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Plant Physiol. (1998) 117: 273-281
Dimethylsulfoniopropionate Biosynthesis in
Spartina
alterniflora1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results & Discussion
References
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The osmoprotectant
3-dimethylsulfoniopropionate (DMSP) occurs in Gramineae and Compositae,
but its synthesis has been studied only in the latter. The DMSP
synthesis pathway was therefore investigated in the salt marsh grass
Spartina alterniflora Loisel. Leaf tissue metabolized
supplied [35S]methionine (Met) to
S-methyl-l-Met (SMM),
3-dimethylsulfoniopropylamine (DMSP-amine), and DMSP. The
35S-labeling kinetics of SMM and DMSP-amine indicated that
they were intermediates and, consistent with this, the
dimethylsulfonium moiety of SMM was shown by stable isotope labeling to
be incorporated as a unit into DMSP. The identity of DMSP-amine, a
novel natural product, was confirmed by both chemical and mass-spectral
methods. S. alterniflora readily converted supplied
[35S]SMM to DMSP-amine and DMSP, and also readily
converted supplied [35S]DMSP-amine to DMSP; grasses that
lack DMSP did neither. A small amount of label was detected in
3-dimethylsulfoniopropionaldehyde (DMSP-ald) when
[35S]SMM or [35S]DMSP-amine was given.
These results are consistent with the operation of the pathway Met 
SMM DMSP-amine DMSP-ald DMSP, which differs from that found
in Compositae by the presence of a free DMSP-amine intermediate. This
dissimilarity suggests that DMSP synthesis evolved independently in
Gramineae and Compositae.
DMSP is a sulfonium betaine accumulated by many marine algae
(Blunden and Gordon, 1986 DMSP is also physiologically important. Like its analog, Gly betaine,
DMSP functions as a compatible solute for enzymes in vitro (Gröne
and Kirst, 1991 The biosynthesis of DMSP from Met has been partially elucidated in the
Compositae W. biflora (Hanson et al., 1994
The DMSP synthesis pathway has also been studied in the marine
macroalga Enteromorpha intestinalis (L.) Link and in three microalgae (Gage et al., 1997 Spartina alterniflora Loisel. and Spartina
patens (Ait.) Muhl. were collected in Florida from coastal marshes
in Crescent Beach and Archie Creek (east Tampa Bay), respectively.
Blocks of soil (3-5 dm3) containing plants were
dug up and transferred to undrained plastic containers. The plants were
then maintained for up to 4 months in a naturally lit greenhouse
(minimum temperature, 18°C) with the water table close to the soil
surface to replace water lost by evapotranspiration; this kept the
salinity close to that of the collection site. Because a high N supply
decreases DMSP levels (Colmer et al., 1996 Chemicals
INTRODUCTION
Top
Abstract
Introduction
Methods
Results & Discussion
References
; Keller et al., 1989) and by certain angiosperms from the Compositae and Gramineae families. The best studied of these are the coastal strand plant Wollastonia
biflora (L.) DC. (Compositae) and the salt marsh grass
Spartina alterniflora Loisel. (Gramineae) (Storey et al.,
1993; Colmer et al., 1996). DMSP is environmentally important as the
main biogenic precursor of atmospheric DMS, which has roles in the
biogeochemical S cycle, in cloud formation, and in acid precipitation
(Malin, 1996). Whereas oceanic DMS fluxes are globally the largest
source of atmospheric DMS, those from salt marshes dominated by
S. alterniflora are 1 to 2 orders of magnitude higher per
unit area and may significantly affect atmospheric S budgets on a
regional or local scale (Steudler and Peterson, 1984; Aneja and Cooper,
1989).
; Nishiguchi and Somero, 1992) and as an osmoprotectant
for bacteria (Mason and Blunden, 1989; Paquet et al., 1994). Consistent
with its having such functions in plants, DMSP has been shown to
accumulate to high levels (
100 mm) in the cytoplasm of
algal cells (Dickson et al., 1980; Dickson and Kirst, 1986) and in
W. biflora chloroplasts (Trossat et al., 1998). There is
also evidence that DMSP is an effective cryoprotectant (Karsten et al.,
1996).
