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Plant Physiol. (1998) 118: 461-469
Overexpression of an Arabidopsis cDNA Encoding a
Sterol-C241-Methyltransferase in Tobacco Modifies the
Ratio of 24-Methyl Cholesterol to Sitosterol and Is
Associated with
Growth Reduction
Hubert Schaller*,
Pierrette Bouvier-Navé, and
Pierre Benveniste
Institut de Biologie Moléculaire des Plantes,
Département d'Enzymologie Cellulaire et Moléculaire,
Institut de Botanique, 28 rue Goethe, 67083 Strasbourg, France
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ABSTRACT |
Higher plants synthesize 24-methyl
sterols and 24-ethyl sterols in defined proportions. As a
first step in investigating the physiological function of this balance,
an Arabidopsis cDNA encoding an
S-adenosyl-L-methionine 24-methylene
lophenol-C241-methyltransferase, the typical plant enzyme
responsible for the production of 24-ethyl sterols, was
expressed in tobacco (Nicotiana tabacum L.) under the
control of a constitutive promoter. Transgenic plants displayed a novel
24-alkyl- 5-sterol profile: the ratio of 24-methyl
cholesterol to sitosterol, which is close to 1 in the wild type,
decreased dramatically to values ranging from 0.01 to 0.31. In
succeeding generations of transgenic tobacco, a high
S-adenosyl-L-methionine 24-methylene lophenol-C241-methyltransferase enzyme activity and,
consequently, a low ratio of 24-methyl cholesterol to sitosterol, was
associated with reduced growth compared with the wild type. However,
this new morphological phenotype appeared only below the threshold
ratio of 24-methyl cholesterol to sitosterol of approximately 0.1. Because the size of cells was unchanged in small, transgenic plants, we
hypothesize that a radical decrease of 24-methyl cholesterol and/or a
concomitant increase of sitosterol would be responsible for a change in
cell division through as-yet unknown mechanisms.
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INTRODUCTION |
Sterol metabolism in the plant cell has features not seen in other
kingdoms: unique enzymatic steps involved in the transformation of
squalene into pathway end products (for review, see Benveniste, 1986 ;
Ourisson, 1994). The occurrence in photosynthetic organisms of sterols
bearing an additional alkyl group at position C24, 24-methyl sterols
and 24-ethyl sterols, has been widely documented (for review, see Nes
and McKean, 1977). Higher plants contain mixtures of
24-alkyl-5-sterols in which 24-methyl
cholesterol is the major 24-methyl sterol, and sitosterol and
stigmasterol are the predominant 24-ethyl sterols (Fig.
1); the R is almost invariable with
respect to species and is therefore controlled.
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| Figure 1.
Simplified biosynthetic pathway of sterols in
higher plants. 24-Methyl cholesterol is present in higher plants as a
mixture of its (24-R)- and (24-S)-epimers
campesterol and dihydrobrassicasterol (Rubinstein et al., 1976; Rendell
et al., 1986) and is a precursor for the synthesis of brassinosteroids.
The dashed arrows indicate more than one biosynthetic step not shown
here.
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The presence of high proportions of 24-ethyl sterols in plant membranes
correlates with their efficiency in developing specific interactions
with plant phospholipids to reinforce and stabilize the bilayer
architecture (Demel and De Kruyff, 1976; Bloch, 1983; Schuler et al.,
1991). The extra carbon atoms are added on sterol intermediates via
SMTs, enzymes that have always been looked at with considerable
interest in terms of the chemistry and biochemistry of their
catalyzed reactions (Rahier et al., 1984; Bladocha and Benveniste,
1985; Rendell et al., 1986; Janssen and Nes, 1992).
