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Plant Physiol. (1999) 119: 1517-1526 Brassinosteroid/Sterol Synthesis and Plant Growth as Affected by lka and lkb Mutations of Pea1
Department of the Science of Plant and Animal Production, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan (T.N.); Department of Bioproductive Science, Utsunomiya University, Utsunomiya 320-8505, Japan (Y.K., M.F.); Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943-8512, Japan (S.T.); Department of Plant Science, University of Tasmania, G.P.O. Box 252C, Hobart, Tasmania 7001, Australia (J.B.R.); and Department of Biosciences, Teikyo University, Utsunomiya 320-8551, Japan (Y.T.)
The dwarf pea (Pisum
sativum) mutants lka and lkb are
brassinosteroid (BR) insensitive and deficient, respectively. The dwarf phenotype of the lkb mutant was rescued to wild type by
exogenous application of brassinolide and its biosynthetic precursors.
Gas chromatography-mass spectrometry analysis of the endogenous
sterols in this mutant revealed that it accumulates
24-methylenecholesterol and isofucosterol but is deficient in their
hydrogenated products, campesterol and sitosterol. Feeding
experiments using 2H-labeled 24-methylenecholesterol
indicated that the lkb mutant is unable to isomerize
and/or reduce the
Steroid hormones play important roles in growth and development of
various organisms. These include the sex hormones glucocorticoids and
mineral corticoids in animals, the molting hormones ecdysteroids in
insects and crustaceans, and an antheridiogen, antheridiol, in the
microorganism Achlya bisexualis. Grove et al. (1979)
The roles of three genes in BR biosynthesis have been determined using
dwarf mutants of Arabidopsis, det2
(d-etiolated 2) (Li et al., 1996 In the case of pea, the stunted phenotypes of the lka,
lkb, lkc, and lk mutants are not fully
reversed by treatment with traditional plant hormones such as GA and
auxin (Reid and Ross, 1989 Plant Materials
Authentic BRs and Sterols
2H Standards 26,27-2H6 labeling of brassinolide, castasterone, typhasterol, and teasterone were reported by Takatsuto and Ikekawa (1986) . [2H6]3-Dehydroteasterone was synthesized as
reported by Yokota et al. (1994),
[2H6]6-deoxocastasterone (melting point,
238°C), [2H6]6-deoxotyphasterol (melting
point, 224°C), and [2H6]6-deoxoteasterone
(melting point, 227°C) were synthesized from [2H6]castasterone,
[2H6]typhasterol, and
[2H6]teasterone, respectively, according to
the method of Mori et al. (1984).
[2H6]3-Dehydro-6-deoxoteasterone (melting
point, 167°C) was synthesized from
[2H6]6-deoxoteasterone as described by Yokota
et al. (1994). [26,27-2H6]Campestanol was
supplied by Dr. S. Fujioka (RIKEN, Saitama, Japan).
[25,26,27-2H7]-24-Methylenecholesterol,
[26,27-2H6]24-methyldesmosterol, and
[26,27-2H6]campesterol
were synthesized by the procedures of Takatsuto et al. (1998).
GC-SIM GC-SIM, in the electron impact mode (70 eV), was carried out on a JMS AX 505 instrument (JEOL) fitted with either a DB-5 or DB-1 column (0.25 mm × 15 m; 0.25-mm film thickness; J & W Scientific, Folsom, CA). The carrier gas was He at a flow rate of 1 mL min 1, the injection port temperature was
260°C, and the samples were introduced by splitless injection. The
column oven temperature was programmed to 170°C for 1.5 min,
increased to 280°C at 37°C min1, and then
increased to 300°C at 1.5°C min1.
Effects of Brassinolide, Its Precursors, and Sterols on Growth Brassinolide precursors in 5 µL of ethanol containing 0.15% Tween 20 were applied to the fourth internode when the third leaf was almost fully expanded about 8 d after planting in a growth cabinet. Sterols were mixed with a fractionate lanolin (Mitchell and Livingston, 1968), and the lanolin paste was applied to the fourth
internode. Control seedlings were treated with the solvent only. After
3 d the lengths of the fourth and fifth internodes were measured.
