Plant Physiol. (1998) 117: 397-405
Differential Expression of the
S-Adenosyl-L-Methionine Synthase Genes
during Pea Development1
Lourdes Gómez-Gómez2 and
Pedro Carrasco*
Departament de Bioquímica i Biologia Molecular, Universitat
de València, Dr. Moliner 50, Burjassot, València,
E-46100 Spain
 |
ABSTRACT |
Two
genes coding for S-adenosyl-L-methionine
synthase (SAMS, EC 2.5.1.6) were previously isolated from pea
(Pisum sativum) ovaries. Both SAMS genes
were highly homologous throughout their coding regions but showed a
certain degree of sequence divergence within the 5
and the 3
untranslated regions. These regions have been used as gene-specific
probes to analyze the differential expression of SAMS1
and SAMS2 genes in pea plants. The ribonuclease protection assay revealed different expression patterns for each individual gene. SAMS1 was strongly expressed in nearly
all tissues, especially in roots. SAMS2 expression was
weaker, reaching its highest level at the apex. Following pollination,
SAMS1 was specifically up-regulated, whereas
SAMS2 was expressed constitutively. The up-regulation of
SAMS1 during ovary development was also observed in
unpollinated ovaries treated with auxins. In unpollinated ovaries an
increase in SAMS1 expression was observed as a
consequence of ethylene production associated with the emasculation
process. In senescing ovaries both SAMS1 and
SAMS2 genes showed increased expression. Ethylene
treatment of unpollinated ovaries led to an increase in the
SAMS1 mRNA level. However, SAMS2
expression remained unchangeable after ethylene treatment, indicating
that SAMS2 induction during ovary senescence was not
ethylene dependent. SAMS mRNAs were localized by in situ
hybridization at the endocarp of developing fruits and in the ovules of
senescing ovaries. Our results indicate that the transcriptional
regulation of SAMS genes is developmentally controlled
in a specific way for each gene.
| |
INTRODUCTION |
SAMS (EC 2.5.1.6) is a key enzyme in plant metabolism, catalyzing
the biosynthesis of SAM from Met and ATP. SAM is a precursor for the
biosynthesis of ethylene (Yang and Hoffman, 1984
) and polyamines (Heby
and Persson, 1990) and is involved in methylation reactions (Tabor and
Tabor, 1984). SAMS cDNA clones have been isolated from
several plants (Izhaki et al., 1995). Recently, two genes from pea
(Pisum sativum), SAMS1 and SAMS2, were
cloned in our laboratory (Gómez-Gómez and Carrasco, 1996).
In all of the characterized systems, a multigene family encodes SAMS
enzymes. The existence of different SAMS genes has generally
been ascribed to the metabolic importance of SAM (Thomas and
Surdin-Kerjan, 1987; Peleman et al., 1989b). Moreover, it has been
suggested that some of the SAMS genes would be expressed
constitutively, whereas others would be specifically regulated by
developmental and/or environmental factors strictly controlled
according to the requirement for SAM (Boerjan et al., 1994).
Marked variations in the levels of SAMS mRNA have been
observed at different stages of plant development (Woodson et al., 1992; Boerjan et al., 1994; Izhaki et al., 1995) and in different plant
tissues (Peleman et al., 1989a; Dekeysen et al., 1990). SAMS
expression is also increased by a variety of environmental factors,
including salt stress (Espartero et al., 1994; Van Breusegem et al.,
1994), fungal and bacterial elicitors (Somssich et al., 1989; Gowri et
al., 1991; Kawalleck et al., 1992), ozone exposure (Tuomainen et al.,
1996), and mechanical stimuli (Espartero et al., 1994; Kim et al.,
1994), all of which are also known to induce ethylene biosynthesis
(Abeles et al., 1992). Hormonal regulation of SAMS has also been
reported in wheat embryos (Mathur et al., 1992) and dwarf mutants of
pea (Mathur et al., 1993), in which treatment with
GA3 induced two additional isoenzymes of SAMS, and in tomato roots, in which treatment with ABA induced the
accumulation of SAMS transcripts (Espartero et al., 1994).
In this paper we report a detailed study of the individual expression
of SAMS genes, their developmental regulation, and the patterns of their response to auxins and ethylene in unpollinated pea
ovaries, a convenient system with which to study both the regulation of
senescence and the induction of fruit set by plant-growth regulators.
Removing stamens 2 d before anthesis avoids self-pollination. Intact, unpollinated ovaries start to senesce naturally about 3 DPA,
but treatment of unpollinated ovaries with different plant-growth regulators on the equivalent to the day of anthesis (d 0) prevents senescence and promotes fruit development
(García-Martínez and Carbonell, 1980). The pea
SAMS gene family consists of two genes. Analysis of the
expression of the SAMS genes during ovary senescence and
fruit development showed that SAMS transcript levels were up-regulated by auxins during fruit setting and by ethylene during ovary senescence (Gómez-Gómez and Carrasco, 1996). However, the high degree of identity made it impossible to differentiate the
expression of each individual SAMS gene.
Using RPA and gene-specific probes, we now show that the
SAMS1 gene is highly expressed during fruit development and
senescence and is also expressed in pea ovaries following treatment
with ethylene. Furthermore, we report that SAMS2 gene
expression is reduced during auxin-induced parthenocarpic fruit
development and that its expression during ovary senescence is not
induced by ethylene. Spatial localization by in situ hybridization
showed that SAMS mRNAs are differentially accumulated in the
endocarp of developing ovaries, suggesting a role for SAM in pod wall
development. We conclude that SAMS genes in pea are
differentially regulated during ovary development and senescence.
| |
MATERIALS AND METHODS |
Pea (Pisum sativum L. cv Alaska) plants were grown as
previously described (Carbonell and García-Martinez, 1985).
