Plant Cell Physiol. 38(11): 1291-1294 (1997)
JSPP © 1997
Short Communication
Identification of Brassinosteroids That Appear to Be Derived from
Campesterol and Cholesterol in Tomato Shoots
Takao Yokota!>3, Takahito Nomura2 and Masayoshi Nakayama1'4
1
2
Department ofBiosciences, School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320 Japan
Department of the Science of Plant and Animal Production, Tokyo University of Agriculture and Technology, 3-8-1 Harumi, Fuchu,
Tokyo, 183 Japan
To obtain information about the biosynthesis of brassinosteroids (BRs) in tomato shoots, we examined endogenous BRs by gas chromatography-mass spectrometry. We
identified two C M BRs, namely, castasterone and 6-deoxocastasterone, and a C27 BR, 28-norcastasterone. Our findings suggest that the major BRs in tomato are derived from
campesterol and cholesterol.
Key words: Brassinosteroids — Castasterone — 6-Deoxocastasterone — Lycopersicon esculentum — 28-Norcastasterone — Tomato.
Brassinosteroids (BRs) are steroidal plant hormones
that are widely distributed in higher plants. When applied
exogenously, BRs have various physiological effects on the
growth and development of plants (Sakurai and Fujioka
1993). The activities of BRs can be distinguished from
those of other plant hormones, such as auxins and gibberellins (Sasse 1990, Yokota 1997). The essential roles of
BRs in many aspects of plant growth, in particular cell elongation, have been demonstrated by the discovery of BRdeficient dwarf mutants of Arabidopsis thaliana, namely,
det2 (Li et al. 1996), cpd/cbb3 (Szekeres et al. 1996,
Kauschmann et al. 1996) and dim/cbbl (Takahashi et al.
1995, Kauschmann et al. 1996), and of Pisum sativum,
namely, Ikb (Nomura et al. 1997) and Ik (Yokota et al. unpublished). These findings were supported by the identification of BR-insensitive mutants of A. thaliana, namely,
bril/cbb2 (Clouse et al. 1996, Kauschmann et al. 1996) and
of P. sativum, namely, Ika (Nomura et al. 1997). Tomato
(Lycopersicon esculentum) is an important crop plant with
well-studied genetic background and a number of tomato
mutants with various growth characteristics that include
dwarfism have been isolated (Karssen et al. 1987, Reid
Abbreviations: BMB, bismethaneboronate; BR, brassinosteroid; GC-MS, gas chromatography-mass spectrometry; ODS, octadecyl silica; SIM, selected ion monitoring.
3
To whom correspondence should be addressed.
4
Present address: National Research Institute of Vegetables, Ornamental Plants and Tea, 360 Kusawa, Ano, Mie, 514-23 Japan.
1986). Thus, tomato seems to be a promising plant material
to investigate the biosynthesis and physiological roles of
BRs and, indeed, the dwarf (d) gene of tomato was suggested recently to have a putative defect in the biosynthesis
of BRs (Bishop et al. 1996). To obtain basic information
about the biosynthesis of the endogenous BRs in tomato,
we analyzed the shoots of normal tomato plants by GCMS/SIM.
Seedlings of tomato (Lycopersicon esculentum cv.
Eifuku) were purchased locally and grown in a greenhouse
under natural light conditions. Ninety-nine seedlings were
harvested at soil level when the seventh leaf had expanded
(2.6 kg). The average height of the seedlings was 33 cm.