Effects of Brassinolide on Sterol Content The procedure of brassinolide application was the same as described above, except that the surface of the expanding third leaf was treated with 100 ng of brassinolide dissolved in 10 µL of the solvent. After 2 d the third leaves were removed, and the apical portions (each with 20 shoots) were harvested by cutting at the third node and used for sterol analysis.Metabolism of 2H-Sterols Seeds of the lkb mutant were surface-sterilized with sodium hypochlorite solution (0.5% active chlorine) and were placed in 100-mL conical flasks (one seed per flask) containing 15 mL of Murashige and Skoog medium (JRH Biosciences, Lenexa, KS). The seeds were grown for 6 d at 25°C under a 16-h light and 8-h dark regime on a reciprocal shaker (60 rpm); then ethanol solutions (40 µL) of [2H7]24-methylenecholesterol (40 µg) or [2H6]24-methyldesmosterol (40 µg) were added to the conical flasks and incubated for an additional 3 d under the same conditions. Roots and shoots were separated and subjected to sterol analysis.Preparation of Microsomal and Soluble Fractions All operations were done at 4°C. Ten fresh segments (approximately 5 g) excised at the sixth node from 23-d-old seedlings were homogenized by a Polytron (Kinematica, Littau/Luzern) with 20 mL of 0.1 M Tris-HCl buffer, pH 7.8, containing 0.5 M Suc and 1 mM EDTA, and the homogenate was filtered through four layers of gauze. The filtrate was centrifuged at 6,000g for 15 min, and the supernatant was then centrifuged at 100,000g for 90 min according to the methods described by Gachotte et al. (1995). The pellet and supernatant obtained by
100,000g centrifugation, which represent microsomal membrane
and soluble cellular fractions, respectively, were subjected to sterol
analysis.
Extraction and Purification of BRs from 49-d-Old Seedlings The methanol extracts of 49-d-old shoots excised at soil levels (Table I) were spiked with 2H6-labeled internal standards, 0.3 µg of [2H6]brassinolide, 0.5 µg each of [2H6]castasterone and [2H6]typhasterol, and 1 µg each of [2H6]3-dehydroteasterone, [2H6]6-deoxocastasterone, [2H6]6deoxotyphasterol, [2H6]3-dehydro-6-deoxoteasterone, and [2H6]6-deoxoteasterone before reduction to an aqueous residue. The aqueous residue was partitioned against chloroform. The chloroform phase was washed with 0.5 M K2HPO4 buffer, pH 9.0, evaporated to dryness, and partitioned between hexane and 80% methanol. The hexane and 80% methanol phases were used for analysis of sterols and BRs, respectively.
Quantitative Analysis of BRs Random aliquots of extracts derived from pooled plant materials were analyzed by GC-SIM in duplicates. BRs were converted to either MBs or BMBs with pyridine that contains methaneboronic acid (2 mg mL1) at 70°C for 30 min. Typhasterol,
teasterone, 6-deoxotyphasterol, and 6-deoxoteasterone were
further trimethylsilylated to yield MB-TMSi derivatives. The
2H0/2H6
ions monitored were m/z 528/534 (M+), 374/374 and
155/161 for brassinolide BMB, m/z 512/518 (M+),
358/358 and 155/161 for castasterone BMB, m/z 544/550
(M+), 529/535 and 515/521 for typhasterol MB-TMSi
and teasterone MB-TMSi, m/z 470/476
(M+), 316/316 and 155/161 for 3-dehydroteasterone
MB, m/z 498/504 (M+), 273/273 and 155/161 for
6-deoxocastasterone BMB, m/z 530/536 (M+),
440/446 and 215/215 for 6-deoxotyphasterol MB-TMSi and
6-deoxoteasterone MB-TMSi, and m/z 456/462 (M+),
231/231, and 155/161 for 3-dehydro-6-deoxoteasterone MB. The contents
of BRs were calculated from the peak area ratios of 2H0
and 2H6 M+ ions.
