Antisense Repression of Hexokinase 1 Leads to an Overaccumulation of Starch in Leaves of Transgenic Potato Plants But Not to Significant Changes in Tuber Carbohydrate Metabolism

Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants). Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers. However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period. Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content. These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis.     

Control of potato tuber dormancy and sprouting by expression of sense and antisense genes of pyrophosphatase in potato

Inorganic pyrophosphate (PPi) is an enzyme involved in sugar metabolism in potato tubers. In our previous study, we isolated an inorganic pyrophosphatase (PPase) gene from potato and obtained the transgenic potato plants transformed with the sense and antisense PPase genes respectively. In the present experiment, the physiological indexes, tuber dormancy, and sprouting characteristics of the transgenic potatoes were analyzed and evaluated. The result showed that the PPase activity and the inorganic phosphate content of tubers were lower in the antisense transgenic plant lines but were higher in the sense transgenic plant lines, compared with wild-type tubers. Soluble sugars, such as glucose, fructose and sucrose increased in transgenic plants that had overexpression of the sense PPase gene, but decreased in the antisense transgenic plant lines, compared with wild-type tubers. Tuber sprouting time of the antisense transgenic plants were delayed for 2 and 3 weeks and reached the 100 % sprouting rate only after 14 and 16 weeks storage compared with the wild-type when tubers are stored under 25 and 4 °C, respectively. In contrast, tuber sprouting time of the sense transgenic plants was earlier by approximately 2 weeks than that of wild-type tubers under these storage temperatures.     

Evidence that SNF1-related kinase and hexokinase are involved in separate sugar-signalling pathways modulating post-translational redox activation of ADP-glucose pyrophosphorylase in potato tubers

We recently discovered that post-translational redox modulation of ADP-glucose pyrophosphorylase (AGPase) is a powerful new mechanism to adjust the rate of starch synthesis to the availability of sucrose in growing potato tubers. A strong correlation was observed between the endogenous levels of sucrose and the redox-activation state of AGPase. To identify candidate components linking AGPase redox modulation to sugar supply, we used potato tuber discs as a model system. When the discs were cut from growing wild-type potato tubers and incubated for 2 h in the absence of sugars, redox activation of AGPase decreased because of a decrease in internal sugar levels. The decrease in AGPase redox activation could be prevented when glucose or sucrose was supplied to the discs. Both sucrose uptake and redox activation of AGPase were increased when EDTA was used to prepare the tuber discs. However, EDTA treatment of discs had no effect on glucose uptake. Feeding of different glucose analogues revealed that the phosphorylation of hexoses by hexokinase is an essential component in the glucose-dependent redox activation of AGPase. In contrast to this, feeding of the non-metabolisable sucrose analogue, palatinose, leads to a similar activation as with sucrose, indicating that metabolism of sucrose is not necessary in the sucrose-dependent AGPase activation. The influence of sucrose and glucose on redox activation of AGPase was also investigated in discs cut from tubers of antisense plants with reduced SNF1-related protein kinase activity (SnRK1). Feeding of sucrose to tuber discs prevented AGPase redox inactivation in the wild type but not in SnRK1 antisense lines. However, feeding of glucose leads to a similar activation of AGPase in the wild type and in SnRK1 transformants. AGPase redox activation was also increased in transgenic tubers with ectopic overexpression of invertase, containing high levels of glucose and low sucrose levels. Expression of a bacterial glucokinase in the invertase-expressing background led to a decrease in AGPase activation state and tuber starch content. These results show that both sucrose and glucose lead to post-translational redox activation of AGPase, and that they do this by two different pathways involving SnRK1 and an endogenous hexokinase, respectively.     

Alteration of the Amount of the Chloroplast Phosphate Translocator in Transgenic Tobacco Affects the Distribution of Assimilate between Starch and Sugar

Tobacco plants (Nicotiana tabacum L.) transformed with sense and antisense constructs of a cDNA encoding the tobacco phosphate-triose phosphate-3-phosphoglycerate translocator (phosphate translocator) were shown to contain altered amounts of phosphate translocator mRNA and protein. Phosphate translocator activity in intact chloroplasts isolated from transformed plants showed a 15-fold variation, from 20% of the wild-type activity in antisense transformants to 300% of the wild-type activity in sense transformants. However, the maximal rates of photosynthesis and the rates of photosynthetic carbon assimilation in ambient CO2 showed no consistent differences between transformants. Starch content was decreased by 20% and total soluble sugars were increased by 20% in leaves of antisense transformants compared to sense transformants. The 40% decrease in the ratio of starch to total soluble sugars in antisense transformants relative to sense transformants indicates that distribution of assimilate between starch and sugar had been altered. However, the amount of sucrose in the leaves was unchanged. The changes in total soluble sugars were accounted for completely by changes in glucose and fructose, suggesting the existence of a homeostatic mechanism for maintaining sucrose concentrations in the leaves at the expense of glucose and fructose.     

