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First published online March 20, 2003; 10.1104/pp.102.015933
Plant Physiol, April 2003, Vol. 131, pp. 1843-1854
Functional Analysis of Cystathionine
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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In plants, metabolic pathways leading to methionine (Met)
and threonine diverge at the level of their common substrate,
O-phosphohomoserine (OPHS). To investigate the regulation of this
branch point, we engineered transgenic potato (Solanum
tuberosum) plants affected in cystathionine 
-synthase (CgS),
the enzyme utilizing OPHS for the Met pathway. Plants overexpressing
potato CgS exhibited either: (a) high transgene RNA levels and 2.7-fold
elevated CgS activities but unchanged soluble Met levels, or (b)
decreased transcript amounts and enzyme activities (down to 7% of
wild-type levels). In leaf tissues, these cosuppression lines revealed
a significant reduction of soluble Met and an accumulation of OPHS.
Plants expressing CgS antisense constructs exhibited reductions in
enzyme activity to as low as 19% of wild type. The metabolite contents
of these lines were similar to those of the CgS cosuppression lines.
Surprisingly, neither increased nor decreased CgS activity led to
visible phenotypic alterations or significant changes in protein-bound
Met levels in transgenic potato plants, indicating that metabolic flux
to Met synthesis was not greatly affected. Furthermore, in vitro feeding experiments revealed that potato CgS is not subject to feedback
regulation by Met, as reported for Arabidopsis. In conclusion, our
results demonstrate that potato CgS catalyzes a near-equilibrium reaction and, more importantly, does not display features of a pathway-regulating enzyme. These results are inconsistent with the
current hypothesis that CgS exerts major Met metabolic flux control in
higher plants.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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The sulfur-containing amino acid
Met, a crucial metabolite in plant cells, is required not only as a
protein component, but also as a precursor for polyamine, ethylene, and
biotin biosynthesis (Ravanel et al., 1998
) and for
secondary metabolites such as S-adenosyl-Met (SAM), the
major one-carbon donor in both prokaryotes and eukaryotes (for review,
see Tabor and Tabor, 1984). Another Met derivative, S-methyl-Met (SMM), is believed to play an important role in
sulfur transport (Bourgis et al., 1999) and the control
of intracellular SAM levels in plants (Ranocha et al.,
2001; Fig. 1). Three enzymatic steps are involved in de novo Met biosynthesis (for review, see Matthews, 1999). The first enzyme, cystathionine
-synthase (CgS; EC 4.2.99.9), catalyzes the formation of
the thioether L-cystathionine by
-replacement of the phosphate group of
O-phosphohomoserine (OPHS) for the nucleophilic sulfhydryl
group provided by Cys (Thompson et al., 1982;
Ravanel et al., 1995, 1998).
Cystathionine is subsequently converted to homocysteine and finally to
Met. Biochemical studies have provided evidence that the first two
enzymes involved in Met biosynthesis are located in the chloroplasts
(Wallsgrove et al., 1983), whereas the third occurs
exclusively in the cytosol (Eichel et al., 1995;
Petersen et al., 1995; Zeh et al., 2002). In potato (Solanum tuberosum), two CgS cDNAs
(StCgS1 and StCgS2) encoding putative
chloroplastidial isoforms, as judged by deduced amino acid sequences,
have been isolated and characterized (Hesse et al.,
1999; Riedel et al., 1999). Comparison of the
coding regions revealed 92.7% similarity (84.3% identity) at the
amino acid level. Even at the nucleotide level, both cDNAs show a high
identity (83.9%) with the highest divergence in the 5'- and
3'-untranslated regions.
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Considering its importance to various cellular processes, the size of
the soluble Met pool is likely to be subject to tight control
mechanisms. Although not elucidated in detail yet, two major control
elements are of particular interest: Excess amounts of Met (or related
metabolites) are known to reduce the stability of CgS mRNA
and, thus, decrease concomitant enzyme activity in Lemna
paucicostata and Arabidopsis. Feeding studies with Met resulted in
decreased CgS activity in L. paucicostata (Thompson
et al., 1982; Giovanelli et al., 1985). The
characterization of mto1 Arabidopsis mutants, in which CgS
modifications result in transcripts resistant to Met-dependent
degradation, suggests that CgS is autoregulated at the
posttranscriptional level, presumably via a mechanism involving the
N-terminal region of the AtCgS protein (Inaba et al.,
1994; Chiba et al., 1999; Ominato et al.,
2002). Mto1 mutants accumulate high levels of
soluble Met (up to 40-fold) in young rosette leaves. Notably, these
levels decrease during flowering at the same time levels rise in
flowers, thus indicating a spatial and developmental regulation of the
size of the soluble Met pool in Arabidopsis. Moreover, studies on
CgS-overexpressing Arabidopsis plants revealed that overaccumulation of
Met (and SMM) was found to inversely correlate with CgS levels only in
tissues of flowering stage plants and not in young plants, suggesting
additional and even more important factors contributing to the
regulation of the size of the Met pool during plant development
(Kim and Leustek, 2000).
As a second major control feature, the efficiency of Met formation is
known to be strictly controlled by competition between CgS and Thr
synthase (TS) for their common substrate OPHS. The enzymatic activity
of plant TS is strongly stimulated by SAM, the end product of the
competing pathway (Giovanelli et al., 1984, 1985; Curien et al., 1996). Because
Km values of fully activated TS for OPHS
have been shown to be 250- to 500-fold lower than those of CgS
(Ravanel et al., 1998), carbon flux is directed into the
Thr branch when Met and, hence, SAM levels are high. Characterization of transgenic and mutant plants altered in the CgS to TS ratio has
provided important evidence for the essential function of this
competition for the flow of carbon into either Met or Thr synthesis.