The harvested materials were extracted with methanol and
the extract was reduced to an aqueous residue in vacuo
prior to partitioning between chloroform and water. The
chloroform phase was evaporated to dryness and partitioned between 80% methanol and hexane. The 80% methanol
phase was evaporated to dryness and partitioned between
ethyl acetate and 0.5 M K2HPO4 buffer. The ethyl acetate
phase was then analyzed. Four-fifth portion of the ethyl
acetate phase was fractionated on a column of silica gel
(Wako gel C-300; Wako Pure Chemical, Osaka) that was
eluted with chloroform, with chloroform that contained
1.5%, 3%, 5%, 7%, 10%, 20%, 40% and 60% methanol
and finally with pure methanol. Using the rice lamina inclination bioassay (Yokota et al. 1996), we detected biological activity in the fractions that had been eluted with 3%
and 5% methanol in chloroform. These fractions were combined and purified on a column of Sephadex LH-20 (bed
volume, 500 ml; Pharmacia Fine Chemicals, Uppsala) that
was eluted with a mixture of methanol and chloroform (4 :
1, v/v), with collection of 10-ml fractions. Biologically active fractions (nos. 34-36) were combined, dissolved in
methanol and percolated through a short column of diethylamino silica. The eluate was passed through a short column
of octadecyl silica (ODS), which was then washed with
methanol. The eluate was purified on a column of Senshu
Pak ODS-3251-D (8 mm i.d. x 250 mm; Senshu Kagaku,
Tokyo). The column was eluted at a flow rate of 2.5 ml
min" 1 at 40° C with the solvent system programmed as
follows: 0 to 20 min, 45% acetonitrile; 20 to 40 min, 45%
1291
1292
Brassinosteroids in tomato shoots
140
120
Brassinolide (0.001 ppm)
«
E
8
§ 60-1
40-
Control
20-
10
15
20
25
30
35
40
45
Retention time (min)
Fig. 1 Separation of endogenous brassinosteroids in tomato
shoots by reversed-phase HPLC. Biological activities (rice lamina
bending angles) were determined by the rice lamina inclination
assay. Fractions collected for analysis by GC-MS are indicated by
bars labeled A through E. The zones indicated correspond to the
elution zones of authentic brassinolide and 28-norcastasterone
(A), castasterone (B), teasterone (C), typhasterol (D), and 6-deoxocastasterone (E).
to 100% acetonitrile; 40 to 45 min, 100% acetonitrile. Fractions were collected over 1-min intervals. Appropriately
combined fractions were analyzed by GC-MS after derivatization to BMBs or methaneboronate-trimethylsilyl ethers.
Identification of BRs was based on full-scan mass spectra
and Kovats retention indices (Yokota et al. 1996). For quantitation, one-fifth of the ethyl acetate phase, mentioned
above, was combined with [2H6]-brassinolide (200 ng),
[2H6]-castasterone (500 ng) and [2H6]-6-deoxocastasterone
(1,000 ng) as internal standards (for synthesis, see Mori et
al. 1984, Takatsuto and Ikekawa 1986, Nomura et al. 1997)
and purified as described above. After ODS-HPLC, the
fractions associated with BRs were analyzed by GC-SIM
under the same conditions as for GC-MS. The [2H0] and
[2H6] ions that we monitored to quantify individual BRs
were m/z 512 and m/z 518 (M + ions of castasterone BMB)
and m/z 498 and m/z 504 (M + ions of 6-deoxocastasterone
BMB). No internal standard was available for 28-norcasta-
sterone, so that its level was determined from a calibration
curve constructed from the ratio of the peak areas of the
molecular ion (m/z 498) of 28-norcastasterone BMB and
molecular ion (m/z 534) of [2Hd-brassinolide BMB.
Reversed-phase HPLC of the partially purified extract
of tomato shoots gave rise to two major biologically active
fractions, numbers 14 and 21/22 (Fig. 1). We expected that
fraction 14 would contain brassinolide in view of the retention time, but no trace of brassinolide was detected. Instead, 28-norcastasterone was identified in this fraction
(Table 1). Fraction 21/22, in which castasterone, a direct
precursor of brassinolide was expected to be present, contained castasterone exclusively (Table 1). Thus, the major
biologically active BRs in tomato shoots were shown to be
castasterone and 28-norcastasterone. No or very weak biological activity was found in fractions 30-32, 35-37 and 3840, with retention times that corresponded to teasterone,
typhasterol and 6-deoxocastasterone, respectively. 6Deoxocastasterone was identified in fractions 38-40 (Table
1), but neither teasterone nor typhasterol was detected in
fractions with appropriate retention times. Quantitative
analysis by GC-SIM with deuterated internal standards indicated that castasterone and 6-deoxocastasterone, were
quantitatively the major BRs in tomato shoots (Table 1).
The level of 28-norcastasterone was one order of magnitude lower than that of castasterone.
