The Plant Journal (2011) 67, 1–12
doi: 10.1111/j.1365-313X.2011.04567.x
FEATURED ARTICLE
Rice CYP734As function as multisubstrate and
multifunctional enzymes in brassinosteroid catabolism
Tomoaki Sakamoto1,*, Ayami Kawabe2, Asako Tokida-Segawa1, Bun-ichi Shimizu2,†, Suguru Takatsuto3, Yukihisa Shimada4,
Shozo Fujioka5 and Masaharu Mizutani6
1
Institute for Advanced Research, Nagoya University, Nagoya, Aichi 464-8601, Japan,
2
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan,
3
Department of Chemistry, Joetsu University of Education, Joetsu, Niigata 943-8512, Japan,
4
RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan,
5
RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan, and
6
Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan
Received 22 December 2010; revised 25 February 2011; accepted 4 March 2011; published online 26 April 2011.
*
For correspondence (fax +81 561 38 5786; e-mail [email protected]).
†
Present address: Faculty of Life Science, Toyo University, Izumino 1-1-1, Itakura, Gunma 374-0193, Japan.
SUMMARY
Catabolism of brassinosteroids regulates the endogenous level of bioactive brassinosteroids. In Arabidopsis
thaliana, bioactive brassinosteroids such as castasterone (CS) and brassinolide (BL) are inactivated mainly by
two cytochrome P450 monooxygenases, CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/SHK1; CYP734A1/BAS1
inactivates CS and BL by means of C-26 hydroxylation. Here, we characterized CYP734A orthologs from Oryza
sativa (rice). Overexpression of rice CYP734As in transgenic rice gave typical brassinosteroid-deficient
phenotypes. These transformants were deficient in both the bioactive CS and its precursors downstream of
the C-22 hydroxylation step. Consistent with this result, recombinant rice CYP734As utilized a range of C-22
hydroxylated brassinosteroid intermediates as substrates. In addition, rice CYP734As can catalyze hydroxylation and the second and third oxidations to produce aldehyde and carboxylate groups at C-26 in vitro. These
results indicate that rice CYP734As are multifunctional, multisubstrate enzymes that control the endogenous
bioactive brassinosteroid content both by direct inactivation of CS and by the suppression of CS biosynthesis
by decreasing the levels of brassinosteroid precursors.
Keywords: brassinosteroid catabolism, cytochrome P450 monooxygenase, substrate specificity, successive
oxidation, rice.
INTRODUCTION
Brassinosteroids are endogenous phytohormones that
are involved in the regulation of various growth and developmental processes in higher plants, such as cell and stem
elongation, dark-adapted morphogenesis (skotomorphogenesis), responses to environmental stress, and differentiation of tracheary elements (reviewed in Clouse and Sasse,
1998; Krishna, 2003; Sasse, 2003). Brassinosteroids are
believed to enhance crop production. In Oryza sativa (rice),
for example, brassinosteroids induce disease resistance
(Nakashita et al., 2003) and abiotic stress tolerance (Koh
et al., 2007), and, interestingly, overproduction and deficiency of brassinosteroids both increase grain yield,
although by different mechanisms (Morinaka et al., 2006;
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd
Sakamoto et al., 2006; Wu et al., 2008). These findings
suggest the feasibility of genetic improvement of crop production by the modulation of brassinosteroid metabolism
and the contents of bioactive brassinosteroids.
The major pathway for brassinosteroid biosynthesis has
been established in Arabidopsis thaliana, and a number of
dwarf mutants have been identified (reviewed in Bishop,
2007). Among the six major enzymes involved in brassinosteroid biosynthesis, four are cytochrome P450 monooxygenases (P450s): in rice, C-22 hydroxylase is encoded by
CYP90B2/OsDWARF4 and CYP724B1/D11 (Sakamoto et al.,
2006); C-23 hydroxylase is encoded by CYP90D2/D2 and
CYP90D3 (TS and MM, unpublished results); and C-6
1
2 Tomoaki Sakamoto et al.
oxidase is encoded by CYP85A1/OsDWARF (Hong et al.,
2002; Mori et al., 2002). CYP90As, encoded by CYP90A3/
OsCPD1 and CYP90A4/OsCPD2, are also believed to be
involved in brassinosteroid biosynthesis in rice, although
their catalytic function has not been clarified (Sakamoto and
Matsuoka, 2006).
Catabolism of brassinosteroids is another important factor that regulates the endogenous levels of bioactive brassinosteroids. In A. thaliana, inactivation of brassinosteroids
is also catalyzed by P450s, and two genes, CYP734A1/BAS1
and CYP72C1/SOB7/CHI2/SHK1, have been identified (Neff
et al., 1999; Turk et al., 2003, 2005; Nakamura et al., 2005;
Takahashi et al., 2005). The biochemical function of
CYP72C1/SOB7/CHI2/SHK1 has not yet been clarified;
however, CYP734A1/BAS1 inactivates castasterone (CS)
and brassinolide (BL) by means of C-26 hydroxylation (Turk
et al., 2003). CYP734A1/BAS1 orthologs have been identified
in Solanum lycopersicum (tomato), in which at least one
of two genes, CYP734A7, encodes a C-26 hydroxylase of
brassinosteroids and is probably involved in brassinosteroid
catabolism (Ohnishi et al., 2006a).
By using the genes for these brassinosteroid biosynthetic
or catabolic enzymes in breeding programs, it should
become possible to control the level of bioactive brassinosteroids and thereby increase crop productivity. We
attempted to identify rice orthologs of CYP734A1/BAS1
and CYP72C1/SOB7/CHI2/SHK1 to enhance our understanding of brassinosteroid metabolism in rice. On the basis
of our results, we discuss the evolution of the genes for
brassinosteroid catabolic enzymes in rice.
