mammalian
enzyme activity. This glutamate is changed to a lysine in
the det2-1 and det2-6 alleles, both of which are very severe
despite being simple missense mutations. The first step in
the BR biosynthetic pathway is the reduction of campes-
terol to campestanol, which is strikingly similar to the
conversion of testosterone to dihydrotestosterone by the
mammalian 5c~-reductase. This suggests that DET2 cata-
lyzes the conversion of campesterol to campestanol.
Direct proof that det2 is a BR-deficient mutant came from
feeding experiments (Li et aL, 1996). Brassinotide rescued
the Det2 phenotype to wild-type in a dose-dependent
manner both in the light and the dark. In contrast, neither
gibberellins nor auxin rescued the mutant phenotype. The
fact that DET2 is required for BR biosynthesis and loss-of-
function mutations in DE72 lead to multiple alterations in
developmental pathways is convincing genetic evidence
that BRs are essential for normal plant growth. Besides
contributing to cell elongation, a well-known BR effect,
it appears that BR levels play a role in other etiolated
developmental responses, as well as leaf development,
apical dominance, flowering time, fertility and senescence
in the light.
Further genetic evidence for the essential role BR plays
in plant development comes from recent work at the
Max Planck Institute. Screening an Arabidopsis T-DNA
insertional mutant collection for plants defective in hypo-
cotyl elongation in the dark identified a recessive mutation
called cpd (constitutive 12hotomorphogenesis and
dwarfism), with a phenotype similar to det2 (but non-
allelic) (Szekeres et al., 1996). In the dark, cpd displayed
the typical de-etiolated response while in the light the
mutation resulted in an extreme dwarf (20- to 30-fold
smaller than wild-type, see Figure 2b), with dark green,
rounded epinastic leaves; chloroplast development in
roots; altered stomatal density; thick transverse files of
cellulose microfibrils in hypocotyl epidermal cells; unequal
division of the cambium producing extra phloem cells at
the expense of xylem; and male sterility due to the inability

of pollen to elongate. Transcript levels of stress-related
genes such as chalcone synthase, alcohol dehydrogenase,
lipoxygenase and heat shock genes were elevated in the
cpd mutant, while expression of the pathogenesis-related
genes PR1, 2 and 5 was severely reduced.
Using the T-DNA as a tag, the CPD gene was cloned and
found to encode a protein with 50-90% sequence identity
with conserved domains of microsomal cytochrome P450
monooxygenases (Szekeres et al., 1996). Furthermore, the
sequence has some homology to the specific domains
of steroid hydroxylases, including rat testosterone-16c~-
hydroxylase (24% identity) and human progesterone-21c~-
hydroxylase (19% identity). Therefore, it appears that the
CPD gene encodes another enzyme in BR biosynthesis,
this time involving a hydroxylation step. When the cpd
mutant was grown in the presence of C-23 hydroxylated
BR precursors (Figure 1) including teasterone, 3-dehydro-
teasterone, typhasterol and castasterone, it was completely
rescued to wild-type phenotype in light or darkness. How-
ever, cathasterone and its precursors, which lack a hydroxyl
at C-23, failed to rescue the cpd mutant, suggesting that
the CPD protein acts in the hydroxylation of cathasterone
to teasterone during BR biosynthesis. The overlapping
phenotypes of det2 and cpd provides confirmation of the
role of BR in the developmental aspects mentioned above.
The alteration of microfibril orientation, expression of
stress-related genes and altered xylem development in the
cpd mutant provides genetic confirmation of previous
physiological studies that implicated BRs in these pro-
cesses (Clouse and Zurek, 1991; Iwasaki and Shibaoka,
1991; Mayumi and Shibaoka, 1995; Wilen et al., 1995).
The Altmann group isolated three dwarf mutants, named
cabbage1 (cbbl), cabbage2 (cbb2) and cabbage3 (cbb3),
during a screen of transposon-mutagenized Arabidopsis
(Kauschmann et aL, 1996). Genetic analysis showed that
cbb3 was allellic to cpd while cbbt was an allele of dwfl,
the original Feldmann T-DNA dwarf (Feldmann etal., 1989),
which itself is allelic to dim, another de-etiolated dwarf
that is thought to control cell elongation by regulation of
a specific tubulin gene (Takahashi et al., 1995). A very
careful study of cbb responses to phytohormones and
their antagonists showed that auxins, auxin antagonists,
gibberellins, ethylene-releasing compounds, ethylene
inhibitors, cytokinins and jasmonic acid could not rescue
the cbb phenotypes or cause a cbb mutant phenocopy of
the wild-type. Moreover, only active brassinosteroids and
not any other structurally related sterol, could rescue the
dwfl (cbbl,dim) and cbb3(cpd) mutant phenotype to wild-
type. They also showed that in the cbb mutants there was
a lack of the BR-regulated TCH4 gene expression which
was restored in cbbl and cbb3 upon BR treatment. The
cbb2 mutant could not be rescued by BR treatment and
appears to be BR-insensitive (see below). Szekeres et al.
