Download A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits

Plant Physiol. (1996) 1 1 1: 671-678
A Brassinosteroid-lnsensitive Mutant in Arabidopsis thaliana
Exhibits Multiple Defects in Growth and Development'
Steven D. Clouse*, Mark Langford, and Trevor C. McMorris
Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695 (S.D.C.);
Pharmingen Co., 10975 Torreyana Road, San Diego, California 921 21 (M.L.); and Department of Chemistry,
University of California, 9500 Gilman Drive, San Diego, California 92093 (T.C.M.)
1979), unequivocal proof that BRs are essential for plant
growth and development has so far been unavailable. Furthermore, the molecular mechanism of BR action is currently unclear. 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). We and others have shown
that BR regulates gene expression in Glycine max (Clouse
and Zurek, 1991; Clouse et al., 1992; Zurek and Clouse,
1994; Zurek et al., 1994) and Arabidopsis thaliana (Clouse et
al., 1993; Xu et al., 1995) and that this modulation of specific
gene expression is correlated with cell elongation, but information on a BR receptor is lacking.
The use of mutants of A . thaliana that are deficient in or
insensitive to plant hormones has been invaluable in dissecting the molecular mechanisms of ABA, auxin, ethylene,
and GA action (Estelle and Klee, 1994; Finkelstein and
Zeevart, 1994; Ecker, 1995). Hormone-deficient mutants
usually result from lesions in genes encoding hormone
biosynthetic enzymes and are rescued to wild-type phenotype by treatment with the hormone. For example, the gal,
ga2, and ga3 mutants of A. thaliana are dwarfs that are
restored to wild-type height by spraying with GA (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 critica1 enzyme in GA biosynthesis (Sun and Kamiya, 1994). Hormone-insensitive mutants may show the same phenotype as hormone-deficient
mutants but are not rescued by hormone treatment.
Hormone-insensitive mutants usually result from lesions
in genes encoding the hormone receptor or elements of the
signal transduction pathway. The ETRl locus of A. tkaliana
was identified by a screen for ethylene resistance (Bleecker
et al., 1988), and positional cloning of the ETRl gene with
subsequent characterization showed that the ETRl locus
encoded an ethylene receptor (Chang et al., 1993).
Our goal is to identify the BR receptor and other
components of the BR signal transduction pathway using
Brassinosteroids are widely distributed plant compounds that
modulate cell elongation and division, but little is known about the
mechanism of action of these plant growth regulators. To investigate brassinosteroids as signals influencing plant growth and development, we identified a brassinosteroid-insensitive mutant in Arabidopsis thaliana (L.) Henyh. ecotype Columbia. The mutant,
termed bril, did not respond to brassinosteroids in hypocotyl elongation and primary root inhibition assays, but it did retain sensitivity
to auxins, cytokinins, ethylene, abscisic acid, and gibberellins. The
bril mutant showed multiple deficiencies in developmental pathways that could not be rescued by brassinosteroid treatment, including a severely dwarfed stature; dark green, thickened leaves;
male sterility; reduced apical dominance; and de-etiolation of darkgrown seedlings. Cenetic analysis suggests that the Bril phenotype
is caused by a recessive mutation in a single gene with pleiotropic
effects that maps 1.6 centimorgans from the cleaved, amplified,
polymorphic sequence marker DHSl on the bottom of chromosome
IV. The multiple and dramatic effects of mutation of the BRIl locus
on development suggests that the BRIl gene may play a critical role
in brassinosteroid perception or signal transduction.
BRs are plant-growth-promoting natural products with
structural similarities to animal steroid hormones (Mandava, 1988). When BRs are applied exogenously at nanomolar to micromolar levels in a number of test systems,
they have a marked effect on cell elongation and/or proliferation that is distinct from that of auxins, cytokinins,
and GAs, although they interact with these plant hormones
and also with environmental signals, such as light and
temperature, in complex ways (Adam and Marquardt,
1986). BRs are found throughout the plant kingdom and
have been shown by microchemical techniques to occur
endogenously at levels sufficient to promote, in planta, the
physiological effects observed in bioassay systems (Sasse et
al., 1992). Taken together, these facts suggest that BRs are
endogenous plant-growth regulators and may serve as one
of the signals controlling plant growth and development
(Sasse, 1991).
Despite the large body of physiological data gathered
since the first publication of a BR structure (Grove et al.,
'
This work was supported in part by grant no. 92-03404 from
the U.S. Department of Agriculture, National Research Initiative
Competitive Grants Program (S.D.C.).
* Corresponding author; e-mail [email protected]; fax
1-919 -515-2505.
Abbreviations: BR, brassinosteroid; CAI'S, cleaved, amplified,
polymorphic sequences; Col-O, Columbia ecotype; EBR, 24epibrassinolide; EMS, ethylmethane sulfonate; Ler, Landsberg
erecta ecotype; SSLP, simple sequence length polymorphism; XET,
xyloglucan endotransglycosylase.
671
672
Clouse et al.
a mutational analysis in A . thaliana. BRs have a range of
physiological effects, but promotion of stem elongation
is perhaps the best characterized (Clouse et al., 1992).
