The sucrose analog palatinose leads to a stimulation of sucrose degradation and starch synthesis when supplied to discs of growing potato tubers

In the present paper we investigated the effect of the sucrose (Suc) analog palatinose on potato (Solanum tuberosum) tuber metabolism. In freshly cut discs of growing potato tubers, addition of 5 mM palatinose altered the metabolism of exogenously supplied [U-14C]Suc. There was slight inhibition of the rate of 14C-Suc uptake, a 1.5-fold increase in the rate at which 14C-Suc was subsequently metabolized, and a shift in the allocation of the metabolized label in favor of starch synthesis. The sum result of these changes was a 2-fold increase in the absolute rate of starch synthesis. The increased rate of starch synthesis was accompanied by a 3-fold increase in inorganic pyrophosphate, a 2-fold increase in UDP, decreased UTP/UDP, ATP/ADP, and ATP/AMP ratios, and decreased adenylate energy charge, whereas glycolytic and Krebs cycle intermediates were unchanged. In addition, feeding palatinose to potato discs also stimulated the metabolism of exogenous 14C-glucose in favor of starch synthesis. In vitro studies revealed that palatinose is not metabolized by Suc synthases or invertases within potato tuber extracts. Enzyme kinetics revealed different effects of palatinose on Suc synthase and invertase activities, implicating palatinose as an allosteric effector leading to an inhibition of Suc synthase and (surprisingly) to an activation of invertase in vitro. However, measurement of tissue palatinose levels revealed that these were too low to have significant effects on Suc degrading activities in vivo. These results suggest that supplying palatinose to potato tubers represents a novel way to increase starch synthesis.     

Subcellular pyrophosphate metabolism in developing tubers of potato (Solanum tuberosum)

PPi has previously been implicated specifically in the co-ordination of the sucrose–starch transition and in the broader context of its role as co-factor in heterotrophic plant metabolism. In order to assess the compartmentation of pyrophosphate (PPi) metabolism in the potato tuber we analysed the effect of expressing a bacterial pyrophosphatase in the amyloplast of wild type tubers or in the cytosol or amyloplast of invertase-expressing tubers. The second and third approaches were adopted since we have previously characterized the invertase expressing lines to both exhibit highly altered sucrose metabolism and to contain elevated levels of PPi (Farré et al. (2000a) Plant Physiol 123:681) and therefore this background rendered questions concerning the level of communication between the plastidic and cytosolic pyrophosphate pools relatively facile. In this study we observed that the increase in PPi in the invertase expressing lines was mainly confined to the cytosol. Accordingly, the expression of a bacterial pyrophosphatase in the plastid of either wild type or invertase-expressing tubers did not lead to a decrease in total PPi content. However, the expression of the heterologous pyrophosphatase in␣the cytosol of cytosolic invertase-expressing tubers led to strong metabolic changes. These results are discussed both with respect to our previous hypotheses and to current models of the compartmentation of potato tuber metabolism.     

Effect of chlorocholine chloride sprays on the carbohydrate composition and activities of sucrose metabolising enzymes in potato (Solanum tuberosum L.)

Chlorocholine chloride (CCC) was sprayed on a potato crop 25 days after sowing (DAS) at 5 day intervals for a total of 7 sprays. Activity of sucrose synthase (SS) in the sucrose cleavage direction was many fold higher than that of acid invertase in all the tissues. The activity of alkaline invertase was negligible. A sharp decline in the starch content of stolons of the CCC-sprayed crop was observed between 60 DAS and 70 DAS. This could divert the carbon towards tubers and thus enhancing its availability for starch synthesis. The CCC-treated crop, in general, had higher SS (cleavage) activity in stem, stolons and tubers. A higher sucrose content in the stem of the CCC-treated crop could be due to the high sucrose phosphate synthase (SPS) activity observed in this plant part. In tubers of CCC-treated crops a higher SS (cleavage) activity along with a high sucrose content in tubers during the active tuber filling stage could lead to better availability of UDP-glucose for its conversion to glucose-1-phosphate, which could enter into the amyloplast leading to higher starch content. High SPS activity in tubers of CCC-treated plants ensures that reducing sugars formed are reconverted efficiently to sucrose. The efficiency of developing tubers from CCC-sprayed plants to convert 14C sucrose fed through stolons into starch was about 2.5 times more than in the control.     

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.     

