The onset of sucrose accumulation in cold-stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose-phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase

These experiments investigate events involved in triggering sugar accumulation in the cold in tubers of Solanum tuberosum L. cv. Desirée. Sugar content, ¹⁴C-glucose metabolism, metabolite levels and activities of sucrose phosphate synthase (SPS) and starch-degrading enzymes were followed after transfer to 4°C. (i) Net sucrose accumulation began between 2 and 4 d. By 10 d, reducing sugars were also increasing. From 20 d onwards, sugar accumulation slowed. Sucrose fell, but reducing sugars continued to increase. (ii) To measure unidirectional sucrose synthesis, U-[¹⁴C]glucose was injected into tubers after various times at 4°C. The tubers were then incubated for 6 h. After 1 d at 4°C, both the absolute and the relative (expressed as a percentage of the metabolized label) rates of sucrose synthesis decreased compared to those at 20°C. Between 2 and 4 d at 4°C, labelling of sucrose increased 3-fold, to over 60% of the metabolized label. This high rate was maintained for up to 50 d in cold storage. When tissue slices were incubated with 2.5 mol m^?3 U-[¹⁴C]glucose, the rate of labelling of sucrose in slices from 6 d cold-stored material was higher than in slices from warm-stored material, irrespective of whether the incubation occurred at 4°C or at 20°C. (iii) Hexose-phosphates increased during the first day after transfer to 4°C. Their levels fell during the next 3 d, as sucrose synthesis increased. They then rose (until 20 d) and fell, in parallel with the rise and decline of sucrose levels. UDPglucose remained unaltered during the first 4 d, and then increased and decreased in parallel with sucrose. (iv) SPS activity assayed in optimal conditions and the total amount of SPS protein did not change. However, when assayed in the presence of phosphate and limiting substrate concentrations, activity rose 3–5-fold between 2 and 4 d. (v) Amylases and phosphorylases were investigated using zymograms to separate isoforms. Phosphorylases did not change. Between 2 and 4 d at 4°C, a new amylolytic activity appeared. (vi) Estimates of the specific activity of the phosphorylated intermediates and the absolute rate of sucrose synthesis (calculated from the ¹⁴C-labelling data and metabolite analysis) showed that changed kinetic properties of SPS and decreased levels of hexose-phosphate are accompanied by a 6–8-fold stimulation of sucrose synthesis. They also show that the final level of sugar is partly determined by a cycle of sugar synthesis and degradation. (vii) It is concluded that the onset of sugar accumulation in cold-stored tubers is initiated by a change in the kinetic properties of SPS and the appearance of a new amylolytic activity. It is discussed how other factors, including hexose-phosphate levels and subcellular compartmentalization, could also influence the final levels of sugars by altering the balance of sugar synthesis and remobilization.     

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.     

Altered metabolic fluxes result from shifts in metabolite levels in sucrose phosphorylase-expressing potato tubers