Unpollinated ovaries were obtained by removing petals and stamens from
flowers 2 d before anthesis. Only the first and second flowers of
each plant were used for the experiments. Samples were obtained from (a) pollinated ovaries; (b) untreated, unpollinated ovaries from emasculated flowers (including presenescent and senescent ovaries); (c)
parthenocarpic ovaries, which were unpollinated and derived from
emasculated flowers treated on d 0 with a 2,4-D solution (20 µL of
100 µg mL
1 2,4-D in 0.1% Tween-80); and (d)
unpollinated ovaries from emasculated flowers treated on d 0 with 10 µL L1 ethylene. After the plants were
harvested, samples to be used for RNA isolation were frozen in liquid N
and stored at 
80°C until used.
Ethylene production was measured by placing ovaries at different stages
of development in 5-mL gas-tight containers for 0.5 h, after which
time the headspace gas was assayed for ethylene by GC. The gas
chromatograph (model GC-14BPFSC, Shimadzu, Columbia, MD) was equipped
with an activated alumina column and a flame-ionization detector. An
ethylene standard was used for calibration of concentration and
retention time.
RNA Isolation and RPA
RNA was prepared from frozen plant material following the method
of Prescott and Martin (1987) and quantified by measuring the
A260. The integrity of rRNA was verified by
electrophoresis through 1% (w/v) agarose gels containing 2.2 M formaldehyde. RPA (Lee and Costlow, 1987) was performed
using [
-32P]UTP strand-specific radiolabeled
probes generated from a 124-bp fragment of the 5 portion of the
SAMS1 cDNA and a 423-bp fragment containing the 3 end of
the SAMS2 cDNA (Gómez-Gómez and Carrasco, 1996).
The sequences selected to generate the probes were different enough in
both pea SAMS genes to ensure probe specificity. To synthesize the
SAMS1 and SAMS2 antisense RNA probes, both
SAMS cDNAs were cloned in pGEM-1 (Promega), and the plasmids
were linearized with PstI and PvuII and
transcribed in vitro with T7 and Sp6 RNA polymerase, respectively. Ten
micrograms of total RNA was hybridized with 55,000 dpm, 760 and 250 attomoles of SAMS1- and SAMS2-labeled riboprobes,
respectively.
Following hybridization, digestion buffer was added and unhybridized
RNA was removed by digestion with a mixture of RNase-A and RNase-T1 at
30°C for 30 min following the directions of the manufacturer
(Boehringer Mannheim). Adding 20% SDS and proteinase K and incubating
at 37°C for 15 min stopped the digestion. After phenol extraction and
ethanol precipitation, RNAs were separated on a 6%
polyacrylamide-sequencing gel. An aliquot of undigested RNA probe was
run as a marker for the size of the transcript. A quantitative measure
of transcript abundance was obtained using a radioanalytical imaging
system (InstantImager 2024, Packard Instruments, Meriden, CT).
Radioactivity was directly measured on the gel. The data were corrected
for the number of uracil residues within each probe, and the specific
activity of the probes was calculated to determine the attomoles of
mRNA according to the following equation:
where cpm* is the counts per minute counted by the
imager and corrected according to the reference date of the
[32P]UTP, E is the efficiency of the
counting, S is the specific activity of the
[-32P]UTP (800 Ci
mmol1), and N is the number of
uracil residues in the probe. All values are the means of three
independent experiments. Autoradiography of the gel was independently
obtained on radiographic film (XAR-5, Kodak) using an intensifying
screen at 80°C.
In Situ RNA Hybridization
Ovaries were harvested from flowers at various stages of
development, fixed, embedded in paraffin, and tissue-sectioned as described previously (Vercher et al., 1984). In situ hybridization was
carried out essentially as described by Duck (1994). Tissue samples
were cut into 10-µm sections and placed on replicated slides coated
with poly-L-Lys. The sections were dewaxed, hydrated, and
treated with HCl, proteinase K, and acetic anhydride.
Digoxigenin-labeled sense and antisense riboprobes were generated by in
vitro transcription of a pGEM-1 template containing a 400-bp insert
with the 5 region of the SAMS1 cDNA
(Gómez-Gómez and Carrasco, 1996). Plasmids were linearized,
and digoxigenin-11-UTP was incorporated using either T7 or SP6
polymerase according to the instructions of the manufacturer
(Boehringer Mannheim).
Slides were hybridized for 30 h at 45°C in 50% formamide, 30 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1× Denhardt's solution, 10% dextran sulfate,
100 mM DTT, 500 µg/mL denatured salmon-sperm DNA, 150 µg mL1 yeast tRNA, and 0.2 µg
mL1 digoxigenin-labeled probe. Following
hybridization, slides were treated with RNase-A to remove nonhybridized
probe and washed once at room temperature in 2× SSC for 30 min, once
at room temperature in 1× SSC for 30 min, and once at 37°C in 0.5×
SSC for 30 min. Hybridization of the riboprobes was detected with
anti-digoxigenin antibody conjugated to alkaline phosphatase and
visualized by color development following the manufacturer's
directions (Boehringer Mannheim).
| |
RESULTS |
Differential Expression of SAMS Genes in Pea Plants
Previous work in our laboratory showed that SAMS
transcripts were present in vegetative and reproductive tissues.