Employing the inability to elongate stems in the presence
of BR as a screen for BR insensitivity was found to be
impractical for technical reasons (S.D. Clouse, M. Langford, T.C. McMorris, unpublished data). However, like
auxin, BR often inhibits primary root elongation even
though shoot growth is stimulated by hormone application (Roddick and Juan, 1991). The axrl (Lincoln et al.,
1990), axr2 (Wilson et al., 1990), and auxl (Pickett et al.,
1990) auxin-resistant mutants and the ckrllein2 (Su and
Howell, 1992; Ecker, 1995) cytokinin-resistant mutant
were selected because of the ability of the mutant plants
to elongate roots in the presence of hormone concentrations that inhibited root growth in wild-type A . thaliana.
We have previously shown that BRs exhibit a similar
inhibitory effect on Arabidopsis root growth and that
EMS-mutagenized plants can be selected that are insensitive to this response (Clouse et al., 1993). We recently
presented a preliminary report on the first BR-insensitive mutant in A . thaliana, bril, that has a severely
dwarfed phenotype and reduced apical dominance and
is male-sterile (Clouse and Langford, 1995). In this paper
we provide additional genetic and physiological analysis
of bril.
MATERIALS AND M E T H O D S
Screening of Mutagenized Plants
M, seeds of A. thaliana (Col-O background) mutagenized
with EMS were obtained from Lehle Seeds (San Antonio,
TX). The M, seeds were supplied in bulked parenta1 groups
of 8,000 seeds representing 1,000 M, parents. M, seeds
(approximately 71,000 total) were sterilized by treatment
with absolute ethanol(l0 min) followed by a 30% commercial bleach solution containing 0.1% Triton X-100 (20 min).
Seeds were then rinsed five times in a large volume of
sterile water and suspended in 0.2% agarose, and between
100 and 200 seeds were pipetted along straight lines in 150
x 100 mm Petri plates containing 1% agar, 2% SUC,and
standard mineral salts (Estelle and Somerville, 1987). EBR
was synthesized as previously described (McMorris and
Patil, 1993) and added to the medium after autoclaving at
either 10-6 or 10p7 M final concentration. The plates were
placed vertically in a growth chamber at 23°C with a 16-h
light/8-h dark cycle at 50 PE m-' s-l intensity. After 6 d of
incubation, M, plants, whose roots elongated substantially
(>9 mm), were picked and grown to maturity to produce
M, seeds. A large number of M, progeny from individual
M, picks were then compared to wild type for their ability
to elongate roots in the presence of 10-7 M EBR. M, plants
that were male-sterile, such as bril, were backcrossed to
wild-type Col-O to generate the M,, which was selfed to
create a segregating M, population. EBR insensitivity of the
mutant phenotype was confirmed in M, individuals followed by a second round of backcrossing and selfing to
generate M, mutant individuals for additional analysis.
Plant Physiol. Vol. 11 1 , 1996
Hormone Sensitivity Assays
Because bril is male-sterile, pure stocks of bril seed were
not available. Therefore, segregating F, progeny from a
backcross of bril X Col-O were used for a11 experiments.
Excess F, seeds were sterilized and plated on agar medium
as described above with the exception that plates were
incubated for 3 d at 4°C in the dark to increase uniform
germination before transfer to the growth chamber. A11
hormones were added after autoclaving and cooling of the
media to <55"C. Root length of a11 seedlings was measured
to the nearest 0.1 mm after 7 or 8 d using a dissecting scope
with a stage micrometer, and plants were tagged individually and grown in soil for 2 months to determine phenotype. Data points represent 20 measurements 2 SE for those
plants determined to be bril, compared with 20 measurements 5 SE for Col-O seedlings treated in the same manner.
Ethylene Experiments
Seeds were sterilized as described above and plated on
1% agar containing mineral salts (Estelle and Somerville,
1987) and 2% SUC,pH 5.8, poured into the bottom of
1-quart Mason jars whose lids were fitted with rubber
septums. Jars were wrapped in aluminum foi1 and placed
at 4°C for 3 d. Ethylene was injected to a final concentration
of 1 p L / L and the jars were placed at 23°C for 4 d. Ethylene, CO,, and O, were monitored daily by GC. Final measurements of root and hypocotyl length were made using a
dissecting microscope with a stage micrometer.
Plant Growth
Plants for crossing and phenotypic analysis were grown
in growth chambers or shaking liquid culture as previously
described (Clouse et al., 1993).
Chromosome Mapping
A single plant showing the Bril phenotype (Col-O background) was pollinated with Ler pollen yielding a fertile F,
hybrid, which was selfed to generate a segregating F,
population of 70 mutant and 212 wild-type individuals.
DNA was isolated from individual mutant plants by placing the entire plant in a microfuge tube with a small
amount of liquid nitrogen, followed by grinding with a
plastic pestle. After sublimation of the nitrogen, 500 p L of
buffer (100 mM Tris, 100 mM Na,EDTA, 250 mM NaC1, pH
8.0) were added and grinding was continued. Fifty microliters of 10% sarkosyl were then added, followed by 5 pL of
proteinase K (10 mg/mL), and the tubes were incubated at
55°C for approximately 1 h. After centrifugation (14,000
rpm, 10 min), the supernatant was extracted twice with
pheno1:chloroform:isoamyl alcohol (24:24:1, v / v), and 0.35
volumes of 5 M NaCl and 2 volumes of 100% ethanol were
added to the final aqueous phase to precipitate DNA free
from polysaccharides (Mettler, 1987). The pelle'ts were
washed twice in 75% ethanol and resuspended in 100 pL of
sterile water, and 1.5 PL were used per 2 0 - ~ LPCR reaction.