Two distinct SMTs are responsible for the two methyl transfers involved
in the conversion of cycloartenol into sitosterol. As shown in Figure
1, cycloartenol is the natural substrate of the first methylation
reaction (SMT1), which produces 24-methylene cycloartenol (Malhotra and
Nes, 1971; Wojciechowski et al., 1973); 24-methylene lophenol is the
natural substrate of the second methylation reaction (SMT2), which
produces 24-ethylidene lophenol (Fonteneau et al., 1977). The
occurrence of two distinct SMTs in plants was recently reinforced after
cDNAs were cloned from Arabidopsis (Husselstein et al.,
1996), soybean (Shi et al., 1996), maize (Grebenok et al., 1997), and
tobacco (Bouvier-Navé et al., 1997); indeed, alignment of the
deduced amino acid sequences indicated that they are distributed into
two families (Bouvier-Navé et al., 1997). Moreover, expression of
plant SMTs in the yeast null mutant erg6 (Gaber et al.,
1989), in which the gene encoding the
S-adenosyl-L-Met-zymosterol C24-methyltransferase has been knocked out, restored the synthesis of
ergosterol in the case of SMTs from maize (Grebenok et al., 1997) and
tobacco (Bouvier-Navé et al., 1998) and of ergosterol and
24-ethyl sterols in the case of SMT from Arabidopsis (Husselstein et
al., 1996).
Further studies in the latter case showed that in a delipidated
microsomal preparation of the complemented yeast, the catalytic efficiency of the expressed SMT was 17 times higher using 24-methylene lophenol as a substrate than when cycloartenol was used
(Bouvier-Navé et al., 1997). This clearly indicates that in the
yeast membrane environment this Arabidopsis SMT operates as a
24-methylene lophenol-C241-methyltransferase
(Fig. 1, SMT2), catalyzing the second methyl transfer, which is
responsible for the plant cell's capability to produce 24-ethyl
sterols.
As a first step in investigating in more detail the physiological
function of 24-methyl- and 24-ethyl sterols and the regulation of their
respective concentrations in the plant cell, transgenic tobacco
expressing an Arabidopsis SMT2 under control of a strong constitutive promoter was generated. We report that a dramatic decrease
in the amount of 24-methyl cholesterol and a concomitant increase in
sitosterol are associated with reduced growth in transgenic plants.
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MATERIALS AND METHODS |
Plasmid Constructs
A 1.45-kb XhoI-SmaI fragment containing an
Arabidopsis cDNA encoding a
sterol-C241-methyltransferase was excised
from the plasmid pSK411 (Husselstein et al., 1996) and cloned into the
XhoI-SmaI opened binary vector pFB8 (Atanassova
et al., 1995). Correct insertion of the 1.45-kb fragment was determined
by restriction of plasmid DNA extracted from Escherichia
coli DH5 cultures transformed with the ligation mixture.
Recombinant pFB8 derivatives, referred to as p35S-SMT2, and
the corresponding void plasmid, referred to as p35S, were introduced
into Agrobacterium tumefaciens LBA 4404 by triparental mating using pRK2013 in E. coli HB101 as a helper plasmid
(Bevan, 1984).
Plant Transformation and Genetic Analysis
Tobacco (Nicotiana tabacum L. var Xanthi) was
transformed with p35S-SMT2 and p35S via A. tumefaciens according to a modification of the method reported by
Horsch et al. (1985). Leaf pieces were co-cultivated with an A. tumefaciens culture on Murashige and Skoog medium supplemented
with Glc (30 g/L), ANA (0.1 mg/L), and 6-benzylaminopurine (1 mg/L) for 2 d and were then
transferred onto the same medium supplemented with kanamycin (100 mg/L)
as the plant-selective agent and cefotaxime (500 mg/L) to prevent further bacterial growth. Regenerated shoots were rooted on Murashige and Skoog medium with a half-reduced concentration of
NH4NO3 and supplemented
with Suc (30 g/L), kanamycin (200 mg/L), and cefotaxime (200 mg/L). In
vitro cultures were grown under a 16-h light period at 24°C and an
8-h dark period at 20°C. Tobacco plants were transferred to soil and
grown under standard greenhouse conditions.