; James et al.,
1995) and Ratibida pinnata (Vent.) Barnhart (Paquet et al., 1995), and its subcellular compartmentation has been established (Trossat et al., 1996). The only known intermediates in this pathway are SMM and DMSP-ald (Fig. 1). Although
conversion of SMM to DMSP-ald entails the loss of both the amino and
carboxyl groups, there is no evidence that this occurs via DMSP-amine
or any other stable intermediate, and
15N-labeling data support a mechanism in which
SMM is transaminated and decarboxylated by the same enzyme or by a
transaminase-decarboxylase complex (Rhodes et al.,
1997). Most, if not all, angiosperms synthesize SMM
(Giovanelli et al., 1980; Bezzubov and Gessler, 1992), and many have
dehydrogenases that can mediate oxidation of DMSP-ald to DMSP (Trossat
et al., 1997; Vojtechová et al., 1997). It is therefore the
conversion of SMM to DMSP-ald that is special to DMSP synthesis.
View larger version (14K):
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Figure 1.
Known steps in DMSP biosynthesis in W. biflora (Compositae) and in the marine alga E. intestinalis. The enantiomers of chiral compounds are
indicated. No intermediate between SMM and DMSP-ald has been identified
in W. biflora (James et al., 1995 ).
; Summers et al., 1998). It proceeds via
the intermediates 4-methylthio-2-oxobutyrate,
4-methylthio-2-hydroxybutyrate, and DMSHB and therefore has no steps in
common with the Compositae pathway (Fig. 1). This dissimilarity shows
that DMSP synthesis evolved independently in algae and in the ancestors
of Compositae. It also suggests that the pathway in Gramineae might
differ from that in Compositae because these families stand far apart
phylogenetically, their progenitors having diverged more than 100 million years ago (Crane et al., 1995). This prompted us to examine the
DMSP synthesis route in S. alterniflora. We found that it is
not the same as either the algal or the Compositae pathway. Although it resembles the latter in having SMM and DMSP-ald intermediates, it
differs in the key SMM DMSP-ald step.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results & Discussion
References
), no fertilization was
given. Other species were fertilized and not salinized: wheat
(Triticum aestivum L. cv Florida 310), maize (Zea
mays L. cv NK 508), Cortaderia selloana Aschers. & Graebn., and Oplismenus compositus Beauv. were greenhouse grown; Pennisetum purpureum Schumach PI 300086 was grown in
a shade house; and Cynodon dactylon Pers. cv Floratex and
Bambusa glaucescens (Willd.) Sieb. ex Munro cv Stripestem
were field grown. DMSP was assayed in leaf samples (30 mg fresh weight)
by a GC method (Paquet et al., 1994). Autumn fern (Dryopteris
erythrosora [Eaton] Kuntze) was purchased locally and its fronds
used to prepare an acetone powder with Met decarboxylase activity
(Stevenson et al., 1990).
). The product (yield, 75%) was isolated using
Dowex-50 as described above and lyophilized; purity was 98%,
as determined by TLC and TLE. DMSP-ald iodide was synthesized as
described previously (James et al., 1995). DMSP hydrochloride was
obtained from Research Plus, Inc. (Bayonne, NJ).
Labeled Compounds
[35S]Met (44 GBq µmol
1, NEN-DuPont) was mixed with Met to give
the desired specific activity; for experiments with leaf tissue it was
then treated with Dowex-1 (formate) and Dowex-50
(NH4+) to remove acidic and
basic impurities, respectively. [35S]SMM (370 kBq nmol1) was synthesized by chemical
methylation of [35S]Met, as described by Gage
et al. (1997). To prepare [35S]DMSP-amine (165 kBq nmol1),
[35S]Met (25 nmol) was first decarboxylated by
incubating (1 or 2 h, 37°C) in 0.1 mL of 0.2 m
succinate-NaOH buffer, pH 5.0, containing 1 mm pyridoxal
5
-phosphate and 11 mg of D. erythrosora acetone powder. The
reaction mixture was then applied to 1-mL Dowex-1 (OH) and BioRex-70 (H+)
columns arranged in series. After washing both columns with water,
[35S]methylthiopropylamine was eluted from the
BioRex-70 column with 5 mL of 1 n HCl and lyophilized. It
was then treated (110°C, 4 h) with 0.3 mL of 6 n HCl
containing 50 µmol of MeOH to give
[35S]DMSP-amine, which was isolated by TLC on
0.25-mm silica-gel G plates (Machery-Nagel, Düren, Germany)
developed with MeOH:acetone:concentrated HCl (90:10:4, v/v). The
overall radiochemical yield from [35S]Met was
24%; radiochemical purity was 99%, as determined by TLC and TLE.
d- and l-DMSHB (37 kBq
nmol1) were prepared as described by
Summers et al. (1998), and
[methyl-14C]DMSP-ald (0.41 kBq
nmol1) was prepared as described by James
et al. (1995). [35S]DMSP was isolated from
S. alterniflora leaf sections that had been given
[35S]Met.