Analysis of Sterols Random aliquots of extracts derived from pooled plant materials were analyzed by GC-SIM in duplicates. Plant tissues were extracted with methanol:chloroform (4:1, v/v), whereas the microsomal membrane pellet was extracted with methanol:dichloromethane (1:2, v/v). In the case of 49-d-old seedlings, the hexane-soluble phase was obtained as described above for sterol analysis. The extracts were partitioned between ethyl acetate and 0.5 M K2HPO4 buffer, pH 9.0, and the organic phases were used for sterol analysis. Sterol extracts equivalent to 100 mg fresh weight of tissue were spiked with 1 µg of [2H6]campestanol as an internal standard and saponified with 1 N sodium hydroxide in methanol at 80°C for 1.5 h. The hydrolysate was partitioned between chloroform and water. The chloroform phase was evaporated to dryness, redissolved in chloroform, and then passed through a short silica gel (Wakogel C-300) column. The eluate was trimethylsilylated at room temperature and subjected to GC-SIM. The levels of sterols were determined using calibration curves constructed from the ratios of the M+ peak area of [2H6]campestanol TMSi (m/z 480) to those of cholesterol TMSi (m/z 458), 24-methylenecholesterol TMSi (m/z 470), campesterol/24-epicampesterol TMSi (m/z 472), campestanol/24-epicampestanol TMSi (m/z 474), stigmasterol TMSi (m/z 484), sitosterol TMSi (m/z 486), sitostanol TMSi (m/z 488), and isofucosterol TMSi (m/z 484). Campesterol and 24-epicampesterol (22-dihydrobrassicasterol), as well as campestanol/24-epicampestanol, were analyzed as a mixture because they were not resolved by GC.
Levels of BRs in lkb Plants Are Reduced, Whereas Those in lka Plants Are Not Nomura et al. (1997) demonstrated that 36-d-old lkb
plants contain significantly lower levels of brassinolide,
castasterone, and 6-deoxocastasterone than WT plants. To locate the
defective biosynthetic step, the levels of the endogenous BRs situated
in the early sections of the biosynthetic pathway were analyzed in extracts from shoots of 49-d-old lkb plants by GC-SIM. BRs
(Table II) were rigorously identified
because the relative intensities of the M+ ion
and two daughter ions for each molecule (see ``Materials and Methods'') were consistent with those of the corresponding
2H internal standard (data not shown). The
contents of BRs, which were calculated from the peak area ratios of
2H0 and
2H6
M+ ions, are shown in Table II. In the case of
6-oxo-BRs, the levels of castasterone and typhasterol were 23% and
15%, respectively, of the WT levels. The castasterone level was
significantly greater than that for the 36-d-old plants, according to
previously published results (Nomura et al., 1997). In keeping with
this, the size of 49-d-old plants relative to the WT (Table I) was
greater than that previously described for 36-d-old plants. It seems
that environmental and/or age differences modified the levels of BRs,
thereby affecting plant growth. There was no substantial difference in
the levels of the earlier precursors, 3-dehydroteasterone
and teasterone. Brassinolide was not present at the detectable
levels in any genotype. As for 6-deoxo-BRs, the levels of
6-deoxocastasterone and 6-deoxotyphasterol were 7% and 29%,
respectively, of the WT levels, whereas no large reduction was
observed in the levels of the earlier precursors, 3-dehydro-6-deoxoteasterone and 6-deoxoteasterone (Table II).
Brassinolide and Its Biosynthetic Precursor BRs Rescue the Dwarf Phenotype of lkb Plants Previously, Nomura et al. (1997) applied 6-oxo-BRs, including
brassinolide, castasterone, typhasterol, 3dehydroteasterone, and
teasterone to the third leaf of lkb plants, and this
resulted in increased elongation of the fourth and fifth internodes.
