Investigations into the role of the plastidial peptide methionine sulfoxide reductase in response to oxidative stress in Arabidopsis

Peptidyl Met residues are readily oxidized by reactive oxygen species to form Met sulfoxide. The enzyme peptide Met sulfoxide reductase (PMSR) catalyzes the reduction of Met sulfoxides back to Met. In doing so, PMSR is proposed to act as a last-chance antioxidant, repairing proteins damaged from oxidative stress. To assess the role of this enzyme in plants, we generated multiple transgenic lines with altered expression levels of the plastid form of PMSR (PMSR4). In transgenic plants, PMSR4 expression ranged from 95% to 40% (antisense) and more than 600% (overexpressing lines) of wild-type plants. Under optimal growing conditions, there is no effect of the transgene on the phenotype of the plants. When exposed to different oxidative stress conditions-methyl viologen, ozone, and high light-differences were observed in the rate of photosynthesis, the maximum quantum yield (Fv/Fm ratio), and the Met sulfoxide content of the isolated chloroplast. Plants that overexpressed PMSR4 were more resistant to oxidative damage localized in the chloroplast, and plants that underexpressed PMSR4 were more susceptible. The Met sulfoxide levels in proteins of the soluble fraction of chloroplasts were increased by methyl viologen and ozone, but not by high-light treatment. Under stress conditions, the overexpression of PMSR4 lowered the sulfoxide content and underexpression resulted in an overall increase in content.     

Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine gamma-synthase cDNA

To investigate the role of cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of cystathionine beta-lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.     

Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione

The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1. 30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5'-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.     

The influence of cytosolic phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPC) on potato tuber metabolism

The aim of this work was to investigate the importance of cytosolic phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPC) in potato carbohydrate metabolism. For this purpose, the cytosolic isoform of phosphorylating GAPC was cloned and used for an antisense approach to generate transgenic potato plants that exhibited constitutively decreased GAPDH activity. Potato lines with decreased activities of phosphorylating GAPC exhibited no major changes in either whole-plant or tuber morphology. However, the levels of 3-phosphoglycerate were decreased in leaves of the transformants. A broad metabolic phenotyping of tubers from the transformants revealed an increase in sucrose and UDPglucose content, a decrease in the glycolytic intermediates 3-phosphoglycerate and phosphoenolpyruvate but little change in the levels of other metabolites. Moreover, the transformants displayed no differences in cold sweetening with respect to the wild type. Taken together these data suggest that phosphorylating GAPC plays only a minor role in the regulation of potato metabolism. The results presented here are discussed in relation to current models regarding primary metabolism in the potato tuber parenchyma.     

Altered plastidic ATP/ADP-transporter activity influences potato ( Solanum tuberosum L.) tuber morphology, yield and composition of tuber starch

The metabolic function of the plastidic ATP/ADP transporter (AATP) in heterotrophic plastids was examined in transgenic potato plants that exhibited increased or decreased amounts of the protein. Altered mRNA levels correlated with activities of the plastidic ATP/ADP transporter. Potato tubers with decreased plastidic ATP/ADP transporter activities exhibited reduced starch contents whereas sense lines accumulated increased amounts of tuber starch. Starch from wild-type tubers had an amylose content of 18.8%, starch from antisense plants contained 11.5–18.0% amylose, whereas starch from sense plants had levels of 22.7–27.0%. The differences in physiological parameters were accompanied with altered tuber morphology. These changes are discussed with respect to the stromal ATP supply during starch biosynthesis.     