[C2H3,C2H3]SMM,
[13CH3,C2H3]SMM,
and
[C2H3,C2H3]DMSP
were prepared as described by Hanson et al. (1994).
Metabolism of Precursors by Leaf Tissue
Sections (about 10 × 5 mm) cut from leaves at or near full expansion were given shallow incisions spaced 1 to 2 mm apart on the whole abaxial surface. For most experiments, 0.2-g batches of sections were then incubated (cut surface down) in 6-cm Petri dishes on a 4.25-cm circle of Whatman no. 1 filter paper containing 0.5 or 1.0 mL of precursor solution. For C. dactylon, 0.2-g batches of shoot tips with four or five leaves were used. For the [35S]SMM pulse-chase experiment, eight sections were incubated on 4 cm2 of paper in 200 µL of [35S]SMM solution and then transferred to 1 mL of water for the chase. For experiments in which DMSP-ald was analyzed, two leaf sections were incubated in 30 µL of solution. Incubation was at 25 ± 2°C, with rotary agitation at 75 rpm, in fluorescent light (PPFD, 150 µE m2
s1); water was added to replace that lost by
evapotranspiration. Sections were rinsed before extraction or transfer
to chase media as follows: for time-course experiments, for 15 s
in water; when DMSP-ald was to be analyzed, for 15 s in 10 mL of a 105 m solution of the
corresponding unlabeled compound; and for other 35S-labeling experiments, for 5 to 15 min in 10 mL of a 104 m solution of the
corresponding unlabeled compound. The washings were pooled with the
remaining incubation medium, and a sample was counted to determine
35S uptake. For experiments with
[13CH3,C2H3]SMM,
DMSP for fast-atom-bombardment MS analysis was isolated as described by
Hanson et al. (1994). For all other experiments, the extraction
procedures, the ion-exchange-fractionation methods, and the TLC and TLE
systems used were as described by James et al. (1995).
MS
DMSP was analyzed without derivatization by fast-atom-bombardment MS, using the hexaethylene glycol/K+ nonofluorobutylsulfonate matrix described previously (Hanson et al., 1994). SMM and DMSP-amine were analyzed without derivatization by
MALDI-MS; the instrumentation and procedures were as described by
Trossat et al. (1998), except that fluorosilicic acid was omitted from
the matrix used to analyze DMSP-amine.
Computer Modeling of 35S-Labeling Data
The computer model used was that described by Mayer et al. (1990),
except that programs were written in Microsoft Visual Basic 5.0. Programs used a 0.06-min iteration interval to generate simulations of
the labeling patterns of metabolites, partitioning pools into metabolically active and inactive (storage) components, and varying the
fluxes between pools until the match between observed and simulated
data was satisfactory.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Methods
Results & Discussion
References
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Metabolism of [35S]Met, [35S]SMM, and [35S]DMSHB
A diagnostic distinction between the algal- and Compositae-type DMSP synthesis pathways is that if radiotracer Met is supplied, the major labeled intermediate is DMSHB in the former and SMM in the latter (Hanson et al., 1994; Gage et al., 1997). We therefore fed
[35S]Met to S. alterniflora leaf
tissue and monitored the labeling of DMSHB, SMM, and DMSP
(Fig. 2A). As in Compositae, no
35S was detected in DMSHB, but SMM acquired label
rapidly and lost it as the [35S]Met was
metabolized, as expected for an intermediate. However, unlike the
pattern in Compositae, DMSP-amine acquired and lost 35S in a way that suggested that it too could be
an intermediate (Fig. 2B). DMSP-amine
[(CH3)2S+CH2CH2CH2NH3+]
is the decarboxylation product of SMM. The
[35S]DMSP-amine pool was far smaller than the
[35S]SMM pool, with its peak
35S content (between 1 and 6 h) being about
2.5% that of SMM (Fig. 2); two similar experiments gave peak values of
2.9 and 5.0% (not shown).