6-Deoxocastasterone, however, was inactive. In the current study, the
fourth internodes of 8-d-old seedlings were treated with BRs to
minimize transportation effects, and under these conditions all of the
6-oxo-BRs and 6-deoxo-BRs exhibited activity when applied in doses of 1 µg and less (Fig. 2). Overall, it is
apparent that the biological activities of the BRs tend to increase
with their proximity to brassinolide in the biosynthetic pathway, as
reported by Yokota and Mori (1992) and Fujioka et al. (1995).
Campesterol and campestanol failed to promote the internode elongation
when applied at high doses in a lanolin paste (Fig. 2). This may
reflect low-efficiency conversion to brassinolide and/or poor uptake by
the plant tissues.
Sterol Content of the Shoots and Seeds Is Drastically Altered in lkb, but Not in lka The levels of sterols in the shoots of 49-d-old plants used for BR analysis and mature seeds of WT, lka, and lkb were analyzed quantitatively by GC-MS, and the data obtained are shown in Table III. All of the sterols listed were rigorously identified by full-scan MS.
Conversion of [25,26,27-2H7]24-Methylenecholesterol to [26,27-2H6]Campesterol Is Blocked in the lkb Mutant Since campesterol has been reported to be synthesized from 24-methylenecholesterol via 24-methyldesmosterol (Fig. 3; Goodwin, 1985; Benveniste, 1986),
[25,26,27-2H7]24-methylenecholesterol
was added to the media in which 6-d-old WT and lkb seedlings
had been aseptically grown. After 3 d data were obtained by
analyzing roots but not by analyzing shoots: It appears that sterols
absorbed by the roots that were submerged in the medium did not move to
the shoots. The roots of the WT plants were found to contain similar
levels (0.5 µg g1 fresh weight) of
[25,26,27-2H7]24-methylenecholesterol
and
[26,27-2H6]campesterol,
but no
[26,27-2H6]24-methyldesmosterol
was present (Fig. 4). In contrast, the roots of the lkb plants contained
[25,26,27-2H7]24-methylenecholesterol
(0.5 µg g1 fresh weight), but
[26,27-2H6]campesterol
and
[26,27-2H6]24-methyldesmosterol
were not detected. Unexpectedly, after feeding
[26,27-2H6]24-methyldesmosterol to the WT and
lkb tissues, neither this compound nor its expected
metabolite,
[26,27-2H6]campesterol,
was present in root extracts. One of the possible reasons for this
result is that
[26,27--2H6]24-methyldesmosterol
might be rapidly metabolized to other products before reaching the
proper reaction site.
Brassinolide-Treated lkb Plants Had Lower Levels of Endogenous Sterols The effect of exogenous brassinolide on the levels of endogenous sterols was investigated because it was suspected that the abnormal sterol content of the lkb plants might be normalized by exogenous brassinolide. WT seedlings (8 d old) were treated with 100 ng of brassinolide. After 2 d elongation was enhanced, and this was accompanied by an increase in the fresh weight of the shoot. The brassinolide treatment also increased the total sterol content per shoot in proportion to the growth increase, although when expressed on a fresh-weight basis this effect was not evident (Table IV). A similar result was obtained in lka plants (Table IV).
The lkb Mutant Has an Altered Sterol Composition in the Membrane A microsomal membrane fraction and a soluble fraction were obtained from 23-d-old WT, lka, and lkb shoots by centrifugation at 100,000g. The microsomal fractions sedimented at 100,000g are composed of a mixture of vesicles originating from various membrane types (Hartmann and Benveniste, 1987). Sterol compositions in these fractions and whole shoots are
shown in Table V. The microsomal membranes of WT contained sitosterol and stigmasterol as the
major sterols, whereas those of the lkb mutant contained
isofucosterol and sitosterol as the principal components. In the WT
membranes the level of sitosterol was increased 1.4-fold compared with
that of the whole shoots. In the lkb membrane the levels of
sitosterol and campesterol/24-epicampesterol were elevated 25- and
4-fold, respectively, compared with those of the whole shoots. The
levels of cholesterol in the microsomal fraction, soluble fraction, and whole shoots of the lkb mutant were nearly comparable to
those of the WT. Surprisingly, the sterol compositions of the WT and lkb soluble fractions were indistinguishable. Sterol
compositions in all samples obtained from lka seedlings
closely resembled those of the WT.