Succeeding generations of transgenic plants were scored for kanamycin
resistance according to two procedures: some of the seeds were
germinated on Murashige and Skoog medium supplemented with 300 mg/L
kanamycin; others were grown on Murashige and Skoog medium and then
used to produce cotyledon-derived calli using callus-inducing medium
(Bourgin et al., 1979) supplemented with 100 mg/L kanamycin.
Integration of the transgene into the plant genome was determined by
PCR on chromosomal DNA prepared according to the method of Krysan et
al. (1996). Two different reactions were carried out with 50 ng of
template DNA and 2 units of Dynazyme (Finnzymes Oy, Espoo,
Finland) in the conditions recommended by the manufacturer. The
first reaction used primers G1 (sense, 5 -GCCGGGATCCATCGCAGCGTAATGC-3) and G2 (antisense, 5-GCCTCCCTGCTGCGGTTT TTCACCG-3) for the
amplification of a 1298-bp internal fragment of the uidA
gene carried on the pFB8 vector (Atanassova et al., 1995); the second
reaction used primers A1 (sense, 5-GGCTCAATCTCCGCCGAGAAAGTCC-3) and
A2 (antisense, 5-CTCTCCTCCGGTGACTCCGG-3) for the amplification of a
965-bp internal fragment of the SMT2 cDNA.
Transgene Expression Analysis
Total RNAs from leaf material of 2-month-old greenhouse-grown
plants were extracted according to the method of Goodall et al. (1990).
Northern analysis was done by separating 10-µg RNA samples on
formaldehyde gels as described by Sambrook et al. (1989) and blotting
them onto nylon filters (Hybond-N+, Amersham).
Randomly primed (Stratagene) 32P-labeled probes
polymerized from the SMT2 cDNA 411 (Husselstein et al.,
1996) were hybridized to the filters as recommended by the
manufacturer. Filters were then washed in 0.2× SSC containing 0.1%
SDS at 65°C before autoradiography. RT-PCR reactions were started
with 5 µg of total RNA, primer A2, and 200 units of RT (Moloney
murine leukemia virus from BRL) with reagents and conditions as listed
by the manufacturer. RT products were amplified in standard PCR
conditions with primers A1 and A2. PCR products were further analyzed
by Southern hybridization (Sambrook et al., 1989) with the
SMT2 probe described above.
SMT Enzymatic Assay
Isolation of membranes from tobacco leaves was carried out as
follows: 12 g of leaf tissue was ground in a 0.2 M
KH2PO4 buffer (pH 7.5)
containing 0.35 M sorbitol, 10 mM
Na2EDTA·2H2O, 5 mM MgCl2·6H2O, 40 g/L PVP,
and 10 mM dithioerythritol. The supernatant fraction
resulting from a 15-min centrifugation at 10,000g was centrifuged for 1 h at 100,000g. The membrane fraction
was then resuspended in a 0.1 M Tris-HCl buffer (pH 7.5)
containing 1 mM  -mercaptoethanol and 20% (v/v)
glycerol. Microsomal proteins were quantified by the Bio-Rad protein
assay using BSA as a standard.
A radiochemical assay was set up based on that reported by Fonteneau et
al. (1977). A standard assay for tobacco SMT2 consisted of 0.1 M Tris-HCl at pH 7.5, 1 mM -mercaptoethanol,
20% glycerol (v/v), 0.1% Tween 80 (w/v), 30 µM sterol
substrate, 100 µM
[methyl-3H]-adenosyl-Met (475,000 cpm,) and 0.8 mg/mL microsomal proteins. The mixture was incubated at
30°C for 45 min, and then the reaction was stopped by adding 100 µL
of 12% (w/v) ethanolic KOH. Sterol carriers were added before neutral
lipids were extracted with n-hexane. Sterols were purified
by TLC (two runs of dichloromethane), and the band corresponding to
4-methyl sterols was scraped off (RF = 0.3)
and collected into scintillation vials containing 10 mL of
liquid-scintillation cocktail (Ready Organic, Beckman). Radioactivity
was determined in a liquid-scintillation counter (Packard Instruments,
Downers Grove, IL).