|
Evidence from Stable Isotope Labeling that SMM and DMSP-amine Are
Intermediates
Labeling Kinetics of DMSP-amine during and after a Pulse of
[35S]SMM
[35S]SMM Metabolism in Grasses That Do Not
Accumulate DMSP
[35S]DMSP-amine Metabolism in S. alterniflora and Grasses Lacking DMSP
S. alterniflora leaf sections (0.2 g fresh weight)
were incubated for 4 h with 18.5 kBq (0.15 nmol) of
[35S]SMM, with or without a 5-µmol trapping pool of
unlabeled DMSP-amine. The size of the DMSP-amine pool in the tissue was
estimated in a parallel experiment in which leaf sections were
incubated with 5 µmol (18.5 kBq) of [35S]DMSP-amine for
4 h. The values shown are per 0.2 g fresh weight and have
been corrected for recovery. The experiment was repeated using an
8-h incubation time, with similar results.
Evidence for DMSP-ald as an Intermediate
Modeling of DMSP Synthesis in S. alterniflora
We have shown that the DMSP synthesis pathway in S. alterniflora is unlike that in marine algae and resembles that in
Compositae by having SMM and DMSP-ald as intermediates. However, it
differs crucially in that SMM is converted to DMSP-ald via a pool of
free DMSP-amine (Fig. 8). The presence of
a free DMSP-amine intermediate is supported by the labeling kinetics of
DMSP-amine when [35S]Met or
[35S]SMM is fed, by the formation of
[C2H3,C2H3]DMSP-amine
from
[C2H3,C2H3]SMM,
and by the ready conversion of supplied DMSP-amine to DMSP. That
DMSP-amine is taken up but does not trap label coming from SMM can be
explained by its relatively poor access to the metabolic pool and by
high capacity for the conversion of DMSP-amine to DMSP.
Received November 26, 1997;
accepted February 1, 1998.
Abbreviations:
DMS, dimethylsulfide.
DMSHB, 4-dimethylsulfonio-2-hydroxybutyrate.
DMSP, 3-dimethylsulfoniopropionate.
DMSP-ald, 3-dimethylsulfoniopropionaldehyde.
DMSP-amine, 3-dimethylsulfoniopropylamine.
MALDI-MS, matrix-assisted
laser-desorption ionization MS.
MeOH, methanol.
SMM, S-methyl-l-Met.
TLE, thin-layer
electrophoresis.
We thank Dr. Peter Stiling for identifying and
collecting S. patens.
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View this table:
Table I.
Conversion of supplied SMM but not DMSHB to DMSP by
S. alterniflora leaf sections
Leaf sections (0.2 g fresh weight) were incubated for 24 h with a
tracer dose (0.2-1.0 nmol) of [35S]SMM or
[35S]DMSHB. The amount of 35S was 19.2 to
19.6 kBq in all cases. Uptake was calculated from the disappearance of
35S from the medium. The experiment was repeated using
100-nmol doses of 35S precursors, with very similar
results. The values shown are per 0.2 g fresh weight and have been
corrected for recovery.
SMM DMSP.
However, because Met and SMM are potentially interconvertible via the
SMM cycle (Mudd and Datko, 1990), they are also consistent with the
sequence SMM 
Met DMSP. To distinguish between these
alternatives, leaf segments were given SMM labeled with
13C in one methyl group and with
2H in the other, and the DMSP formed was
analyzed. Only
[13CH3,C2H3]DMSP
was detected (Table II). This shows that
SMM was converted to DMSP directly, not via Met, because conversion via
Met would give
13CH3,13CH3-,
13CH3,C2H3-,
and
C2H3,C2H3-labeled
species of DMSP in a 1:2:1 ratio. These results therefore confirm that
SMM is an intermediate of DMSP synthesis in S. alterniflora. They also indicate that flux through the SMM cycle in this plant is
small compared with the flux to DMSP.
View this table:
Table II.
Labeling of DMSP synthesized by S. alterniflora
leaf sections given
[13CH3,C2H3]SMM
Leaf sections (0.2 g fresh weight) were incubated with 5.0 µmol of
[13CH3,C2H3]SMM
(experimental samples) or unlabeled SMM (controls) for 24 or 48 h.