Castasterone and 6-deoxocastasterone, which we identified in extracts of tomato, are CM BRs with a methyl
group at 24S. A group of such BRs is distributed in a wide
range of plants and is known to be synthesized from
campesterol (Fujioka and Sakurai 1997, Sakurai and Fujioka 1997, Yokota 1997). Castasterone is synthesized via
two biosynthetic pathways (Fig. 2): the early C6-oxidation pathway, in which castasterone is produced from
campesterol via teasterone and typhasterol (Suzuki et al.
1994, 1995); and the late C6-oxidation pathway, in which
castasterone is synthesized from campesterol via 6-deoxoteasterone, 6-deoxotyphasterol and 6-deoxocastasterone
(Choi et al. 1996, 1997). The occurrence of 6-deoxocastasterone in tomato tissues suggests that the late C6-oxidation
Table 1 Identification and quantification of brassinosteroids in tomato shoots by GC-MS/SIM
Fraction
no. after
HPLC
Brassinosteroid
identified
Derivative
Kovats
retention
index"
Characteristic ion m/z
(relative intensity, %)
14
28-Norcastasterone
BMB
3,533 (3,533)
498 (M + , 100), 483 (9),
358 (19), 287 (38), 141 (45)
21/22
Castasterone
BMB
3,617(3,618)
38-40
6-Deoxocastasterone
BMB
3,428 (3,429)
512
358
498
288
•
* Kovats retention indices of authentic samples are shown in parentheses.
(M + , 100), 503 (15),
(41), 287 (33), 155 (88)
(M + , 84), 483 (23),
(19), 273 (100), 155 (23)
Endogenous
level
[ng(kgFW)-']
29
210
1,690
Brassinosteroids in tomato shoots
1293
shoots. Although lactonization does enhance the biological
activities of BRs, it remains unclear whether lactonization
of BRs is necessary for their biological activities in plants
(Yokota 1997).
Brassinosteroids stimulate the elongation of tomato
seedlings, and brassinolide is ten-fold more potent in this respect than 28-norbrassinolide (Takatsuto et al. 1983b). In
the rice lamina inclination bioassay, the biological activities of castasterone and brassinolide are approximately tenfold higher than those of 28-norcastasterone and 28-norbrassinolide, respectively (Takatsuto et al. 1983a, Wada et
al. 1983). Thus, castasterone seems to be more important
than 28-norcastasterone in the growth of tomato. The
tomato D gene, the wild type allele of d (dwarf) encodes a
cytochrome P450 enzyme (Bishop et al. 1996). This enzyme
is the first member of CYP85, a new family of P450 enzymes and exhibits considerable similarity to CYP90 of
A. thaliana, which might be involved in hydroxylation
at the C23 position in the biosynthesis of brassinolide
(Szekeres et al. 1996). It remains to be determined whether
the D gene is involved in the synthesis of castasterone and/
or 28-norcastasterone.
The authors thank Ms. M. Mashima for technical assistance.
6-Deoxocastastefone*
Castasterone*
28-Nor castasterone*
Fig. 2 Putative biosynthetic pathways of brassinosteroids in
tomato shoots. Compounds with asterisks were identified in the
present analysis of tomato shoots.
pathway is operative in this plant (Fig. 2). Although we
did not find teasterone and typhasterol in our analysis of
tomato, we cannot exclude the possibility of the presence
of the early C6-oxidation pathway because endogenous
levels of typhasterol and teasterone are generally low.
It is very likely that 28-norcastasterone is synthesized
from cholesterol because 28-norcastasterone and cholesterol have the same structural skeleton. However, no experimental evidence has been obtained relating to its biosynthesis. To date, 28-norcastasterone has been found in as many
as eleven plant species, including tomato (Fujioka and
Sakurai 1997). Thus, it may be important to clarify the biosynthesis of 28-norcastasterone for a full understanding of
controls of plant growth.
In studies of cells of Catharanthus roseus in culture, castasterone was shown to be a direct precursor of
brassinolide, the most potent BR (Suzuki et al. 1994).
Likewise, 28-norcastasterone is very likely to be the precursor of 28-norbrassinolide. However, such a conversion
(lactonization) has rarely been observed in feeding experiments. In the present study, we failed to demonstrate the
presence of brassinolide and 28-norbrassinolide in tomato
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(Received June 3, 1997; Accepted September 4, 1997)