DNA from individual plants showing the wild-type phenotype was isolated in a similar manner except that a small
A Brassinosteroid-lnsensitive Mutant in Arabidopsis thaliana
mortar and pestle was used for grinding whole plants and
the buffer was increased to 5 mL/g tissue. Genotypes of the
F2 wild-type plants were determined by F3 progeny analysis. Linkage to known molecular markers was determined
by SSLP (Bell and Ecker, 1994) and CAPS (Konieczny and
Ausubel, 1993) analysis using primers from Research Genetics (Huntsville, AL).
RESULTS
Phenotype and Genetics of the br'(\ Mutant
From a screen of approximately 70,000 EMS-mutagenized
M2 seedlings of A. thalicma, an individual was selected that
showed root elongation in the presence of 10~6 or 10~ 7 M
EBR, a concentration that inhibited the elongation of wildtype Col-0 roots by up to 75%. This mutant consistently
exhibited long roots and very short hypocotyls when placed
on various EBR concentrations and showed a striking phenotype when grown to maturity, which suggested that a fundamental pathway controlling growth and development had
been compromised by the mutation. Figure la shows that the
bril mutant is an extreme dwarf that reaches less than 10% of
the height of wild-type Col-0 plants grown under the same
conditions. Two-month-old bril plants did not exceed 1.5 cm,
whereas Col-0 plants of the same age were greater than 15 cm
in height. Figure 1, b and c, shows that the leaf morphology of
the bril mutant was distinctly different from that of the wild
type. Rosette leaves were shorter in the mutant than in the
673
wild type, had a thickened, curled appearance, and were
darker green, and their petioles failed to elongate. Preliminary microscopic evaluation of the bril mutant suggests that
reduced cell size rather than cell number is responsible for the
dwarf phenotype (data not shown). Figure Id shows that
4-month-old plants had a bushy appearance, indicating reduced apical dominance when compared to mature wild-type
plants.
bril plants failed to yield viable seeds even when selfpollinated by hand and grown at either 23 or 15 to 18°C.
However, when pollen from Col-0 or Ler wild-type plants
were placed on bril stigmas, siliques containing fertile F,
seeds developed, indicating that the bril mutant is most
likely male-sterile but female-fertile. We have not yet examined the mechanism of male sterility. The F, resembled
wild type in appearance, and selfed F, plants gave an F2
population that segregated in a 3:1 wild-type-to-mutant
phenotypic ratio, suggesting that the Bril phenotype is
caused by a single recessive Mendelian allele. Table I summarizes the F2 phenotypic segregation of a number of bril
X Col-0 and bril X Ler crosses. In all cases examined, those
plants showing the dwarf phenotype also were BR insensitive, as determined by the root-elongation assay. Moreover, after two generations of backcrossing interspersed
with two generations of selfing, there was no segregation
of the dwarf phenotype and BR insensitivity.
To determine the map position of the bril locus, DNA
was isolated from individual F2 progeny of a bril X Ler
cross and analyzed for linkage to SSLP (Bell and Ecker,
Figure 1. Phenotype ot the far/1 mutant, a, Two-month-old mutant (left) and wild-type (right) plants grown in 50-mL
centrifuge tubes in a 23°C growth chamber (16-h light/8-h dark), b and c, Side and top views, respectively, of a 2-month-old
far/I mutant plant, d, The same plant after 4 months. All bars = 1 cm.
Table 1. Phenotypic ratios in segregating Fz populations of b r i l x
COLO or b r i l X Ler crosses
Cross No.
1 (bril
2 (bril
3 (bril
4 (bril
5 (bril
6 (bril
7 (bril
8 (bril
9 (bril
Plant Physiol. Vol. 11 1, 1996
Clouse et ai.
674
X COLO, F2)
X
x
x
X
X
X
Col-O, F),
COLO, F,)
Col-O, F,)
COLO, F),
COI-O, F),
Col-O, F,)
Col-O, F,)
x
x COLO, F,)
Total
10 (bril
Wild Type
480
21 2
234
289
1585
427
661
192
220
4080
X
Ler, F,)
601
Mutant
164
70
87
83
495
127
214
70
103
11310
21 3
Ratio
2.93:l
3.03:l
2.69:l
3.48:l
3.20:l
3.36:l
3.09:l
2.74:l
2.1 4:l
3.1 1 :1
3.03:l
1994) and CAPS (Konieczny and Ausubel, 1993) markers.