A bypass of sucrose synthase leads to low internal oxygen and impaired metabolic performance in growing potato tubers

Plants possess two alternative biochemical pathways for sucrose (Suc) degradation. One involves hydrolysis by invertase followed by phosphorylation via hexokinase and fructokinase, and the other route-which is unique to plants-involves a UDP-dependent cleavage of Suc that is catalyzed by Suc synthase (SuSy). In the present work, we tested directly whether a bypass of the endogenous SuSy route by ectopic overexpression of invertase or Suc phosphorylase affects internal oxygen levels in growing tubers and whether this is responsible for their decreased starch content. (a) Oxygen tensions were lower within transgenic tubers than in wild-type tubers. Oxygen tensions decreased within the first 10 mm of tuber tissue, and this gradient was steeper in transgenic tubers. (b) Invertase-overexpressing tubers had higher activities of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase, and (c) higher levels of lactate. (d) Expression of a low-oxygen-sensitive Adh1-beta-glucuronidase reporter gene construct was more strongly induced in the invertase-overexpressing background compared with wild-type background. (e) Intact transgenic tubers had lower ATP to ADP ratios than the wild type. ATP to ADP ratio was restored to wild type, when discs of transgenic tubers were incubated at 21% (v/v) oxygen. (f) Starch decreased from the periphery to the center of the tuber. This decrease was much steeper in the transgenic lines, leading to lower starch content especially near the center of the tuber. (g) Metabolic fluxes (based on redistribution of (14)C-glucose) and ATP to ADP ratios were analyzed in more detail, comparing discs incubated at various external oxygen tensions (0%, 1%, 4%, 8%, 12%, and 21% [v/v]) with intact tubers. Discs of Suc phosphorylase-expressing lines had similar ATP to ADP ratios and made starch as fast as wild type in high oxygen but had lower ATP to ADP ratios and lower rates of starch synthesis than wild type at low-oxygen tensions typical to those found inside an intact tuber. (h) In discs of wild-type tubers, subambient oxygen concentrations led to a selective increase in the mRNA levels of specific SuSy genes, whereas the mRNA levels of genes encoding vacuolar and apoplastic invertases decreased. (i) These results imply that repression of invertase and mobilization of Suc via the energetically less costly route provided by SuSy is important in growing tubers because it conserves oxygen and allows higher internal oxygen tensions to be maintained than would otherwise be possible.     

Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants

By using barley seeds, developmental changes of ADPglucose (ADPG)-producing sucrose synthase (SS) and ADPG pyrophosphorylase (AGPase) have been compared with those of UDPglucose (UDPG), ADPG, sucrose (Suc) and starch contents. Both ADPG-synthesizing SS and AGPase activity patterns were found to correlate well with those of ADPG and starch contents. Remarkably, however, maximal activities of ADPG-synthesizing SS were found to be several fold higher than those of AGPase throughout seed development, the highest rate of starch accumulation being well accounted for by SS. Kinetic analyses of SS from barley endosperms and potato tubers in the Suc cleavage direction showed similar K(m) values for ADP and UDP, whereas apparent affinity for Suc was shown to be higher in the presence of UDP than with ADP. Moreover, measurements of transglucosylation activities in starch granules incubated with purified SS, ADP and [U-(14)C]Suc revealed a low inhibitory effect of UDP. The ADPG and UDPG contents in the transgenic S-112 SS and starch deficient potato mutant [Zrenner et al. (1995) Plant J. 7: 97] were found to be 35% and 30% of those measured in wild-type plants, whereas both glucose-1-phosphate and glucose-6-phosphate contents were found to be normal as compared with those of wild-type plants. The overall results thus strongly support a novel gluconeogenic mechanism reported previously [Pozueta-Romero et al. (1999) CRIT: Rev. Plant Sci. 18: 489] wherein SS catalyses directly the de novo production of ADPG linked to starch biosynthesis in heterotrophic tissues of plants.     

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers

Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571–581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa1 tubers. Unexpectedly, the rate of degradation of [¹⁴C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20–30% in ppa1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to threefold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30–100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed. Received: 9 July 1997 / Accepted: 3 November 1997     

Expression of a bacterial sucrose phosphorylase in potato tubers results in a glucose-independent induction of glycolysis

Sugars are not only metabolic substrates: they also act as signals that regulate the metabolism of plants. Previously, we found that glycolysis is induced in transgenic tubers expressing a yeast invertase in the cytosol but not in those expressing invertase in the apoplast. This suggests that either the low level of sucrose, the increased formation of cytosolic glucose or the increased levels of metabolites downstream of the sucrose cleavage is responsible for the induction of glycolysis in storage organs. In order to discriminate between these possibilities, we cloned and expressed a bacterial sucrose phosphorylase gene from Pseudomonas saccharophila in potato tubers. Due to the phosphorolytic cleavage of sucrose, formation of glucose was circumvented, thus allowing assessment of the importance of cytosolic glucose – and, by implication, flux through hexokinase – in glycolytic induction. Expression of sucrose phosphorylase led to: (i) a decrease in sucrose content, but no decrease in glucose or fructose; (ii) a decrease in both starch accumulation and tuber yield; (iii) increased levels of glycolytic metabolites; (iv) an induction of the activities of key enzymes of glycolysis; and (v) increased respiratory activity. We conclude that the induction of glycolysis in heterotrophic tissues such as potato tubers occurs via a glucose‐independent mechanism.     