BR Biosynthesis Is Retarded in the lkb Mutant In the biosynthesis of brassinolide, campesterol is first converted to campestanol, which is metabolized to castasterone via either the early or late C-6 oxidation pathway, after which castasterone is converted to brassinolide (Fig. 1). The analysis of endogenous BRs in the present study indicated the presence of components of both the early and late C-6 oxidation pathways in pea (Choi et al., 1997; Table II). Low levels of castasterone,
typhasterol, 6-deoxocastasterone, and 6-deoxotyphasterol in shoots
of 49-d-old lkb plants indicate that both pathways are
affected. However, the lkb plants had levels of
3-dehydroteasterone and teasterone in the early C-6 oxidation pathway,
as well as of 3-dehydro-6-deoxoteasterone and 6-deoxoteasterone in the
late C-6 oxidation pathway, comparable to those of the WT. It is
possible to speculate that the reduction of the 3-oxo group to
3 -hydroxyl function may be blocked in the lkb mutant.
However, this possibility is unlikely because both 3-dehydroteasterone
and 3-dehydro-6-deoxoteasterone are biologically active (Fig. 2),
probably via the conversion to typhasterol and 6-deoxotyphasterol,
respectively (Fig. 1). All of the BRs examined could counteract the
dwarfism of the lkb mutant (Fig. 2), suggesting a blockage
at a very early step in BR biosynthesis, possibly even as far back as
the sterol biosynthesis pathway.
BR Deficiency in the lkb Mutant Is Due to Impaired Synthesis of Campesterol from 24-Methylenecholesterol Sterol compositions of 10-, 23-, and 49-d-old lkb shoots, which are summarized in Tables IV, V, and III, respectively, as well as those of mature seeds (Table III), were almost comparable. Such sterol profiles indicate lesions in the reductive conversion of 24-methylenecholesterol to campesterol and of isofucosterol to sitosterol in shoots and seeds of the lkb mutant. The inability to metabolize [25,26,27-2H7]24-methylenecholesterol to [26,27-2H6]campesterol was confirmed using 6-d-old lkb seedlings. This conversion occurred in WT tissues with a loss of the 2H at C-25, indicating that the 24(28) double bond in
24-methylenecholesterol is first isomerized to a
24(25) double bond, yielding
24-methyldesmosterol, which, in turn, is hydrogenated to campesterol
(Fig. 4), as was postulated by Goodwin (1985) and Benveniste (1986).
Thus, it is possible that the LKB protein has a dual role in that it
catalyzes both isomerization and reduction of the
24(28) double bond in the hydrogenation of
24-methylenecholesterol and isofucosterol. However, we do not exclude
the possibility that the LKB gene product may act as a
regulator influencing gene expression or enzyme activity of one or more
sterol isomerase(s)/reductase(s) or that the LKB gene may
encode either an isomerase or a reductase that act together in a
tightly coupled fashion. Nonetheless, we conclude that the dwarfism of
the lkb mutant is due to BR deficiency caused by blocked
conversion of 24-methylenecholesterol to campesterol. Recently, Klahre
et al. (1998) found that the dwarf dim mutant of
Arabidopsis has the same defect as the lkb mutant.