No linkage between the Bril phenotype and the following
SSLP markers was observed: nga63, nga248, nga280, and
ATPase on chromosome I; nga168 on chromosome 11;
nga126, nga162, and nga6 on chromosome 111; and ctrl,
nga225, ngal51, and ngal06 on chromosome V. The Bril
phenotype, however, was found to be tightly linked to the
CAPS marker DHSl on the bottom of chromosome IV (1
recombinant out of 63 mutant F, plants examined), with a
map distance of 1.61 centimorgans estimated by the
method of Kosambi and 1.59 centimorgans by the method
of Haldane (Koornneef and Stam, 1992).Comparison of the
map position and phenotype of bril with those of other
dwarf and hormone-insensitive Arabidopsis mutants indicates that bril is not allelic to other mutants in the published literature. A collection of unpublished T-DNA
dwarfs is also nonallelic to bril based on map position
or physiological responses to BR (K. Feldmann, personal
communication).
EBR. EBR concentrations greater than 1.0 p~ were not
attempted so as to preserve the limited amounts of the
compound available.
The inhibition of root elongation in wild-type Arabidopsis seedlings seems to be a general effect of most plant
hormones tested (Bleecker et al., 1988; Lincoln et al., 1990;
Pickett et al., 1990; Wilson et al., 1990; Su and Howell, 1992;
Clouse et al., 1993). To determine if the bril mutation
confers insensitivity 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
Col-O and bril seedlings on a number of plant hormones.
Figure 4 shows that concentrations of 2,4-D from 5 to 100
nM resulted in even greater inhibition of bril root elongation than in wild-type, and that at 0.5 and 1.0 p~ the
inhibition was equally severe in both genotypes. Therefore,
the bril mutant retains complete sensitivity to 2,4-D. Figure
5 shows that bril and Col-O are equally sensitive to 0.5 FM
IAA, 6-benzylaminopurine, and kinetin, and that bril is
hypersensitive to 0.5 p~ ABA. bril appears to be slightly
less sensitive to 0.5 WM GA, than Col-O, but nowhere does
it approach the insensitivity observed with 0.5 p~ EBR (cf.
Fig. 2).
To determine the sensitivity of bril to ethylene, darkgrown seedlings were used in an experiment similar to that
described by Bleecker et al. (1988) for the analysis of the
etrl ethylene-insensitive mutant. It is interesting that the
bril mutant grown in the dark in air had short, thickened
hypocotyls and fully opened cotyledons characteristic of
the det2 and cop class of photomorphogenic mutants
(Chory and Susek, 1994). Figure 6 shows that even though
30
CONTROL
0.5, M 24-EPI BR
Hormone Sensitivity of the bril Mutant
The bril mutant was identified by the presence of long
roots when grown on a concentration of EBR that inhibited wild-type root elongation. Figure 2 shows that 0.5
PM EBR caused, on average, 82% inhibition of root elongation in wild-type Col-0 when compared to a buffer
control. In a segregating F, population from a cross of
the bril mutant with wild-type Col-O, those individuals
showing Col-O phenotype gave a similar response to the
wild-type parent with respect to inhibition of root elongation by 0.5 p~ EBR. However, those individuals with
the Bril phenotype were completely insensitive to the
effect of 0.5 ~ L EBR
M
on root elongation. This insensitivity was confirmed over a wide range of EBR concentrations, as shown in Figure 3. Concentrations of EBR
greater than 5.0 IIM resulted in a rapid decrease in root
length of wild-type Col-O seedlings, reaching a maximum inhibition at 0.5 WM. In contrast, bril mutants maintained the same root length on EBR concentrations ranging from 10.0 nM to 0.5 p~ as they did when they were
grown on control medium. An EBR concentration of 1.0
p~ did result in a slight inhibition of bril root elongation, but the mutant roots were still over five times
longer than roots of Col-O seedlings grown on 1.0 p~
WILD-TYPE
COLUMBIA
bril X COL. F2
W.T. PHENOTYPE
bril X COL. Fz
MUTANT PHENOTYPE
Figure 2. Effect of EBR on root length in F, progeny of a bril X Col-O
backcross. Because bril is male-sterile, pure stocks of bril seed were
not available. Therefore, segregating F, progeny from a backcross of
bril X COLO were plated on agar medium with or without 0.5 p~
EBR as described in "Materials and Methods." Root length was
measured under a dissecting microscope after 7 d. The graph represents the mean of 20 measurements t SE. All seedlings with long
roots on EBR showed the bril dwarf phenotype when mature.
A Brassinosteroid-lnsensitive Mutant in Arabidopsis thaliana
----t Columbia
* bril
O
05
1
5
1
5
10
50
100
(w.t.)
(mutant)
500
1000
24-EPIBRASSINOLIDE CONCENTRATION (nM)
Dose-response curve of root elongation on EBR. Segregating F, progeny from a backcross of bril X COLO were plated on agar
medium with the indicated concentration of EBR. Root length of all
seedlings was measured after 7 d and plants were tagged individually
and grown in soil for 2 months to determine phenotype. Data points
represent 20 measurements 2 SE for those plants determined to be
bril, compared with 20 measurements -t SE for COLO seedlings
treated in the same manner.
Figure 3.
roots of bril plants grown in the dark in air had shorter
roots than the wild type, both Col-O and bril root elongation were equally inhibited by approximately 60% upon
treatment with 1.0 pL/L ethylene, indicating that bril retains sensitivity to ethylene. Therefore, of a11 compounds
tested, bril shows marked insensitivity only to BRs.