Arabidopsis plants expressing CgS antisense mRNA revealed a
4- to 7-fold increase in Thr levels accompanied by severe morphological aberrations due to reduced Met synthesis capacity (Gakière
et al., 2000; Kim and Leustek, 2000). More
interesting findings were obtained when TS activity was reduced: An
Arabidopsis mutant (mto2) deficient in TS enzymatic activity
exhibited an accumulation of free Met in young rosette leaves
(20-fold), accompanied by comparably reduced soluble Thr contents (down
to 6%), but not in mature plants (Bartlem et al.,
2000). As a consequence, these results suggest that in young
Arabidopsis plants, the regulation of Met synthesis is mainly dependent
on the dynamic interplay between changing biochemical properties of CgS
and TS (Thompson et al., 1982; Bartlem et al.,
2000) and, moreover, when the CgS to TS ratio is altered in
favor of CgS, autoregulation of CgS alone is not sufficient to maintain
the net rate of Met synthesis.
In contrast to results in Arabidopsis, reducing TS activity in potato plants by using an antisense approach caused a rather disproportional increase of the soluble Met pool (up to 239-fold), as compared with reductions of free Thr levels (up to 55%). Even more interesting, this report also indicated that autogenous regulation of CgS is impaired in potato plants and, hence, might be not conserved among plant species.
In the current article, we aimed to test the assumption that Met production is less dependent on CgS in potato plants than in Arabidopsis. Therefore, we investigated effects of exogenously applied Met on CgS expression and enzymatic activity in different in vitro systems. Moreover, we modulated the ratio of CgS to TS in potato plants by altering the level of CgS transcription using sense and antisense technologies.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Production of Transgenic CgS Sense and Antisense Potato Plants
Transgenic potato plants that overexpress CgS were
created by placing the potato CgS isoform 1 (StCgS1; Riedel et al., 1999) coding sequence
downstream of the cauliflower mosaic virus 35S promoter in the
plant transformation vector pBinAR (Höfgen and Willmitzer,
1990). Fifty independent kanamycin-resistant transformants were
regenerated and used for further analyses based on transcript levels.
From the initial transformants, three transgenic lines were selected
(sCgS lines 11, 16, and 28) that exhibited increases in CgS
mRNA levels (Fig. 2B). Moreover, two
lines were identified that revealed cosuppression of CgS (csCgS lines
33 and 41), resulting in a substantial reduction in its transcript
levels (Fig. 3B).
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To decrease the activity of the CgS, potato plants were transformed with the vector pBinAR harboring a cDNA encoding a partial sequence of the StCgS1 gene in reverse orientation with respect to the cauliflower mosaic virus 35S promoter. After regenerating 75 independent kanamycin-resistant transgenic plant lines, three lines (aCgS 18, 27, and 56) with reduced steady-state levels of CgS mRNA were selected. Decreased transcript amounts of the endogenous CgS were accompanied by the accumulation of the 600-bp truncated antisense transcript (Fig. 4B). Selected overexpression and antisense plants were amplified and grown in five replicates of each line in the greenhouse to confirm transgene expression and to enable a more detailed analysis of the transformants.
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Measurements of CgS Protein Content and Enzyme Activity in Transgenic Potato Lines
Immunoblotting of leaf tissue extracts from sCgS plants unexpectedly revealed that an increase in CgS mRNA expression did not lead to identically increased CgS protein accumulation. In comparison with untransformed control plants, sCgS lines exhibited varying CgS levels ranging from only slight increases in lines 16 and 28 to still weak but distinct CgS protein accumulation in line 11 (Fig. 2C). These moderately elevated protein accumulations gave rise to significant 1.4- to 1.7-fold increases in CgS activity in lines 16 and 28. Line 11 revealed a 2.7-fold enhanced catalytic activity (Fig. 2D), thus confirming the findings of the immunoblot analysis.
CgS antisense and cosuppression lines demonstrated a substantial decrease in CgS protein levels in comparison with control plants (Figs. 4C and 3C). As judged by the immunoblot experiments, the level of CgS protein was correlated to the RNA blot, indicating that repression of CgS transcript led to a reduced availability of the corresponding mRNA for translation. Furthermore, these plants exhibited a comparable decrease in total CgS activity. aCgS lines 18, 27, and 56 exhibited approximate reductions to 19%, 33%, and 51% as compared with wild-type activity (Fig. 4D). Cosuppression of CgS had even stronger effects on enzyme activity, yielding a decrease to 7% and 21% in csCgS lines 41 and 33, respectively (Fig. 3D).
Considering these results, we concluded that overexpression and repression of the CgS gene by antisense inhibition or cosuppression resulted in alterations of CgS protein levels, which were in accordance with the corresponding protein quantities and enzyme activities in respective transgenic lines.
Alteration of CgS Activity Does Not Affect Plant Growth and Development in Potato
With the aim to test potential influences of the changes in CgS activity on plant growth and development, potato wild-type plants and the selected transgenic lines were transferred into soil and cultivated under two different greenhouse conditions. Neither increased nor decreased CgS levels led to visible phenotypic differences when compared with control plants under any of the growth conditions (Figs. 2A, 3A, and 4A). Both wild-type and transgenic plants switch from their vegetative to the reproductive stage of development at the same time and show no detectable deviation in size, number, and total yield of the harvested tubers (data not shown).