(1996) also independently showed that dim could be res-
Brassinosteroids are required for plant growth 5
cued to wild-type by BR treatment. The DWF1 (dim, cbbl)
locus has been cloned and shows homology to an FAD-
dependent oxidase (Mushegian and Koonin, 1995), but
also has a potential nuclear targeting signal that may point
to a regulatory function for the DWF1 protein (Takahashi
et aL, 1995). Therefore the role of dwfl (dim, cbbl) in BR
biosynthesis is not as clear as det2 and cpd (cbb3). The
known alleles of dwfl do not result in as severe a phenotype
as det2 and cpd (cbb3), indicating that BR levels may not
be lowered to the same extent in dwfl as in det2 and cpd
(cbb3). Alternatively, dwfl may result from partial BR
insensitivity that is rescued by the high levels of BR applied
in feeding experiments (Kauschmann et aL, 1996).
The availability of at least two molecular markers for BR
biosynthesis (det2 and cpd) will allow elegant studies of
the temporal and spatial regulation of BR levels in response
to developmental and environmental signals. For example,
the role of DET2 as a negative regulator of de-etiolation
must be expanded to a positive role in cell elongation,
given its function as a BR biosynthetic enzyme. Molecular
probes for DET2 and CPD will allow precise studies of the
role of light on BR levels. Preliminary results on the effect
of light, sugar and cytokinins on CPD expression have
been reported (Szekeres et al., 1996). Chory et al. (1994),
found that cytokinin treatment of wild-type Arabidopsis
could cause a der2 phenocopy in dark-grown plants. The
finding that the CPD gene is downregulated by cytokinin
is consistent with the det2 results and may indicate that
cytokinins negatively control BR biosynthesis (Szekeres
et al., 1996).
BR-insensitive mutants may point to a BR receptor
Hormone insensitivity generally results from one of three
mechanisms:
(i) a mutation in the receptor such that the hormone no
longer binds, or the receptor fails to become activated
even when bound to the ligand;
(ii) the receptor is functional but a mutation in a critical
step in the signal transduction pathway has occurred;
(iii) the applied hormone is not actually the active endo-
genous compound and an enzyme that converts the
applied hormone to the active form has been mutated,
Original views of the mechanism of animal steroid hor-
mone action assumed a single protein receptor/signal
transduction model in which the steroid ligand diffused
through the cell membrane, bound to the steroid receptor
in the cytosol or nucleus, and the receptor/ligand complex
interacted directly with hormone-responsive elements in
the promoters of steroid-regulated genes (Evans, 1988). It
is now known that steroid receptors interact with heat-
shock proteins in the cytosol, are regulated by phosphoryla-
tion and can complex with non-steroidal transcription fac-
6 Steven D. Clouse
tors in the nucleus (Beato et al., 1995). Therefore, while
the steroid signal transduction pathway in animals is more
complex than previously thought, it is still quite short. If
BRs have a similar mechanism of action in plants, a small
set of BR-insensitive mutants will define the entire signal
transduction pathway. Even if the BR pathway differs
from animals and is more complex, isolation of hormone-
insensitive mutants in Arabidopsis is still a very fruitful
approach, as has been amply demonstrated in ethylene
(Ecker, 1995) and abscisic acid (Finkelstein and Zeevart,
1994) signal transduction studies.
We have been screening Arabidopsis for BR-insensitive
mutants for the past several years. The axrl (Lincoln et al.,
1990), axr2 and auxl (Wilson et al., 1990) auxin-resistant
mutants and the ckrl/ein2 (Ecker, 1995; Su and Howell,
1992) cytokinin-resistant mutant were selected by the
ability of the mutant plants to elongate roots in the presence
of hormone concentrations that inhibited root growth in
wild-type Arabidopsis. We have previously shown that
BRs exhibit a similar inhibitory effect on Arabidopsis root
growth and that EMS-mutagenized plants can be selected
which are insensitive to this response (Clouse et al., 1993).
One of these mutants, named bril (brassinosteroid-_insensi-
tive), consistently exhibited long roots and very short
hypocotyls when placed on various BR concentrations and
showed a striking phenotype when grown to maturity that
suggested a fundamental pathway controlling growth and
development had been compromised by the mutation
(Clouse and Langford, 1995). Figure 2(c) shows that the
bril mutant is an extreme dwarf that reaches less than
10% of the height of wild-type plants grown under the same
conditions, and shows the general phenotypic character-
istics of de~2 and cpd. The leaf morphology of the bril
mutant was distinctly different from that of wild-type.