Finally, to determine if the Bril phenotype could be
rescued by hormone treatment, plants were sprayed twice
weekly with 1.0 PM GA, or 1.0 PM EBR, or were grown for
21 d in shaking liquid culture containing 1.0 ~ L GA,
M
or 1.0
PM brassinolide. None of the treatments had any observable effect on the Bril phenotype. Mutant plants retained
the same long roots, short hypocotyls, and dwarf stature
with or without hormone treatment, whereas wild-type
plants showed substantial hypocotyl elongation and inhibition of root growth with GA, or BR treatment (data not
shown).
675
reduced apical dominance, and male sterility are caused by
a mutation in a single gene with pleiotropic effects that also
confers insensitivity to BRs. However, after two backcrosses and two rounds of selfing, it cannot be completely
ruled out that the dwarf phenotype and the BR insensitivity are caused by independent mutations in genes that are
very tightly linked.
Another Arabidopsis dwarf that has a very similar phenotype to bril is det2, a de-etiolated mutant that shows
characteristics of a light-grown plant even when it is grown
in the dark (Chory and Susek, 1994). Very recently, the
DET2 locus has been cloned by chromosome walking and
has been found to have significant homology to a mammalian 5a steroid reductase (Li et al., 1996), an enzyme involved in steroid metabolism. Moreover, the Det2 mutant
phenotype is rescued by BR treatment, indicating that det2
is a BR biosynthetic mutant (Li et al., 1996). The fact that
det2 (a single locus that is nonallelic to bril and that can be
rescued to wild type by BR treatment) has a closely related
phenotype to bril strongly suggests that the single locus
conferring insensitivity to BR is the same locus controlling
the developmental aspects of the Bril phenotype. In the
cases of both det2 and bril, it is likely that the dwarf
phenotype is caused by a lack of BR action, either through
reduced synthesis or reduced response, respectively.
It is not surprising that both a BR-insensitive and a
BR-deficient mutant show a dwarfed growth habit, since
BRs have long been known to promote stem elongation.
BR-promoted elongation of young vegetative tissue has
been observed in mung bean, azuki bean, and pea epicotyls
(Yopp et al., 1981; Gregory and Mandava, 1982; Mandava,
-
40
h
E
E
I
Columbia (w.t.)
bril (mutant)
30
Y
I-
20
Lu
J
ò
O
a!
10
D I SCUSSI ON
The inhibitory effect of BRs on primary root elongation
in wild-type A. thaliana proved to be a facile and reproducible screen for BR insensitivity (Clouse et al., 1993). While
the root assay allowed the initial identification of bril,
mutant plants exhibited a phenotype that extended far
beyond the simple root response to BR, which suggested
that the entire developmental program had been altered.
Analysis of severa1 thousand F, progeny showed no segregation between root elongation in the presence of BR and
the altered developmental morphology. Therefore, it is
most likely that the short stature, altered leaf structure,
O
.
O
.O5
.I
.5
1
5
10
50
.
.
.
100 5 0 0 1000
2,4-D CONCENTRATION (nM)
Figure 4. Dose-response curve of root elongation on 2,4-D. As in
Figure 3 , segregating F, progeny from a backcross of bril X Col-O
were plated on agar medium but with the indicated concentration of
2,4-D replacing EBR. Root length of all seedlings was measured after
7 d and plants were tagged individually and grown in soil for 2
months to determine phenotype. Data points represent 20 measurements t SE for those plants determined to be bril, compared with 20
measurements -t- SE for Col-O seedlings treated i n the same manner.
Clouse et al.
676
100
COLUMEIA (W.T.)
b r i l (MUTANT)
T
T
O
E
80
8
Y
O
5
60
W
O
o!
W
5
I
I-
40
o
2
A
W
ô
s
20
O
FpM'0.5
IAA
FM
BAP
'
0.5pM
KlNETlN
'
0.5 pM
ABA
'
0.5 ,M
=A3
'
100 p M
=A,
Figure 5. Effect of other hormones on root elongation. Segregating F
progeny from a backcross of bril X Col-O were plated on agar
medium as described in Figure 3 with the indicated concentration of
hormone. Root length of all seedlings was measured after 8 d and
plants were tagged individually and grown in soil for 2 months to
determine phenotype. Data points represent 20 measurements t SE
for those plants determined to be bril, compared with 20 measurements 2 SE for Col-O seedlings treated in the same manner.
1988); bean, sunflower, and cucumber hypocotyls (Mandava et al., 1981; Katsumi, 1985); Arabidopsis peduncles
(Clouse et al., 1993); and maize mesocotyls and wheat
coleoptiles (Yopp et al., 1981; Sasse, 1985). We have previously shown that BR is a potent enhancer of epicotyl elongation in soybeans and we have found that BRs modulated
cell-wall mechanical properties and gene expression in this
system (Clouse et al., 1992; Zurek et al., 1994). It is well
known that auxin also promotes stem elongation (Taiz,
1984), and comparisons of auxin- versus BR-stimulated
growth showed that the kinetics of elongation and effects
on gene expression were quite different for BR and auxin
(Clouse et al., 1992; Zurek and Clouse, 1994).