Extraction and Dosage of Sterols
Lipids from about 5 to 10 mg (small-scale qualitative analysis) or
100 to 200 mg (quantitative analysis) of ground, dry material were
extracted at 70°C in dichloromethane:methanol (2:1, v/v). The dried
residue was saponified with 6% (w/v) KOH in methanol at 90°C for
1 h to release the sterol moiety of steryl esters. Sterols were
then extracted with 3 volumes of n-hexane, and an acetylation reaction was performed on the dried residue for 1 h at
60°C in toluene with a mixture of pyridine:acetic anhydride (1:1,
v/v). Steryl-acetates were resolved by TLC using precoated silica
plates (60F254, Merck, Darmstadt, Germany), with one run of
dichloromethane as a single band at RF = 0.5. Purified steryl acetates were separated and identified using a gas
chromatograph (model 8300, Varian, Les Ulis, France) with a
flame-ionization detector and a glass capillary column (wall coated,
open, and tubular; 30 m long; 0.25 mm i.d.; coated with DB1; J & W
Scientific, Folsom, CA) using H2 as a carrier gas
(2 mL/min). The temperature program included a fast increase from
60°C to 230°C (30°C/min) and a slow increase from 230°C to
280°C (2°C/min). Data from the detector were monitored with a
computer program (Star, Varian). Sterol structures were confirmed
by GC-MS (model MD800, Fisons Instruments, Beverly, MA) equipped
with a glass capillary column (WCOT coated with DB5; J & W Scientific)
as described previously (Rahier and Benveniste, 1989). Determination of
the sterol content from microsomes was as indicated above except
for the extraction procedure, which started with the
saponification step.
Measurement of Cell Length
Stem and midrib epidermis from the middle of the fifth internode
and from the middle of the fifth leaf were peeled off from 3-month-old
greenhouse-grown plants. Five cells per epidermis sample were randomly
chosen and their lengths were measured under a microscope with a
micrometer (Zeiss). Cell measurements were done for five plants per
line.
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RESULTS |
Generation of Tobacco Plants Expressing the Arabidopsis SMT2 cDNA
A set of 21 plants randomly chosen from the population of
leaf-disc-derived kanamycin-resistant regenerants were subjected to a
small-scale, rapid sterol analysis to point out any putative new
trait(s). Analysis of leaf material from all 21 regenerants revealed a
novel 24-alkyl-5-sterol composition: a
dramatic decrease in the proportion of 24-methyl sterols and a
concomitant increase in the proportion of 24-ethyl sterols. Gas
chromatograms shown in Figure 2
illustrate this first sterol screen; R (peaks 3-5) was calculated for
each T1 transformant. Wild-type plants and
transgenic controls (p35S) had R values of approximately 1, whereas
p35S-SMT2 tobacco had R values ranging from 0.03 to 0.62 (data not shown).
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| Figure 2.
Gas chromatograms of steryl acetates from leaf
material of primary tobacco transformants 1 month after their transfer
to the greenhouse. A, Wild type; B, p35S-SMT2.
Representative samples are shown. Peak 1, Cholesteryl-acetate; peak 2, 24-methylene cholesteryl-acetate; peak 3, 24-methyl
cholesteryl-acetate; peak 4, stigmasteryl-acetate; peak 5, sitosteryl-acetate; peak 6, isofucosteryl-acetate; peak 7, cycloartenyl-acetate.
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At this point in the study seven of these new lines, as well as one
wild-type line and one p35S control line originating from the same
leaf-disc culture, were chosen for further characterization. The
integration of T-DNA(s) into the genome of kanamycin-resistant tobacco
was demonstrated using internal primers recognizing specifically the
uid A gene (data not shown) or the Arabidopsis
SMT2 cDNA cloned into the T-DNA cassette (Fig.