Rossette leaves were shorter than wild-type, had a
thickened, curled appearance, were darker green and the
petioles failed to elongate. The bril plants also exhibited
delayed maturity and senescence, male sterility and
reduced apical dominance. Furthermore, dark-grown bril
plants had a de-etiolated phenotype similar to der2 and cpd.
To determine whether the bril mutation confers insensi-
tivity specifically to BRs or results in insensitivity to multiple
hormones, as has been observed for axrl, axr2, auxl and
ckrl, we compared root elongation of wild-type and bril
seedlings on a number of plant hormones. The bril mutant
is completely sensitive to auxins, cytokinins, gibberellins,
abscisic acid and ethylene but shows marked insensitivity
to BRs over a broad range of concentrations. Treatment of
bril with gibberellins or BRs could not rescue the pheno-
type. The specificity of the hormone insensitivity of bril,
lends further support to the importance of BRs in growth
and development. Analysis of several thousand F2 progeny
of a bril wild-type cross showed no segregation between
root elongation in the presence of BR and the altered
developmental morphology, suggesting that the bril
developmental phenotype is caused by a mutation in
a single gene with pleiotropic effects that also confers
insensitivity to BRs (Clouse et al., 1996).
A second BR-insensitive mutant, cbb2, has been isolated
that has a very similar phenotype to bril and it was
demonstrated that expression of BR-regulated genes is
absent in both BR-insensitive and BR-deficient mutants
(Kauschmann et al., 1996). It is not surprising that both
BR-insensitive (bril, cbb2) and BR-deficient (det2, cpd)
mutants show a similar dwarfed growth habit since BRs
have long been known to promote stem elongation. In
both cases it is likely that the lack of BR action, either
through reduced synthesis or reduced perception, is
blocking cell elongation, perhaps through lowered expres-
sion of TCH4 and other genes encoding the molecular
components of the cell elongation machinery. Attempts
are underway in two laboratories to clone the BRI1 and
CBB2 loci. The dramatic and pleiotropic effects of these
mutations suggest that BRI1 and CBB2 lie very early in the
signal perception/transduction pathway. Therefore, cloning
and characterization of BRI1 and CBB2 should lead to a
major advance in our understanding of BR action and help
clarify the BR signal transduction pathway.
Conclusion
Figure 1 summarizes what we currently know about BR
mutants. The availability of these new genetic resources
for BR biosynthesis and response will allow studies of
tissue-specific control of BR synthesis and receptivity dur-
ing development. Double mutants of det2, cpd and bril
with photoreceptor mutants such as phyA and phyB (Chory
and Susek, 1994) should clarify the role of light in con-
trolling BR activity, a phenomenon suggested by early
physiological studies (Mandava, 1988). The identification
of BR response elements in the BRU1 and TCH4 genes will
provide tools to analyze specific DNA- or RNA-binding
proteins, thus clarifying the terminal portion of the BR
signal transduction pathway. Furthermore, the cloning of
the BRI1 and CBB2 genes will enhance our understanding
of BR molecular mechanisms. Sequence analysis of these
clones may reveal significant homology with mammalian
steroid receptors or other plant and/or animal transcription
factors. Overexpression of recombinant proteins will allow
binding studies with 3H-BR and the production of anti-
bodies for protein expression studies. Co-expression of the
TCH4 promoter:GUS fusion and a BRI1 gene expression
cassette in the same cell could lead to the type of elegant
experiments that have been performed in animal steroid
research for many years (Evans, 1988).
Constructing sense and antisense transgenic plants with
any of DET2, CPD, BRI1 and CBB2 will reveal much about
the developmental and tissue-specific regulation of these

genes. Cloning of these genes could also have agricultural
importance considering the dramatic control over develop-
mental processes that their gene products seem to exert.
These sequences could be used as probes to isolate the
corresponding homologs in crop species, followed by over-
or underexpression of the genes in transgenic crops to
control growth patterns. An example of this type of
approach can already be found in the use the Arabidopsis
ETR1 gene to clone the ethylene receptor in tomato
(Lanahan et aL, 1994).
The successful application of molecular genetics will
bring brassinosteroids from the periphery to the forefront
of plant hormone research and should rapidly increase our
understanding of these unique regulators of plant growth
and development.
Acknowledgments
The author is supported by the USDA competitive grants
program and the National Science Foundation. Thanks to
Joanne Chow, Csaba Koncz and Thomas Altmann for
providing manuscripts prior to publication.
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