Also of interest is 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, particularly xyloglucans (Albersheim, 1976). XET is an enzyme
that specifically cleaves a xyloglucan chain and transfers
a fragment of that chain to an acceptor xyloglucan (Fry,
1995). Severa1 groups (Smith and Fry, 1991; Nishitani
and Tominaga, 1992; Fry et al., 1992; Fanutti et al., 1993)
have proposed a wall-loosening function for this enzyme
during cell elongation, or it may have other important
roles in elongation such as xyloglucan biosynthesis or
inte- gration of new xyloglucan polymers into the wall
(McQueen-Mason et al., 1993).
We have cloned a BR-regulated gene from soybean
called BRUl that shows high sequence homology to XETs
from other species (Zurek and Clouse, 1994) and whose
Plant Physiol. Vol. 11 1 , 1996
transcript levels are correlated with the extent of BRpromoted stem elongation and increases in plastic extensibility of the cell wall (Zurek et al., 1994).We recently found
that the recombinant BRUl protein has XET activity and
that extractable XET activity in soybean epicotyls is increased by BR treatment (W. Romanow, R. Smith, S.D.
Clouse, unpublished data). Therefore, XETs may play a
role in BR-modulated stem elongation in soybean.
A similar story is unfolding in A. thaliana. TCH4, which
is 70% identical to BRUl at the amino acid level, shows
increased transcript levels in BR-treated Arabidopsis tissue
and has also been found to encode a XET (Xu et al., 1995).
Moreover, transgenic A. thaliana plants harboring TCH4
promoter:GUS fusions show highest reporter gene expression in elongating tissue (Xu et al., 1995). Therefore, a likely
explanation for the dwarf phenotype of bril is that BR
insensitivity results in reduced XET activity (along with
reduced expression of other unidentified molecular components of the cell-elongation machinery), which results in
shortened cells. We are currently examining TCH4 expression and XET activity in the bril mutant.
The specificity of the hormone insensitivity of bril lends
additional support to the importance of BRs in growth and
development. Based on the root-elongation assay, bril is as
sensitive to auxins, GAs, cytokinins, ABA, and ethylene as
wild-type Col-O. Therefore, if insensitivity to BR in the root
assay and the developmental phenotype of bril are caused
by a single gene, it is compelling evidence that BR activity
is essential for growth and development. Furthermore, besides cell elongation, BRs must play a role in male fertility
and apical dominance. Based on early physiological studies, it was suggested that the effects of BR are mediated
through auxin or that BR increases tissue sensitivity to
endogenous auxins (Mandava, 1988). We previously
showed that BR can act independently of auxin in elongating soybean epicotyls (Clouse et al., 1992) and that auxin-
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Figure 6. Effect of ethylene on root elongation. Segregating F, progeny from a backcross of bril X Col-O were plated on agar medium
and placed at 4°C for 3 d. Ethylene or air was t h e n injected to a final
concentration of 1 pL/L and the jars were placed at 23°C for 4 d in
the dark. Final measurements of root length were made using a
dissecting microscope with a stage micrometer. Data points represent the mean of 20 measurements 5 SE.
A Brassinosteroid-lnsensitive Mutant in Arabidopsis thaliana
insensitive mutants such as axrl (Lincoln et al., 1990) and
dgt (Daniels et al., 1989) retained sensitivity to BR (Clouse
et al., 1993; Zurek et al., 1994). The complete sensitivity of
bril to auxin demonstrated here provides additional evidente of the independence of BR and auxin action.
Our primary motivation in studying BR-insensitive
mutants is to identify the BR receptor and clarify the BR
signal transduction pathway. Hormone insensitivity
generally results from one of three mechanisms: (a) a
mutation in the receptor prevents the hormone from
binding, or the receptor fails to become activated even
when bound to the ligand; (b) the receptor i s functional
but a mutation in a critical step in the signal transduction
pathway has occurred; or (c) the applied hormone is not
actually the active endogenous compound and an enzyme that converts the applied hormone to the active
form has been mutated. In view of the proven biological
activity of numerous forms of BRs, including brassinolide, EBR, 28-homobrassinolide, and castesterone (McMorris et al., 1994), and the fact that we have shown
insensitivity of the bril mutant to more than one BR (S.D.
Clouse, M. Langford, T.C. McMorris, unpublished data),
option c is unlikely and bril probably represents a mutation in class a or b. A collaborative effort between the
Chory and Sakurai groups to determine endogenous BRs
in Arabidopsis will help to clarify this issue (J. Chory,
personal communication).
Original views of the mechanism of animal steroid hormone action assumed a single protein receptor 1signal
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 1ligand 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 phosphorylation,
and can complex with nonsteroidal transcription factors in
the nucleus (Beato et al., 1995). Therefore, although 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
that of animals and is more complex, isolation of hormoneinsensitive mutants in Arabidopsis is still a very fruitful
approach, as has been amply demonstrated in ethylene
(Ecker, 1995) and ABA (Finkelstein and Zeevart, 1994) signa1 transduction studies.