Induction of the activity of glycolytic enzymes correlates with enhanced hydrolysis of sucrose in the cytosol of transgenic potato tubers

The aim of this work was to define the metabolic factors which regulate the respiratory pathways in trangenic potato tubers. We previously found that respiration is enhanced in transgenic tubers which express a yeast invertase and a glucokinase from Zymomonas mobilis . In this study we investigated glycolysis in three further transgenic potato lines with profound changes in the mobilization of sucrose. We studied antisense ADPglucose pyrophosphorylase lines which are characterized by a reduction in starch accumulation and a significant build up of sucrose and related metabolic intermediates. We also report the generation of two novel double transgenic lines where the yeast invertase is expressed specifically in tubers of the ADPglucose pyrophosphorylase antisense line, targeted to either the cytosol or apopolast. We evaluated whether the localization of sucrose cleavage had an impact on the glycolytic induction, and assessed if invertase expression in the high-sucrose background had any further effects on glycolysis. We found that induction of the glycolytic enzymes only occurs when the invertase is targeted to the cytosol, and that the extent of this induction was comparable in the wild type and antisenseADPglucose pyrophosphorylase backgrounds. We conclude that the signal regulating glycolysis is directly linked to cytosolic sucrose hydrolysis.     

Evidence against sink limitation by the sucrose-to-starch route in potato plants expressing fructosyltransferases

To investigate whether the route from sucrose to starch limits sink strength of potato tubers, we established an additional storage carbohydrate pool and analyzed allocation of imported assimilates to the different pools. Tuber specific expression of the fructan biosynthetic enzymes of globe artichoke resulted in accumulation of fructans to about 5% of the starch level, but did not increase tuber dry weight per plant. While partial repression of starch synthesis caused yield reduction in wild-type plants, it stimulated fructan accumulation, and yield losses were ameliorated in tubers expressing fructosyltransferases. However, a nearly complete block of the starch pathway by inhibition of sucrose synthase could not be compensated by the fructan pathway. Although fructan concentrations rose, yield reduction was even enhanced, probably because of a futile cycle of fructan synthesis and degradation by invertase, which is induced when sucrose synthase is knocked out. The data do not support a limitation of sink strength by enzyme activities of the starch pathway but point to an energy limitation of storage carbohydrate formation in potato tubers.     

Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum)

Tuberising stolon tips of potato ( Solanum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2 cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present) to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum, ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase. fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

RNA interference-mediated repression of sucrose-phosphatase in transgenic potato tubers (Solanum tuberosum) strongly affects the hexose-to-sucrose ratio upon cold storage with only minor effects on total soluble carbohydrate accumulation

Storage of potato tubers at low temperatures leads to the accumulation of glucose and fructose in a process called 'cold sweetening'. The aim of this work was to investigate the role of sucrose-phosphatase (SPP) in potato tuber carbohydrate metabolism at low temperature (4 degrees C). To this end, RNA interference (RNAi) was used to reduce SPP expression in transgenic potato tubers. Analysis of SPP specific small interfering RNAs (siRNAs), SPP protein accumulation and enzyme activity indicated that SPP silencing in transgenic tubers was stable during the cold treatment. Analysis of soluble carbohydrates showed that in transgenic tubers, cold-induced hexogenesis was inhibited while, despite strongly reduced SPP activity, sucrose levels exceeded wild-type (WT) values four- to fivefold after 34 d of cold treatment. This led to a drastic change in the hexose-to-sucrose ratio from 1.9 in WT tubers to 0.15 to 0.11 in transgenic tubers, while the total amount of soluble sugars was largely unchanged in both genotypes. Sucrose-6(F)-phosphate (Suc6P), the substrate of SPP, accumulated in transgenic tubers in the cold which most likely enables the residual enzyme to operate with maximal catalytic activity in vivo and thus, in the long term, counterbalances reduced SPP activity in the transformants. Northern analysis revealed that cold-induced expression of vacuolar invertase (VI) was blocked in SPP-silenced tubers explaining a reduced sucrose-to-hexose conversion. Suc6P levels were found to negatively correlate with VI expression. A possible role of Suc6P in regulating VI expression is discussed.     