The intensities of the labeled DMSP ions are expressed relative to that
of endogenous unlabeled DMSP (m/z 135, 100%); the endogenous DMSP level was 23 ± 1 µmol g 1
fresh weight (mean ± se, n = 4). All
signals were corrected for background noise from the matrix, and those
at m/z 141 were further corrected for the
contribution from the natural-abundance isotope peaks associated with
[13CH3,C2H3]DMSP
(m/z 139). Because labeled DMSP yields at 24 and
48 h did not differ significantly, results for both times were
pooled. Data are means from eight experimental samples and four
controls, and were subjected to analysis of variance. That all of the
values for control sections given unlabeled SMM are close to 0 confirms the validity of the corrections applied.
View larger version (25K):
[in a new window]
Figure 3.
MALDI-MS analysis of the base fractions from
S. alterniflora leaf segments (0.2 g fresh weight)
incubated for 6 h with 5.0 µmol of unlabeled SMM (A) or
[C2H3,C2H3]SMM (B).
The peaks at m/z 164 and 170 correspond
to the unlabeled SMM and
[C2H3,C2H3]SMM
supplied, respectively, and those at m/z
120 and 126 correspond to unlabeled and
C2H3,C2H3-labeled
DMSP-amine formed during the experiment, respectively. In these
spectra, matrix background peaks are largely suppressed, but residual
signals can be seen at m/z 123, 137, and
146. The relative response of DMSP-amine and SMM in MALDI-MS analyses
under these conditions was approximately 30:1, determined by spiking the samples with known amounts of DMSP-amine and SMM (data not shown).
In leaf segments not given SMM, the intensities of the peaks
corresponding to DMSP-amine and SMM were about 5 and 15%, respectively, of those in A. No peaks attributable to DMSP-amine were
detected in the SMM substrates supplied to the leaf segments.
1 fresh weight. A signal attributable to
endogenous DMSP-amine was detectable by MALDI-MS; it was too small to
quantify accurately, but indicated that the DMSP-amine level was not
more than 20 nmol g1 fresh weight, i.e. far
lower than the SMM level.
Met flux via the SMM cycle, because
little 35S accumulated in free Met at any time
(<0.1 kBq; not shown) and labeling of the insoluble fraction, a
maximum estimate of protein-bound [35S]Met, was
small relative to that of DMSP (Fig. 4A).
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Figure 4.
Labeling kinetics of SMM, DMSP, and insoluble
material (Insol., A) and DMSP-amine (B) in a pulse-chase experiment
with [35S]SMM. Batches of eight S. alterniflora leaf sections (approximately 0.1 g fresh
weight) were supplied with 18.5 kBq (0.16 nmol) of [35S]SMM; after 2.5 h they were rinsed for 15 s
and transferred to water. That more 35S was lost from SMM
during the chase than appeared in other products may be ascribed to
efflux of [35S]SMM present in the apoplast. The inset in
B is an expanded-scale plot of the labeling of DMSP-amine and DMSP at
early times; note that DMSP-amine is initially more heavily labeled
than DMSP.
View this table:
Table III.
[35S]DMSP-amine is not a metabolite
of [35S]SMM in grasses that lack DMSP
Leaf tissue (0.2 g fresh weight) of eight grasses shown not to
accumulate DMSP was incubated for 6 h with 38.9 kBq (0.12 nmol) of
[35S]SMM; S. alterniflora was included as a
benchmark. Uptake was calculated from 35S disappearance
from the medium. The radioactivity values shown are per 0.2 g
fresh weight and have been corrected for recovery. Endogenous DMSP
contents were determined on duplicate 30-mg fresh weight leaf samples.
The comparison between S. alterniflora and S. patens was repeated, with similar results.
). That the DMSP-free grasses
made a little [35S]DMSP agrees with previous
data for T. aestivum and for dicot species that lack DMSP
(James et al., 1995). This seemingly nonspecific DMSP production may be
caused by the tandem action of diamine oxidase and 
-aminoaldehyde
dehydrogenase, both of which occur in grasses and dicots and can attack
substrates with a dimethylsulfonium group (Bardsley et al., 1971;
Awal et al., 1995; Suzuki, 1996; Trossat et al., 1997).
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Figure 5.
Uptake and conversion to [35S]DMSP
of tracer (A) or substrate-level (B) doses of
[35S]DMSP-amine. Leaf tissue (0.2 g fresh weight) of nine
grass species was incubated with 140 ± 15 pmol (tracer) or 5.0 µmol (substrate level) of [35S]DMSP-amine for 6 h.
Uptake was calculated from disappearance of 35S from the
medium. Species are arranged in order of ascending label uptake.