We are engaged currently in the positional cloning of the
BRIl locus, which is greatly facilitated by the recent publication of a complete physical map of chromosome IV
(Schmidt et al., 1995). The dramatic and pleiotropic effects
of the bril mutation suggests that the B X l l gene lies very
early in the signal perceptionl transduction pathway.
Therefore, cloning and characterization of the BRIl locus
should lead to a major advance in our understanding of BR
action. The BR-insensitive (bril) and BR-deficient (def2)
mutants will be valuable resources for further understanding the molecular mode of BR action. Furthermore, the
677
phenotype of these mutants provides strong genetic evidente that BRs are essential for normal plant growth and
development.
ACKNOWLEDCMENTS
Thanks to Gregory Scott for excellent technical assistance and to
Sylvia Blankenship for performing the GC. We also thank Joanne
Chory, Ken Feldmann, Punita Nagpal, and Jason Reed for helpful
discussions.
Received February 7, 1996; accepted April 4, 1996.
Copyright Clearance Center: 0032-0889/96/111 /O671 /OS.
LITERATURE CITED
Adam G, Marquardt V (1986) Brassinosteroids. Phytochemistry
25: 1787-1799
Albersheim P (1976) The primary cell wall. In J Bonner, JE Varner,
eds, Plant Biochemistry, Ed 3. Academic Press, New York, pp
225-274
Beato M, Herrlich P, Schutz G (1995) Steroid hormone receptors:
many actors in search of a plot. Cell 83: 851-857
Bell CJ, Ecker JR (1994)Assignment of 30 microsatellite loci to the
linkage map of Arabidopsis. Genomics 19: 137-144
Bleecker AB, Estelle MA, Somerville C (1988) Insensitivity to
ethylene conferred by a dominant mutation in Arabidopsis thaliunu. Science 241: 1086-1089
Chang C, Kwok SF, Bleecker AB, Meyerowitz EM (1993) Arabidopsis ethylene-response gene ETRI: similarity of product to
two-component regulators. Science 262: 539-544
Chory J, Susek RE (1994)Light signal transduction and the control
of seedling development. In E Meyerowitz, C Somerville, eds,
Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, pp 579-614
Clouse SD, Langford M (1995) A brassinosteroid-insensitive mutant in Arabidopsis thaliana (abstract no. 375). Plant Physiol 108:
s-81
Clouse SD, Langford M, Hall AF, McMorris TC, Baker ME (1993)
Physiological and molecular effects of brassinosteroids on Arabidopsis thuliuna. J Plant Growth Regul 12: 61-66
Clouse SD, Zurek D (1991) Molecular analysis of brassinolide
action in plant growth and development. In HG Cutler, T
Yokota, G Adam, eds, Brassinosteroids: Chemistry, Bioactivity
and Applications. ACS Symposium Series 474. American Chemical Society, Washington, DC, pp 122-140
Clouse SD, Zurek DM, McMorris TC, Baker ME (1992) Effect of
brassinolide on gene expression in elongating soybean epicotyls.
Plant Physiol 100: 1377-1383
Daniels SG, Rayle DL, Cleland RE (1989) Auxin physiology of the
tomato mutant diageotropica. Plant Physiol 91: 804-807
Ecker JR (1995) The ethylene signal transduction pathway in
plants. Science 268: 667-675
Estelle M, Klee H (1994)Auxin and cytokinin. In E Meyerowitz, C
Somerville, eds, Arabidopsis. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, pp 555-578
Estelle MA, Somerville CR (1987) Auxin-resistant mutants of
Arabidopsis witli an altered morphology. Mo1 Gen Genet 206:
200-206
Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240: 889-895
Fanutti C, Gidley MJ, Reid JS (1993) Action of a pure xyloglucan
endo-transglycosylase from the cotyledons of germinated nasturtium seeds. Plant J 3: 691-700
Finkelstein RR, Zeevart JAD (1994) Gibberellin and abscisic acid
biosynthesis and response. In E Meyerowitz, C Somerville, eds,
Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, pp 523-554
Fry SC (1995) Polysaccharide-modifying enzymes in the plant cell
wall. Annu Rev Plant Physiol Plant Mo1 Biol 46: 497-520
678
Clouse et al.
Fry SC, Smith RC, Renwick KF, Martin DJ, Hodge SK, Matthews
KJ (1992) Xyloglucan endotransglycosylase, a new wall-loosening
enzyme activity from plants. Biochem J 282: 821-828
Gregory LE, Mandava NB (1982) The activity and interaction of
brassinolide and gibberellic acid in mung bean epicotyls.
Physiol Plant 54: 239-243
Grove MD, Spencer GF, Rohwedder WK, Mandava N, Worley JF,
Warhen JD (1979) A unique plant growth promoting steroid
from Brassica napus pollen. Nature 281: 216-217
Katsumi M (1985) Interaction of a brassinosteroid with IAA and
GA, in the elongation of cucumber hypocotyl sections. Plant
Cell Physiol 26: 615-625
Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCRbased markers. Plant J 4: 403410
Koornneef M, Stam P (1992) Genetic analysis. In C Koncz, N
Chua, J Schell, eds, Methods in Arabidopsis Research. World
Scientific, Singapore, pp 83-99
Li J, Nagpal P, Vitart V, McMorris TC, Chory J (1996) A role for
brassinosteroids in light-dependent development of Arabidopsis.