Overexpression of CYP734A genes in transgenic rice
To assess the activity of the CYP734A gene products in rice,
the entire coding regions for the rice CYP734As were
amplified by means of RT-PCR using total RNA extracted
from whole seedlings. Because we could not obtain the
full-length cDNA for CYP734A5 by RT-PCR, we ectopically
expressed the other three cDNAs in transgenic rice under the
control of the rice actin1 promoter (McElroy et al., 1990).
All transformants overexpressing the CYP734A cDNA
exhibited abnormal phenotypes, and we categorized them
into two groups on the basis of leaf morphology and gross
morphology. The control transformants that contained the
empty vector were indistinguishable from wild-type rice
plants. They flowered about 90 days after the regeneration
and the plant height reached about 90 cm (Figure 1a). Plants
exhibiting a group-1 phenotype had severe dwarfing (about
10 cm in height) and malformed leaves with twisted leaf
blades (Figure 1b). The leaves of these plants were erect,
and the ratio of leaf sheath length to leaf blade length was
reduced (0.209 0.002, versus 0.601 0.011 in the wild
type). The floral organs did not form and internodes did not
elongate in the group-1 phenotype plants, and they did not
bear any seeds. These phenotypes were indistinguishable
from those of the brassinosteroid-deficient mutant brd1-2
(Figure 1c; Hong et al., 2002). Transformants categorized as
group-2 phenotype formed only abnormal leaves with stiff,
tortuous blades, and their leaf sheaths were scarcely developed (Figure 1d, right plant). These plants did not flower,
exhibit internode elongation, or bear seeds. These pheno-
(a)
(b)
(c)
RESULTS
Isolation of CYP734A1/BAS1-like genes from rice
The predicted amino acid sequences of CYP734A1/BAS1 and
CYP72C1/SOB7/CHI2/SHK1 were used as in silico probes
to screen all available rice DNA databases. Candidate
sequences detected during this process were also used iteratively as search probes. We found four candidate genes that
had relatively high identity with the amino acid sequences
of CYP734A1/BAS: CYP734A2 (locus ID Os02g0204700),
CYP734A4 (Os06g0600400), CYP734A5 (Os07g0647200),
and CYP734A6 (Os01g0388000). We found no orthologs of
CYP72C1/SOB7/CHI2/SHK1 in the rice genome. The deduced
amino acid sequences of CYP734A2, CYP734A4, and
CYP734A6 were very similar (ranging between 70.2
and 82.1% identity), whereas they showed between 58.0 and
58.9% identity with CYP734A5 (Table S1). CYP734A1/BAS1
showed the highest identity with CYP734A6 (62.0%), and
lower identity with CYP734A5 (50.5%; Table S1). All four rice
CYP734As and CYP734A1/BAS1 showed lower identities
with CYP72C1/SOB7/CHI2/SHK1 (ranging between 34.9 and
37.6%; Table S1).
(d)
Figure 1. Phenotypes of transgenic rice plants that overexpress the CYP734A
genes.
(a) Typical morphology of control transgenic rice with wild-type phenotype at
about 90 days after regeneration.
(b) Typical morphology of CYP734A transgenic rice plants with group-1
phenotype at about 90 days after regeneration.
(c) Phenotype of the brassinosteroid-deficient mutant brd1-2.
(d) Typical morphology of CYP734A transgenic rice plants with group-2
phenotype at about 90 days after regeneration (right) and the brassinosteroid-insensitive mutant d61-3 (left).
Bars = 10 cm in (a–c), and 3 cm in (d).
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
Rice brassinosteroid catabolic genes 3
Table 1 Endogenous content of sterols and brassinosteroids in transgenic rice
Content (ng g)1 fresh weight)
Sterol or brassinosteroid intermediate
Control
CYP734A2
CYP734A4
CYP734A6
24-Methylenecholesterol
Campesterol
Campestanol
6-Oxocampestanol
6-Deoxocathasterone
Cathasterone
6-Deoxoteasterone
Teasterone
3-Dehydro-6-deoxoteasterone
3-Dehydroteasterone
6-Deoxotyphasterol
Typhasterol
6-Deoxocastasterone
Castasterone
Brassinolide
1680
58900
1660
61.3
1.02
n.d.
0.97
0.05
4.16
0.14
11.10
0.38
0.46
0.22
n.d.
1110
68600
1520
41.7
0.06
n.d.
0.01
0.01
0.09
n.d.
0.08
n.d.
0.01
0.01
n.d.
1030
50300
1750
49.4
0.07
n.d.
0.01
n.d.
0.06
n.d.
0.12
n.d.
0.01
n.d.
n.d.
1280
72800
2190
63.1
0.27
n.d.
0.03
0.01
0.21
n.d.
0.66
0.01
0.06
0.01
n.d.
n.d., not detected.
types were indistinguishable from those of the brassinosteroid-insensitive mutant d61-3 (Figure 1d, left plant;
Nakamura et al., 2006). About 90% (40 plants) of the
transformants overexpressing CYP734A2 exhibited the
group-2 phenotype and the remaining 10% (five plants)
had the group-1 phenotype. About 55% (23 plants) of the
CYP734A6 transformants exhibited the group-2 phenotype
and the remaining 45% (19 plants) had the group-1 phenotype. In contrast to these two genes, all 38 transformants
that overexpressed CYP734A4 exhibited the group-2 phenotype. These results strongly suggest that overexpression of
CYP734A2, CYP734A4, and CYP734A6 cDNA decreased the
level of bioactive brassinosteroids in the transgenic rice.