Effect of CgS Overexpression on Metabolite Levels in Source Leaves, Sink Leaves, and Flowers
The effect of an increased expression of the CgS gene
on the amounts of Asp-derived amino acids and thiol compounds was
tested. Following the generally believed assumption that the de novo
synthesis of amino acids in higher plants occurs in the chloroplasts,
source leaf tissues were analyzed for soluble metabolites. Met was also measured in sink leaves and flowers of the transformants because the
concentration of soluble Met is presumably temporally and spatially
regulated in Arabidopsis after the onset of the reproductive growth and
accumulates in sink organs, such as the inflorescence apex
(Chiba et al., 1999). However, increases of CgS
activity had no substantial impact on the concentrations of soluble end products and intermediates of the Asp pathway in any of the tested potato tissues. As shown in Table
I, the amounts of Met revealed no
statistically significant alterations in comparison with control plants. Sink leaves of wild-type plants and transgenic sCgS lines 16 and 28 demonstrated an approximately 2-fold increase in soluble Met
levels in comparison with source leaves. Though the Met levels observed
in sink leaves of sCgS line 11 showed a tendency of increase when
compared with those of wild-type plants, there was a high degree of
variability among individuals of this plant line. The free Met pool in
flowers was increased approximately 5-fold when compared with source
leave values, but again no significant differences between wild-type
plants and transgenics were measured. Moreover, neither amounts of Cys,
one of the CgS substrates, nor levels of Thr changed significantly in
the transgenic lines as compared with control plants (Table I). The
same holds true for other pathway related metabolites as Asp, Lys,
homoserine, Ile, and glutathione, which is derived from Cys (data not
shown). The levels of OPHS (second substrate of the CgS),
L-cystathionine, homocysteine (both metabolites
downstream of the CgS reaction), or SMM (a derivative of Met) were
below the detection limit of the analytical methods, even though
respective pure substances could be verified in the same order of
magnitude as other compounds. These findings were confirmed by
analyzing a successive set of plants using gas chromatography/mass spectrometry-based technology according to Roessner et al.
(2000, 2001; data not shown).
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Effect of CgS Decreases on Metabolite Levels in Source Leaves
Decreases of CgS levels provoked substantial reductions in soluble Met pools, as measured in source leaves of the selected antisense and cosuppression lines. Free Met levels were determined to be reduced to 66%, 52%, and 75%, respectively, when compared with levels observed in wild-type plants (Table II). Moreover, reductions of Met levels were even more pronounced in CgS cosuppression lines. They were calculated to be decreased to 46% and 17% in csCgS lines 33 and 41, respectively.
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Furthermore, repression of CgS caused a considerable accumulation of
OPHS, which is the common substrate for Met and Thr biosynthesis in
higher plants, though this was accompanied by a considerable variation
among the individuals of each line. Because this intermediate was
usually not detectable in leaves of wild-type plants, its degree of
accumulation could not be determined in a reasonable manner. Yet,
concentrations of Cys, the second substrate of the CgS, which provides
reduced sulfur for Met synthesis (Hesse and Höfgen,
1998; Saito, 1999), were found to be equal to
the control values in all transgenic lines. Moreover, the accumulation
of OPHS did not lead to statistically significant increases in soluble Thr levels (Table II). The amino acids Lys, Ile, and Ser also revealed
no alterations when compared with the controls (data not shown). These
findings were confirmed by analyzing a successive set of plants using
gas chromatography/mass spectrometry-based technology according to
Roessner et al. (2000, 2001; data not shown).
Manipulation of CgS Does Not Affect the Met Content of Soluble Leaf Proteins
Because reduced soluble Met contents observed in CgS antisense and cosuppression lines might limit its incorporation into proteins, extracts made from source leaves were analyzed with regard to their protein amino acid composition. Protein-bound amino acids were determined by enzymatic hydrolysis of water-soluble proteins followed by HPLC analysis. The proportion of Met found in soluble leaf proteins of transgenic lines repressed in CgS, no matter whether by antisense inhibition or cosuppression, exhibited no significant differences to respective control values. Likewise, contents of protein-bound Met were not significantly altered in CgS overexpression lines (Table III).
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In Potato, CgS mRNA Expression and Enzymatic Activity Is Not Subject to Metabolic Control by Met
It was shown for Arabidopsis that increased amounts of soluble Met
result in reduced levels of CgS transcript (Inaba et
al., 1994; Chiba et al., 1999). These
observations gave grounds for the current hypothesis that Met regulates
its own synthesis through feedback control of CgS mRNA
accumulation (Chiba et al., 1999). In disagreement with
this, increased Met content has no detectable effect on CgS
mRNA levels, CgS protein accumulation, or CgS enzymatic activities in
transgenic TS antisense potato plants (Zeh et al., 2001). To further elucidate the apparent differences between
Arabidopsis and potato concerning the regulation of Met synthesis at
the step of cystathionine formation, two different in vitro experiments were performed to test the stability of the CgS mRNA in
response to Met.
A feeding experiment was carried out using freshly detached compound
leaves of potato wild-type plants that were incubated for 24 h
under constant light conditions in solutions containing various
concentrations of Met. Resulting actual soluble Met pools in the leaves
were determined to be 4.4, 11.7, 14.3, and 91.5 nmol
g
1 fresh weight (Fig.