3A). Expression of the p35S-driven SMT2 cDNA in T1 plants was checked by
RT-PCR. As shown in Figure 3, RT-PCR assays gave conclusive evidence
for the presence of an Arabidopsis-specific SMT2 mRNA in
transgenic tobacco.
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| Figure 3.
Transgene integration and expression in tobacco.
Lane 1, Wild type; lane 2, transgenic p35S control; lanes 3 to 9, transgenic p35S-SMT2 lines A, B, C, D, E, F, and G,
respectively. A, Internal 0.9-kb fragment of the Arabidopsis cDNA
encoding SMT2 cloned into the T-DNA of the plant-transformation vector
was amplified from chromosomal DNA of primary transformants. B, Total
leaf RNA subjected to a RT-PCR reaction using primers specific to the
0.9-kb internal fragment of the Arabidopsis cDNA encoding SMT2. C,
Ethidium bromide-stained gel showing equal amounts of total RNA used in
B. D, Southern hybridization of the PCR products obtained as described
in B with a 32P-radiolabeled SMT2 probe. The
arrows denote the size of the expected and obtained PCR products.
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Sterol Composition of p35S-SMT2 Tobacco
Sterol composition of control and transgenic tobacco plants was
accurately determined on leaf samples from the upper third of the
plants. Results reported in Table I
demonstrate clearly the striking difference between control and
SMT2 lines: the latter show a considerable decrease in the
amount of 24-methyl cholesterol with a concomitant increase of 24-ethyl
sterols (sitosterol) compared with the controls. There were only slight
increases in other 24-ethyl sterols (e.g. stigmasterol). The other
minor variation of the sterol profile of SMT2 tobacco leaves
was a faint decrease in cholesterol compared with control lines. We did
not detect any significant variation in the amount of sterol in
transgenic plants: all contained approximately 2 mg total sterols
g 1 dry weight, the same as in the controls. From these
overall observations, the shift occurring in the sterol profile of
SMT2 plants may be clearly expressed by R. As mentioned
above, R was approximately 1 for control plants, whereas it decreased
radically for the seven T1 SMT2 plants
analyzed, ranging from 0.01 to 0.31.
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Table I.
Total sterol content of tobacco plants transformed
with an Arabidopsis SMT2 cDNA
Analysis was performed on leaf material from T1 plants
grown for 6 weeks in the greenhouse.
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Growth of p35S-SMT2 Tobacco
Growth rates and morphological phenotypes were observed throughout
succeeding generations of transgenic tobacco. During the early stages
of growth (i.e. during germination of seeds in vitro on a nutrient
medium or in soil in the greenhouse), the SMT2 lines exhibited the same morphological phenotype as controls; however, a
reduction in growth of SMT2 seedlings from some transgenic
lines became apparent after 3 weeks of culture (data not shown). When the plants were transferred to standard-size pots in the same greenhouse conditions, we observed a significant size reduction in a
large number of SMT2 plants compared with control plants and
with another set of wild-type-like SMT2 plants. The smallest SMT2 plants were one-half the size of control plants (Fig.
4). The low growth rate of some of the
SMT2 plants was always associated with up to a 2-month delay
in flowering. Delayed flowering tobacco plants displayed a reduced
number of flowers per inflorescence compared with control lines;
however, the morphology and fecundity of flowers were similar to those
of controls (data not shown).
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| Figure 4.
Wild-type and p35S-SMT2 tobacco
plants. Three-month-old plants grown in standard greenhouse conditions
are shown: wild-type plants are on the left and T3 plants
from line D are on the right.
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To describe more precisely the growth-reduction phenotype of the
SMT2 overexpressors, plants from the
T3 generation were compared with the wild type
with respect to plant height, number of internodes, and size of cells.