We compared the endogenous levels of sterols and
brassinosteroids in the control transformants and CYP734A
transformants having the group-2 phenotype by using gas
chromatography–mass spectrometry (GC-MS) analyses
(Table 1). Cathasterone (CT) and BL were not detected in
either the control or the CYP734A transformants, suggesting
that these compounds are minor components of the total
brassinosteroid pool in rice. Although BL was not detected,
another bioactive brassinosteroid (i.e. CS) was detected in
the control, but not in the CYP734A4 transformants, confirming that the CYP734A4 transformants are brassinosteroid-deficient. Levels of the other eight intermediates
downstream of C-22 hydroxylation – 6-deoxocathasterone
(6-deoxoCT), 6-deoxoteasterone (6-deoxoTE), teasterone
(TE), 3-dehydro-6-deoxoteasterone (6-deoxo3DT), 3-dehydroteasterone (3DT), 6-deoxotyphasterol (6-deoxoTY),
typhasterol (TY), and 6-deoxocastasterone (6-deoxoCS) –
were also greatly reduced in the CYP734A4 transformants.
Similar results were observed in the CYP734A2 and
CYP734A6 transformants. These results suggest that overexpression of CYP734A2, CYP734A4, and CYP734A6 increases
the inactivation of C-22 hydroxylated brassinosteroids in
transgenic rice.
Catalytic activities of rice CYP734As
To determine the catalytic function of the rice CYP734A gene
products, we performed in vitro conversion assays using the
recombinant CYP734A proteins produced in insect cells by a
baculovirus expression system. Brassinosteroid intermediates with a diol at the C-22 and C-23 positions of the side
chain were used for the assays, in which the reaction products were converted into 9-phenanthreneboronate derivatives and analyzed by means of HPLC. In the presence of
NADPH, CYP734A2 metabolized 6-deoxo3DT into three new
products with retention times (rt) of 6.5, 8.3, and 9.0 min
(Figure 2a); these products were not identical to the compounds previously found in the brassinosteroid biosynthetic
pathway. Analysis of the products by means of fast-atombombardment mass spectrometry (FAB-MS) revealed that
the product at rt 6.5 min was 29.97 mass units larger than
that of the substrate 6-deoxo3DT; this is consistent with
the theoretical mass of the carboxylate form of 6-deoxo3DT
(Table 2). On the other hand, the observed mass of the
product at rt 8.3 min was 15.99 mass units larger, which was
consistent with the theoretical mass of the hydroxylate form
of 6-deoxo3DT. These results suggested that CYP734A2 is a
multifunctional P450 that catalyzes a three-step successive
oxidation of 6-deoxo3DT to produce an alcohol, an aldehyde, and a carboxylate group. Similarly, three products
were detected in the assays for CYP734A2 with all the 22,23hydroxylated compounds in the brassinosteroid biosynthetic pathway, including CS and BL (Figure S1).
In the case of the brassinosteroid compounds without the
diol side chain, the reaction products were converted into
trimethylsilyl derivatives and analyzed by means of GC-MS.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
4 Tomoaki Sakamoto et al.
fission fragment ion of the derivative of the substrate
22-OH-3-one had a mass-to-charge ratio (m/z) of 187,
whereas those of the product derivatives were predicted
to be m/z 201, 185, and 289, respectively, but there was no
predicted fragment peak with an m/z of 187 (Table 3). These
results suggested that one of the methyl groups at C-26,
C-27, and C-28 in the side chain is consecutively oxidized
into a carboxylated form. The other 22-hydroxylated brassinosteroid compounds were similarly metabolized into three
products by CYP734A2 (Figure S2). Campesterol (CR, Figure S2), campest-4-en-3-one, campest-3-one, and campestanol (CN) were not metabolized at all, suggesting that the
presence of the hydroxyl group at C-22 is crucial for
recognition of the substrate by CYP734A2. These results
indicated that CYP734A2 is a multifunctional and multisubstrate enzyme capable of metabolizing C-22 hydroxylated
compounds in the brassinosteroid biosynthetic pathway.
We also performed conversion assays with CYP734A4
and CYP734A6 under the same conditions. We found that
the CYP734A4 assays gave results similar to those of the
CYP734A2 assays for all the substrates we tested. Interestingly, CYP734A6 acted on a similar array of substrates, but
only two products were detected in each of the conversion
assays. For instance, CYP734A6 metabolized 6-deoxo3DT
into two products, which corresponded to the oxidized
products in alcohol and aldehyde forms found in the
CYP734A2 assay (Figure 2b). No carboxylated product was
detected even after extended incubation, suggesting that
CYP734A6 can catalyze only a two-step oxidation to produce
an aldehyde form.
Determination of the position oxidized by CYP734A2
Figure 2. High-performance LC analysis of products from the rice CYP734A
assay with 6-deoxo3DT as a substrate.
(a) Results of the CYP734A2 assay. Upper panel, –NADPH; lower panel,
+NADPH. The retention times of the substrate and products were: substrate (s),
13.7 min; product (a), 6.5 min; product (b), 8.3 min; and product (c), 9.0 min.
(b) Results of the CYP734A6 assay. Upper panel, –NADPH; lower panel,
+NADPH. The retention times of the substrate and products were: substrate (s),
14.7 min; product (b), 8.4 min; product (c), 9.1 min.
In the assay with (22S,24R)-22-hydroxy-5a-ergostan-3-one
(22-OH-3-one), three products (with retention times of 12.06,
13.38, and 14.48 min) were detected (Figure 3). The C20–C22
To determine the specific position of the hydroxyl group
introduced by CYP734A2, we performed a conversion assay
with CS as a substrate. Castasterone was metabolized into
three products, and one of the products, which corresponded to hydroxylated CS, was identical to the authentic
26-hydroxyCS (26-OHCS) in its GC retention time and mass
spectrum (Figure S3).
Next, d7-6-deoxoCS labeled with deuterium atoms at the
C-25, C-26, and C-27 positions was applied as a substrate.