5A), referring to external Met
concentrations of 0, 0.1, 1, and 5 mM and, hence,
demonstrating the suitability of the test system. Compared with
controls, elevated Met contents did not lead to changes in steady-state
levels of CgS mRNA (Fig. 5B). In agreement with this
finding, corresponding CgS enzyme activities were not found to be
altered, as indicated by their specific activities of 1.37, 1.23, 1.4, and 1.33 nmol min1 mg1
(Fig. 5C).
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In a second approach, CgS mRNA from potato and Arabidopsis, respectively, were synthesized in vitro in a coupled transcription/translation system based on wheat germ extracts. The intention was to test the transcript stability depending on accurately defined Met concentrations independent from uptake and, further, to facilitate a direct comparison of both species. cDNAs coding for StCgS1 and AtCgS, respectively, were transcribed/translated in presence of 0, 0.1, 0.5, 1, and 5 mM Met. RNA-blot analyses revealed that AtCgS mRNA contents showed a distinct decrease when expressed at Met concentrations higher than 0.1 mM (Fig. 6B), whereas transcript levels of the potato CgS were found to be unaffected by Met within the range of the experimental setup (Fig. 6A). By using the firefly luciferase gene as a positive control, it was clear that gene expression was not generally impaired by Met (Fig. 6C). The result of these experiments suggests that in potato plants CgS is not subject to metabolic control by Met.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Recent results from transgenic Arabidopsis plants manipulated in
CgS enzymatic activity levels gave rise to the hypothesis that CgS
exerts major flux control for Met metabolism in Arabidopsis (Gakière et al., 2000, 2002;
Kim and Leustek, 2000; Kim et al., 2002).
This hypothesis is supported by studies indicating that Arabidopsis CgS
is feedback-regulated by Met at the posttranscriptional level
(Chiba et al., 1999; Bartlem et al.,
2000). Using the potato plant as a model system, our aim was to
test whether similar mechanisms can be assigned to other plant species.
Until now, molecular investigations of CgS regulation have been focused
on a stretch of 39 amino acids, encoded by exon 1 of AtCgS
and designated as the MTO1 region, which is believed to act in cis to
destabilize its own transcript in a process that involves Met or
related metabolites (Chiba et al., 1999). In accordance, AtCgS mRNA levels and enzymatic activities are reduced in
the presence of excess Met in Arabidopsis (Inaba et al.,
1994; Chiba et al., 1999; Bartlem et a.,
2000). In contrast, we could show that increasing the soluble
Met pool in potato leaves was not accompanied by changes in levels of
CgS transcript or activity. Even more important, quantities
of potato CgS (StCgS1) mRNA synthesized in vitro
in a transcription/translation system were unaffected by Met even at
concentrations higher than might be encountered in a biological system.
This is interesting with regard to the fact that Arabidopsis
CgS transcript levels markedly decreased when synthesized
under similar conditions, thus indicating that mechanisms determining
AtCgS transcript accumulation in response to Met are
impaired in the potato CgS ortholog. This finding confirms previous results (Zeh et al., 2001).
Although this paradoxical observation is difficult to explain, it
cannot be exclusively attributed to the polypeptide encoded by
CgS exon 1. The amino acid sequence in the MTO1 region is
almost perfectly conserved among plant species, including both potato CgS isoforms, thus indicating a general motif with a functional role
(Chiba et al., 1999; Ominato et al.,
2002). However, it has to be mentioned that the only known
exception is a Val-to-Leu substitution in one of the potato CgS
isoforms (StCgS2). Yet, according to Ominato et al.
(2002), we do not expect that change to have influence on
regulation because it is located near the border of the MTO1 region and
does not involve major chemical or sterical alterations in the amino
acid chain. Moreover, it can be ruled out that differences in
CgS feedback regulation are attributed to species-specific
trans-acting factors because mRNA synthesis conditions were identical
for both CgS cDNA templates.
Our data suggest that additional levels regulating AtCgS
transcript stability involve the nucleotide sequence of the
AtCgS exon 1. In contrast to the highly conserved MTO1 amino
acid sequences, corresponding nucleotide sequences reveal major
differences between Arabidopsis and potato: Homology between
AtCgS and the StCgS1 or StCgS2 cDNA is
rather low (76.9% or 72.6%, respectively) with respect to the MTO1
coding region. Therefore, we postulate that specific DNA and/or RNA
cis-elements contribute to the AtCgS autoregulation. To
follow this argument to its logical conclusion, we propose that
analogous functional cis-elements are absent in potato plants. Interestingly, Amir et al. (2002) recently reported that
the mRNA sequence of the AtCgS exon 1 located near the MTO1
coding region might form stable stem-loop structures as predicted by
computer analysis. Thus, the hypothesis that modifications in the
nucleotide sequence cause the deregulation of the potato CgS messenger
will be crucial to further molecular and biochemical investigations.
To gain further knowledge about the contribution of CgS to the control
of Met synthesis in potato plants, we employed different reverse
genetic approaches to generate transgenic potato plants displaying
increased or decreased CgS enzymatic activity levels. Transgenic potato
plants constitutively overexpressing the StCgS1 gene
(Riedel et al., 1999) showed no visible phenotype,
despite strong increases in CgS transcript and protein accumulation,
associated by up to 2.7-fold increases in CgS enzymatic activities.
