Plant Molecular Biology 25: 217-227, 1994.

© 1994 Kluwer Academic Publishers. Printed in Belgium. 217

Differential accumulation of S-adenosylmethionine synthetase

transcripts in response to salt stress

Joaquin Espartero, Jos6 A. Pintor-Toro and Jos6 M. Pardo*

Instituto de Recursos Naturales y Agrobiologia, C.S.I.C. Apdo. 1052, Sevilla-41080, Spain (* author for
correspondence)

Received 3 December 1993; accepted in revised form 9 February 1994

Key words: AdoMet, Lycopersicon esculentum, salt stress, S A M genes, sequence

Abstract

NaCI stress causes the accumulation of several mRNAs in tomato seedlings. An upregulated c D N A
clone, SAM 1, was found to encode a S-adenosyl-L-methionine synthetase enzyme (AdoMet synthetase).
Expression of the c D N A SAM 1 in a yeast mutant lacking functional S A M genes resulted in high AdoMet
synthetase activity and AdoMet accumulation. We show that tomato plants contain at least four S A M
isogenes. Clones corresponding to isogenes SAM2 and SAM3 have also been isolated and sequenced.
they encode predicted polypeptides 95 ~o and 92 ~o identical, respectively, to the SAMl-encoded AdoMet
Synthetase. RNA hybridization analysis showed a differential response of S A M genes to salt and other
stress treatments. SAM1 and SAM3 mRNAs accumulated in the root in response to NaC1, mannitol
or ABA treatments. SAM1 m R N A accumulated also in leaf tissue. These increases of m R N A level were
apparent as soon as 8 h after the initiation of the salt treatment and were maintained for at least 3 days.
A possible role for AdoMet synthetases in the adaptation to salt stress is discussed.

Introduction donor in the biosynthesis of the polyamines sper-

S-adenosyl-L-methionine (AdoMet) is an impor-
tant metabolite participating in many cellular pro-
cesses. AdoMet is the major methyl group donor
in transmethylation of proteins, nucleic acids,
polysaccharides and fatty acids [42]. It is also an
important effector in the regulation of the biosyn-
thesis of threonine and methionine [ 12]. Decar-
boxylated AdoMet is the propylamino group
midine and spermine [46]. In addition to these
functions, AdoMet participates in metabolic
pathways specific to plant cells: it is a precursor
in ethylene biosynthesis [50] and it is required for
the biosynthesis of the phenylpropanoid constitu-
ents of the cell wall [16]. AdoMet is synthesized
from methionine and ATP by the enzyme AdoMet
synthetase (ATP:L-methionine S-adenosyltrans-
ferase, EC 2.5.1.6). AdoMet synthetase is an

The nucleotide sequence data reported will appear in the GenBank; EMBL and DDBJ Nucleotide Sequence Databases under
the accession numbers Z24741 (SAM1), Z24742 (SAM2) and Z24743 (SAM3).
218
ubiquitous enzyme, well conserved between bac-
teria, yeast, plants and mammals [45]. Genes en-
coding plant AdoMet synthetases have been iso-
lated from Arabidopsis thaliana and carnation [25,
33, 34]. A. thaliana contains two S A M genes that
have similar expression patterns. A chimeric
/%glucuronidase gene fused to the A. thaliana
SAM1 gene promoter showed, both in A. thaliana
and tobacco transgenic plants, a preferential ex-
pression in all vascular tissues, stem scleren-
chyma and root cortex [33, 34]. This expression
pattern led to the proposal that high levels of
AdoMet synthetase are required in lignifying tis-
sues. Accordingly, S A M m R N A and caffeic acid
3-O-methyltransferase (COMT) transcripts, a key
enzyme in lignin biosynthesis, exhibited similar
expression patterns at various stages of develop-
ment in alfalfa [14].
The process of adaptation to salt stress is ac-
companied by many physiological adjustments
that can be monitored as changes in the levels of
numerous proteins and mRNAs [ 18, 40]. An in-
creasing number of genes whose expression is
up-regulated by salt stress have been isolated.
Some of these genes correspond to proteins that
are apparently required because of their presump-
tive protective functions. Examples are the LEA-
or RAB-type proteins [ 13, 29] and the thaumatin-
like protein osmotin [24]. Other genes code for
enzymes that participate in metabolic processes
specifically invoked by salt stress, such as the
biosynthesis of the osmoprotective compounds
pinitol and proline [21, 47]. On the other hand,
genes encoding proteins which have central roles
in cellular metabolism under non-stressful envi-
ronmental conditions, change their expression
upon salt stress. This is the case for phospho-
enolpyruvate carboxylase [9], the 70 kDa subunit
of tonoplast (H+)ATPase [30] and a putative
(CaZ+)ATPase [35, 48]. We report here the mo-
lecular characterization of a gene family encoding
AdoMet synthetase, an essential enzyme in cel-
lular metabolism. The transcripts corresponding
to this gene family accumulate differentially in
response to salt and other stress treatments.
Materials and methods
Plant material and stress treatments
Tomato plants (Lycopersicon esculentum cv.
Rutgers) were germinated and grown as previ-
ously described [ 13]. Stress treatments were given
to 7-8-day old seedlings by moistening the filter
paper with NaC1 (5 g/l) and ABA (10 #M) solu-
tion. Hydroponically grown, 5-6 true-leaved
plants were stressed by adding NaC1 (10g/l),
mannitol (0.3 M) or ABA (50 ~M) directly to the
hydroponic solution; ABA treatments were com-
pleted by spraying 100/~M ABA onto the aerial
parts of the plants because a gradient in the re-
sponse to ABA was observed from roots to leaves
when the hormone was added to the hydroponic
solution [13]. Plants were wounded by clipping
several leaves with dialysis tubing clamps for 24 h.
RNA was extracted from 2-3 pooled plants [ 15 ].
When required, poly(A) + was purified using two
rounds of chromatography in oligo(dT)-ceUulose
columns. Total D N A was obtained from leaves
according to Taylor and Powell [43].
Differential screening, nucleic acid hybridizations
and sequencing
About 10 000 phage o f a c D N A library made from
tomato seedlings treated with 5 g/1 NaC1 and
10/~M ABA [13] were screened by differential
hybridization. [32p]-labelled first-strand cDNAs
synthesized from 0.5/~g poly(A) + RNA, obtained
from seedlings treated with 5 g/1 NaCI and 10 ktM
ABA and from control, untreated seedlings, were
used as probes [38]. Clones up-regulated by NaC1
and ABA treatment were selected and purified by
two more rounds of plating and screening.
Denatured total RNA was separated in agar-
ose-formaldehyde gels. Nucleic acids were trans-
ferred to Hybond-N membranes (Amersham)
and cross-linked with UV light. [32p]-labelled
D N A probes were synthesized with a random
primer labelling kit (Boehringer Mannheim
GmbH). Low-stringency hybridizations were
done at 35 °C in 40~o formamide, 5 x SSC, 2 x

