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Starches extracted from most plant species are phosphorylated. alpha-Glucan water dikinase (GWD) is a key enzyme that controls the phosphate content of starch. In the absence of its activity starch degradation is impaired, leading to a starch excess phenotype in Arabidopsis and in potato leaves, and to reduced cold sweetening in potato tubers. Here, we characterized a transposon insertion (legwd::Ds) in the tomato GWD (LeGWD) gene that caused male gametophytic lethality. The mutant pollen had a starch excess phenotype that was associated with a reduction in pollen germination. SEM and TEM analyses indicated mild shrinking of the pollen grains and the accumulation of large starch granules inside the plastids. The level of soluble sugars was reduced by 1.8-fold in mutant pollen grains. Overall, the transmission of the mutant allele was only 0.4% in the male, whereas it was normal in the female. Additional mutant alleles, obtained through transposon excision, showed the same phenotypes as legwd::Ds. Moreover, pollen germination could be restored, and the starch excess phenotype could be abolished in lines expressing the potato GWD homolog (StGWD) under a pollen-specific promoter. In these lines, where fertility was restored, homozygous plants for legwd::Ds were isolated, and showed the starch excess phenotype in the leaves. Overall, our results demonstrate the importance of starch phosphorylation and breakdown for pollen germination, and open up the prospect for analyzing the role of starch metabolism in leaves and fruits.

A functional screen in Escherichia coli was established to identify potato genes coding for proteins involved in transitory starch degradation. One clone isolated had a sequence very similar to a recently described chloroplast-targeted beta-amylase of Arabidopsis. Expression of the gene in E. coli showed that the protein product was a functional beta-amylase that could degrade both starch granules and solubilized amylopectin, while import experiments demonstrated that the beta-amylase was imported and processed into pea chloroplasts. To study the function of the protein in transitory starch degradation, transgenic potato plants were generated where its activity was reduced using antisense techniques. Analysis of plants reduced in the presence of this beta-amylase isoform showed that their leaves had a starch-excess phenotype, indicating a defect in starch degradation. In addition, it was shown that the antisense plants degraded only 8-30% of their total starch, in comparison with 50% in the wild type, over the dark period. This is the first time that a physiological role for a beta-amylase in plants has been demonstrated.

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.

GDP-mannose pyrophosphorylase (GMPase, EC 2.7.7.22) catalyses the synthesis of GDP-D-mannose and represents the first committed step in the formation of all guanosin-containing sugar nucleotides found in plants which are precursors for cell wall biosynthesis and, probably more important, the synthesis of ascorbate. A full-length cDNA encoding GMPase from S. tuberosum was isolated. Transgenic potato plants were generated in which the GMPase cDNA was introduced in antisense orientation to the 35S promoter. Transformants with reduced GMPase activity were selected. Transgenic plants were indistinguishable from the wild-type when held under tissue culture conditions, however, a major change was seen 10 weeks after transfer into soil. Transgenic plants showed dark spots on leaf veins and stems with this phenotype developing from the bottom to the top of the plant. In case of the line with the strongest reduction, all aerial parts finally dried out after 3 months in soil, in contrast to the wild-type plants which did not start to senesce at this time. This coincides with a reduction of ascorbate contents in the transgenic plants, which is in agreement with the recently proposed pathway of ascorbate biosynthesis. Furthermore, leaf cell walls of the transgenic potato plants had mannose contents that were reduced to 30-50% of the wild-type levels, whereas the composition of tuber cell walls was unchanged. The glycosylation pattern of proteins was unaffected by GMPase inhibition, as studied by affinoblot analysis.

We have used top-down metabolic control analysis to investigate the control of carbon flux through potato (Solanum tuberosum) plants during tuberisation. The metabolism of the potato plant was divided into two blocks of reactions (the source and sink blocks) that communicate through the leaf apoplastic sucrose pool. Flux was measured as the transfer of 14 C from CO 2 to the tuber. Flux and apoplastic sucrose concentration were varied either by changing the light intensity or using transgenic manipulations that specifically affect the source or sink blocks, and elasticity coefficients were measured. We have provided evidence in support of our assumption that apoplastic sucrose is the only communicating metabolite between the source and sink blocks. The elasticity coefficients were used to calculate the flux control coefficients of the source and sink blocks, which were 0.8 and 0.2, respectively. This work suggests that the best strategy for the manipulation of tuber yield in potato will involve increases in photosynthetic capacity, rather than sink metabolism.

To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.