Science 272: 398-401
Lincoln C, Britton JH, Estelle M (1990) Growth and development
of the a x r l mutants of Arabidopsis. Plant Cell 2: 1071-1080
Mandava NB (1988) Plant growth-promoting brassinosteroids.
Annu Rev Plant Physiol Plant Mo1 Biol 39: 23-52
Mandava NB, Sasse JM, Yopp JH (1981) Brassinolide, a growthpromoting steroidal lactone. 11. Activity in selected gibberellin
and cytokinin bioassays. Physiol Plant 53: 453-461
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G,
Umesono K, Blumber B, Kastner P, Mark M, Chambon P,
Evans RM (1995) The nuclear receptor superfamily: the second
decade. Cell83: 835-839
McMorris TC, Patil PA (1993) Improved synthesis of 24epibrassinolide from ergosterol. J Org Chem 58: 2338
McMorris TC, Patil PA, Chavez RG, Baker ME, Clouse SD (1994)
Synthesis and biological activity of 28-homobrassinolide and
analogs. Phytochemistry 3 6 585-589
McQueen-Mason SJ, Fry SC, Durachko DM, Cosgrove DJ (1993)
The relationship between xyloglucan endotransglycosylase and
in-vitro cell wall extension in cucumber hypocotyls. Planta 190:
327-331
Mettler IJ (1987) A simple and rapid method for minipreparation
of DNA from tissue cultured plant cells. Plant Mo1 Biol Rep 5:
346-349
Nishitani K, Tominaga R (1992) Endo-xyloglucan transferase, a
nove1 class of glycosyltransferase that catalyzes transfer of a
segment of xyloglucan molecule to another xyloglucan molecule. J Biol Chem 267: 21058-21064
Pickett FB, Wilson AK, Estelle M (1990) The auxl mutation of
Arabidopsis confers both auxin and ethylene resistance. Plant
Physiol94: 1462-1466
Plant Physiol. Vol. 11 1, 1996
Roddick JG, Juan M (1991) Brassinosteroids and root development. In HG Cutler, T Yokota, G Adam, eds, Brassinosteroids:
Chemistry, Bioactivity and Applications. ACS Symposium
Series 474. American Chemical Society, Washington, DC, pp
231-245
Sasse JM (1985) The place of brassinolide in the sequential response to plant growth regulators in elongating tissue. Physiol
Plant 63: 303-308
Sasse JM (1991)The case for brassinosteroids as endogenous plant
hormones. In HG Cutler, T Yokota, G Adam, eds, Brassinosteroids: Chemistry, Bioactivity and Applications. ACS Symposium Series 474. American Chemical Society, Washington, DC,
p p 158-166
Sasse JM, Yokota T, Taylor PE, Griffiths PG, Porter QN, Cameron DW (1992) Brassinolide-induced elongation. In C Karssen,
L van Loon, D Vreugdenhil, eds, Progress in Plant Growth
Regulation. Kluwer Academic, Dordrecht, The Netherlands, pp
319-325
Schmidt R, West J, Love K, Lenehan Z, Lister C, Thompson H,
Bouchez, Dean C (1995) Physical map and organization of Arabidopsis thaliana chromosome 4. Science 270: 480-483
Smith RC, Fry SC (1991) Endotransglycosylation of xyloglucans in
plant cell suspension cultures. Biochem J 279: 529-535
Su W, Howell SH (1992) A single genetic mutant, c k r l , defines
Arabidopsis mutants in which root growth is resistant to low
concentrations of cytokinin. Plant Physiol99: 1569-1574
Sun T, Goodman HM, Ausubel FM (1992) Cloning the Arabidopsis G A 1 locus by genomic subtraction. Plant Cell 4: 119-128
Sun T, Kamiya Y (1994) The Arabidopsis GA1 locus encodes the
cyclase ent-kaurene synthetase A of gibberellin biosynthesis.
Plant Cell 6: 1509-1518
Taiz L (1984) Plant cell expansion: regulation of cell wall mechanical properties. Annu Rev Plant Physiol 35: 585-657
Wilson AK, Pickett FB, Turner JC, Estelle M (1990) A dominant
mutation in Arabidopsis confers resistance to auxin, ethylene and
abscisic acid. Mo1 Gen Genet 222: 377-383
Xu W, Purugganan MM, Polisensky DH, Antosiewicz DM, Fry
SC, Braam J (1995) Arabidopsis TCH4, regulated by hormones
and the environment, encodes a xyloglucan endotransglycosylase. Plant Cell 7: 1555-1567
Yopp JH, Mandava NB, Sasse JM (1981) Brassinolide, a growthpromoting steroidal lactone. I. Activity in selected auxin bioassays. Physiol Plant 53: 445-452
Zurek DM, Clouse SD (1994) Molecular cloning and characterization of a brassinosteroid-regulated gene from elongating soybean epicotyls. Plant Physiol 104: 161-170
Zurek DM, Rayle DL, McMorris TM, Clouse SD (1994) Investigation of gene expression, growth kinetics and wall extensibility
during brassinosteroid-regulated stem elongation. Plant Physiol
104: 505-513