Denhart's solution, 0.1~o SDS, 50 mM Tris/HC1
pH 7.4. Final washes were at 45 ° C in 0.2 x S SC,
0.1 ~o SDS. For high stringency, filters were hy-
bridized at 42 °C in 50~o formamide and washed
at 65 °C in 0.1 x SSC, 0.1~o SDS. Hybridiza-
tion signals from northern blots were quantified
by densitometric scanning of autoradiograms.
Relative signal intensities were normalized by re-
probing the blots with the G Eco RI fragment
from the radish 18S r R N A gene [ 10]. To use the
D N A and RNA blots in successive hybridiza-
tions, probes were stripped out as recommended
by manufacturer.
For the isolation of cognate S A M genes and
cDNAs, the tomato seedlings c D N A library (see
above) and a tomato genomic library constructed
in 2EMBL3 (kindly provided by Olga del Pozo)
were screened at low stringency with the [32p]_
labelled SAM 1 c D N A insert. Positive clones were
isolated and characterized by cross-hybridization
at high stringency and by restriction mapping.
D N A sequencing was performed after subclon-
ing internal restriction fragments into pBluescript
plasmids (Stratagene), and by generation of uni-
directional deletions with exonuclease III (Nested
Deletion Kit from Pharmacia). Sequences were
obtained from both strands using single- or
double-stranded templates with a T7 D N A poly-
merase sequencing kit from Promega. Sequence
analysis and comparisons were performed with a
VAX computer using the U W G C G software
package [ 11 ].
AdoMet synthetase expression in yeast, biochemical
activity and AdoMet pool determinations
A 1440 bp Eco RI-Eco RV fragment containing
the entire coding region of c D N A SAM1 was
cloned into the unique Xho I site of the yeast ex-
pression vector IB61 by blunt-end ligation. The
resulting plasmid was named pYTSAM1. Plas-
mid IB61 contains the promoter and transcrip-
tional termination signal for the PMA1 (plasma
membrane ATPase) gene of Saccharomyces cer-
evisiae [39] cloned into the vector YEp351 [ 17].
The AdoMet auxotroph yeast strain CC435 [44],
219
a saml sam2 double mutant, was transformed
with pYTSAM1 by the procedure of Ito etal.
[22]. For biochemical determinations, exponen-
tially growing yeast cells were harvested and
broken with glass beads in 75 m M Tris/HC1
pH 8.0, 1 0 m M MgCI2, 100mM KC1, 5 mM
2-mercaptoethanol, 0 . 1 m M PMSF. AdoMet
synthetase activity was measured in extraction
buffer without PMSF, in the presence of 2 mM
L-methionine (supplemented with [35S]-L-me-
thionine) and 20 mM Mg-ATP [6]. Protein con-
centration was measured by the method of Brad-
ford [3]. For determination of AdoMet cellular
pools, cells of CC435 (pYTSAM 1) and a control
strain ($288C) were grown in YPD medium to
absorbance 1.0 at 600 nm, washed with distilled
water by repeated centrifugations, and boiled for
15 min. The AdoMet content of the soluble frac-
tions were measured by HPLC as described [27].
Yeast manipulations and growth media followed
standard procedures [37].
Results
Isolation of a AdoMet synthetase cDNA
A c D N A library made from NaCl- and ABA-
treated tomato seedlings m R N A was screened by
differential hybridization, c D N A probes were de-
rived from poly(A) + RNA obtained from seed-
lings treated with 5 g/1NaC1 and 10 #M ABA and
from non-treated seedlings. One clone giving a
strong hybridization signal that was further in-
creased by salt and ABA treatment was selected
for detailed analysis. The complete nucleotide se-
quence of the c D N A insert was obtained from
both strands and compared to the GenBank/
EMBL data bank. The putative protein encoded
by the c D N A showed extensive sequence simi-
larity to AdoMet synthetases from bacteria [26],
yeast [44], mammals [19, 20] and plants [25, 34].
Therefore, the c D N A isolated was named SAM 1.
Expression of SAM1 cDNA in yeast
To confirm that the c D N A SAM1 actually en-
coded a functional AdoMet synthetase homo-
220