As reported in a previous paper (Plant, Cell and Environment 24, 357–365, 2001), introduction of sucrose phosphorylase into the cytosol of potato results in increased respiration, an inhibition of starch accumulation and decreased tuber yield. Herein a more detailed investigation into the effect of sucrose phosphorylase expression on tuber metabolism, in order to understand why storage and growth are impaired is described. (1) Although the activity of the introduced sucrose phosphorylase was low and accounted for less than 10% of that of sucrose synthase its expression led to a decrease in the activities of enzymes of starch synthesis relative to enzymes of glycolysis and relative to total amylolytic activity. (2) Incubation of tuber discs in [¹⁴C]glucose revealed that the transformants display a two‐fold increase of the unidirectional rate of sucrose breakdown. However this was largely compensated by a large stimulation of sucrose re‐synthesis and therefore the net rate of sucrose breakdown was not greatly affected. Despite this fact major shifts in tuber metabolism, including depletion of sucrose to very low levels, higher rates of glycolysis, and larger pools of amino acids were observed in these lines. (3) Expression of sucrose phosphorylase led to a decrease of the cellular ATP/ADP ratio and energy charge in intact growing tubers. It was estimated that at least 30% of the ATP formed during respiration is consumed as a result of the large acceleration of the cycle of sucrose breakdown and re‐synthesis in the transformants. Although the absolute rate of starch synthesis in short‐term labelling experiments with discs rose, starch synthesis fell relative to other fluxes including respiration, and the overall starch content of the tubers was lower than in wild‐type tubers. (4) External supply of amino acids to replace sucrose as an osmoticum led to a feed‐back inhibition of glycolysis, but did not restore allocation to starch. (5) However, an external supply of the non‐metabolizable sucrose analogue palatinose – but not sucrose itself – stimulated flux to starch in the transformants. (6) The results indicate that the impaired performance of sucrose phosphorylase‐expressing tubers is attributable to decreased levels of sucrose and increased energy consumption during sucrose futile cycling, and imply that sucrose degradation via sucrose synthase is important to maintain a relatively large sucrose pool and to minimize the ATP consumption required for normal metabolic function in the wild type.     

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.     

Carbohydrate metabolism during postharvest ripening in kiwifruit

Mature fruit (kiwifruit) of Actinidia deliciosa var. deliciosa (A. Chev.), (C.F.) Liang and Ferguson cv. Haywood (Chinese gooseberry) were harvested and allowed to ripen in the dark at 20° C. Changes were recorded in metabolites, starch and sugars, adenine nucleotides, respiration, and sucrose and glycolytic enzymes during the initiation of starch degradation, net starch-to-sucrose conversion and the respiratory climacteric. The conversion of starch to sucrose was not accompanied by a consistent increase in hexose-phosphates, and UDP-glucose declined. The activity of sucrose phosphate synthase (SPS) measured with saturating substrate rose soon after harvesting and long before net sucrose synthesis commenced. The onset of sugar accumulation correlated with an increase in SPS activity measured with limiting substrates. Throughout ripening, until sucrose accumulation ceased, feeding [¹⁴C] glucose led to labelling of sucrose and fructose, providing evidence for a cycle of sucrose synthesis and degradation. It is suggested that activation of SPS, amplified by futile cycles, may regulate the conversion of starch to sugars. The respiratory climacteric was delayed, compared with net starchsugar interconversion, and was accompanied by a general decline of pyruvate and all the glycolytic intermediates except fructose-1,6-bisphosphate. The ATP/ ADP ratio was maintained or even increased. It is argued that the respiratory climacteric cannot be simply a consequence of increased availability of respiratory substrate during starch-sugar conversion, nor can it result from an increased demand for ATP during this process.Abbreviations Frul,6bisP fructose-1,6-bisphosphate - Frul,6Pase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - SPS sucrose phosphate synthase - UDPGlc uridine 5'-diphosphoglucose We thank Professor G. Costa, University of Udine and Flavia Succhi, University of Bologna for their help in obtaining the fruit in Italy. E.A.M. was the recipient of a travel grant through the NZ/German Technological Agreement.     

Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana

Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi-limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose-1,5-bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose-P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end-product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP-glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards starch synthesis. These responses are presumably triggered when sucrose synthesis is decreased in the night, as well as by day.     

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).     

Tuber-specific expression of a yeast invertase and a bacterial glucokinase in potato leads to an activation of sucrose phosphate synthase and the creation of a sucrose futile cycle