The data in Table II show that the
growth-reduction trait was stably transmitted through generations.
These data also show that, whatever the stem height of a plant, the
average number of expanded leaves per plant remained stable at
approximately 21; therefore, growth reduction could be calculated, on
average, as a 36% internode length reduction compared with wild-type
plants. It is crucial to point out that the length of stem epidermis
cells and midrib epidermis cells was not significantly different in SMT2 plants compared with wild-type plants; therefore, the
growth reduction reported here is not due to a reduction of the cell length but rather to a reduced number of cells per unit length. Finally, overexpression of SMT2 in T3
plants was verified by northern analysis (Fig.
5), and the proportion of 24-methyl- to
24-ethyl sterols was determined (Table II).
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Table II.
Size measurements on 3-month-old greenhouse-grown
p35S-SMT2 plants (vegetative stage) from T3 generations and
corresponding mean sterol composition
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| Figure 5.
Northern analysis. Lane 1, Wild-type tobacco line;
lanes 2 to 4, transgenic p35S-SMT2 plant lines A, D, and
E, respectively. Ten micrograms of total leaf RNA from T3
plants was hybridized with a probe derived from the Arabidopsis
SMT2. The arrow denotes the SMT2 1.4-kb
transcript. The ethidium bromide-stained gel demonstrates equivalent
RNA quantities loaded in each lane.
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Modification of the Proportions of 24-Methyl- and 24-Ethyl
Cholesterol and Growth Effect
To establish that a dramatic decrease in 24-methyl cholesterol
and/or an increase in sitosterol may be associated with reduced plant
growth and size, individuals within five independent
T2 families were checked on the basis of the
following three parameters: the height of the stem, the R, and the
24-methylene
lophenol-C241-methyltransferase activity
monitored in microsomes from leaf tissue. The population of transgenic
plants was grown in the greenhouse after the wild-type individuals from
a given T2 family were counterselected in vitro
on a germination medium containing 200 mg/L kanamycin. Data collected
from the analysis of 2-month-old plants are shown in Table
III. The overall results assigned to
wild-type or transgenic control plants define a pool of controls having
an SMT2 activity of approximately 0.8 nmol mg1
protein h1, an R value of
approximately 1 in microsomes from leaf tissue and from the total
sterol fraction, and a stem height of approximately 60 cm.
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Table III.
SMT2 enzyme assay, sterol profile, and plant size
in T2 generations of p35S-SMT2 lines
Values in parentheses are data collected from the same plants after
seed setting.
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When control data were compared with the values obtained for the 20 T2 plants, four important points appeared. First,
the SMT2 activity measured in leaf microsomes from each individual was
increased up to 14-fold over the mean control value; this increase was
greater than 5-fold for 60% of the analyzed population. Second, the R
measured in total lipid extracts decreased to values ranging from 0.02 to 0.18. These values may be distributed into two groups: one
comprising the lowest values ranging from 0.02 to 0.05 and
corresponding to the highest SMT2 levels and the other ranging from
0.09 to 0.18, which includes plants displaying relatively low R values
and corresponding to SMT2 levels found below the limit of 5-fold the
control values. Third, when plant growth was monitored in terms of stem
height, the first group of plants displaying the most dramatic change
in sterol metabolism were those exhibiting a growth reduction matching
values from 10% to 50% of the size of control plants; the second
group of plants, although showing an important decrease in R but
relatively moderate when compared with the extreme values (0.02-0.05
found in the first group), had a growth rate very close to that of the
controls. These measurements were fully validated when sizes of plants
having completed their growth and flowering were recorded, along with
their R factor taken from a total leaf sterol extract: the lower the R
value, the smaller the plant (Table III). Fourth, the R from a leaf
sterol extract was without exception in the analyzed plant population almost identical to that measured in the corresponding microsomal fraction, which contains the cell membranes in which sterols are localized.