logue, a 1440 bp Eco RI-Eco RV fragment com-
prising the entire coding region was subcloned
into plasmid IB61. This construct (pYTSAM1)
resulted in the expression of SAM 1 from the con-
stitutive yeast plasma membrane (H +)ATPase
PMA1 gene promoter. The yeast strain CC435, a
saml sam2 double mutant which requires
AdoMet for growth, was transformed with plas-
mid pYTSAM1. CC435(pYTSAM1) transfor-
mants were unable to form normal colonies in
selective synthetic medium plates (YNB), but they
developed healthy colonies when spread on rich
medium or synthetic medium supplemented with
methionine 20/~g/ml. These results indicated that
a complementation of the AdoMet auxotrophy of
CC435 cells had occurred but the expression of
c D N A SAM1 induced a new auxotrophy for me-
thionine. Measurements of AdoMet synthetase
activities in cell-flee protein extracts demon-
strated a 19-fold higher specific activity in
CC435(pYTSAM1) than in the wild-type
yeast strain $288C (specific activities were
4.7 and 0.25 nmol AdoMet per minute per mg
protein, respectively). Furthermore, the cellular
pool of AdoMet was 12-fold higher in
CC435(pYTSAM1) than in S'288C (2.4 versus
0.2 nmol per ml of culture). Together, these re-
sults confirmed that the tomato AdoMet syn-
thetase encoded by the c D N A SAM 1 was func-
tionally active in yeast cells.
Southern analysis of the SAM gene family
To estimate the number of genes encoding SAM
proteins, total D N A of tomato plants was di-
gested with various restriction endonucleases and
subjected to Southern blot analysis. Blotted re-
striction fragments were hybridized to [32p]_
labelled SAM 1 c D N A under low-stringency con-
ditions. Besides the restriction fragments that
gave strong signals in the autoradiogram, a set of
bands hybridizing weakly indicated that there
were D N A sequences homologous, but distinct,
to SAM1. Hence, we used the SAM1 probe and
the same relaxed hybridization conditions to iso-
late more cognate SAM cDNAs from the seed-

Fig. l. D N A blot analysis of the S A M gene family. Ten/~g of total D N A from tomato, digested with Eco RI (E), Eco RV (V), Nco I
(N) or Stu I (S), and 18 pg of S A M 1 c D N A (C) were electrophoresed, blotted and hybridized successively to probes derived from
S A M 1 (panels A and B), S A M 2 (panel C) and S A M 3 (panel D), under low (panel A) or high stringency (panels B, C and D) (see
Materials and methods). Arrowheads in panel A indicate hybridizing restriction fragments that were not assigned to S A M 1 , S A M 2
or S A M 3 genes.
 221

ling library. Fifty-five positive c D N A clones were under low stringency was hybridized successively
obtained out of 100 000 p.f.u, screened. They were to SAM1, SAM2 and SAM3 c D N A probes,
grouped in three classes according to cross- using high stringency that ensured gene-specific
hybridization under high-stringency conditions: hybridizations. The results (Fig. 1) clearly showed
50 c D N A clones belonged to the same class as that all except one of the restriction fragments
c D N A SAM1, whereas the five remaining iso- previously seen at low stringency (panel A) could
lates were named SAM2 (one clone) and SAM3 be assigned to either SAM1, SAM2 or SAM3
(four clones). This nomenclature is arbitrary and genes for every endonuclease used, suggesting that
not based on relatedness to previously described the AdoMet synthetase gene family is composed
SAM genes in other organisms. The nucleotide by at least four members. Attempts to isolate the
sequences of c D N A s SAM2 and SAM3 con- putative SAM4 gene from a tomato genomic
firmed that they encoded new AdoMet synthetase library were unsuccessful, as we could only iso-
proteins (see below). late clones corresponding to SAM1 and SAM2
The Southern blot probed with c D N A SAM 1 genes (data not shown).

1 50
SAM1 METFLFTSESVNEGHPDKLCDQISDAVLDACLEQDPESKVACETCTKTNL
SAM2 ...................... V ......... A .................
SAM3 .. .................... V...I ...................... M

51 i00
SAM1 VMVFGEITTKAIVDYEKIVRDTCRNIGFVSDDVGLD~NCKVLVYIEQ~
SAM2 ........... NI ........... E ..... P ......... R...N .....
SAM3 ........... T ............ G ..... A ............. N .....

i01 150
SAM1 PDIA~GVHGHLTKRPEEIGAGDQGH~GYATDETPELMPLSHVLATKLGA
SAM2
S~3 iiiiiiiii[iiiiiiiiiiii[[iiiiiiiiiiiiiiilT~iiiiii[i
151 200
SAM1 R L T E V R K N=G T=C A W L R P D G K T Q V T V E Y S N D N G A M V P I =
RVHTVLISTQHDET =__
SAM2 ........... S .............. H ........ L ..............
SAM3 K ....... K..P .............. K .......................

201 250
SAM1 VTNDEIARDLKEHV~KPVI~EKYLDENTIFHLNPSGRFVIGGPHGDAGLT
SAM2
SAM3 .... Q..Q ............ A .............................

251 300
SAM1 GRKIII~TYGGWGAHGGGAFSGKDPTKVDRSGAYIVR_QAA_KSIVASGLAR
SAM2 ............................................. N ....
SAM3 .......................................... V .......