Soluble acid invertase determines the hexose-to-sucrose ratio in cold-stored potato tubers

Cold storage of potato (Solanum tuberosum L.) tubers is known to cause accumulation of reducing sugars. Hexose accumulation has been shown to be cultivar-dependent and proposed to be the result of sucrose hydrolysis via invertase. To study whether hexose accumulation is indeed related to the amount of invertase activities, two different approaches were used: (i) neutral and acidic invertase activities as well as soluble sugars were measured in cold-stored tubers of 24 potato cultivars differing in the cold-induced accumulation of reducing sugars and (ii) antisense potato plants with reduced soluble acid invertase activities were created and the soluble sugar accumulation in cold-stored tubers was studied. The cold-induced hexose accumulation in tubers from the different potato cultivars varied strongly (up to eightfold). Large differences were also detected with respect to soluble acid (50-fold) and neutral (5-fold) invertase activities among the different cultivars. Although there was almost no correlation between the total amount of invertase activity and the accumulation of reducing sugars there was a striking correlation between the hexose/sucrose ratio and the extractable soluble invertase activitiy. To exclude the possibility that other cultivar-specific features could account for the obtained results, the antisense approach was used to decrease the amount of soluble acid invertase activity in a uniform genetic background. To this end the cDNA of a cold-inducible soluble acid invertase (EMBL nucleicacid database accession no. X70368) was cloned from the cultivar Desirée, and transgenic potato plants were created expressing this cDNA in the antisense orientation under control of the constitutive 35S cauliflower mosaic virus promotor. Analysis of the harvested and cold-stored tubers showed that inhibition of the soluble acid invertase activity leads to a decreased hexose and an increased sucrose content compared with controls. As was already found for the different potato cultivars the hexose/sucrose ratio decreased with decreasing invertase activities but the total amount of soluble sugars did not significantly change. From these data we conclude that invertases do not control the total amount of soluble sugars in coldstored potato tubers but are involved in the regulation of the ratio of hexose to sucrose.The authors are grateful to Heike Deppner and Christiane Prüßner for tuber harvest and technical assistance during the further analysis. We thank Andrea Knospe for taking care of tissue culture, Birgit Schäfer for patient photographic work, Hellmuth Fromme and the greenhouse personnel for attending plant growth and development and Astrid Basner for elucidating the sequence of clone INV-19. The work was supported by the Bundesministerium für Forschung und Technologie (BMFT).     

Effect of antisense repression of the chloroplast triose-phosphate translocator on photosynthetic metabolism in transgenic potato plants

The introduction of an antisense DNA into transgenic potato (Solanum tuberosum L.) plants decreased the expression of the chloroplast triose-phosphate translocator and lowered its activity by 20–30%. With plants propagated from tubers, the effect of the transformation on photosynthetic metabolism was analysed by measuring photosynthesis, the formation of leaf starch, and the total and subcellular metabolite contents in leaves. Although the transformants, in contrast to those propagated from cell cultures, did not differ from the wild-type plants in respect to rates of photosynthesis, plant appearance, growth and tuber production, their photosynthetic metabolism was found to be severely affected. The results show that the decrease in activity of the triose-phosphate translocator in the transformants caused a fourfold increase in the level of 3-phosphoglycerate and a corresponding decrease in inorganic phosphate in the stromal compartment, resulting in a large increase in the synthesis of starch. Whereas during a 12-h day period wild-type plants deposited 43% of their CO₂ assimilate into starch, this value rose to 61–89% in the transformants. In contrast to the wild-type plants, where the rate of assimilate export from the leaves during the night period was about 75% of that during the day, the export rate from leaves of transformants appeared to be much higher during the night than during the day. As the mobilisation of starch occurs in part hydrolytically, resulting in the formation of glucose, the triose-phosphate translocator loses its exclusive function in the export of carbohydrates from the chloroplasts when the photoassimilates are temporarily deposited as starch. It appears that by directing the CO₂ assimilates mainly into starch, the transformants compensate for the deficiency in triose-phosphate translocator activity in such a way that the productivity of the plants is not affected by the transformation.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - 3-PGA 3-phosphoglycerate - Rubisco ribulose,1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - trioseP triose phosphate - WT wild type The able technical assistance of Mrs. K. Wildenberger and Mrs. A. Großpietsch is gratefully acknowledged. This work has been supported by the Bundesminister für Forschung und Technologie.     

Phytochrome A resets the circadian clock and delays tuber formation under long days in potato

Transgenic potatoes (Solanum tuberosum) with either increased (sense transformants) or reduced (antisense transformants) phytochrome A (phyA) levels were used, in combination with specific light treatments, to investigate the involvement of phyA in the perception of signals that entrain the circadian clock. Far-red or far-red plus red light treatments given during the night reset the circadian rhythm of leaf movements in wild-type plants and phyA over-expressors, but had little effect in phyA under-expressors. Far-red light was also able to reset the rhythm of leaf movement in wild-type Arabidopsis thaliana but was not effective in mutants without phyA. Blue light was necessary to reset the rhythm in phyA-deficient potato plants. Resetting of the rhythm by far-red plus red light was only slightly affected in transgenic plants with reduced levels of phytochrome B. The production of tubers was delayed by day extensions with far-red plus red light, but this effect was reduced in transgenic lines deficient in phyA. We conclude that phyA is involved in resetting the circadian clock controlling leaf movements and in photoperiod sensing in light-grown potato plants.     