Abnormal Sterol Compositions in the lkb Mutant May Damage Functions of Membrane In an attempt to examine the effect of brassinolide on the sterol composition, we found that sterol synthesis was retarded in 10-d-old lkb seedlings treated with brassinolide, despite the dwarf phenotype being counteracted with internode elongation being promoted strongly (Table IV). The suppression of sterol synthesis suggests that membranes of the lkb mutant may have an unusual sterol composition, which may, in turn, affect adversely the activity of membrane-bound enzymes (Nes, 1977; Benveniste, 1986; Hartmann, 1998),
including those involved in sterol biosynthesis. The membranes of
23-d-old lkb shoots were most unusual in that they contain isofucosterol as the major sterol (64% in weight), in contrast to the WT and lka membranes in which sitosterol is the major
component (52% in weight). Surprisingly, the sitosterol content in the
lkb membranes increased to 25%, although sitosterol in the
whole shoot accounts for only 1% of the total sterols (Table V). A
similar increase was also observed in the campesterol/24-epicampesterol content. It is well known that sterols affect membrane fluidity (Hartmann, 1998). Sitosterol and campesterol/24-epicampesterol have
been found to be the most efficient sterols for restricting the
mobility of fatty acyl chains in soybean phosphatidylcholine bilayers
and appear to be very active in reducing the water permeability of the
soybean membranes (Schuler et al., 1991). Thus, the partial recovery in
the sitosterol and campesterol/24-epicampesterol level in the
lkb membranes could be due to homeostatic control restoring sterol composition and hence membrane functions. The finding that the
soluble fraction of the lkb mutant has the same composition as WT also may be due to homeostatic control, although the role of
sterols in the soluble fraction seems to be undefined (Gachotte et al.,
1995).
The Level of Castasterone May Be Controlled by a Feedback Mechanism Although the lka mutant has a phenotype similar to the lkb mutant, it is not BR deficient (Table II) and its dwarfism is not counteracted by exogenous brassinolide (Nomura et al., 1997). Furthermore, the sterol compositions of the lka
mutant are almost identical with those of the WT (Tables III-V). Such
evidence suggests that the lka mutant has a defect in the
perception or signal transduction pathway of BRs.
* Corresponding author; e-mail yokota{at}nasu.bio.teikyo-u.ac.jp; fax 81-28-627-7187. Received September 21, 1998;
accepted December 22, 1998.
Abbreviations: BMB, bismethaneboronate. BR, brassinosteroid. MB, monomethaneboronate. SIM, selected ion monitoring. TMSi, trimethylsilyl ether. WT, wild type.
We thank Akihiko Saito for the bioassay of BRs. We also thank Dr. Alan Crozier (Institute of Biochemical and Life Sciences, University of Glasgow, UK) and Dr. Gerard Bishop (The Institute of Physics and Chemistry, Wako, Japan) for providing helpful comments to improve this manuscript.
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  G. M. Symons, J. J. Ross, C. E. Jager, and J. B. Reid Brassinosteroid transport J. Exp. Bot., January 1, 2008; 59(1): 17 - 24. [Abstract] [Full Text] [PDF]
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 C. E. Jager, G. M. Symons, T. Nomura, Y. Yamada, J. J. Smith, S. Yamaguchi, Y. Kamiya, J. L. Weller, T. Yokota, and J. B. Reid Characterization of Two Brassinosteroid C-6 Oxidase Genes in Pea Plant Physiology, April 1, 2007; 143(4): 1894 - 1904. [Abstract] [Full Text] [PDF]
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T. Nomura, M. Ueno, Y. Yamada, S. Takatsuto, Y. Takeuchi, and T. Yokota Roles of Brassinosteroids and Related mRNAs in Pea Seed Growth and Germination Plant Physiology, April 1, 2007; 143(4): 1680 - 1688. [Abstract] [Full Text] [PDF]
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 N. FUKUTA, K. FUKUZONO, H. KAWAIDE, H. ABE, and M. NAKAYAMA Physical Restriction of Pods Causes Seed Size Reduction of a Brassinosteroid-deficient Faba Bean (Vicia faba) Ann. Bot., January 1, 2006; 97(1): 65 - 69. [Abstract] [Full Text] [PDF]
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R. Radchuk, V. Radchuk, W. Weschke, L. Borisjuk, and H. Weber Repressing the Expression of the SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE Gene in Pea Embryo Causes Pleiotropic Defects of Maturati |
Top
Abstract
Introduction
-reductase that
hydrogenates the (24R)-24-methylcholest-4-en-3-one
intermediate involved in the conversion of campesterol to campestanol
(Fujioka et al., 1997