Fluxes were investigated in growing tubers from wild-type potato (Solanum tuberosum L. cv. Desiree) and from transformants expressing a yeast invertase in the cytosol under the control of the tuber-specific patatin promoter either alone (EC 3.2.1.26; U-IN2-30) or in combination with a Zymomonas mobilis glucokinase (EC 2.7.1.2; GK3-38) by supplying radiolabelled [¹⁴C]sucrose, [¹⁴C]glucose or [¹⁴C]fructose to tuber discs for a 90-min pulse and subsequent chase incubations of 4 and 12 h, and by supplying [¹⁴C]fructose for 2 h and 4 h to intact tubers attached to the mother plant. Contrary to the expectation that this novel route for sucrose degradation would promote starch synthesis, the starch content decreased in the transgenic lines. Labelling kinetics did not reveal whether this was due to changes in the fluxes into or out of starch. However, they demonstrated that glycolysis is enhanced in the transgenic lines in comparison to the wild type. There was also a significant stimulation of sucrose synthesis, leading to a rapid cycle of sucrose degradation and resynthesis. The labelling pattern indicated that sucrose phosphate synthase (SPS; EC 2.4.1.14) was responsible for the enhanced recycling of label into sucrose. In agreement, there was a 4-fold and 6-fold increase in the activation status of SPS in U-IN2-30 and GK3-38, respectively, and experiments with protein phosphatase inhibitors indicated that this activation involves enhanced dephosphorylation of SPS. It is proposed that this activation of SPS is promoted by the elevated glucose 6-phosphate levels in the transgenic tubers. These results indicate the pitfalls of metabolic engineering without a full appreciation of the metabolic system and regulatory circuits present in the tissue under investigation. Received: 21 July 1998 / Accepted: 5 December 1998     

Decreased amount of reducing sugars in transgenic potato tubers and its influence on yield characteristics

This work focuses on the comparison of field characteristics and amounts of reducing sugars in cold-stored tubers of transgenic plants derived from two potato cultivars. The bacterial gene coding for phosphofructokinase under the tuber-specific promoter was used to support the glycolysis in stored tubers. While the tubers from untransformed control plants steadily accumulated reducing sugars during cold storage, the tubers from transformed plants regardless the genotype were characterized by subsequent decrease in the sugar content. After long period of cold storage the greatest reduction in the reducing sugar content was by more than 60 % compared to control. Before the storage, however, the content of reducing sugars was in 80 % of transgenic lines higher than in control ones. The plants evaluated in field trials for their appearance showed any changes in growth characteristics in about 25 % of the transgenic lines. Despite the introduced modification of sugar metabolism the yield of transgenic plants with normal appearance did not differ significantly from the yield of control plants.     

In vitro and in vivo inhibition of sucrose synthesis by sucrose

The inhibitory effects of sucrose on rates of sucrose synthesis by sucrose phosphate synthase (SPS) from the maize scutellum and on net rates of sucrose production in maize scutellum slices from added glucose or fructose were studied. Scutellum extracts were prepared by freezing and thawing scutellum slices in buffer. The extracts contained SPS and sucrose phosphate phosphatase, but were free of sucrose synthase. SPS activity was calculated from measurement of UDP formation in the presence of UDPG, fructose-6-P and sucrose. The ranges of metabolite concentrations used were those estimated to be in scutellum slices after incubation in water or fructose for periods up to 5 hr. UDPG and fructose-6-P also were added at concentrations that saturated SPS. At saturating substrate levels, sucrose inhibition of SPS was less than that when tissue levels of substrates were used. With tissue levels of substrates and sucrose concentrations up to ca 166 mM, sucrose inhibitions of sucrose synthesis in vitro by SPS were similar to those observed in vivo. However, as the sucrose concentration rose above 166 mM, SPS activity was not inhibited further, whereas there was a further sharp decline in sucrose production by the slices. It is concluded that sucrose synthesis in vivo is controlled by sucrose inhibition of SPS over a considerable range of internal sucrose concentrations.     

Effect of temperature on invertase, invertase inhibitor, and sugars in potato tubers

The accumulation of reducing sugars in potato tubers exposed to low temperatures occurs with concomitant formation of the enzyme invertase. During the initial period of cold treatment when reducing sugars increase rapidly, invertase formation proceeds until the level of enzyme exceeds that of an endogenous macromolecular invertase inhibitor, resulting in a basal invertase activity. As the rate of sugar accumulation decreases and the sugar level becomes nearly constant, total invertase decreases, the basal activity disappears, and a low excess of inhibitor develops. On transfer of cold-stored tubers to warmer temperatures, sugars and invertase decrease sharply and a large excess of inhibitor develops. These changes in sugars, invertase and inhibitor occur reversibly when the tubers are subjected to alternating temperatures.     

Contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis is decreased by water stress in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch and cell-wall synthesis in tissue slices of growing potato (Solanum tuberosum L. cv. Desirée) tubers. Based on the analysis of fluxes and metabolites, Geigenberger et al. (1997, Planta 201: 502–518) proposed that water deficits up to −0.72 MPa stimulate sucrose synthesis, leading to decreased starch synthesis as a result of the resulting decline of phosphorylated metabolite levels, whereas more-severe water deficits directly inhibit the use of ADP-glucose. Potato plants with decreased expression of adenosine 5′-diphosphoglucose pyrophosphorylase (AGPase) have been used to test the prediction that the contribution of AGPase to the control of starch synthesis should decrease in severely water-stressed tuber material. Freshly cut slices from wild-type and antisense tubers were incubated at a range of mannitol concentrations (20, 300 and 500 mM) and the metabolism of [¹⁴C]glucose was analysed. A 86–97% reduction of AGPase activity led to a major but non-stoichiometric inhibition of starch accumulation in intact growing tubers attached to the plant (40–85%), and an inhibition of starch synthesis in non-stressed tuber slices incubated in 20 mM mannitol (60–80%). The inhibition of starch synthesis was accompanied by a 2- to 8-fold increase in the levels of sugars in intact tubers and a 2- to 3-fold stimulation of sucrose synthesis in tuber slices, whereas respiration and cell-wall synthesis were not significantly affected. The strong impact of AGPase on carbon partitioning in non-stressed tubers and tuber slices was retained in slices subjected to moderate water deficit (300 mM mannitol, corresponding to −0.72 MPa). In discs incubated in 500 mM mannitol (corresponding to −1.2 MPa) this response was modified. A 80–97% reduction of AGPase resulted in only a 0–40% inhibition of starch synthesis. Further, the water stress-induced stimulation of sucrose synthesis was abolished in the transformants. The results provide direct evidence that the contribution of AGPase to the control of starch synthesis can be modified by environmental factors, leading to a lower degree of control during severe water deficits. There was also a dramatic decrease in the labelling of cell-wall components in wild-type tuber slices incubated with 300 or 500 mM mannitol. The water stress-induced inhibition of cell-wall synthesis occurred independently of AGPase expression and the accompanying changes in starch and sucrose metabolism, indicating a direct inhibition of cell-wall synthesis in response to water stress. Received: 24 February 1999 / Accepted: 28 May 1999     

苹果中磷酸蔗糖合酶家族基因的表达特性及其与蔗糖含量的关系

磷酸蔗糖合酶(sucrose phosphate synthase,SPS)是植物中蔗糖合成的主要限速酶,影响植物的生长发育和果实中蔗糖的含量。为探明苹果中SPS基因家族特性及其在蔗糖合成中的作用,该研究从苹果基因组中分离了MdSPS家族基因,分析了它们的进化关系以及mRNA表达特性与酶活性和蔗糖含量的关系。结果显示:(1)在苹果基因组中有8个SPS家族基因表达,它们分别属于双子叶植物的3个SPS亚家族。(2)荧光定量PCR分析显示,苹果C类的MdSPS6基因和A类的MdSPS1a/b基因是苹果中表达丰度最高的SPS基因成员,其中MdSPS6在苹果成熟果中表达丰度最高,其次是成熟叶片,而MdSPS1a/b在不积累蔗糖的幼果中表达丰度最高。(3)在果实发育过程中,除MdSPS1a/b之外,其它5个苹果MdSPS家族基因均随果实的生长表达丰度增加,与SPS活性和蔗糖含量明显呈正相关关系。研究表明,C类家族MdSPS6是苹果果实发育后期和叶片中蔗糖合成的主要SPS基因。