SAMS mRNA was detected at higher levels in roots and stems
than in leaves when the SAMS1-coding sequence was used as a
probe for northern analysis (Gómez-Gómez and Carrasco,
1996). To determine whether both SAMS genes contributed to
this expression pattern, RPA (Lee and Costlow, 1987) was performed with
RNA samples from different tissues. RPA was carried out using 10 µg
of total RNA, as described in ``Materials and Methods''. Antisense
RNA probes of 124 and 436 bp, respectively, were synthesized from cDNA
clones for SAMS1 and SAMS2 and labeled with [-32P]UTP. No RNase protection was observed
after hybridizing the SAMS gene-specific probes to yeast tRNA or human
RNA (Fig. 1).
View larger version (28K):
[in this window]
[in a new window]
| Figure 1.
RPA of SAMS1 transcripts. Control lanes
for the SAMS1 RNA probe are: lane 1, undigested probe; lane
2, SAMS1 RNA probe hybridized to yeast tRNA; lane 3, SAMS1 RNA probe hybridized to total human RNA; and lanes
4 to 6, RNA (5, 10, and 15 µg, respectively) from pollinated pea
ovaries 5 DPA. All were hybridized to the SAMS1 gene-specific RNA probe, and RPA was performed as described in ``Materials and Methods''. The counts per minute detected in the
samples are shown under each band. Radioactivity was measured using a
radioanalytical imaging system (see ``Materials and Methods'').
|
|
Quantification of RPA of increasing RNA amounts showed the linearity of
the method (Fig. 1). As shown in Figure
2, expression of SAMS1 mRNA
was strong in most tissues, especially in roots, apices, and petals; a
little lower in stems, flower buds, and mature leaves; and lowest in
young leaves and tendrils. By contrast, SAMS2 was expressed
preferentially in the apex and was almost absent from vegetative
tissues such as young and mature leaves and tendrils (Fig. 2). In
general, changes in SAMS1 mRNA level were more significant
than changes in SAMS2 mRNA. RPA of SAMS2 transcripts gave rise to two bands, because the local denaturation of
the 3 end of the probe located at the poly(A+)
tail of the mRNA. Quantification of the counts and correction by the
specific activity of the probes showed that in all tissues SAMS2 transcripts were expressed at lower levels than
SAMS1 transcripts (Fig. 2B).
View larger version (67K):
[in this window]
[in a new window]
| Figure 2.
RPA of SAMS transcripts in pea
tissues. A, Ten micrograms of total RNA extracted from different
tissues of pea plants was hybridized to SAMS
gene-specific RNA probes, and RPA was performed. F, Floral buds (7,
5, and 3, d 7, 5, and 3 before anthesis, respectively); L, leaves
(Y, young; M, mature); A, apex; S, stem; R, root; T, tendrils; and P,
petals. B, Quantification of the SAMS mRNAs at the
stages shown in A. SAMS transcript levels were quantified using a radioanalytical imaging system. Light gray bars,
SAMS1; dark gray bars, SAMS2. The data were corrected for the number of
uracil residues within each probe.
|
|
Expression of SAMS Genes during Fruit Development
Pea ovaries ranging from 2 d before to 6 DPA were used to
analyze the differential expression of the SAMS genes during
fruit development. RPA analysis showed different expression patterns for SAMS1 and SAMS2 genes during fruit
development (Fig. 3). Before d 0, SAMS1 was expressed at a higher level than SAMS2
mRNA, which was almost undetectable (Fig. 3). Immediately after
pollination, SAMS1 transcript level decreased and remained
invariable up to 3 DPA, then increased suddenly on 4 DPA, and stayed
high through 6 DPA (Fig. 3). On the contrary, SAMS2 level
increased after anthesis and reached its maximum after 24 h, which
was maintained for the following 3 d (Fig. 3).
View larger version (53K):
[in this window]
[in a new window]
| Figure 3.
Temporal expression of SAMS genes
during fruit setting. A, Ten micrograms of total RNA extracted from pea
ovaries at different stages of development were hybridized to
SAMS gene-specific RNA probes, and RPA was performed.
d2 to d6, Days with respect to anthesis. B, Quantification of the
SAMS mRNAs at the stages shown in A. SAMS
transcript levels were quantified using a radioanalytical imaging
system. Light gray bars, SAMS1; dark gray bars, SAMS2. The data were
corrected for the number of uracil residues within each probe.
|
|
Analysis of SAMS expression in pods and seeds during fruit
development showed opposite gradients of SAMS1 mRNA
expression. SAMS1 mRNA accumulated during seed development
(Fig. 4). On the contrary,
SAMS1 mRNA level in the pod decreased from 4 to 10 DPA, when
the pod began to dry (Fig. 4). In the meantime, SAMS2 levels varied in pods in a way similar to SAMS1 but were present at
almost undetectable levels in seeds during fruit development (Fig. 4).
View larger version (59K):
[in this window]
[in a new window]
| Figure 4.
Analysis of SAMS transcripts in pea
pods and ovules during fruit development. Ten micrograms of total RNA
extracted from pea pods and seeds from pollinated pea ovaries at
different stages of development was hybridized to SAMS
gene-specific RNA probes, and RPA was performed. d4 to d12, Days with
respect to anthesis.
|
|
Treatment of emasculated ovaries with auxins induced parthenocarpic
development, generating fruits without seeds. Previous work in our
laboratory showed that SAMS transcripts accumulate only in
parthenocarpic ovaries generated by auxin application in a way similar
to that produced by pollination (Gómez-Gómez and Carrasco,
1996). We analyzed the expression of the individual SAMS
genes during this process. RPA using SAMS1 and
SAMS2 antisense RNA probes showed a differential expression
pattern for both genes during auxin-induced parthenocarpy (Fig.