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.     

Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein

The expression patterns of plant defense genes encoding osmotin and osmotin-like proteins imply a dual function in osmotic stress and plant pathogen defense. We have produced transgenic potato (Solanum commersonii Dun.) plants constitutively expressing sense or antisense RNAs from chimeric gene constructs consisting of the cauliflower mosaic virus 35S promoter and a cDNA (pA13) for an osmotin-like protein. Transgenic potato plants expressing high levels of the pA13 osmotin-like protein showed an increased tolerance to the late-blight fungus Phytophthora infestans at various phases of infection, with a greater resistance at an early phase of fungal infection. There was a decrease in the accumulation of osmotin-like mRNAs and proteins when antisense transformants were challenged by fungal infection, although the antisense transformants did not exhibit any alterations in disease susceptibility. Expression of pA13 sense and antisense RNAs had no effect on the development of freezing tolerance in transgenic plants when assayed under a variety of conditions including treatments with abscisic acid or low temperature. These results provide evidence of antifungal activity for a potato osmotin-like protein against the fungus P. infestans, but do not indicate that pA13 osmotin-like protein is a major determinant of freezing tolerance.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and ¹³C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.Abbreviations CaMV cauliflower mosaic virus - NMR nuclear magnetic resonance We gratefully acknowledge Dr. Ulrich Eder (Schering AG, Berlin, Germany) for performing ¹³C-NMR spectroscopy, and Dr. Susanne Hoffmann-Benning (Institut für Genbiologische Forschung) for introducing us to immunohistochemistry. We thank Jessyca Dietze for plant transformations, Birgit Burose for taking care of greenhouse plants, and Antje Voigt for photographic work.     

Antisense inhibition of cytosolic phosphorylase in potato plants (Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate 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.     

Growth and tuberization of transgenic potato plants expressing sense and antisense sequences of Cu/Zn superoxide dismutase from lily chloroplasts

Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed reactive oxygen species, possibly via the GA biosynthesis pathway.     

Transgenic potato plants with altered expression levels of chloroplast NADP-malate dehydrogenase: interactions between photosynthetic electron transport and malate metabolism in leaves and in isolated intact chloroplasts

The contribution of the malate valve in the regulation of steady-state photosynthesis was studied in transgenic potato (Solanum tuberosum L. cv Désirée) plants with altered expression of plastidic NADP-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82). Mutant plants were obtained after transformation with the homologous Nmdh gene in antisense orientation, or with the Nmdh gene from pea (Pisum sativum L.) in sense orientation. A total number of nine stable sense and antisense lines with 10% or 30%, and 400% of wild-type NADP-MDH capacity were selected. Intact chloroplasts were isolated from leaves of wild-type and mutant plants. In chloroplasts from sense transformants the increased enzyme amount was activated as in wild-type chloroplasts, but increased rates of oxaloacetate-dependent malate formation were only measured upon partial uncoupling. In contrast, chloroplasts from antisense transformants produced only little malate upon oxaloacetate addition. Measurements with intact leaves during steady-state photosynthesis yielded no differences in gas-exchange parameters and chlorophyll fluorescence. The leaf malate content was unchanged in NADP-MDH underexpressors, but twice as high in overexpressing plants. The altered NADP-MDH expression clearly influences the redox state of ferredoxin, especially in low light. Furthermore, the malate valve can successfully compete for electrons with cyclic electron flow, but the conditions under which this occurs are quite artificial. Received: 14 February 1998 / Accepted: 12 May 1998     

Normal starch content and composition in tubers of antisense potato plants lacking D-enzyme (4-α-glucanotransferase)

Transgenic potato (Solanum tuberosum L.) plants were created with sense and antisense copies of the potato D-enzyme (disproportionating enzyme; EC␣2.4.1.25) cDNA linked to patatin and cauliflower mosaic virus 35 S promoters, and screened for D-enzyme activity in tubers. Transformants with sense constructs mostly had wild type D-enzyme activity but two plants had only about 1% wild-type activity. Transformants with antisense constructs had activity ranging from 90% to about 1% of wild type. Three 35 S antisense plants with very low activity were analysed in detail. Western blot analysis showed that D-enzyme was present in greatly reduced amounts in tubers and in leaves, whereas plastidic starch phosphorylase (EC 2.4.1.1) was unaffected. The lack of D-enzyme resulted in slow plant growth but development was otherwise apparently normal. Furthermore, the starch content of tubers was not appreciably altered in amount, proportion of amylose, molecular weight of debranched amylopectin, or branch chain length, despite the lack of D-enzyme. These results do not indicate a direct requirement for D-enzyme in the synthesis and accumulation of storage starch in tubers. The results are discussed in terms of the known reactions catalysed by D-enzyme and possible involvement of D-enzyme in starch metabolism. Received: 12 November 1997 / Accepted: 23 December 1997     

Decreased expression of sucrose phosphate synthase strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.