Metabolite analyses demonstrated that soluble Met contents were
unchanged in source leaves, sink leaves, and flowers when compared with that of control plants, thus indicating that in vivo achieved increases
in CgS activity did not lead to an improved metabolite flux toward Met
synthesis. This assumption is strongly supported by the analysis of
protein-bound Met levels, which were found to be generally unaltered in
leaf tissues of lines overexpressing CgS. Although leaves of sCgS lines
revealed moderate increases of soluble Met levels, this was
statistically insignificant. Because leaf metabolites are known to
display large variations (up to 40%) among equivalent samples
(Fiehn et al., 2000; Roessner et al.,
2000; 2001; Maimann et al., 2001;
Zeh et al., 2001), these changes might simply reflect
biological variability and, thus, do not allow further interpretation.
The finding that free or bound leaf Met contents are immune to enhanced
CgS activity levels was rather unexpected. If potato CgS were to
function as a flux-controlling enzyme, even minor increases should
enhance the metabolite flux toward Met synthesis and/or its
accumulation. Such an effect has been observed in Arabidopsis and
tobacco (Nicotiana tabacum): Increases in free Met
levels (up to 40-fold) and SMM contents (up to 25-fold) were obtained
as a result of expressing either full-length or N-terminal truncated
AtCgS proteins, respectively (Gakière et al.,
2002; Hacham et al., 2002; Kim et al.,
2002). As a simple explanation, one might assume that
substrates required for cystathionine synthesis are not available in
sufficient amounts to cope with increased CgS activity. Yet, this
explanation is rather unlikely because precursors of the Met pathway
have been proven to be generally not limiting for Met biosynthesis in
potato plants (Zeh et al., 2001). The fact that none of
the CgS overexpression lines showed significant alterations in soluble
Asp, homoserine, or Cys levels corroborates this interpretation. The
lack of Met accumulation indicates that potato CgS is present at levels
exceeding requirements to provide the overall flux of the pathway and,
therefore, is not limiting the rate of Met synthesis in potato plants.
With the intention of inhibiting both known potato CgS isoforms
(Hesse et al., 1999; Riedel et al., 1999)
in a single transgenic approach, we used a partial sequence of the
StCgS1 cDNA showing a high homology of 86.9% to the
StCgS2 sequence for antisense expression. Analysis of
antisense and cosuppression lines revealed clearly decreased levels of
CgS mRNA, protein, and enzymatic activities. Cosuppression of
CgS had even stronger effects on activity, resulting in
merely 7% residual activity in leaves. Surprisingly, none of these
transgenic lines showed visible morphological changes, even during
developmental stages with higher demands for Met (for example, the
onset of reproductive growth). This is even more impressive with regard
to analyses of concomitant free amino acid compositions, revealing that
inhibition of CgS was accompanied by substantial and statistically
significant decreases in leaf-soluble Met levels. The expectation was
that a reduced capacity to synthesize Met would lead to abnormal
phenotypes such as severe stunting of growth and an inability to
flower, as reported for Arabidopsis CgS antisense plants displaying
reductions in CgS activity comparable with those we observed
(Gakière et al., 2000; Kim and Leustek,
2000). Though decreased Met levels were consistently observed
among potato plants suppressed in CgS, our data also revealed large
variations among soluble leaf Met contents between different sets of
plants, as indicated by wild-type Met levels shown in Tables I and II.
Yet, similar observations have been described by Zeh et al.
(2001). This phenomenon might be attributed to slight
differences in the developmental stage among analyzed sets of plants or
to minor variations in growth conditions in the greenhouse. However,
our results still suggest that the bottlenecks resulting from antisense inhibition and cosuppression were effective. Although this
interpretation seems to be straight forward because it may also explain
the accumulation of the CgS substrate OPHS, it does not account for the
observed maintenance of protein-bound Met in CgS antisense and
cosuppression lines. The most likely interpretation of this intriguing
finding is that decreasing CgS activity does not cause a reduced
metabolite flow toward Met synthesis in potato. As a consequence, we
have to assume that Met continues to be available for further
biochemical processes. At first sight, this striking behavior appears
to show, paradoxically, that potato CgS functions primarily to control the metabolic state of the Met pathway and not to control the rate of
the corresponding metabolite flux. However, considering that
perturbations of enzyme activities bare the risk of introducing artificial control points into a metabolic system (Kacser et
al., 1995; Fell, 1997), we suggest that potato
CgS catalyzes a near equilibrium reaction under wild-type conditions
(as already indicated by the analysis of the CgS-overexpressing
plants). Given that, CgS has a rather low flux control coefficient for
Met in potato plants
a finding that strongly contradicts the generally
accepted model developed for Arabidopsis.