5). In parthenocarpic ovaries,
SAMS1 mRNA levels accumulate after 3 DPA (Fig. 5) in a way
similar to that observed after pollination (Fig. 3). On the contrary,
SAMS2 mRNA levels were lower than those observed in
pollinated ovaries (Fig. 5).
View larger version (28K):
[in this window]
[in a new window]
| Figure 5.
Effect of 2,4-D treatment on d 0 on
SAMS expression in unpollinated pea ovaries. A, Ten
micrograms of total RNA extracted from 2,4-D-treated pea ovaries was
hybridized to SAMS gene-specific RNA probes, and RPA was
performed. d3 to d5, Days with respect to anthesis. B, Quantification
of the SAMS mRNAs at the stages shown in A. SAMS transcript levels were quantified using a
radioanalytical imaging system. Light gray bars, SAMS1; dark gray bars,
SAMS2. The data were corrected for the number of uracil residues within each probe.
|
|
Expression of SAMS Genes in Senescing Pea Ovaries
Since ethylene levels are related to the senescence of plant
organs (Davies and Grierson, 1989) and because SAM is involved in
ethylene biosynthesis, we determined ethylene production in pea ovaries
(Fig. 6A) and compared it with
SAMS expression in ovaries that had been pollinated (Fig.
3), 2,4-D-treated (Fig. 5), or emasculated (Fig. 6B). To induce
senescence, flowers were emasculated 2 d before anthesis.
Unpollinated ovaries grow slightly until the equivalent of 3 DPA, and
then enter a degenerative process that ends with the death of the
organ. A strong increase in ethylene production was immediately
detected in ovaries emasculated 2 d before anthesis (Fig. 6A).
View larger version (30K):
[in this window]
[in a new window]
| Figure 6.
Ethylene levels and temporal expression of
SAMS genes during ovary senescence. A, Ethylene levels
were determined in emasculated ovaries from 2 d before anthesis
(at the time of emasculation) to 6 DPA.  , Ovaries allowed to be
pollinated;  , emasculated ovaries; and  , emasculated ovaries
treated with 2,4-D on d 0. d-2 to d5, Days with respect to anthesis. B,
Ten micrograms of total RNA extracted from the same samples used to
measure ethylene was hybridized to SAMS gene-specific
RNA probes, and RPA was performed. C, Quantification of the
SAMS mRNAs at the stages shown in A. SAMS
transcript levels were quantified using a radioanalytical imaging
system. White bars, SAMS1; black bars, SAMS2. The data were corrected
for the number of uracil residues within each probe.
|
|
Because of the wounding caused by the emasculation process, this
ethylene increase was accompanied by an increase in the level of
SAMS1 mRNA with no change in the expression of
SAMS2 (Fig. 6B). Later, on the equivalent of 4 DPA, there
was a slight increase in ethylene production that could be associated
with ovary senescence (Fig. 6A). There was a transcript accumulation of
SAMS1 mRNA at 4 DPA that was associated with the onset of
senescence (Fig. 6, B and C). Thereafter, a decline in SAMS1
mRNA abundance was associated with the advanced stages of ovary
senescence. The SAMS2 mRNA level remained almost unchanged
in emasculated ovaries but was slightly induced at the onset of ovary
senescence (Fig. 6B).
We examined the effect of exogenous ethylene on SAMS1 and
SAMS2 gene expression in emasculated pea ovaries on d 0. As
shown in Figure 7, the levels of
SAMS1 mRNA increased in the ovaries in response to ethylene
treatment. The level of SAMS1 mRNA after 24 h of
treatment was the same as the level in senescing ovaries (Figs. 5 and
6). In contrast to SAMS1 mRNA, SAMS2 mRNA
remained almost constant, which is consistent with the lack of
stimulation of this gene during the emasculation process.
View larger version (20K):
[in this window]
[in a new window]
| Figure 7.
Effect of ethylene treatment on
SAMS expression in unpollinated pea ovaries. Treatment
was made on d 0, and ovaries were collected 24 h later. A, Ten
micrograms of total RNA extracted from ethylene-treated pea ovaries was
hybridized to SAMS gene-specific RNA probes, and RPA was
performed. Lane A, Air; lane E, after ethylene treatment. B,
Quantification of the SAMS mRNAs at the stages shown in
A. SAMS transcript levels were quantified using a
radioanalytical imaging system. White bars, SAMS1; black bars, SAMS2.
The data were corrected for the number of uracil residues within each
probe.
|
|
Spatial Distribution of SAMS Genes in Pea Ovaries
Previous reports have described the expression of
SAMS-GUS reporter genes in roots, stems, and leaves of
Arabidopsis (Peleman et al., 1989a) and tobacco (Peleman et al., 1989b)
and in leaves of rice (Dekeysen et al., 1990). We determined the
spatial distribution of SAMS genes at different stages of
ovary development and senescence. In situ hybridization was performed
using SAMS1 (Gómez-Gómez and Carrasco, 1996)
sense and antisense RNA probes. Hybridization of longitudinal (Fig.