301 350
SAM1 BCIV~VSYAIGVPEPLS~VDTYG~GKIPDREILKIVKENFDFR~GMMSI
SAM2 .............................. K . . . N . . . . . . . . . . . . I..

SAM3 ............ A .......... K..T...KD..VLI .............

351 393
SAM1 N L- D -L K R G G N R R F L K T A A Y G H F G=R D D P D F T W E W K P L K =W E K P Q D= =

SAM2 .... L .... G ............................ D..EA

SAM3 .... L .... Y.YQ .................. T..V..---.KA

Fig. 2. Amino acid sequence comparison of predicted polypeptides for SAM1, SAM2 and SAM3 genes. Sequences were aligned
using the PILEUP program of the U W G C G software package. Amino acid differences predicted for SAM2 and SAM3 polypep-
tides are shown beneath the SAM1 sequence. Underlined residues in SAM1 indicate fully conserved amino acids in all the known
sequences of AdoMet synthetases. Dashes in SAM3 represent a gap.
222

Tomato AdoMet synthetase isoforms

We sequenced full-length c D N A clones of SAM1

and SAM3 genes (1479 bp and 1455 bp, respec-
tively) and a 1651 bp Eco RI-Eco RV fragment
containing the SAM2 gene isolated from the to-
mato genomic library. No introns were found in
the SAM2 genomic sequence. The nucleotide se-
quence of the coding region of c D N A SAM 1 was
83 ~o and 80 ~o identical to those of the gene SAM2
and the c D N A SAM3, respectively. SAM2- and
SAM3-coding regions were 77~o identical. No
significant homology existed between flanking
sequences. The comparison of deduced amino
acid sequences of the tomato AdoMet synthetase
isoforms showed that they are highly similar pro-
teins (Fig. 2). SAM1 and SAM2 are the most
closely related protein isoforms: both have 393 Fig. 3. RNA gel blot analysis of S A M transcripts in control
amino acid residues which are 95 ~o identical (only and NaCl-stressed tomato plants. Total RNA (20 #g) purified
20 amino acid substitutions, 9 of them being con- from root, stem and leaves of control plants (C) or maintained
in 10 g/1 NaCI for 24 h (S) was separated by denaturing form-
servative changes). The protein isoform deduced
aldehyde electrophoresis, transferred to nylon membrane and
for the SAM3 gene is 390 residues long and is hybridized with SAM1, SAM2, SAM3 and 18S rRNA gene
92~o and 91.3~o identical to SAM1 and SAM2 probes under conditions of high stringency. SAMI, SAM2 and
proteins, respectively. The three deduced poly- SAM3 mRNA levels are not directly comparable, because
peptides contain all the amino acid sequences different exposure times were necessary to obtain similar hy-
bridization signals.
fully conserved in other AdoMet synthetases from
bacteria to humans (Fig. 2).

of SAM1 m R N A s was higher in stem (Fig. 3),

Differential accumulation of SAM transcripts in
response to stress
Northern blot analysis was used to investigate the
steady-state levels of S A M m R N A s in roots,
stems and leaves from control and stressed to-
mato plants. SAM1, SAM2 and SAM3 c D N A
probes were used to detect their corresponding
m R N A s under gene-specific hybridizations (see
Fig. 1). All three probes detected m R N A species
1.6 kb in size, in agreement with their c D N A s
sizes (Fig. 3). The different exposure times nec-
essary to obtain similar autoradiogram signals in-
dicated that the m R N A abundance varied greatly
between the members of the gene family. SAM1
m R N A was the most abundant transcript, and
SAM2 m R N A the least abundant of the three
mRNAs. In control plants, the steady-state levels
whereas SAM3 is preferentially expressed in aerial
parts and SAM2 m R N A is more abundant in the
root.
We examined the effect of NaC1 stress in the
expression of the S A M gene family. Treatment
with 10 g/1 NaC1 for 24 h resulted in a differential
response of S A M genes (Fig. 3). Salt stress pro-
moted a significant accumulation of SAM1 and
SAM3 transcripts in roots (10- and 7-fold, respec-
tively). SAM1 m R N A level also increased 2-fold
in leaves of stressed plants, whereas SAM3
m R N A level dropped 7-fold when compared to
control leaves. SAM2 did not clearly respond to
salt stress.
Genes may be induced by salt stress as an early
response aimed to overcome the initial salt and
osmotic shocks or as the result of a long-term
adaptation to new growth conditions. Hence, we