CYP734A2 metabolized d7-6-deoxoCS into three products,
and these metabolites were separated and collected by
means of HPLC. The metabolites corresponding to the
aldehyde and carboxylate forms were found to have m/z
values of 469.38 and 507.36, respectively, in the highresolution mass spectra (HRMS) analysis (Table 4). The
observed values were consistent with the theoretical m/z
values of d5-6-deoxoCS-CHO and d4-6-deoxoCS-COOH, respectively. These results suggest that two or three deuterium
atoms in either the C-26 or the C-27 methyl group were
displaced by successive oxidation. Taken together, these
results suggest that CYP734A2 catalyzes the oxidation of
brassinosteroids to produce C-26 oxidized metabolites.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
Rice brassinosteroid catabolic genes 5
Table 2 Fast-atom-bombardment mass spectrometry (FAB-MS) data for the reaction products with 6-deoxo3DT as the substrate and CYP734A2
as the enzyme
Compound
a
6-Deoxo3DT
6-Deoxo3DT-OHa
6-Deoxo3DT-CHOb
6-Deoxo3DT-COOHa
+
M
M+
[M+Na]+
M+
Theoretical m/z
Observed m/z
Error (p.p.m. per mmu)
Composition
618.4252
634.4201
469.3294
648.3994
618.4257
634.4178
469.3304
648.3990
+2.0/+1.2
–2.5/–1.6
+2.2/+1.0
+0.6/+0.4
C42 H55 O3 B
C42 H55 O4 B
C28 H46 O4 Na
C42 H53 O5 B
a
9-Phenanthreneboronate derivative.
Free form.
mmu, millimass units.
b
the brassinosteroid biosynthetic pathway. This hypothesis
is consistent with the content of the endogenous brassinosteroid intermediates that we observed in the transgenic
rice that overproduced CYP734As.
Expression of CYP734A genes in wild-type and mutant rice
Figure 3. Total ion chromatogram of products from the CYP734A2 assay with
22-OH-3-one as a substrate. Upper panel, –NADPH; lower panel, +NADPH.
The retention times of the substrate and products were: substrate (s),
10.28 min; product (a), 12.06 min; product (b), 13.38 min; product (c),
14.48 min.
Substrate specificity of CYP734A2
The present results clearly demonstrated that, in contrast
to CYP734A1/BAS1 and tomato CYP734A7, which selectively
metabolize CS and BL, rice CYP734A2 could metabolize
various brassinosteroid intermediates. Thus, we determined
the substrate specificity of CYP734A2 by determining the
relative activities for the brassinosteroid intermediates with
a diol at the C-22 and C-23 positions (Table 5). Among the
compounds that we tested, 6-deoxo3DT provided the best
match for CYP734A2, with another early intermediate, 22,23dihydroxycampesterol, also preferred. On the other hand,
CS and BL were not highly suitable substrates for CYP734A2
(with relative activities of 39 and 16%, respectively). These
results suggested that rice CYP734As metabolize endogenous brassinosteroid intermediates during the early steps of
Quantitative reverse-transcriptase PCR (qRT-PCR) analysis
revealed that all four rice CYP734A genes were expressed
at different levels in different organs (Figure 4). CYP734A2,
CYP734A4, and CYP734A6 were expressed in all the organs
of wild-type rice that we tested, including the vegetative
shoot apices, leaf sheaths, leaf blades, elongating internodes, roots, and panicles at flowering time, whereas
CYP734A5 expression was not observed in the leaves or
internodes, was present at nearly undetectable levels in the
shoot apices, leaf sheaths, and panicles, and was expressed
at the highest level in the roots.
Previous observations in A. thaliana indicate that the
expression of CYP734A1/BAS1 was up-regulated by the
application of brassinosteroid (Goda et al., 2002; Tanaka
et al., 2005). Thus, we examined whether such feedback
regulation also occurs in rice. We also determined the
expression of genes for the brassinosteroid biosynthetic
enzyme (OsDWARF; Hong et al., 2002; Mori et al., 2002) and
for the brassinosteroid receptor (OsBRI1; Yamamuro et al.,
2000) as controls. Quantitative RT-PCR analysis revealed that
the expression of all four rice CYP734As was rapidly
increased by BL treatment (Figure 5a), whereas it was
down-regulated in brassinosteroid-deficient brd1-2 (a lossof-function mutant of OsDWARF; Figure 5b). The level of
CYP734A2 transcripts decreased in brassinosteroid-insensitive d61-3 (a loss-of-function mutant of OsBRI1), but the
expression of the other three CYP734A genes increased in
the d61-3 mutant.
Genetic evidence has uncovered a role for brassinosteroids in the control of skotomorphogenesis, the process
by which etiolated seedlings rapidly grow an exaggerated
hypocotyl in the dark so they can reach the soil surface. In
previous research, brassinosteroid-deficient or brassinosteroid-insensitive mutants did not show an elongated phenotype and developed similarly to plants grown in the light
(Clouse et al., 1996; Szekeres et al., 1996). Expression of
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
6 Tomoaki Sakamoto et al.
Table 3 Gas chromatography-MS data for the products obtained in the CYP734A2 assay
Substrate
Product
22-OH-CR
22-OH-CR-CHO
22-OH-CR-OH
22-OH-CR-COOH
22-OH-3-one
22-OH-3-one-CHO
22-OH-3-one-OH
22-OH-3-one-COOH
Retention time (min)
Characteristic ions m/z (relative intensity percentage)
10.043
11.730
12.995
14.040
10.281
12.060
13.380
14.480
385 (0.2), 343 (0.2), 295 (1.4), 187 (81.7), 97 (100)
343 (12), 295 (2.4), 201 (100), 111 (53.7)
343 (0.8), 295 (0.8), 185 (10.6), 113 (100)
343 (0.2), 295 (0.6), 289 (50.8), 199 (60.8), 143 (100)
374 (7.4), 345 (1.6), 313 (2.0), 271 (3.6), 187 (65.4), 97 (100)
374 (26.1), 271 (100), 201 (88.6), 111 (61.7)
374 (2.0), 345 (0.3), 313 (1.3), 271 (2.4), 185 (10), 113 (100)
313 (0.8), 289 (48.6), 273 (1.2), 199 (48.0), 143 (100)
Table 4 High-resolution mass spectra data for the reaction products with d7-6-deoxoCS as the substrate and CYP734A2 as the enzyme
Compound
a
d7-6-deoxoCS
d6-6-deoxoCS-OHb
d5-6-deoxoCS-CHOb
d4-6-deoxoCS-COOHb
+
M
n.d.