8c) and transverse (Fig. 8d) sections of pollinated ovaries on d 0 with an antisense SAMS probe
revealed a general pattern of expression in all living cells,
especially in vascular tissue, the ovule, and ovary epidermal cells.
Control for cell density, which was accomplished by staining a
longitudinal section with toluidine blue, showed that higher
SAMS expression was not correlated with greater cell density
(Fig. 8a).
View larger version (126K):
[in this window]
[in a new window]
| Figure 8.
(Figure appears on facing page.)
In situ localization of SAMS mRNAs in pea ovaries.
Pollinated and unpollinated ovaries at different stages of development were sectioned and hybridized to an antisense SAMS1
probe. a, b, and c, Longitudinal sections of a pollinated pea ovary at
anthesis stained with toluidine blue (a) or hybridized with a
SAMS sense probe (b) or a SAMS antisense
probe (c). d to h, Transverse sections of a pollinated pea ovary at
anthesis hybridized with a SAMS antisense probe (d), at
3 DPA hybridized with a SAMS sense probe (e) or a
SAMS antisense probe (f), and at 5 DPA hybridized with a
SAMS sense probe (g) or a SAMS antisense
probe (h). i, Detail of a transverse section of a pollinated pea ovary
5 DPA hybridized with a SAMS antisense probe. j,
Transverse section of a pollinated pea ovary at 5 DPA, including an
ovule, hybridized with a SAMS antisense probe. k and l,
Transverse sections of an emasculated pea ovary at the equivalent to 4 DPA hybridized with a SAMS sense probe (k) and a
SAMS antisense probe (l). Bar in i = 200 µm; bars in all other panels = 100 µm. e, Endocarp; m, mesocarp; ov,
ovule; and vc, vascular cell.
{/ANNT;152064n;;11968n;1056n}
|
|
In pollinated ovaries on 3 DPA, SAMS was expressed mostly in
the endocarp and vascular bundles (Fig. 8f). This pattern of expression
was reinforced 5 DPA (Fig. 8, h-j), when the transcripts were present
in cells associated with the vascular tissue of developing seed and pod
mesocarp. Moreover, at this stage of development, SAMS mRNAs
were particularly abundant in endocarp cells. In senescing, unpollinated ovaries 4 DPA, SAMS mRNAs were localized in
vascular tissues and also in the aborted ovules (Fig. 8l). No signal
was detected when a sense probe was used to hybridize sections at different stages (Fig. 8, b, e, g, and k).
| |
DISCUSSION |
SAMS genes have been considered to be "housekeeping
genes" that are expected to be expressed constitutively in all
tissues. Previous work showed that SAMS transcripts were
induced in developing pea ovaries in response to pollination and auxins
and also during ovary senescence (Gómez-Gómez and Carrasco,
1996). However, northern analysis was not able to determine whether the
genes were biologically equivalent or whether they were differentially regulated. In this work we used RPA with gene-specific probes to
distinguish the expression pattern of each SAMS gene. The
steady-state levels of mRNA, corresponding to both SAMS1 and
SAMS2 genes, were monitored for different pea tissues at
different developmental stages and in response to different plant
hormones.
Our studies demonstrate that SAMS1 and SAMS2
genes are individually regulated, with their transcripts accumulating
to varying degrees among different tissues and at different stages of
ovary development or senescence. SAMS1 mRNA was found to be
expressed at higher levels than SAMS2 in all tissues. In
fact, the SAMS1 expression pattern was found to be the same
as that obtained by northern analysis using a probe able to hybridize
both SAMS1 and SAMS2 genes
(Gómez-Gómez and Carrasco, 1996). Nonetheless, the expression pattern for SAMS2 was quite different, although
its contribution to the pool of SAMS mRNAs was much lower. A
similar relationship between SAMS genes has been found in
tomato, in which transcripts corresponding to the sam1 gene
were the most abundant, although the expression pattern for each gene
was different in each tissue (Espartero et al., 1994).
In contrast, the two Arabidopsis SAMS genes show a similar
expression pattern (Peleman et al., 1989b): SAMS1 was highly
expressed in roots, apex, and petals and also accumulated to
significant levels in the other tissues examined; preferential
expression of SAMS2 was found in the apex. The expression of
both genes in this tissue could be due to polyamine biosynthesis, which
is required for cell proliferation in dividing tissues (Evans and
Malmberg, 1989), and to the biosynthesis of macromolecules associated
with the meristematic activity of this tissue.
In the apex SAM could be decarboxylated and used for the
biosynthesis of spermidine and spermine. This would agree with the high
levels of Arg decarboxylase mRNA and enzymatic activity detected in the
pea apex (Pérez-Amador and Carbonell, 1995; Pérez-Amador et
al., 1995). In floral buds both mRNAs change their levels coordinately according to the developmental stage of the organ. A developmental stage pattern has also been observed during corolla development in
petunia (Izhaki et al., 1995) and has been related to the rapid growth
of the tissue (Weiss and Halevy, 1989).
Expression of SAMS Genes in Fruits Is Developmentally
Regulated
Before the day of anthesis, coinciding with a period of elevated
cell division rate, SAMS1 was highly expressed, whereas
SAMS2 mRNA was almost undetectable. At the time of anthesis,
when cell division was about to conclude, the relationship between
SAMS1 and SAMS2 expression changed.