investigated the changes in SAM m R N A content
in tomato plants over different periods of salt
stress. Plants were transferred to 10 g/l NaC1 and
total R N A was extracted from root, stem, and
leaves, after 8 h of treatment, when plants still
showed symptoms of salt shock-induced wilting;
after 24h, when wilting diminished; and after
72 h, well after plants had recovered turgor.
Gene-specific m R N A levels were determined by
high-stringency hybridizations to SAM 1, SAM2
and SAM3 probes and densitometric scanning of
the autoradiograms. Figure 4 shows a graphic
representation of the results obtained. Samples
corresponding to 24 h gave results comparable to
those shown in Fig. 3, although the extent of
m R N A content changes were distinct. The in-
creased accumulation of SAMI m R N A in root
and leaves and that of SAM3 transcripts in roots
were observable at 8 h, and augmented with time.
On the contrary, the decreases of SAM2 and
SAM3 m R N A contents in leaves were transient
responses. Their relative levels returned to con-
trol values after 24 h, correlating with the recov-
3 6
2 [] 8h,
[] 24h.
C
• 72h.
-2
-3
-4
-5
R S L R S L R S L
SAM1 SAM2 SAM3
Fig. 4. Time-course of changes in S A M transcripts levels in
NaCl-stressed plants. Tomato plants were transferred to hy-
droponic solution containing 10 g/l NaC1 and collected after
indicated times. R N A was purified from root (R), stem (S) and
leaves (L), and 12#g were electrophoresed in agarose-
formaldehyde gels, blotted onto nylon membrane and hybrid-
ized with SAM1, SAM2 and SAM3 probes under high strin-
gency. R N A loading was normalized by hybridization with a
radish 18S rRNA gene probe. Hybridizations signals were
quantified by densitometry. The ordinate indicates the number
of times that the R N A level increases (positive values) or
decreases (negative values) relative to control samples ob-
tained from non-treated plants (baseline C, value i).
223
ery of turgor. N o significant changes of SAM
m R N A levels were noticed in stem of NaC1-
stressed plants.
The effect of salt stress on the expression of
SAM genes could be due to a combination of ion
toxicity and osmotic stress. In addition, osmotic
stress has been shown to induce the synthesis of
ABA that in turn activates a large number of
osmotically regulated genes [41]. Hence, we in-
vestigated whether changes in SAM m R N A s
levels were also induced by water stress and ABA
treatment. Both, mannitol (0.3 M) and ABA
(50/~M) induced an up-regulation of SAM1 and
SAM3 transcripts similar to that of NaC1 (Fig. 5).
However, quantitative differences in m R N A ac-
cumulation were noticed. In the root, NaC1 was
more effective than mannitol and ABA in elicit-
ing the accumulation of SAM1 and SAM3
mRNAs. As observed for NaC1, mannitol and
ABA treatments also resulted in a down-
regulation of SAM2 and SAM3 m R N A levels in
leaves. These decreases were still significant after
24 h, in contrast to the shorter response elicited
by NaC1. Furthermore, mannitol and ABA treat-
ments resulted in a down regulation of SAM2
5
4
• NaCl
[] Mannitoi
[] ABA
[] Wounding
c • I
-2
-3
R S L R S L R S L
SAM1 SAM2 SAM3
Fig. 5. S A M m R N A level changes in plants subjected to vari-
ous stresses. Tomato plants were transferred to hydroponic
solution supplemented with 10 g/1 NaC1, 0.3 M mannitol or
50/~M ABA, or wounded by clipping their leaves. After 24 h,
R N A was purified from root (R), stem (S) and leaves (L) and
processed as described in Fig. 4. The ordinate indicates the
number of times that R N A level increases (positive values) or
decreases (negative values) relative to control samples ob-
tained from untreated plants (baseline C, value 1), in response
to indicated treatments.
224
m R N A in the root, an effect not observed with
NaC1.
Recent reports have shown that S A M tran-
script levels are influenced by fungal-elicitor treat-
ments in alfalfa and parsley cell cultures [ 14, 23],
suggesting a role for AdoMet synthetase in plant
defense against pathogen attack. Because wound-
ing increases expression of most plant defense-
related genes, we also investigated the response of
the S A M gene family to wounding. Only small
changes in steady-state levels of S A M m R N A s
contents were observed, except for the significant
S A M 3 m R N A increase found in root (Fig. 5).
Discussion
Differential screening for tomato c D N A s up-
regulated by salt stress resulted in the isolation of
the c D N A SAM1. We have demonstrated that
this c D N A encodes a functional AdoMet syn-
thetase enzyme by complementation of a yeast
mutant deficient in AdoMet synthetase activity.
Expression o f c D N A SAM1 resulted in a 19-fold
higher specific enzymatic activity to that found in
wild-type S. cerevisiae cells. This over-expression
relieved the CC435 mutant of AdoMet auxotro-
phy but induced a methionine auxotrophy. The
biosynthesis of methionine and AdoMet in S. cer-
evisiae are under negative regulation by AdoMet,
both by inhibition of the activity of biosynthetic
enzymes, and by transcriptional repression of
their genes [ 5, 28, 44]. Our results suggest that the
expression of the plant AdoMet synthetase en-
zyme represses the biosynthesis of methionine in
transformed yeast cells, probably as a result of the
large increase in AdoMet (12-fold over the wild-
type cells). Although it has been reported that the
plant AdoMet synthetase is feedback inhibited by
its product [12], the enzyme encoded by the to-
mato c D N A SAM 1 does not appear to be inhib-
ited by AdoMet. Alternatively, other unknown
factors modulating the regulation o f A d o M e t syn-
thetases by its product may not recognize the
plant enzyme when expressed in yeast cells.
Southern blot analysis of total D N A revealed
the presence of at least four genes encoding
AdoMet synthetases in tomato. SAM1, S A M 2
and S A M 3 genes are expressed in seedlings (their
c D N A s were isolated from a seedling library),
and in 5-6 true-leaved plants (Fig. 3). Although
the relative abundance of different S A M tran-
scripts was not measured, it appeared from
northern blot signals that SAM1 is the most abun-
dant transcript in tomato plants, thus probably
accounting for the majority of the AdoMet syn-
thetase activity. Because of the lack of a specific
probe, we do not know if the putative S A M 4 gene
is expressed. A. thaliana plants have two S A M
genes that share similar expression patterns, both
showing a preferential expression in stems and
roots [34]. In contrast, the tomato S A M tran-
scripts show different relative abundances in dis-
tinct organs (Fig. 3).
The AdoMet synthetase activity has been long
regarded as a 'housekeeping' function, providing
a methyl group donor (AdoMet) for the numer-
ous transmethylation reactions that take place
into the cell. However, the abundance of S A M
transcripts change significantly in response to
fungal elicitor treatment in alfalfa and parsley [ 14,
23], and during the ethylene-dependant senes-
cence of carnation petals [49]. In addition, we
show that NaC1, osmotic stress and ABA treat-
ment induce changes in S A M m R N A levels in
both a gene- and organ-specific manner. S A M 2
and S A M 3 m R N A levels decreased in leaves upon
salt stress, but the time course of this reduction
indicated that they were transient responses that
might correlate with salt-induced wilting. Their
relative abundance were minimal at 8 h and re-
turned to control values at 24 and 72 h, coinci-
dent with the recovery of turgor in plants trans-
ferred to NaC1. Mannitol and ABA treatments
elicited similar changes in m R N A levels that per-
sisted after 24 h, suggesting that ABA might be
involved in these responses. In contrast, the in-
crease in S A M m R N A abundance upon salt
stress was a sustained response, with maximal
accumulation at 72 h after the onset of stress.
This persistence, and the fact that mannitol and
ABA also elicited a similar pattern of transcript
accumulation, suggest that AdoMet synthetases
were required for plant adaptation to salt and