Species are as follows: SP, S. patens; SA, S. alterniflora; CS, C. selloana; ZM, Z. mays; CD, C. dactylon; TA, T. aestivum; BG, B. glaucescens; PP, P. purpureum; and OC, O. compositus. All species
except S. alterniflora lacked detectable DMSP (Table
III). Data for S. alterniflora are means + se for four similar experiments; those for other species
are single determinations.
View this table:
Table IV.
DMSP-amine does not act as a trapping pool for
label from [35S]SMM
View this table:
Table V.
Labeling of DMSP-ald from [35S]SMM or
[35S]DMSP-amine in S. alterniflora
Pairs of leaf segments were supplied with a pulse of
[35S]SMM (77 kBq, 0.99 nmol) or
[35S]DMSP-amine (35 kBq, 1.33 nmol) for 3 h, rinsed
for 15 s, and then transferred to water for a 9-h chase period.
DMSP-ald was analyzed as the corresponding alcohol after reduction with
NaBH4 (see ``Materials and Methods''); a correction was
made for traces of alcohol detectable in controls that were not treated
with NaBH4. That more 35S was lost from SMM or
DMSP-amine during the chase than appeared in DMSP may be ascribed
primarily to efflux of unabsorbed label from the apoplast. The values
shown are per pair of segments and have been corrected for recovery.
The experiment was repeated, with similar results.
DMSP flux.
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Figure 6.
A model of DMSP synthesis from Met in
S. alterniflora, based on computer-assisted analyses of
the data shown in Figures 2 and 4 and an additional pulse-chase
experiment with [35S]Met (not shown). Values in boxes are
mean pool sizes (nmol g 1 fresh weight) and those next to
arrows are mean flux rates (nmol min1 g1
fresh weight). Values in parentheses are se. The DMSP pool
size shown is based on the experimental values given in Tables II and III because modeling did not permit estimation of this parameter (modeling permitted estimation only of the amount of label that accumulated in DMSP, which is independent of the pool size assumed). The rates of [35S]SMM and [35S]Met uptake
from the apoplast (assumed to equilibrate rapidly with the medium)
apply only to the pulse phase of the pulse-chase experiments. During
the chase the uptake rates were reduced by 20- to 40-fold, depending on
the dilution of the medium plus apoplastic precursor pools achieved
during the chase. For the experiment shown in Figure 2, which did not
involve a chase, the [35S]Met uptake rate was assumed to
be 0.192 nmol min1 g1 fresh weight
initially, and thereafter to decline in direct proportion to the size
of the exogenous [35S]Met pool (see Fig. 2A, inset). For
all experiments it was necessary to postulate that there was a large
endogenous flux of (unlabeled) homocysteine to Met, SMM, DMSP-amine,
and DMSP, and that portions of the SMM and DMSP-amine pools were
sequestered in "storage" compartments. These storage pools were
postulated to be in slow equilibrium with the "metabolically
active" pools that participated as intermediates in DMSP synthesis.
Labeling of DMSP-ald (an intermediate between DMSP-amine and DMSP) was
not considered.
); in
this case, the metabolic pools of SMM and DMSP-amine would be
chloroplastic and the storage pools extrachloroplastic (cytosolic
and/or vacuolar). For DMSP-amine, which is doubly positively charged at
cellular pH, another possible basis for the storage component is
reversible adsorption to negative charges on macromolecules, as occurs
with di- and polyamines (Kumar et al., 1997, and refs. cited therein).
1 fresh weight)
encompasses the experimental value of approximately 150 nmol
g1 fresh weight. The model-estimated total
DMSP-amine pool has a mean value of about 7% of the SMM pool, which is
broadly consistent with the MALDI-MS estimates (see "Evidence from
Stable Isotope Labeling that SMM and DMSP-amine are Intermediates").
When expressed per day, the model-estimated flux rate to DMSP is 2.36 µmol d1 g1 fresh
weight, which is equivalent to about 10% of the total DMSP pool.