SAMS1 mRNA expression decreased, and SAMS2 mRNA
expression increased slightly 24 h after anthesis and remained
almost constant through 6 DPA. The analysis of SAMS2 expression in pods and seeds showed that the expression detected in
fruits was almost completely restricted to the pod. In pollinated ovaries, the most dramatic increase in SAMS1 expression was
detected 4 DPA. SAMS1 expression was mainly due to the level
of SAMS1 mRNA in the pod and was localized primarily to
cells of the presclerenchyma zone of the endocarp. At this time the
differentiation of the endocarp cells into sclerenchyma fibers starts
(Vercher et al., 1984).
The accumulation of SAMS1 mRNA in cells under lignification
would suggest that SAMS1 is involved in the biosynthesis of
the secondary cell wall. During this process SAM is needed for
methylation of lignin monomers before polymerization (Higuchi, 1981).
At 8 DPA, when the pod reached its maximum length, SAMS1
expression decreased. Then, as the pod began to dry out, the amount of
dry matter declined. Simultaneously, the seeds enlarged rapidly and increased in fresh weight by accumulation of dry matter, partly at the
expense of the pod wall (Eeuwens and Schwabe, 1975). During pea seed
development the synthesis and amount of storage and metabolic proteins
increases (Bewley and Black, 1994). SAMS1 mRNA also
increased, suggesting the participation of SAM in macromolecule
biosynthesis as a methyl group donor. Moreover, the presence of
stored SAMS mRNA in wheat embryos (Mathur et al., 1991)
indicates that SAMS1 mRNA could remain in the dry seed and
then be reutilized upon subsequent hydration. mRNAs coding for enzymes
essential for intermediary metabolism and proteins essential for the
successful completion of germination have been found in the stored or
conserved messages in dry seeds (Kermade, 1990).
SAMS Genes Are Differentially Regulated in
Parthenocarpic Developing Ovaries
The expression of SAMS1 and SAMS2 genes was
investigated in parthenocarpic pea ovaries induced by auxin treatment.
The weight of these ovaries is lower than that of the pollinated
ovaries, but at the histological and physiological levels the
developmental pattern of the ovaries is the same (Vercher and
Carbonell, 1991). Northern analysis showed that in pollinated and
auxin-induced parthenocarpic ovaries the expression pattern of
SAMS genes was the same (Gómez-Gómez and
Carrasco, 1996). However, the individual analysis of SAMS
transcripts showed that SAMS1 mRNA was induced in
auxin-treated unpollinated ovaries as well as in pollinated ovaries,
which suggests that similar mechanisms are involved in the
transcriptional control of the SAMS1 gene.
On the contrary, SAMS2 expression was dramatically reduced
in parthenocarpic fruits. This could be due to the lack of pollination and/or fertilization in the parthenocarpic ovaries that may provide the
stimulus for SAMS2 mRNA induction, which began at anthesis in pollinated ovaries. In addition, since auxins induce the
parthenocarpic development of the ovaries, generating fruits without
seeds, the lack of induction of SAMS2 mRNA could be
associated with factors originating in the seeds and translocated to
the pod, where they induce SAMS2 transcription.
SAMS Gene Expression during Ovary Senescence
The involvement of ethylene in pea ovary senescence has already
been reported (Gómez-Gómez and Carrasco, 1996). During
senescence, the increase in SAMS gene expression was
dependent on ethylene production. In the present study we showed that
ethylene increases in unpollinated pea ovaries as a consequence of the
emasculation process and during senescence. Both of these increases in
ethylene production are accompanied by increases in SAMS
expression. In both cases SAMS1 was expressed at higher
levels, whereas the SAMS2 transcript accumulated only at the
onset of ovary senescence and was less abundant than SAMS1
mRNA. Because only the SAMS1 gene was induced by ethylene,
accumulation of SAMS1 mRNA after emasculation and during
ovary senescence could be associated with an increase in ethylene
biosynthesis.
Increases in SAMS gene expression in response to mechanical
stimuli have been reported in wounded Arabidopsis leaves (Kim et al.,
1994) and tomato roots, in which only one of the three SAMS
genes was induced (Espartero et al., 1994). However, induction of
SAMS2 mRNA at the onset of senescence was ethylene
independent and could have been age related. Leaf senescence in
Arabidopsis (Grbic and Bleecker, 1995) and leaf senescence and ripening
in tomato (Picton et al., 1993) are developmental processes that are
regulated by the interplay between putative age-related factors and
ethylene. In unpollinated pea ovaries, these age-related factors could
determine the sensitivity of ovary tissue to ethylene and the lack of
sensitivity of ovary tissue to plant-growth substances 5 d after
emasculation (García-Martínez and Carbonell, 1980).
The expression of SAMS genes in unpollinated pea ovaries at
the equivalent of 4 DPA was localized specifically in cells of the
embryo sac and in vascular cells, which were differentiating at this
time (Vercher et al., 1989) and in which SAM is necessary for lignin
biosynthesis. Expression of other genes related to the senescence
process has also been observed during the differentiation of vascular
cells (Granell et al., 1992; Nadeau et al., 1993; Tang et al., 1994), a
process related to senescence (Jones and Dangl, 1996).
In conclusion, SAMS1 and SAMS2 mRNAs are
differentially expressed during ovary development in pea. Ethylene
induction of SAMS1 mRNA and the lack of induction of
SAMS2 mRNA by ethylene-treated and parthenocarpic fruits
suggest the existence of different factors controlling SAMS
gene transcription. These findings raise the question of how this
separate action is established and maintained. Future research will
focus on the mechanism regulating SAMS1 and SAMS2
expression during ovary development in pea.
| |
FOOTNOTES |
1
This research was supported by grant no.