osmotic stresses, and that ABA might participate
in the up-regulation of S A M transcripts.
Because of the variety of cellular processes in
which AdoMet is involved, it is difficult to assign
a definite role for AdoMet synthesis in plant
stress. Nevertheless, a hypothesis can be drawn
from the use of AdoMet in plants. AdoMet syn-
thetase is preferentially expressed in tissues un-
dergoing lignification under non-stressful environ-
mental conditions [33, 34]. Because lignin
monomers need to be methylated before polymer-
ization, AdoMet may be consumed in cells that
produce large amounts of lignin [16]. The coor-
dinate induction by fungal elicitors of AdoMet
synthetase with S-adenosylmethionine:caffeic
acid 3-O-methyltransferase (COMT) and S-
adenosyl-L-homocysteine hydrolase (SHH)
mRNAs, enzymes that are required for cell wall
formation, strongly support the hypothesis of an
increased demand of AdoMet for lignin biosyn-
thesis [14, 23]. Furthermore, AdoMet is also
needed for methylation of other derivatives of the
phenylpropanoid pathway, such as isoflavones
and chalcones [14]. The abundance of those
compounds and other cell wall proteinaceus con-
stituents and enzymes is modified in plants by
various stresses, including water stress (which is
an effect of high salinity) [2, 4, 7, 32]. Accord-
ingly, the physical properties of the cell wall
change at low water potentials [31 ]. Also, accel-
erated lignification occurs in roots of water
stressed sorghum and salt stressed maize plants
[1, 8]. We suggest that the expression of S A M
genes may be induced upon salt stress because a
greater demand of AdoMet is imposed by
increased cell wall synthesis or modification, es-
pecially in roots where the highest accumulation
of S A M transcripts was found.
Other explanations are also possible, although
less likely. Increased ethylene biosynthesis is a
general response of all plant tissues to many en-
vironmental stresses, including water stress [50].
Because the main control step in ethylene pro-
duction is the conversion of AdoMet to ACC, it
could be argued that an accelerated synthesis of
ethylene might demand a greater supply of
AdoMet. However, Yang and Hoffman [ 50] have
225
reasoned that it is unlikely that AdoMet syn-
thetase becomes a rate-limiting enzyme in ethyl-
ene biosynthesis. Further, massive ethylene bio-
synthesis in carnation was not accompanied by
an increase in S A M m R N A abundance [49].
AdoMet also participates in the biosynthesis of
the polyamines spermidine and spermine [46].
However, polyamine biosynthesis is regulated
mainly through arginine decarboxylase and S-
adenosylmethionine decarboxylase, and decar-
boxylated AdoMet constitutes the rate-limiting
factor in the synthesis of spermidine and sper-
mine. In addition, Priebe and Jager [36] did not
find significant changes of polyamines in several
salt stressed plants. Therefore, it seems unlikely
that polyamine biosynthesis accounts for the in-
duction of S A M transcripts in salt stressed to-
mato.
In conclusion, our results show that S A M genes
are responsive to salt stress and other related
treatments in a gene- and organ-specific manner.
Current studies are aimed to correlate m R N A
abundance with protein content and to the his-
tochemical localization of SAM proteins in salt
stressed plants.
Acknowledgements
We thank Dr F. Portillo for providing the plas-
mid IB61, Dr Y. Surdin-Kerjan for the yeast
strain CC435, Dr E. Martinez-Force for the
analysis of AdoMet pools, and Imelda Mendoza
for technical assistance. We also thank Drs
J. Jordano, A. Aguilera and K.G. Raghothama
for critical reading of the manuscript. This work
was supported by Grant AGR91-858 from
CICYT to J.M.P.
References
1. Azaizeh H, Steudle E: Effects of salinity on water trans-
port of excised maize (Zea mays L.) roots. Plant Physiol
97:1136-1145 (1991).
2. Bozarth CS, Boyer JS: Cell wall proteins at low water
potentials. Plant Physiol 85:261-267 (1987).
3. Bradford MM: A rapid and sensitive method for the
226
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem 72:
248-254 (1976).
4. Gassab GI, Varner JE: Cell wall proteins. Annu Rev
Plant Physiol Plant Mol Biol 39:321-353 (1988).
5. Cherest H, Surdin-Kerjan Y, Antoniewski J, Robinson-
Szulmajster H: S-adenosyl methionine-mediated repres-
sion of methionine biosynthetic enzymes in Saccharomy-
ces cerevisiae. J Bact 114:928-933 (1973).
6. Chou TC, Lombardini JB: A rapid assay procedure for
ATP:L-methionine adenosyl transferase. Biochim Bio-
phys Acta 276:399-406 (1972).
7. Creelman RA, Mullet JE: Water deficit modulates gene
expression in growing zones of soybean seedlings. Analy-
sis of differentially expressed cDNAs, a new fl-tubulin