M+
[M+Na]+
Theoretical m/z
Observed m/z
Error (p.p.m. per mmu)
Composition
643.4789
472.4035
469.3816
507.3600
643.4803
n.d.
469.3816
507.3591
+2.1/+1.4
n.d.
+0.1/+0.1
)1.8/)0.9
C42 1H50 D7 O4 B
n.d.
C28 1H43 D5 O5
C28 1H44 D4 O6 Na
a
9-Phenanthreneboronate derivative.
Free form.
mmu, millimass units; n.d., not detected.
b
Table 5 Substrate specificity of CYP734A2
Compound
Relative
activity (%)a
22,23-Dihydroxycampesterol
22,23-Dihydroxy-4-en-3-one
6-Deoxoteasterone
6-Deoxo-3-dehydroteasterone (6-deoxo3DT)
6-Deoxotyphasterol
Typhasterol
6-Deoxocastasterone
Castasterone (CS)
Brassinolide (BL)
90
75
55
100
73
38
51
39
16
a
The value obtained using 6-deoxo-3-dehydroteasterone as the
substrate was arbitrarily set at 100.
CYP734A2, CYP734A4, and CYP734A5 was up-regulated
in dark-grown seedlings, whereas the level of CYP734A6
transcripts decreased (Figure 5b).
DISCUSSION
The concentration of bioactive phytohormones is tightly
controlled by the regulation of both their biosynthesis
and their catabolism. Therefore, characterization of catabolic
enzymes is an important part of understanding phytohormone metabolism. Inactivation of bioactive brassinosteroids such as CS and BL, as well as their biosynthetic
precursors, occurs through various reaction processes,
including epimerization of 2- and 3-hydroxy groups followed
by glucosylation or acylation, hydroxylation of C-20 and
successive side-chain cleavages, glucosylation of the C-23
hydroxy group, and hydroxylation of C-25 or C-26 followed
by glucosylation (Bajguz, 2007). In A. thaliana, a UDPglucosyltransferase (UGT73C5) catalyzes 23-O-glucosylation
of CS and BL (Poppenberger et al., 2005). Two P450s,
CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/SHK1, also act in
the inactivation of brassinosteroids by means of different
enzymatic activities (Turk et al., 2005). Biochemical analyses
revealed that CYP734A1/BAS1 is a C-26 hydroxylase that
utilizes CS and BL as substrates.
Our results showed that there are four rice CYP734A
genes, but there appears to be no CYP72C1/SOB7/CHI2/
SHK1 ortholog in the rice genome. It is noteworthy that
at least three rice CYP734As (CYP734A2, CYP734A4, and
CYP734A6) utilized a broad range of C-22 hydroxylated
brassinosteroid intermediates as substrates (summarized
in Figure 6). In addition, CYP734A2 catalyzed the oxidation
of 22-hydroxy-cholesterol, suggesting that rice CYP734As
can metabolize both C27-brassinosteroids and C28-brassinosteroids. Consistent with this result, the CYP734A-overproducing rice transformants showed the greatest decrease
in levels of the endogenous brassinosteroid intermediates
downstream of the C-22 hydroxylation step. These results
suggest that rice CYP734As control the endogenous bioactive brassinosteroid content not only by direct inactivation
of CS by C-26 oxidation (as in A. thaliana), but also by the
suppression of CS biosynthesis through decreases in CS
precursors.
Interestingly, some rice CYP734As (CYP734A2 and
CYP734A4) can catalyze not only the hydroxylation of C-22
hydroxylated brassinosteroids but also the second and third
oxidations to produce an aldehyde and a carboxylate group
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
Rice brassinosteroid catabolic genes 7
Figure 4. Relative mRNA level of CYP734As in
various organs of wild-type rice.
Values represent the ratio between each
CYP734A and the corresponding ubiquitin levels.
Bars indicate standard deviation from the mean
(n = 3).
Figure 5. Regulation of CYP734A gene expression.
(a) Changes in the expression level of CYP734As
in wild-type rice seedlings after the 100 nM
brassinolide (BL) treatment. The value obtained
from the seedlings just before the treatment
was arbitrarily set at 1.0. Bars indicate standard
deviation from the mean (n = 3).
(b) Wild-type seedlings were grown under continuous light (Light) or complete darkness (Dark).
The brassinosteroid-deficient mutant brd1-2
and the brassinosteroid-insensitive mutant d613 were grown under continuous light. The value
obtained from the wild-type seedlings grown
under continuous light was arbitrarily set at 1.0.
Bars indicate standard deviation from the mean
(n = 3).
at C-26 in vitro (Figure 6). Although hydroxylation at C-25
and C-26 and subsequent glucosylation were observed in
24-epi-brassinolide exogenously applied to cultured tomato
cells (Hai et al., 1995; Winter et al., 1997), the metabolic
processes that follow the successive oxidations are
unknown. Previously, demethylation of BL at C-26 was
observed when BL was applied to Marchantia polymorpha
cells (Kim et al., 2000), and a cell-free tomato solution also
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
8 Tomoaki Sakamoto et al.
Figure 6. Schematic diagram of catabolism of C-22 hydroxylated brassinosteroid by rice CYP734As.