PB92-0018-C02-02 from Dirección General de Investigación
Científica y Tecnológica (Spain) to P.C. L.G.G. was
funded by the Conselleria d'Educació i Ciència de la
Generalitat Valenciana.
2
Present address: Friedrich Miescher-Institut,
Postfach 2543, CH-4002 Basel, Switzerland.
*
Corresponding author; e-mail pedro.carrasco{at}uv.es; fax
34-6-3864635.
Received November 28, 1997;
accepted February 26, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DPA, days postanthesis.
RPA, RNase protection
assay.
SAM, S-adenosyl-L-Met.
SAMS, S-adenosyl-L-Met synthase.
| |
ACKNOWLEDGMENTS |
The authors are grateful to Drs. A. Molowny and C. López-García for their assistance with tissue preparation
for the in situ hybridization experiments and to R. Martínez-Pardo and A. Villar for their technical assistance at
the greenhouse.
| |
LITERATURE CITED |
Abeles FB, Morgan PW, Saltveit ME (1992) Regulation of
ethylene production by internal, environmental, and stress factors.
In FB Abeles, PW Morgan, ME Salveit, eds, Ethylene in Plant
Biology, Ed 2. Academic Press, New York, pp 56-119
Bewley JD, Black M (1994) Seed development and maturation.
In JD Bewley, M Black, eds, Seed, Physiology of Development
and Germination, Ed 2. Plenum Press, New York, pp 35-115
Boerjan W,
Bauw G,
Van Montagu M,
Inzé D
(1994)
Distinct phenotypes generated by overexpression and suppression of S-adenosyl-L-methionine reveal developmental patterns of gene silencing in tobacco.
Plant Cell
6:
1401-1414
[Abstract]
Carbonell J,
García-Martinez JL
(1985)
Ribulose-1,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum.
Planta
164:
534-539
Davies KM,
Grierson D
(1989)
Identification of cDNA clones for tomato (Lycopersicom esculentum Mill). mRNAs that accumulate during ripening and leaf senescence.
Planta
179:
73-80
[CrossRef]
Dekeysen RA,
Claes B,
De Rycke R,
Mabets M,
Van Montagu M,
Caplan A
(1990)
Transient gene expression in intact and organized tissues.
Plant Cell
2:
591-602
[Abstract/Free Full Text]
Duck NB
(1994)
RNA in situ hybridisation in plants.
In
SB Gelvin,
RA Schilperoort,
eds, Plant Molecular Biology Manual, Ed 2.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 1-13
Eeuwens CJ,
Schwabe WW
(1975)
Seed and pod wall development in Pisum sativum, L. in relation to extracted and applied hormones.
J Exp Bot
26:
1-14
[Abstract/Free Full Text]
Espartero J,
Pintor-Toro JA,
Pardo JM
(1994)
Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress.
Plant Mol Biol
25:
217-227
[CrossRef][ISI][Medline]
Evans TP,
Malmberg RL
(1989)
Do polyamines have roles in plant development?
Annu Rev Plant Physiol Plant Mol Biol
40:
235-269
[ISI]
García-Martínez JL,
Carbonell J
(1980)
Fruit-set of unpollinated ovaries of Pisum sativum L. Influence of plant growth regulators.
Planta
147:
451-456
Gómez-Gómez L,
Carrasco P
(1996)
Hormonal regulation of S-adenosylmethionine synthetase transcripts in pea ovaries.
Plant Mol Biol
30:
821-832
[CrossRef][ISI][Medline]
Gowri G,
Bugos R,
Campbell W,
Maxwell C,
Dixon R
(1991)
Stress responses in alfalfa (Medicago sativa).
Plant Physiol
97:
7-14
[Abstract/Free Full Text]
Granell A,
Harris N,
Pisabarro AG,
Carbonell J
(1992)
Temporal and spatial expression of a thiol protease gene during pea ovary senescence, and its regulation by gibberellin.
Plant J
2:
907-915
[ISI][Medline]
Grbic V,
Bleecker A
(1995)
Ethylene regulates the timing of leaf senescence in Arabidopsis.
Plant J
8:
595-602
[CrossRef][ISI]
Heby O,
Persson L
(1990)
Molecular genetics of polyamine synthesis in eukaryotic cells.
Trends Biochem Sci
15:
153-158
[CrossRef][ISI][Medline]
Higuchi T
(1981)
Biosynthesis of lignin.
In
W Tanner,
FA Loewurs,
eds, Encyclopedia of Plant Physiology, Vol 138, New Series, Plant Carbohydrates II.
Springer-Verlag, Berlin, pp 194-224
Izhaki A,
Shoseyov O,
Weiss D
(1995)
A petunia cDNA encoding S-adenosyl-methionine synthase.
Plant Physiol
108:
841-842
[Medline]
Jones AM,
Dangl JL
(1996)
Logjam at the styx: programmed cell death in plants.
Trends Plant Sci
1:
114-119
[CrossRef][ISI]
Kawalleck P,
Plesch G,
Halhbrock K,
Somssich IE
(1992)
Induction of S-adenosyl-L-methionine synthetase and S-adenosyl-L-homocysteine hydrolase mRNAs in cultured cells and leaves of Petroselium crispum.
Proc Natl Acad Sci USA
89:
4713-4717
[Abstract/Free Full Text]
Kermade AR
(1990)
Regulatory mechanisms involved in the transition from seed development to germination.