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DISCUSSION |
We have generated transgenic tobacco plants expressing an
Arabidopsis cDNA encoding SMT2 under control of a strong constitutive promoter, and examined the biosynthetic and biological features of this
new material over succeeding generations.
Tobacco plants expressing the SMT2 cDNA were characterized
by a dramatic drop in the amount of 24-methyl sterols and a concomitant increase in the amount of 24-ethyl sterols. Analysis of the overall sterol profiles indicated that aside from minor variations, the major
change occurring in the transgenic material was a strong shift from
24-methyl cholesterol to sitosterol; therefore, the R was calculated.
Whereas R was approximately 1 in control lines (wild-type, transgenic
controls), it ranged from 0.01 to 0.31 in p35S-SMT2 lines.
The amount of other 24-ethyl sterols found in tobacco remained almost
unchanged, particularly stigmasterol, which is thought to be the
product of the desaturation at C22 of sitosterol (Fig. 1; for
review, see Grünwald, 1975; Benveniste, 1986, and refs.
therein). Although in plants a sterol-22-desaturase has not yet been
characterized in terms of enzymatic activity or sequence information,
it is tempting to speculate about its features. These would be either a
rate-limiting enzymatic step in the pathway or a highly regulated point
in the course of plant development for as yet unknown reasons. In
summary, from the biosynthetic point of view, transgenic tobacco lines
characterized in this work represent the first case, to our knowledge,
of a stable modification of the sterol content whereby only two
molecular species, 24-methyl cholesterol and sitosterol, are affected
by a selective action on SMT2 enzyme activity. Although powerful
inhibitors of plant SMTs do exist (Rahier et al., 1980; Schmitt et al.,
1981), these molecules are not selective regarding SMT1 and SMT2.
SMT2 is located at a branching point in the sterol pathway and governs
the final concentrations of 24-methyl cholesterol and sitosterol in the
plant cell. In this study we have evaluated the biological effect of
SMT2 overexpression, which results in a modified R in
transgenic tobacco. Our results show that a dramatic decrease in R is
associated with a reduction of plant growth. Precisely, plants
exhibiting the highest SMT2 activity and therefore the lowest R value
are those affected in growth: their height is reduced up to one-half
the size of the wild type, their leaves are smaller, and they exhibit
delayed flowering and produce fewer flowers compared with the wild
type.
This biological effect was not linear with respect to R but was clearly
below the threshold R value of approximately 0.1, suggesting that
tobacco may tolerate a variable composition of 24-methyl- and 24-ethyl
sterols in a range of R values between 0.1 and 1 without consequences
on its development. Since growth is hampered below the threshold value
of R, one could speculate that the isolation of primary transformants
(on the basis of reporter gene expression selection) having an R value
equal or close to 0 was counterselected. Growth reduction can be due to
a reduction in cell length, a reduction in the number of cells, or a
combination of both. Data collected from a population of
T3 plants, for which the height reduction
corresponded to a 36% reduction of the mean internode length, clearly
showed that the cell length was not affected in SMT2 tobacco
compared with the wild type. Therefore, these data indicate that the
reduction in plant stature may have been due to a decrease in cell
number. Although it is difficult to determine the effects on cell
growth by looking at only the final size of cells, we hypothesize that
it is the cell division frequency (the meristem activity) that is
reduced in transgenic SMT2 tobacco. Based on these
observations, we propose that a radical decrease in the concentration
of 24-methyl cholesterol and/or an equivalent increase in that of
sitosterol may be responsible for the reduced growth rate.