gene, and expression of genes encoding cell wall proteins.
Plant Mol Biol 17:591-608 (1991).
8. Cruz RT, Jordan WR, Drew MC: Structural changes and
associated reduction of hydraulic conductance in roots of
Sorghum bicolor L. following exposure to water deficit.
Plant Physiol 99:203-212 (1992).
9. Cushman JC, Meyer G, Michalowski CB, Schmitt JM,
Bohnert H J: Salt stress leads to differential expression of
two isogenes of phosphoenolpyruvate carboxylase during
crassulacean acid metabolism induction in the common
ice plant. Plant Cell 1:715-725 (1989).
10. Delcasso-Tremousaygue D, Grellet F, Panabieres F,
Ananiev ED, Delseny M.: Structural and transcriptional
characterization of the external spacer of a ribosomal
RNA nuclear gene from a higher plants. Eur J Biochem
172:767-776 (1988).
11. Devereux J, Haeberli P, Smithies O: A comprehensive set
of sequence analysis programs for the VAX. Nucl Acids
Res 12:387-395 (1984).
12. Giovanelli J, Mudd SH, Datko AH: Sulfur amino acids
in plants. In: Miflin BJ (ed) The Biochemistry of Plants:
A Comprehensive Treatise, vol. 5, Amino Acids and De-
rivatives, pp. 453-505. Academic Press, New York
(1980).
13. Godoy JA, Pardo JM, Pintor-Toro JA: A tomato c D N A
inducible by salt stress and abscisic acid: nucleotide se-
quence and expression pattern. Plant Mol Biol 15: 695-
705 (1990).
14. Gowri G, Bugos RC, Campbell WH, Maxwell CA, Dixon
RA: Stress responses in Alfalfa (Medicago sativa L.). Mo-
lecular cloning and expression of S-adenosyl-L-methion-
ine: caffeic acid 3-O-methyltransferase, a key enzyme in
lignin biosynthesis. Plant Physiol 97:7-14 (1991).
15. Hall TC, Ma Y, Buchbinder BU, Pyne JW, Sun SM,
Bliss FA: Messenger RNA for G1 protein of french bean
seeds: cell-free translation and product characterization.
Proc Natl Acad Sci USA 75:3196-3200 (1978).
16. Higuchi T: Biosynthesis of lignin. In: Tanner W, Loewus
FA (eds) Plant Carbohydrates II. Encyclopedia of Plant
Physiology, New Series Vol 13B, pp. 194-224. Springer-
Verlag, Berlin (1981).
17. Hill JE, Myers AM, Koerner TJ, TzagoloffA: Yeast/
E. coli shuttle vectors with multiple unique restriction
sites. Yeast 2:163-167 (1986).
18. Ho TY, Mishkind ML: The influence of water deficits on
mRNA levels in tomato. Plant Cell Envir 14:67-75
(1991).
19. Horikawa S, Sasuga J, Shimizu K, Ozasa H, Tsukada K:
Molecular cloning and nucleotide sequence of c D N A en-
coding the rat kidney S-adenosylmethionine synthetase. J
Biol Chem 265:13683-13686 (1990).
20. Horikawa S, Tsukada K: Molecular cloning and nucle-
otide sequence of cDNA encoding the human liver
S-adenosylmethionine synthetase. Biochem Int 25:81-90
(1991).
21. Hu CA, Delauney AJ, Delauney AJ, Verma DPS: A bi-
functional enzyme (A-pyrroline-5-carboxylate synthetase)
catalyzes the first two steps in proline biosynthesis in
plants. Proc Natl Acad Sci USA 89:9354-9358 (1992).
22. Ito H, Fukuda I, Murata K, Kimura A: Transformation
of intact yeast cells treated with alkali cations. J Bact 153:
163-168 (1983).
23. Kawalleck P, PleschG, Hahlbrock K, Somssich I: In-
duction by fungal elicitor of S-adenosyl-L-methionine
synthetase and S-adenosyl-L-homocysteine hydrolase
mRNAs in cultured cells and leaves of Petroselinum
crispum. Proc Natl Acad Sci USA 89:4713-4717 (1992).
24. King GJ, Turner VA, Hussey CE, Wurtele ES, Lee SM:
Isolation and characterization of a tomato c D N A clone
which codes for a salt-induced protein. Plant Mol Biol 10:
401-412 (1988).
25. Larsen PB, Woodson WR: Cloning and nucleotide se-
quence ofa S-adenosylmethionine synthetase cDNA from
carnation. Plant Physiol 96:997-999 (1991).
26. Markham JD, DeParasis J, Gatmaitan J: The sequence
of MetK, the structural gene for S-adenosylmethionine
synthetase in Escherichia coli. J Biol Chem 259: 14505-
14507 (1984).
27. Martinez-Force E, Benitez T: Separation of O-phthalal-
dehyde derivatives of amino acids of the internal pool of
yeast by reverse-phase liquid chromatography. Biotech-
nol Tech 5:209-214 (1991).
28. Mountain HA, BystrOm AS, Larsen JT, Korch C: Four
major transcriptional responses in the methionine/
threonine biosynthetic pathway of Saccharomyces cerevi-
siae. Yeast 7:781-803 (1991).
29. Mundy J, Chua NH: Abscisic acid and water-stress in-
duce the expression of a novel rice gene. EMBO J 7:
2279-2286 (1988).
30. Narasimhan ML, Binzel ML, Perez-Prat E, Chen Z, Nel-
son DE, Singh NK, Bressan RA, Hasegawa PM: NaC1
regulation of tonoplast ATPase 70-kilodalton subunit
mRNA in tobacco cells. Plant Physio197:562-568 (1991).
31. Nonami H, boyer JS: Wall extensibility and cell hydraulic
conductivity decrease in enlarging stem tissues al low
water potentials. Plant Physiol 93:1610-1619 (1990).
32. Oliveira DE, Seurinck J, Inz6 D, Van Montagu M, Bot-