CRC Crit Rev Plant Sci
9:
155-195
Kim CS,
Kwak JM,
Nam HG,
Kim K,
Cho BH
(1994)
Isolation and characterization of two cDNAs that are rapidly induced during the wound response of Arabidopsis thaliana.
Plant Cell Rep
13:
340-343
Lee JJ,
Costlow NA
(1987)
A molecular titration assay to measure transcript prevalence levels.
Methods Enzymol
152:
633-648
[ISI][Medline]
Mathur M,
Saluja D,
Sachar RC
(1991)
Post-translational regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos.
Biochim Byophys Acta
1078:
161-170
[Medline]
Mathur M,
Satpthy M,
Sachar RC
(1992)
Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.
Biochim Biophys Acta
1137:
161-170
Mathur M,
Sharma N,
Sachar RC
(1993)
Differential regulation of S-adenosylmethionine synthetase isozymes by gibberellic acid in dwarf pea epicotyls.
Biochim Biophys Acta
1162:
283-290
[Medline]
Nadeau JA,
Zhang XS,
Nair H,
ONeill SD
(1993)
Temporal and spatial regulation of 1-aminocyclopropane-1-carboxylate oxidase in the pollination-induced senescence of orchid flowers.
Plant Physiol
103:
31-39
[Abstract]
Peleman J,
Boerjan W,
Engler G,
Seurinck J,
Botterman J,
Alliotte T,
Van Montagu M,
Inzé D
(1989a)
Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosyl-methionine synthetase.
Plant Cell
1:
81-93
[Abstract/Free Full Text]
Peleman J,
Saito K,
Cottyn B,
Engler G,
Seurinck J,
Van Montagu M,
Inzé D
(1989b)
Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana.
Gene
84:
359-369
[CrossRef][ISI][Medline]
Pérez-Amador MA,
Carbonell J
(1995)
Arginine decarboxylase and putrescine oxidase in ovaries of Pisum sativum L.
Plant Physiol
107:
865-872
[Abstract]
Pérez-Amador MA,
Carbonell J,
Granell A
(1995)
Expression of arginine decarboxylase is induced during early fruit development and in young tissues of Pisum sativum L.
Plant Mol Biol
28:
997-1009
[CrossRef][ISI][Medline]
Picton S,
Barton SL,
Bouzayen M,
Hamilton AJ,
Grierson D
(1993)
Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene.
Plant J
3:
469-481
Prescott A,
Martin C
(1987)
A rapid method for the quantitative assessment of levels of specific mRNAs in plants.
Plant Mol Biol Rep
4:
219-224
Somssich JE,
Bollmann J,
Hahlbrock L,
Kombrink E,
Schultz W
(1989)
Differential early activation of defence-related genes in elicitor-treated parsley cells.
Plant Mol Biol
12:
227-234
Tabor CW,
Tabor H
(1984)
Methionine adenosyltransferase (S-adenosylmethionine synthetase) and S-adenosylmethionine decarboxylase.
Adv Enzymol
56:
251-282
Tang X,
Gomes AMTR,
Bhatia A,
Woodson WR
(1994)
Pistil-specific and ethylene-regulated expression of 1-aminocyclopropane-1-carboxylate oxidase genes in petunia flowers.
Plant Cell
6:
1227-1239
[Abstract]
Thomas D,
Surdin-Kerjan Y
(1987)
SAMS1, the structural gene for one of the S-adenosylmethionine synthetases in Saccharomyces cerevisiae.
J Biol Chem
262:
16704-16709
[Abstract/Free Full Text]
Tuomainen J, Betz C, Ernst D, Langebartels C, Sandermann H Jr,
Kangasjärvi J (1996) Ozone affects the ethylene biosynthetic
pathway at both biochemical and mRNA levels. NATO Advanced Research
Workshop. In Biology and Biotechnology of the Plant Hormone
Ethylene. Chania, Greece, p 33
Van Breusegem F,
Dekeyser R,
Gielen J,
Van Montagu M,
Kaplan A
(1994)
Characterization of a S-adenosylmethionine synthase gene in rice.
Plant Physiol
105:
1463-1464
[CrossRef][ISI][Medline]
Vercher Y,
Carbonell J
(1991)
Changes in the structure of ovary tissues and in the ultrastructure of mesocarp cells during ovary senescence or fruit development induced by plan growth substances in Pisum sativum.
Physiol Plant
81:
518-526
[CrossRef]
Vercher Y,
Carrasco P,
Carbonell J
(1989)
Biochemical and histochemical detection of endoproteolytic activities in ovary senescence or fruit development in Pisum sativum.
Physiol Plant
76:
405-411
Vercher Y,
Molowny A,
López C,
García-Martínez JL,
Carbonell J
(1984)
Structural changes in the ovary of Pisum sativum L. induced by pollination and gibberellic acid.
Plant Sci Lett
36:
87-91
[CrossRef]
Weiss D,
Halevy AH
(1989)
Stamens and gibberellin in the regulation of corolla pigmentation and growth in Petunia hibrida.
Planta
179:
89-96
Woodson WR,
Park KY,
Drory A,
Larsen PB,
Wang H
(1992)
Expression of ethylene biosynthetic pathway transcripts in senescing carnation flowers.
Plant Physiol
99:
526-532
[Abstract/Free Full Text]
Yang SF,
Hoffman NE
(1984)
Ethylene biosynthesis and its regulation in higher plants.
Annu Rev Plant Physiol
35:
155-189
[CrossRef][ISI]
Top
Abstract
Introduction