We should now investigate two hypotheses about the physiological
relationship between such a novel sterol balance and its effect on
growth. First, the new sterol content could modify the physical
properties of the plasma membrane, which may affect cell growth. Some
clues about this aspect may be found in our previous work, in which
treatment of plants or plant cell cultures with post-squalene sterol
biosynthesis inhibitors and selection of mutants resistant to some of
these drugs indicated that replacement of most of the pathway end
products (24-alkyl-5-sterols) with sterol
intermediates was highly toxic (Maillot-Vernier et al., 1990; Schaller
et al., 1994). Moreover, tobacco lines overproducing sterols by means
of an increased 3-hydroxy-3-methyl glutaryl CoA reductase activity were
shown to regulate the concentration of free sterols present in
membranes (Schaller et al., 1995). Likewise, a functional analysis of
the physical properties of artificial plant membrane systems sets
sitosterol at a given concentration as the optimal plant membrane
reinforcer among various sterols (Schuler et al., 1991). This aspect in
planta is very poorly documented; however, Crèvecoeur et al.
(1992) reported physical modifications of the plasma membrane and
modification of its sterol content in meristematic cells during the
transition from vegetative to floral meristem.
The second hypothesis is that the new sterol content in plants
overexpressing SMT2 could affect the biosynthetic interface between sterols and brassinosteroids. The role of the latter in plant
growth and development has been recently demonstrated through molecular
genetics (Clouse, 1996; Kauschmann et al., 1996; Li et al., 1996;
Szekeres et al., 1996; Li and Chory, 1997; Choe et al., 1998), which
were preceded by solid biochemical studies (for review, see Adam and
Marquardt, 1986; Mandava, 1988; Suzuki et al., 1995; Schmidt et al.,
1997). Altogether, these data show that 24-methyl cholesterol is the
sterol precursor for the biosynthesis of brassinolide. It is of course
tempting to suggest that a severe decrease in the amount of 24-methyl
cholesterol would affect the production of brassinosteroids in
concentrations required to fulfill normal development. However, it has
been shown that all brassinosteroid-biosynthetic mutants characterized
so far in Arabidopsis (which are rescued by exogenous brassinosteroids)
display a dwarfed phenotype caused by the lack of cell elongation. This
is true of det2, which is blocked in the reduction of
3-dehydro-4(5)-campestenol to 3-dehydro
campestanol (Fujioka et al., 1997); of dwf4, which is
impaired in the C22-hydroxylation of campestanol (Choe et al.,
1998); and of cpd, which presents a defect in the C23-hydroxylation of cathasterone (Szekeres et al., 1996). Likewise, Nomura et al. (1997) reported a blockage of brassinosteroid synthesis in the pea lkb mutant, in which considerable reduction of
brassinolide and brassinosteroid intermediates were associated with a
50% reduction of the cell length. These overall data indicate that
brassinosteroids are essential to controlling plant development and
especially cell elongation (Clouse, 1996; Kauschmann et al., 1996).
We report here a reduced plant stature likely due to a reduction in
cell number without alteration of cell size. This seems to contrast
with what would be expected from a modification of the brassinosteroid
pathway. In this respect, further investigations challenging the
brassinosteroid profile of SMT2 tobacco will clarify this
point. Finally, this novel plant material bearing a unique and stable
modification of the post-squalene sterol pathway represents a starting
point for investigation of the expression of SMT genes in
higher plants.
| |
FOOTNOTES |
*
Corresponding author; e-mail schaller{at}medoc.u-strasbg.fr;
fax 3-88-35-84-84.
Received March 30, 1998;
accepted July 9, 1998.
| |
ABBREVIATIONS |
Abbreviations:
R, ratio of 24-methyl cholesterol to sitosterol.
RT, reverse transcriptase.
SMT, sterol-C-methyltransferase.
SMT1, S-adenosyl-L-Met cycloartenol
C24-methyltransferase.
SMT2, S-adenosyl-L-Met 24-methylene
lophenol-C241-methyltransferase.
| |
ACKNOWLEDGMENTS |
We thank Drs. R. Atanassova and M. Legrand (IBMP,
Strasbourg) for the gift of the plant expression vector pFB8. We warmly acknowledge B. Bastian for typing the manuscript and A. Hoeft for GS-MS
analysis.
| |
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Abstract
Introduction