Numbers in brassinolide indicate the carbon number (C-25, C-26, and C-27).
Figure 7. CYP734A subfamily from various plants. Phylogenetic tree of the
CYP734As from various plant species.
The tree was rooted by using CYP72C1 as the outgroup gene. Bar, 0.1 amino
acid substitutions per site.
converted CS into 26-norCS by means of C-26 demethylation
(Kim et al., 2004). It seems that oxidation and subsequent
decarboxylation at C-26 should occur before demethylation.
In our conversion assays, we did not detect any demethylated product. This indicates that rice CYP734As do not
catalyze C-26 demethylation; however, our results do not
rule out the possibility that other enzymes are involved in
decarboxylation in rice. Because of technical difficulties,
we could not confirm the unique catalytic activities of rice
CYP734As in vivo. However, we believe that further analyses
will support the conclusion that we reached from our in vitro
analyses.
Our results indicate that rice CYP734As are multifunctional and multisubstrate P450s, which catalyze the consecutive
oxidation of various C-22 hydroxylated brassinosteroids.
There are some examples of multifunctional and/or multisubstrate P450s in the primary and secondary metabolism,
such as sterol C-14 demethylase (CYP51G) in sterol biosynthesis (Kahn et al., 1996), ent-kaurene oxidase (CYP701A)
and ent-kaurenoic acid 7a-hydroxylase (CYP88A) in gibberellin biosynthesis (Helliwell et al., 1998, 2001), CYP79s
catalyzing successive N-oxidations of amino acids to form
aldoximes (Bak et al., 2006), and abietadienol/abietadienal
oxidase (CYP720B1) in diterpene resin acid biosynthesis
(Ro et al., 2005). The consensus amino acid sequence motif
(A/G)GX(D/E)TT in the I-helix of most P450s are known to
be involved in the substrate interaction and the oxygen
activation, and in particular, the conserved Asp/Glu and Thr
residues in the motif play a crucial role in the hydrogen-bond
network that enables the protonation of the distal O2 to
promote the heterolytic scission of the O–O bond. Some
P450s catalyzing unusual reactions have a unique substitution in the conserved motif (Mizutani and Sato, 2011).
In rice CYP734A2, CYP734A4, and CYP734A6, the conserved
Asp/Glu residue is replaced by glutamine, suggesting that
this amino acid substitution may be crucial for CYP734A
activity. In the brassinosteroid biosynthetic pathway,
hydroxylation at C-22 and C-23 positions occurs at several
intermediate steps, and CYP90B, CYP90C, CYP90D, and
CYP724B are multisubstrate enzymes towards brassinosteroid intermediates (Fujita et al., 2006; Ohnishi et al., 2006b,c).
Furthermore, Arabidopsis CYP85A2 catalyzes the C-6 oxidation and lactonization reactions of teasterone, typhasterol,
and castasterone (Katsumata et al., 2008). Thus the brassinosteroid biosynthetic pathway constitutes a complicated
metabolic grid, which provides a broad range of potential
bioactive brassinosteroids. The multifunctional and multisubstrate properties of rice CYP734As will enable the
regulation of the endogenous content of bioactive brassinosteroids.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
Rice brassinosteroid catabolic genes 9
The CYP734A subfamily is found in various plant
species, including gymnosperms and angiosperms, but
not mosses and ferns. This indicates that the CYP734A
subfamily is widely distributed among vascular plants.
Phylogenetic analysis revealed that CYP734As are divided
into different clades between monocot and dicot species
(Figure 7). Duplication of CYP734A has also occurred in
tomato, but tomato CYP734As function as a C-26 hydroxylase that utilizes CS and BL as substrates, as in the case
of CYP734A1/BASI (Ohnishi et al., 2006a). These results
suggest that the rice CYP734As acquired their novel
function in brassinosteroid inactivation after the differentiation of monocots and dicots, and then increased their
number in the rice genome. Three CYP734A genes in Zea
mays (CYP734A-Zm1, CYP734A-Zm2, and CYP734A-Zm3)
are clustered with rice CYP734A6, CYP734A4, and
CYP734A2, respectively (Figure 7). This suggests that
CYP734As were duplicated before speciation in the monocots. In contrast to this finding, the CYP85A gene has been
duplicated in A. thaliana and tomato but not in rice. The
rice CYP85A enzyme catalyzes the C-6 oxidation of
6-deoxoCS to produce CS, a bioactive brassinosteroid
(Hong et al., 2002; Kim et al., 2008), whereas one of the
two enzymes in A. thaliana and tomato, AtCYP85A2 and
LeCYP85A3, has evolved to catalyze a Baeyer–Villiger
oxidation at C-6 to form BL, the most biologically active
brassinosteroid, from 6-deoxoCS through CS (Kim et al.,
2005; Nomura et al., 2005). These findings suggest that
divergent evolutionary processes have occurred in brassinosteroid metabolism.
Expression of all four CYP734A genes was up-regulated
by the BL treatment and down-regulated in the brassinosteroid-deficient mutant brd1-2, suggesting that the levels of
endogenous bioactive brassinosteroids control the expression of these genes, as was the case for CYP734A1/BAS1
in A. thaliana. However, the levels of CYP734A4, CYP734A5,
and CYP734A6 transcripts increased in d61-3 (a loss-offunction mutant of the rice brassinosteroid receptor gene,
OsBRI1). In d61-3, lack of the brassinosteroid signal stimulates feedback up-regulation of the brassinosteroid biosynthetic enzyme gene, OsDWARF (Figure 5b), resulting in
accumulation of bioactive CS in the OsBRI1 mutant
(Nakamura et al., 2006). In the same context, lack of the
brassinosteroid signal should stimulate down-regulation of
brassinosteroid catabolic enzyme gene expression. Indeed,
CYP734A2 transcripts decreased in d61-3. However, the
expression levels of CYP734A4, CYP734A5, and CYP734A6
were up-regulated in d61-3. One possible explanation for
this phenomenon is that accumulated bioactive CS in d61-3
sensed by OsBRI1 paralogs (OsBRL1 and OsBRL3) stimulates up-regulation of these CYP734As, because several lines
of evidence indicate that OsBRL1 and OsBRL3 are partly
involved in the detection of brassinosteroids in rice
(Nakamura et al., 2006).