DMSP-amine and DMSP-amine DMSP fluxes in two additional
labeling experiments. The first experiment was that described in Table
IV, with and without a trapping pool of DMSP-amine. The results with no
trapping pool were satisfactorily accommodated by using flux and pool
size values that fell within the ranges given in Figure 6. When
DMSP-amine was supplied, the model gave a good fit to experimental data
if (a) the exogenous DMSP-amine was postulated to enter the DMSP
synthesis pathway by passing via the storage pool, (b) the DMSP-amine
storage pool expanded massively from approximately 5 to 2700 nmol
g1 fresh weight, (c) the DMSP-amine metabolic
pool expanded from 4 to 75 nmol g1 fresh
weight, and (d) the DMSP-amine DMSP flux in-creased from 1 to 3.78 nmol min1 g1 fresh
weight. These assumptions seem biologically reasonable; note that
assumption (a) is consistent with exogenously supplied DMSP-amine
passing through a cytosolic (storage) pool en route to a chloroplastic
(metabolic) pool. The model thus provides an acceptable quantitative
explanation for the failure of added DMSP-amine to trap label from SMM.
1 fresh weight coupled with an increase in the
SMM DMSP-amine flux from 1 to 4.8 nmol min1
g1 fresh weight. This resulted in an expansion
of the metabolic DMSP-amine pool from 4 to 100 nmol
g1 fresh weight and an increase in the
DMSP-amine DMSP flux from 1 to 4.4 nmol
min1 g1 fresh weight.
DMSP-amine flux, and between the DMSP-amine
metabolic pool size and the DMSP-amine DMSP flux (Fig.
7). Such saturable kinetics are to be
expected given that the fluxes are enzyme mediated, and the fact that
they are observed supports the validity of the model. Double-reciprocal
plots gave apparent Km values for SMM and
DMSP-amine of 310 and 5.8 nmol g1 fresh weight,
respectively. These values cannot be converted to concentrations
because the size of the compartment(s) containing the metabolic pools
of SMM and DMSP-amine are unknown.
View larger version (15K):
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Figure 7.
Model-derived relationships between metabolic pool
sizes and fluxes for SMM and the SMM DMSP-amine flux (A) and for
DMSP-amine and the DMSP-amine DMSP flux (B). Each data point
corresponds to metabolic pool size and flux values obtained using the
model shown in Figure 6. The experimental data input for the model came from Figures 2 and 4, from Table IV, and from the two additional experiments described in the text. FW, Fresh weight.
CONCLUSION
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Figure 8.
DMSP synthesis from Met in S. alterniflora and species of Compositae. Many flowering plants
can carry out the S-methylation of Met to SMM and the
oxidation of DMSP-ald to DMSP. Only the conversion of SMM to DMSP-ald
is unique to DMSP synthesis.
; Trossat et al., 1997), it is
the ability to convert SMM to DMSP-ald that distinguishes plants that
produce DMSP from those that do not (Fig. 8). Because S. alterniflora and members of Compositae evidently differ in how
they carry out this key conversion, it seems likely that their DMSP
pathways had independent evolutionary origins. DMSP synthesis may thus
have evolved at least three times, once each in algae, dicots, and
monocots. With respect to evolution of the pathway in S. alterniflora, two novel enzymes would presumably be required: an
SMM decarboxylase and a DMSP-amine oxidase, dehydrogenase, or
aminotransferase. In this regard, it is worth noting that SMM is an
analog of S-adenosyl-l-Met, and that
S-adenosyl-l-Met decarboxylases are ubiquitous
(Kumar et al., 1997). Likewise, DMSP-amine is an analog of a diamine,
and diamine oxidases occur widely in plants (Suzuki, 1996). It is
therefore easy to imagine how novel enzymes able to decarboxylate SMM
and to oxidize DMSP-amine might have originated.
*
Corresponding author; e-mail adha{at}gnv.ifas.ufl.edu; fax
1-352-392-6479.
FOOTNOTES
1
This work was supported in part by the National
Science Foundation (grant nos. IBN-9514336 to A.D.H. and IBN-9628750 to
D.A.G.) and by an endowment from the C.V. Griffin, Sr., Foundation.
Mass spectral data were acquired at the Michigan State
University-National Institutes of Health (NIH) Mass Spectrometry
Facility, which is supported in part by the NIH, National Center for
Research Resources (grant no. RR 00484). This is University of Florida
Agricultural Experiment Station journal series no. R-06248.
ABBREVIATIONS
ACKNOWLEDGMENT
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results & Discussion
References
-dimethylsulphoniopropionate, glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis.
Planta
167:
536-543
[CrossRef] -dimethylsulfoniopropionate.
Cryobiology
29:
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[CrossRef][Medline] -aminoaldehydes.
Plant Physiol
113:
1457-1461
[Abstract]
Copyright Clearance Center: 0032-0889/98/117/0273/09
© 1998 American Society of Plant Physiologists
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