Impact of invertase overexpression on cell size, starch granule formation and cell wall properties during tuber development in potatoes with modified carbon allocation patterns

Transgenic potato tubers that overexpressed either a cytosolic or an apoplastic invertase in the wild type or AGPase antisense background were used to analyse the effect of invertase activity on cell expansion, starch granule formation and turgor pressure during tuber development. Although the transgenic plants did not develop a visible phenotype in aerial regions the size and number of tubers were significantly modified in the various lines. Transmission electron and light microscopy were performed to monitor starch grain size and number, cell size and cell wall thickness. Water potential, osmotic pressure, and indirectly, turgor pressure were determined during the final stages of tuber development. Glucose levels were high in transgenic tubers that overexpressed a yeast-derived invertase. The number of starch grains per cell was almost identical in all transgenic lines. However, the amount of starch was modified in the transgenics as compared to the wild type. As expected, the size of starch grains was reduced in all lines that expressed an AGPase antisense mRNA. These results indicate that invertase activity and glucose levels do not affect initiation of starch grain formation during the early stages of tuber development, but growth of starch corns in the later stages of tuber maturation.     

Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques

Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning.     

Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis

Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H(+)-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1(Pro):IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal β-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.     

Temporally regulated expression of a yeast invertase in potato tubers allows dissection of the complex metabolic phenotype obtained following its constitutive expression

The constitutive cytosolic expression of a yeast (Saccharomyces cerevisiae) invertase within potato (Solanum tuberosum) tubers has previously been documented to produce a dramatic metabolic phenotype in which glycolysis, respiration and amino acid synthesis are markedly enhanced at the cost of starch synthesis. These transgenic lines were further characterised by a massive cycle of sucrose degradation and resynthesis via sucrose-phosphate synthase. We have recently developed a B33 patatin driven alc gene construct allowing tight chemical control of gene expression following supply of acetaldehyde with minimal pleiotropic effects of the inducing agent on metabolism. This construct was used for chemical induction of the yeast invertase gene after 10-weeks growth to dissect the complex metabolic phenotype obtained after constitute expression. Inducible expression led to increased invertase activity within 24 h in well-defined areas within growing tubers. Although the sucrose levels were reduced, there was no effect on the levels of starch whilst levels of many amino acids decreased. Labelling experiments revealed that these lines exhibited increased rates of sucrose cycling, whereas rates of glycolysis and of starch synthesis were not substantially changed. From these results we conclude that sucrose cycling is stimulated in response to a short-term increase in the rate of sucrose mobilisation, providing evidence for a role of sucrose cycling as a buffering capacity that regulates the net rate of sucrose usage. In contrast, the dramatic increase in hexose-phosphate levels and the switch from starch synthesis to respiration seen on the constitutive expression of the invertase was not observed in the inducible lines, suggesting that this is the result of cumulative pleiotropic effects that occurred when the transgene was expressed throughout development.     

Sucrose metabolism in plastids

The question whether sucrose (Suc) is present inside plastids has been long debated. Low Suc levels were reported to be present inside isolated chloroplasts, but these were argued to be artifacts of the isolation procedures used. We have introduced Suc-metabolizing enzymes in plastids and our experiments suggest substantial Suc entry into plastids. The enzyme levansucrase from Bacillus subtilis efficiently synthesizes fructan from Suc. Targeting of this enzyme to the plastids of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants leads to high-level fructan accumulation in chloroplasts and amyloplasts, respectively. Moreover, introduction of this enzyme in amyloplasts leads to an altered starch structure. Expression of the yeast invertase in potato tuber amyloplasts results in an 80% reduction of total Suc content, showing efficient hydrolysis of Suc by the plastidic invertase. These observations suggest that Suc can enter plastids efficiently and they raise questions as to its function and metabolism in this organelle.     

Sucrose-to-Starch Metabolism in Tomato Fruit Undergoing Transient Starch Accumulation

Immature green tomato (Lycopersicon esculentum) fruits undergo a period of transient starch accumulation characterized by developmental changes in the activities of key enzymes in the sucrose (Suc)-to-starch metabolic pathway. Activities of Suc synthase, fructokinase, ADP-glucose (Glc) pyrophosphorylase, and soluble and insoluble starch synthases decline dramatically in parallel to the decrease in starch levels in the developing fruit. Comparison of "maximal" in vitro activities of the enzymes in the Suc-to-starch pathway suggests that these same enzymes are limiting to the rate of starch accumulation. In contrast, activities of invertase, UDP-Glc pyrophosphorylase, nucleoside diphosphate kinase, phosphoglucoisomerase, and phosphoglucomutase do not exhibit dramatic decreases in activity and appear to be in excess of starch accumulation rates. Starch accumulation is spatially localized in the inner and radial pericarp and columella, whereas the outer pericarp and seed locule contain little starch. The seed locule is characterized by lower activities of Suc synthase, UDP-Glc pyrophosphorylase, phosphoglucomutase, ADP-Glc pyrophosphorylase, and soluble and insoluble starch synthases. The outer pericarp exhibits comparatively lower activities of ADP-Glc pyrophosphorylase and insoluble starch synthase only. These data are discussed in terms of the developmental and tissue-specific coordinated control of Suc-to-starch metabolism.