Seedlings grown in complete darkness showed the etiolation and unusual internode elongation known as skotomorphogenesis. In contrast, rice mutants with deficiencies
in brassinosteroid biosynthesis or detection had a
de-etiolated phenotype without internode elongation
(Yamamuro et al., 2000; Hong et al., 2002, 2003). These
findings indicate that the brassinosteroid-related rice mutants have a deficiency in skotomorphogenesis similar to
that reported in A. thaliana. In the dark-grown seedlings,
expression of OsDWARF and OsBRI1 was up-regulated
(Figure 5b), suggesting that biosynthesis and sensitivity to
brassinosteroids both increase in rice plants under complete
darkness. This is a likely explanation for the up-regulation of
CYP734A2, CYP734A4, and CYP734A5 expression. However,
the level of CYP734A6 transcripts decreased in dark-grown
seedlings even when they were treated with exogenous BL.
Interestingly, the expression level of CYP734A1/BAS1 was
slightly reduced in dark-grown A. thaliana seedlings (Turk
et al., 2005). These results suggest that expression of
CYP734A1/BAS1 and CYP734A6 is regulated in a lightdependent manner. Because phytochrome B acts as a
negative regulator of brassinosteroid-regulated growth and
development processes in rice (Jeong et al., 2007), regulation of CYP734A6 transcription may occur downstream of
phytochrome B signal transduction.
EXPERIMENTAL PROCEDURES
Isolation of rice CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/
SHK1 orthologs
We performed a BLAST search using the predicted amino acid
sequences encoded by CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/
SHK1 as probes against the rice DNA databases, using the methods
of Sakamoto et al. (2004). The entire coding region for the CYP734A
genes was amplified by means of RT-PCR using total RNA extracted
from whole seedlings (O. sativa L. ‘Nipponbare’). Reverse transcriptase PCR was performed with the oligonucleotide primers
shown in Table S2. Amplified fragments were cloned into pBluescript II SK (Stratagene, http://www stratagene.com/), and their
sequences were determined.
Phylogenetic analyses
A phylogenetic tree of CYP734As was reconstructed by the neighbor-joining (NJ) method (Saitou and Nei, 1987) on the basis of
Kimura’s two-parameter distances (Kimura, 1980). PHYLIP (Felsenstein, 1989) was used to perform the phylogenetic reconstruction.
Bootstrap values were estimated (with 1000 replicates) to assess the
relative support for each branch (Felsenstein, 1985). All positions
containing alignment gaps were eliminated in pairwise sequence
comparisons in the NJ analyses. The alignment used to produce the
phylogeny is shown in Figure S4.
Plasmid constructs and plant transformation
The whole coding regions for CYP734A2, CYP734A4, and CYP734A6
were inserted between the rice actin1 promoter and the nopaline
synthase polyadenylation signal of the hygromycin-resistant binary
vector pAct-Hm2. This vector is modified from pBI-H1 (Ohta et al.,
1990) so that it contains a rice actin promoter. The resulting
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
10 Tomoaki Sakamoto et al.
Campesterol was purchased from Tama Biochemical Co. (http://
www.tama-bc.co.jp/), and brassinolide and castasterone were purchased from Fuji Chemical Industries, Ltd (http://www.fujichemical.co.jp/english/index.html). Other brassinosteroid compounds
were chemically synthesized in our laboratory by the methods of
Ohnishi et al. (2006a).
et al., 1989), and 20 lL of the sample was analyzed by using
HPLC apparatus equipped with a SunFire C18 column (20 mm
length · 4.6 mm internal diameter; Waters, http://www.waters.
com/). Fluorescence detection was performed with excitation at a
wavelength of 305 nm and emission at 350 nm. The column
temperature was kept constant at 40C, and the flow rate of the
mobile phase was 1.0 ml per min. The binary gradient elution
system consisted of distilled water (A) and acetonitrile (B) using the
following gradient: 70% (B) from 0 to 2 min, 70 to 95% (B) from 2 to
5 min, 95% (B) from 5 to 15 min, and 70% (B) from 15 to 18 min. To
determine the substrate specificities, we used the sum of the peak
areas of the three catabolites to estimate the total activities of the
three products. A JEOL JMS-700 was used to perform FAB-MS
analysis of the reaction products.
For brassinosteroids without a diol side chain, the reaction
products were converted into trimethylsilyl derivatives and analyzed by means of GC-MS, as follows. The residue left behind after
evaporation of the organic phase was treated with 10 ll of
N-methyl-N-trimethylsilyltrifluoroacetamide at 80C for 30 min.
The derivatized products were analyzed by means of GC-MS, as
described by Fujita et al. (2006).
Heterologous expression in a baculovirus/insect cell system
Substrate specificity of CYP734A2
Rice CYP734A cDNAs were cloned as BamHI–XhoI fragments in
the pFastBac1 vector (Invitrogen, http://www.invitrogen.com/), and
were then used to generate the corresponding recombinant Bacmid
DNAs by transformation of Escherichia coli strain DH10Bac (Invitrogen). Preparation of the recombinant baculovirus DNAs that
contained CYP734A cDNA and transfection of Sf9 (Spodoptera
frugiperda 9) cells were carried out according to the instructions
of the manufacturer (Invitrogen). Heterologous production of the
CYP734A proteins in Sf9 cells and spectrophotometric analysis were
carried out as described by Saito et al. (2004).