Based on the presented work on potato CgS, we deduced an alternative
model for the regulation of de novo Met synthesis in higher plants: The
crux of this model is that potato CgS is not the flux-determining step
of the pathway because it shows no feedback regulation and demonstrates
a low flux control coefficient for Met synthesis. The same holds true
for the enzymes downstream of the CgS, namely CbL and MS, because they
do not increase the flow of metabolites when overexpressed in potato
(Maimann et al., 2000; Nikiforova et al.,
2002). As the remaining regulatory feature, we propose that the
balancing of the fluxes into the Met and Thr branches is almost
exclusively executed by TS activity levels in potato, whereas carbon
skeletons are distributed mainly due to CgS/TS competition in
Arabidopsis. Our finding that changing the CgS to TS ratio by
manipulating CgS activity levels does not affect flux toward Met (and
Thr) in potato supports this interpretation. Even more evidence is
provided by previous studies, in which expression of TS from
Escherichia coli leads to 5-fold increased Thr levels and
reduced Met contents in transgenic solanaceous plants (Muhitch, 1997). In as much, our hypothesis could explain why suppression of the potato TS gave rise to a much lower molar decrease in leaf Thr
contents than increase in soluble Met (Zeh et al.,
2001), whereas impaired TS activity caused an accumulation of
Met at the expense of nearly equimolar reductions in Thr levels in
rosette leaves of young Arabidopsis mto2 mutant plants
(Bartlem et al., 2000). Last but not least, sizes of
soluble Met and Thr pools in Solanaceae seem to be strongly mediated by
the availability of carbon resources. For that reason, enhancing the
overall carbon flux toward Met and Thr synthesis by expressing a
feedback-insensitive Asp kinase caused up to 9-fold rises in Thr in
transgenic tobacco plants (Shaul and Galili, 1992;
Galili, 1995). The observation that Met levels remained
constant under these conditions might reflect that the common substrate
OPHS is preferably channeled toward Thr production in wild-type plants
(Thompson et al., 1982).
Yet, how can all these differences in pathway regulation be reconciled?
There is no doubt that important regulatory features remain to be
uncovered. Nevertheless, our data demonstrate that de novo synthesis of
Met in potato is much more flexible than in Arabidopsis and, as a
consequence, that potato plants can tolerate large variations in the
soluble Met pool. Reasons for this could be attributed to adaptive
differences between plant species. Ben-Tzvi Tzchori et al.
(1996) pointed out that Asp, which provides the carbon skeleton
for Met and Thr, might be more available in Solanaceae than in
Arabidopsis. Considering a relatively short timescale from germination
to seed formation, Arabidopsis might utilize strict flux control to
avoid inefficient consumption of resources. Because potato plants
exhibit longer growth periods mainly adjusted to generate tubers as
sink organs in their later growth phase, such efficient mechanisms for
flux control or regulation of metabolite pools, respectively, may not
be necessary. Therefore, our results contribute to the hypothesis that
Met metabolic flux control mechanisms are not ubiquitous among all
plant species (Miron et al., 2000; Zeh et al.,
2001; Amir et al., 2002; Galili and
Höfgen, 2002).
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Generation of Transgenic Potato Lines
Potato (Solanum tuberosum cv Désirée;
Saatzucht Lange AG, Bad Schwartau, Germany) CgS (designated
StCgS1; GenBank accession no. AF082891; Riedel et
al., 1999) was cut from pBluescript SK as a
truncated 1.2-kb EcoRI fragment and digested with Klenow enzyme from Escherichia coli DNA polymerase I to
generate blunt ends. The fragment was ligated in reverse orientation
with respect to the 35S promoter into the vector pBinAR-Kan
(Höfgen and Willmitzer, 1990) previously cut with
SmaI to provide an antisense construct for plant
transformation. To create a construct for stable overexpression of
CgS, the full-length StCgS1 cDNA (1.8 kb)
was cut from pBluescript SK as a
BamHI/Asp718 fragment and cloned into the
same sites of the vector pBinAR-Kan. Transformation of potato plants
was carried out by Agrobacterium tumefaciens-mediated
gene transfer (Rocha-Sosa et al., 1989) using the strain
C58C1/pGV2260 (Deblaere et al., 1985) as described by
Dietze et al. (1995). Transgenic plants were selected on
kanamycin-containing medium (10 mg L1) supplemented with
casein hydrolysate (200 mg L1). The resulting transgenic
lines were transferred into soil and grown in the greenhouse at 20°C
with a light/dark rhythm of 16 h/8 h. Transformants were screened for
changes in CgS transcript levels by RNA-blot analyses of leaf tissues.
Standard techniques were essentially executed as described by
Sambrook et al. (1989).
Plant Cultivation
Transgenic CgS-overexpressing and antisense plants were
propagated in tissue culture along with potato wild-type plants and transferred into soil after 2 weeks of cultivation. The rooted shoots
were planted in small pots and grown in the phytotron with a light
regime of 200 to 250 µmol s1 m1 (16 h/8
h) under a hood to retain high air humidity. After 2 weeks, plants were
transferred into pots with a diameter of 20 cm and cultivated in a
greenhouse providing nearly natural light conditions with an
approximately 16-h-light/8-h-dark period plus natural sunlight. Light
intensity and temperature were dependent on environmental conditions,
but light did not fall below 250 to 300 µmol photons m2
s1, and temperature did not sink below 18°C.
Alternatively, plants were grown in a seasonally (March-September)
used "summer greenhouse" providing only natural light and
temperature conditions. Leaf material was harvested from
greenhouse-grown plants after approximately 8 weeks of cultivation,
before the onset of flowering. Leaf discs were excised from tissues of
similar developmental stage. Transition to the reproductive stage could
usually be observed only in plants older than 10 weeks. In accordance,
flowers were collected from 10- to 12-week-old plants. All plant
material was sampled between 10 and 12 AM and immediately
frozen in liquid nitrogen before storage at 
80°C.