Enhanced formaldehyde detoxification by overexpression of glutathione-dependent formaldehyde dehydrogenase from Arabidopsis

The ADH2 gene codes for the Arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism in eukaryotes. In the present work, we have investigated the potential role of FALDH in detoxification of exogenous formaldehyde. We have generated a yeast (Saccharomyces cerevisiae) mutant strain (sfa1Delta) by in vivo deletion of the SFA1 gene that codes for the endogenous FALDH. Overexpression of Arabidopsis FALDH in this mutant confers high resistance to formaldehyde added exogenously, which demonstrates the functional conservation of the enzyme through evolution and supports its essential role in formaldehyde metabolism. To investigate the role of the enzyme in plants, we have generated Arabidopsis transgenic lines with modified levels of FALDH. Plants overexpressing the enzyme show a 25% increase in their efficiency to take up exogenous formaldehyde, whereas plants with reduced levels of FALDH (due to either a cosuppression phenotype or to the expression of an antisense construct) show a marked slower rate and reduced ability for formaldehyde detoxification as compared with the wild-type Arabidopsis. These results show that the capacity to take up and detoxify high concentrations of formaldehyde is proportionally related to the FALDH activity in the plant, revealing the essential role of this enzyme in formaldehyde detoxification.     

Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning

Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content. Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%). Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.     

Antisense Inhibition of Threonine Synthase Leads to High Methionine Content in Transgenic Potato Plants

Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality.     

Homeostatic regulation upon changes of enzyme activities in the Calvin cycle as an example for general mechanisms of flux control. What can we expect from transgenic plants?

In order to explain the mechanisms of Calvin-cycle regulation, the general properties of metabolic systems under homeostatic flux control are analyzed. It is shown that the main characteristic point for an enzyme in such a system can be the value of a sharp transition from some constant homeostatic flux to a limitation by this enzyme. A special method for the quantitative treatment of the experimental dependence of a metabolic flux such as photosynthesis on enzyme content is developed. It is pointed out that reactions close to a thermodynamic equilibrium under normal conditions can considerably limit the homeostatic fluxes with a decrease of the enzyme content. Calvin-cycle enzymes are classified as non-limiting, near-limiting and limiting. The deduced rules for the regulation of a homeostatic metabolic pathway are used to explain the data obtained for transgenic plants with reduced activities of Calvin-cycle enzymes. The role of compensating mechanisms that maintain the photosynthesis rate constant upon the changes of enzyme contents is analyzed for the Calvin cycle. The developed analysis explains the sharp transitions between limiting and non-limiting conditions that can be seen in transgenic plants with reduced content of some Calvin-cycle enzymes, and the limiting role of such reversible enzymes as aldolase, transketolase and others. The attempt is made to predict the properties of plants with increased enzyme contents in the Calvin cycle.     

Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.     

Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6-phosphate

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.     

珠子参中法尼基焦磷酸合酶(FPS)对皂苷生物合成的影响研究

珠子参作为名贵中草药已有上千年应用历史,珠子参皂苷是珠子参的主要活性成分,由达玛烷型和齐墩果烷型三萜皂苷组成。目前,对珠子参皂苷的生物合成了解甚少,有必要开展与皂苷合成相关的基础研究工作。已有报道表明,法尼基焦磷酸合酶（FPS）可能是植物中皂苷合成的关键酶。本研究克隆了珠子参FPS基因（PjFPS）的cDNA序列,并对其进行了生物信息学分析。基于PjFPS序列构建了珠子参过表达载体pCAMBIA2300s-PjFPS,将其转化到珠子参细胞中,成功获得阳性转基因细胞;测定了阳性细胞系中PjFPS基因的相对表达量、FPS酶活、皂苷以及植物甾醇含量的变化。结果表明,与野生型珠子参细胞相比,PjFPS转基因珠子参细胞系中,PjFPS基因的相对表达量、FPS酶活以及皂苷含量均有不同程度的提高。而且,由于皂苷合成代谢流的增加,也促进了相关重要关键酶基因的表达。在效果最好的阳性细胞中,PjFPS基因的相对表达量、FPS酶活、皂苷含量分别为对照的12、4和3倍;同时,转基因细胞系的植物甾醇含量也有所升高。本研究通过对皂苷合成途径关键酶基因的调控实现了对皂苷生物合成的调节,为获得高效、稳定的珠子参皂苷合成调控技术提供理论参考和依据。