Microsomal fractions of the insect cells that expressed the
CYP734As were obtained from the infected cells (300 ml of
suspension-cultured cells). Infected cells were washed with phosphate-buffered saline buffer and suspended in buffer A, which
consisted of 20 mM potassium phosphate (pH 7.25), 20% (v/v)
glycerol, 1 mM EDTA, and 1 mM DTT. The cells were sonicated,
and cell debris was removed by centrifugation at 10 000 g for
15 min. The supernatant was further centrifuged at 100 000 g
for 1 h, and the pellet was homogenized with buffer A to provide
the microsomal fractions. The microsomal fractions were stored at
)80C before the enzyme assay described in the next section.
The CYP734A2 assays (250 ll) were performed with various 22,23hydroxylated brassinosteroids at a concentration of 20 lM. Reactions were initiated by the addition of NADPH and incubation at
30C for 30 min. This incubation period afforded <20% consumption of each substrate. Reactions were terminated by the addition
of 250 ll of ethyl acetate. The reaction products were extracted
three times with 250 ll of ethyl acetate and converted to 9-phenanthreneboronate derivatives. The amounts of the reaction
products were estimated by HPLC on the basis of the fluorescence
of phenanthrene. Because CYP734A2 metabolized each of the
22,23-hydroxylated brassinosteroids into three oxidized products
by successive oxidation, the value obtained from the sum of the
peak areas of the three products was used to estimate the overall
conversion rate of each substrate. Relative activity was calculated
by comparing the value from each substrate with that from
6-deoxo-3-dehydroteasterone.
construct was introduced into A. tumefaciens strain EHA105, and
Agrobacterium-mediated transformation of rice (O. sativa L.
‘Nipponbare’) was performed as described by Hiei et al. (1994).
Transgenic plants were selected on media containing 50 mg L)1
hygromycin.
Analysis of endogenous brassinosteroid levels
Shoots from control plants (created by means of transformation
with an empty vector) and from transgenic rice that overexpressed
CYP734A were harvested 4 weeks after regeneration. Brassinosteroids were then extracted, purified and quantified by the methods
of Hong et al. (2003).
Chemicals
Analysis of CYP734A activities
The activities of CYP734A were reconstituted by mixing each of the
CYP734A-containing microsomes with purified A. thaliana NADPHP450 reductase (Mizutani and Ohta, 1998). The reaction mixture
consisted of 100 mM potassium phosphate (pH 7.25), 50 pmol ml)1
recombinant P450 protein, 0.1 unit per ml NADPH-P450 reductase,
and 20 lM of brassinosteroids. Reactions were initiated by the addition of 1 mM NADPH, and were carried out at 30C for 30 min. We
also produced controls by using the same reaction mixture, but
without NADPH (the ‘)NADPH’ treatment). The reaction products
were extracted three times with half the original solution’s volume
of ethyl acetate. The organic phase was collected and evaporated.
When brassinosteroids with a diol at the C-22 and C-23 positions
of the side chain were applied to the assays, the reaction products
were converted into 9-phenanthreneboronate derivatives and analyzed by means of high-performance liquid chromatography (HPLC)
using the following procedure: The residue left behind after
evaporation of the organic phase was treated with 1 mg ml)1
9-phenanthreneboronic acid in pyridine at 80C for 30 min (Gamoh
Gene expression analysis
To determine the organ specificity of CYP734A gene expression, we
separately prepared total RNA from various organs of wild-type rice
(O. sativa L. ‘Nipponbare’). Seeds of the wild-type, mutants, and
transformants were sterilized in 1% NaClO and sown on MS medium. Seedlings were grown in a growth chamber for 2 weeks under
continuous light. To investigate the effects of the brassinosteroids,
seedlings of wild-type rice (2 weeks old) were treated with 100 nM
BL. Total RNA was extracted from the whole seedlings. Singlestrand cDNAs were synthesized by using an Advantage RT-for-PCR
kit (Clontech, http://www.clontech.com/). Quantitative RT-PCR was
performed with an iCycler iQ real-time PCR system (Bio-Rad Laboratories, http://www.bio-rad.com/). Expression levels were normalized against the values obtained for the rice ubiquitin gene, which
was used as an internal reference. The primer sequences are listed
in Table S3.
ACKNOWLEDGEMENTS
TS was supported by the Program for Improvement of the Research
Environment for Young Researchers, from the Special Coordination
Funds for Promoting Science and Technology, commissioned by
the Ministry of Education, Culture, Sports, Science and Technology
(MEXT) of Japan; and by a Grant-in-Aid for Young Scientists (A)
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12
Rice brassinosteroid catabolic genes 11
(no. 19688001) from MEXT. SF was supported by a Grant-in-Aid for
Scientific Research (B) (no. 19380069) from MEXT. MM was supported by a Grant-in-Aid for Scientific Research (C) (no. 18580091)
from MEXT; and by the Program for Promotion of Basic and Applied
Researches for Innovations in Bio-oriented Industry (BRAIN).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. High-performance LC analysis of the products from the
rice CYP734A2 assay with 22,23-hydroxylated brassinosteroid s as
substrates.
Figure S2. Total ion chromatogram of products from the CYP734A2
assay with 22-hydroxylated brassinosteroid s as substrates.
Figure S3. Gas chromatography retention time and mass spectrum
of products from the CYP734A2 assay with castasterone as
substrate.
Figure S4. Multiple alignment of amino acid sequences of the
CYP734A subfamily from various plants.
Table S1. Amino acid sequence identities among CYP734A
enzymes.
Table S2. Primer sequences used in CYP734A cDNA cloning.
Table S3. Primer sequences used in expression analysis.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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Accession numbers: CYP734A2 (AB488666), CYP734A4 (AB488667), CYP734A5 (AB488668), CYP734A6 (AB488669).
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1–12