terman J: Differential expression of five Arabidopsis genes
encoding glycine-rich proteins. Plant Cell 2:427-436
(1990).
33. Peleman J, Boerjan W, Engler G, Seurinck J, Botter-
man J, Alliotte T, Van Montagu M, Inze D: Strong cel-
lular preference in the expression of a housekeeping gene
of Arabidopsis thaliana encoding S-adenosylmethionine
synthetase. Plant Cell 1:81-93 (1989).
34. Peleman J, Saito H, Cottyn B, Engler G, Seurinck J, Van
Montagu M, Inze D: Structure and expression analyses
of the S-adenosylmethionine synthetase gene family in
Arabidopsis thaliana. Gene 84:359-369 (1989).
35. Perez-Prat E, Narasimhan ML, Binzel ML, Botella M,
Chen Z, Valpuesta V, Bressan RA, Hasegawa PM: In-
duction of a putative C a 2 + - A T P a s e mRNA in NaCI-
adapted cells. Plant Physiol 100:1471-1478 (1992).
36. Priebe A, J~iger HJ: Effect of NaCI on the levels of pu-
trescine and related polyamines in plants differing in salt
tolerance. Plant Sci Lett 12:365-369 (1978).
37. Rothstein R: Cloning in yeast. In: Glover DM (ed) DNA
cloning, vol. II, pp. 45-66. IRL Press, Oxford (1985).
38. Schwartz-Sommer Z, Gierl A, Cuypers H, Peterson PA,
SaedlerH: Plant transposable elements generate the
DNA sequence diversity needed in evolution. EMBO J 4:
591-597 (1985).
39. Serrano R, Kielland-Brandt MC, Fink GR: Yeast plasma
membrane ATPase is essential for growth and has ho-
mology with (Na+/K ÷ ), K +, and Ca2+-ATPases. Nature
319:689-693 (1986).
40. Singh NE, Handa AK, Hasegawa PM, Bressan RA: Pro-
teins associated with adaptation of cultured tobacco cells
to NaC1. Plant Physiol 79:126-137 (1985).
41. Skriver K, Mundy J: Gene expression in response to ab-
scisic acid and osmotic stress. Plant Cell 2:503-512
(1990).
227
42. Tabor CW, Tabor H: Methionine adenosyltransferase
(S-adenosylmethionine synthetase) and S-adenosylme-
thionine decarboxylase. Adv Enzymol 56:251-282
(1984).
43. Taylor B, Powell A: Isolation of plant DNA and RNA.
Focus 4 : 4 - 6 (1982).
44. Thomas D, Rothstein R, Rosenberg N, Surdin-Kerjan Y:
SAM2 encodes the second methionine S-adenosyl trans-
ferase in Saccharomyces cerevisiae: physiology and regu-
lation of both enzymes. Mol Cell Biol 8:5132-5139
(1988).
45. Thomas D, Surdin-Kerjan Y: The synthesis of the two
S-adenosyl-methionine synthetases is differently regulated
in Saccharomyces cerevisiae. Mol Gen Genet 226: 224-
232 (1991).
46. Tiburcio AF, Kaur-Sawhney R, Galston AW: Polyamine
metabolism. In: Miflin BJ, Lea PJ (eds) The Biochemis-
try of Plants: A Comprehensive Treatise, vol. 16, Inter-
mediary Nitrogen Metabolism, pp. 283-325. Academic
Press, Orlando, FL (1990).
47. Vernon DM, Bohnert HJ: A novel methyl transferase
induced by osmotic stress in the facultative halophyte
Mesembryanthemum crystallinum. EMBO J 11: 2077-
2085 (1992).
48. Wimmers LE, Ewing NN, Bennett AB: Higher plant
Ca2+-ATPase: primary structure and regulation of
mRNA abundance by salt. Proc Natl Acad Sci USA 89:
9205-9209 (1992).
49. Woodson WR, Park KY, Drory A, Larsen PB, Wang H:
Expression of ethylene biosynthetic pathway transcripts
in senescing carnation flowers. Plant Physiol 99:526-532
(1992).
50. Yang SF, Hoffman NE: Ethylene biosynthesis and its
regulation in higher plants. Annu Rev Plant Physiol 35:
155-189 (1984).