Extraction and Analysis of Soluble Amino Acids
Soluble amino acids were determined following a modified
protocol from Scheible et al. (1997). Leaf tissues
(about 100 mg per plant) were ground to a fine powder in liquid
nitrogen in a bead mill and extracted three times for 20 min at 80°C:
once with 400 µL of 80% (v/v) aqueous ethanol (buffered with 2.5 mM HEPES-KOH, pH 7.5) and 10 µL of 20 µM
L-nor-Val (as an internal standard), once with 400 µL of
50% (v/v) aqueous ethanol (buffered as before) and once with 200 µL
of 80% (v/v) aqueous ethanol. Between the extraction steps, the
samples were centrifuged for 10 min at 14,000 rpm, and the supernatants
were collected. The combined ethanol/water extracts were stored at
20°C or directly subjected to RP-HPLC using an ODS column (Hypersil
C18; 150- × 4.6-mm i.d.; 3 µm; Knauer GmbH, Berlin)
connected to an HPLC system (Dionex, Idstein, Germany). Amino
acids were measured by precolumn derivatization with OPA in combination
with fluorescence detection (Lindroth and Mopper,
1979) as described by Ravanel et al. (1996). Peak areas were integrated by using Chromeleon 6.30 software
(Dionex) and subjected to quantification by means of calibration curves made from standard mixtures.
Extraction and Analysis of Soluble Thiol Compounds
Individual soluble thiols were determined as the sum of their
reduced and oxidized forms. One hundred milligrams of fresh ground leaf
material (see above) was added to 100 mg of
polyvinylpolypyrrolidone (previously washed with 0.1 M HCl) and 1 mL of 0.1 M HCl. The samples were
shaken for 60 min at room temperature. After centrifugation (15 min at
14,000 rpm; 4°C), the supernatants were frozen at 20°C until
reduction/derivatization. Thiols were reduced by incubating 120 µL of
the extracts with 200 µL of 0.25 CHES-NaOH (pH 9.4) and 70 µL of
freshly prepared 10 mM dithiothreitol for 40 min at
RT. According to Fahey et al. (1981), thiols were
derivatized for 15 min in the dark after adding 10 µL of 25 mM mBrB to each sample. The reaction was stopped by the
addition of 220 µL of 100 mM methanesulfonic acid and
incubation for 30 min in the dark. After centrifugation (15 min at
14,000 rpm; 4°C), the supernatants were submitted to RP-HPLC
analysis. The separation of thiols was performed according to
Blaszczyk et al. (2002) using an ODS column (Eurosphere
C18; 200- × 4.6-mm i.d.; 5 µm; Knauer GmbH) and a Dionex
HPLC system. Mixed standards treated exactly as the sample supernatants
were used as a reference for the quantification of Cys and
glutathione content.
Analysis of Protein-Bound Amino Acids
The determination of protein bound amino acids was carried using
HPLC analysis after proteolytically cleaving soluble leaf proteins with
Pronase (Roche, Manheim, Germany). The suitability of this
method was tested by hydrolyzing different quantities of soluble leaf
proteins. Released amino acids were linearly correlated to
corresponding protein amounts within the tested range (10-400 µg of
total proteins). Moreover, the digestion of bovine serum albumin
revealed an amino acid composition according to published data
(Brown, 1975). Protein concentrations were determined
according to Bradford (1976).
About 250 mg of fresh ground leaf material (see above) was extracted in 1 mL of 50 mM HEPES (pH 7.5), 10 mM CaCl2, and 0.1% (v/v) Triton X-100. Aliquots of 500 µL were desalted using pre-equilibrated NAP-5 columns (Amersham Pharmacia Biotech, Freiburg, Germany). Protein hydrolysis was performed by utilizing extract volumes containing 200 µg of protein and 1 unit of Pronase for each sample in a total volume of 800 µL of 50 mM HEPES (pH 7.5) and 10 mM CaCl2. To enable efficient degradation, proteins were digested at 37°C for 2 d. Amino acids were then extracted by adding 400 µL of methanol and 200 µL of chloroform to 500 µL of each reaction mix. After centrifugation (10 min at 14,000 rpm; 4°C), the upper, aqueous phase was subjected to amino acid analysis via HPLC as described above. Concurrently, three samples containing no protein extract were treated in the same manner to quantify the background resulting from Pronase self-digestion.
RNA- and Protein-Blot Analysis
Total potato leaf RNA was prepared according to Logemann
et al. (1987). Thirty micrograms of total RNA was loaded per
lane on denaturing 1.5% (w/v) agarose gels containing 15%
(w/v) formaldehyde. Gels were blotted to nylon membranes,
hybridized under stringent conditions with specific radioactively
labeled cDNA-probes. To examine mRNA expression of both isoforms of the
StCgS, we utilized a 1.2-kb internal
EcoRI fragment of the StCgS1 cDNA
(Riedel et al., 1999) showing high homology (86.9%) to
the corresponding StCgS2 sequence (Hesse et al.,
1999). Arabidopsis CgS mRNA contents were
analyzed using a 1.25-kb SacI fragment of the
AtCgS cDNA (Kim and Leustek, 1996). The
level of gene expression was estimated from resulting x-ray films.
Protein-blot analyses were performed as described by Maimann et
al. (2000) using the polyclonal antibodies described there.
CgS Assay
CgS activity was measured following the protocol provided by
Zeh et al. (2001), which is based on the method
described by Ravanel et al. (1995). CgS activity
measurements were carried out using desalted protein extracts (100 µg
total protein) made from source leaf tissues of 8-week-old
greenhouse-grown plants utilizing OPHS (ChiroBlock GmbH, Wolfen,
Germany) and L-Cys as the physiological substrates of the
CgS. Product formation (L-cystathionine) was determined
after 30 min of incubation according to the protocol for the HPLC
analysis of amino acids.
Feeding Experiments with Detached Potato Compound Leaves
Feeding experiments were carried out using compound leaves
directly cut from 8-week-old greenhouse-grown potato plants. To avoid
xylem embolism, the compound leaves were cut again under a buffer
solution (10 mM MES and 1 mM EDTA [pH 6.5])
and then immediately transferred to fresh buffer containing 0.1, 1, and 5 mM dissolved Met. Detached potato compound leaves were
incubated in buffer without Met as a control. After 24 h of
incubation under constant light conditions (120 µmol m2
s1), leaf discs were taken from subterminal leaflets and
analyzed for soluble Met, CgS mRNA levels, and CgS
enzyme activities as described above.
Feeding Experiments Using an in Vitro Transcription and Translation System
The full-length AtCgS cDNA was generated from
Arabidopsis (Columbia-0) rosette leaf mRNA templates by utilizing the
First Strand cDNA Synthesis Kit (Pharmacia). PCR was performed using oligonucleotide primer covering the flanking regions of the AtCgS coding region and proof reading Pfu DNA polymerase
(Stratagene, Heidelberg, Germany). Sequence analysis of the
1.7-kb PCR product revealed identity with the published
AtCgS sequence (CGS1; Kim and Leustek,
1996; GenBank accession no. U43709). The AtCgS cDNA was ligated into the pBlueScript SK (Stratagene) via
BamHI/Asp718 restriction sites introduced
by PCR amplification and subjected to further analysis.
In vitro transcription and translation of the full-length cDNA of the StCgS1 or AtCgS, respectively, was performed using the TNT coupled wheat germ system (Promega, Madison, WI). Reaction mixtures were prepared by utilizing an amino acid mixture lacking Met supplied by the manufacturers. Final concentrations of 0.1, 0.5, 1, and 5 mM Met were adjusted by adding appropriate quantities of this amino acid to each reaction mixture separately. Moreover, respective cDNAs were expressed in the absence of Met. After incubation at 30°C for 90 min, reactions were stopped by freezing the samples in liquid nitrogen. StCgS and AtCgS transcript levels were subsequently determined via RNA-blot analysis as previously described. To ensure that the application of Met was not limiting the transcription rate and/or transcript stability in general, expression analysis of the firefly luciferase gene (provided by the manufacturer) was performed as a positive control.
Statistical Analysis
The Student's t test method (Microsoft Excel 2000, Microsoft, Redmond, WA) was routinely used to determine the significance of differences between means of data sets. Mean values were compared under the assumption that the variances of both ranges of data are unequal; it is referred to as a heteroscedastic t test. Differences between data sets were regarded as significant in case probabilities of error were below 5% (P < 0.05).
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ACKNOWLEDGMENTS |
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We wish to thank Romy Ackermann for performing the potato transformations, the gardeners for excellent greenhouse work, and Josef Bergstein for photographical assistance. We thank Prof. Mark Stitt, his technical assistant Regina Feil, and Dr. Malcolm Hawkesford for their support during metabolite analyses. We would like to thank Dr. Stefanie Maimann and Dr. Michaela Zeh for providing the antibodies for CbL and MS. Moreover, we wish to thank Dr. Bertrand Gakière for fruitful discussions and Megan McKenzie for carefully editing this manuscript. We are grateful to Prof. Lothar Willmitzer for supporting this work.
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FOOTNOTES |
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Received October 10, 2002; returned for revision November 6, 2002; accepted January 3, 2003.
1 This work was supported by European FP4 (no. Bio-4CT-97-2182) and FP5 (no. QLRT-2000-00103) project grants and by the Max-Planck-Society.
* Corresponding author; e-mail hesse{at}mpimp-golm.mpg.de; fax 49-331-567-8250.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.015933.
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LITERATURE CITED |
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|
TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
|
|---|
-synthase and threonine synthase operate in concert to regulate carbon flow towards methionine in plants.
Trends Plant Sci
7: 153-156[CrossRef][Web of Science][Medline]-synthase mRNA stability in Arabidopsis.
Science
286: 1371-1374-synthase in Arabidopsis thaliana leads to increased levels of methionine and S-methylmethionine.
Plant Physiol Biochem
40: 119-126-synthase cDNA.
C R Acad Sci III
323: 841-851[Medline]-synthase plays an important role in methionine metabolism.
Plant Physiol
128: 454-462-synthase in potato (Solanum tuberosum L.) (PGR 99-161).
Plant Physiol
121: 1057-synthase in Arabidopsis leads to accumulation of soluble methionine and S-methylmethionine.
Plant Physiol
128: 95-107-synthase from Arabidopsis thaliana.
Plant Mol Biol
32: 1117-1124[CrossRef][Web of Science][Medline]-synthase in Arabidopsis thaliana produces partial methionine auxotrophy and developmental abnormalities.
Plant Sci
151: 9-18[CrossRef]
-lyase activity in transgenic potato plants does not force metabolite flow towards methionine.
Planta
214: 163-170[Web of Science][Medline]-lyase in plant growth and development.
Plant J
23: 747-758[CrossRef][Web of Science][Medline]-synthase gene in Arabidopsis.
J Biol Chem
277: 36380-36386-synthase from spinach chloroplasts.
Arch Biochem Biophys
316: 572-584[CrossRef][Web of Science][Medline]-synthase from potato.
Plant Biol
1: 638-644-synthase, O-phosphohomoserine sulfhydrylase, and O-acetyl sulfhydrylase.
Plant Physiol
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-Synthase in Genetically
Engineered Potato Plants
