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.     

Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes.

Transgenic potato plants were created in which the expression of ADP-glucose pyrophosphorylase (AGPase) was inhibited by introducing a chimeric gene containing the coding region of one of the subunits of the AGPase linked in an antisense orientation to the CaMV 35S promoter. Partial inhibition of the AGPase enzyme was achieved in leaves and almost complete inhibition in tubers. This resulted in the abolition of starch formation in tubers, thus proving that AGPase has a unique role in starch biosynthesis in plants. Instead up to 30% of the dry weight of the transgenic potato tubers was represented by sucrose and up to 8% by glucose. The process of tuber formation also changed, resulting in significantly more tubers both per plant and per stolon. The accumulation of soluble sugars in tubers of antisense plants resulted in a significant increase of the total tuber fresh weight, but a decrease in dry weight of tubers. There was no significant change in the RNA levels of several other starch biosynthetic enzymes, but there was a great increase in the RNA level of the major sucrose synthesizing enzyme sucrose phosphate synthase. In addition, the inhibition of starch biosynthesis was accompanied by a massive reduction in the expression of the major storage protein species of potato tubers, supporting the idea that the expression of storage protein genes is in some way connected to carbohydrate formation in sink storage tissues.     

The contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis in potato tubers

The aim of this work was to investigate the extent to which starch synthesis in potato (Solanum tuberosum L.) tubers is controlled by the activity of ADPglucose pyrophosphorylase (EC 2.7.7.27; AGPase). In order to do this, fluxes of carbohydrate metabolism were measured in tubers that had reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. Reduction in AGPase activity led to a reduction in starch accumulation, and an increase in sucrose accumulation. The control coefficient of AGPase on starch accumulation in intact plants was estimated to be around 0.3. The fluxes of carbohydrate metabolism were measured in tuber discs from wild-type and transgenic plants by investigating the metabolism of [U-¹⁴C]glucose. In tuber discs, the control coefficient of AGPase over starch synthesis was estimated as 0.55, while the control coefficient of the enzyme over sucrose synthesis was −0.47. The values obtained suggest that AGPase activity exerts appreciable control over tuber metabolism in potato. Received: 24 February 1999 / Accepted: 8 April 1999     

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.     

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.     

Constitutive expression of cell wall invertase genes increases grain yield and starch content in maize

Grain size, number and starch content are important determinants of grain yield and quality. One of the most important biological processes that determine these components is the carbon partitioning during the early grain filling, which requires the function of cell wall invertase. Here, we showed the constitutive expression of cell wall invertase–encoding gene from Arabidopsis, rice (Oryza sativa) or maize (Zea mays), driven by the cauliflower mosaic virus (CaMV) 35S promoter, all increased cell wall invertase activities in different tissues and organs, including leaves and developing seeds, and substantially improved grain yield up to 145.3% in transgenic maize plants as compared to the wild‐type plants, an effect that was reproduced in our 2‐year field trials at different locations. The dramatically increased grain yield is due to the enlarged ears with both enhanced grain size and grain number. Constitutive expression of the invertase‐encoding gene also increased total starch content up to 20% in the transgenic kernels. Our results suggest that cell wall invertase gene can be genetically engineered to improve both grain yield and grain quality in crop plants.     

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.     

Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity

We showed recently that antisense plants with decreased activity of the plastidic ATP/ADP-transporter protein exhibit drastically reduced levels of starch and a decreased amylose/amylopectin ratio, whereas sense plants with increased activity of the transporter possessed more starch than wild-type plants and an increased amylose/amylopectin ratio. In this paper we investigate the effect of altered plastidic ATP/ADP-transporter protein expression on primary metabolism and granule morphology in more detail. Tuber tissues from antisense and sense plants exhibited substantially increased respiratory activity compared with the wild type. Tubers from antisense plants contained markedly increased levels of free sugars, UDP-Glc, and hexose phosphates, whereas phosphoenolpyruvate, isocitrate, ATP, ADP, AMP, UTP, UDP, and inorganic pyrophosphate levels were slightly decreased. In contrast, tubers from sense plants revealed a slight increase in adenine and uridine nucleotides and in the levels of inorganic pyrophosphate, whereas no significant changes in the levels of soluble sugars and metabolites were observed. Antisense tubers contained 50% reduced levels of ADP-Glc, whereas sense tubers contained up to 2-fold increased levels of this sole precursor for starch biosynthesis. Microscopic examination of starch grain morphology revealed that the size of starch grains from antisense tubers was substantially smaller (50%) compared with the wild type. The large starch grains from sense tubers appeared of a more angular morphology, which differed to the more ellipsoid shape of wild type grains. The results suggest a close interaction between plastidial adenylate transport and starch biosynthesis, indicating that ADP-Glc pyrophosphorylase is ATP-limited in vivo and that changes in ADP-Glc concentration determine starch yield, as well as granule morphology. Possible factors linking starch synthesis and respiration are discussed.     

Effect of modified carbon allocation on turgor, osmolality, sugar and potassium content, and membrane potential in the epidermis of transgenic potato (Solanum tuberosum L.) plants

The effects of modification in sugar concentrations on turgor pressure and membrane potential in epidermal leaf cells of transgenic potato (Solanum tuberosum cv. Desirée) plants were studied. Measurements of turgor pressure were performed by insertion of a micro pressure probe. Osmolality and sugar concentrations were determined by micro analysis of single cell extracts. Membrane potentials and cell diameters were calculated from repeated, computer-controlled scans with voltage-sensitive microelectrodes. Epidermal cells of sucrose transporter antisense plants showed a more than 100% elevation in osmolality and turgor pressure compared to the wild-type. As a consequence, cell diameters were enhanced in this transgenic line. However, membrane potentials were only slightly reduced in sucrose transporter antisense plants. In addition to sucrose transporter antisense lines, transgenic plants that were reduced in their capacity to accumulate starch due to antisense inhibition of the chloroplastic fructose-1,6-bisphosphatase (FBPase) were investigated. These antisense plants maintained membrane potential and turgor pressure comparable to the wild-type.     

Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

Adenine nucleotides are of general importance for many aspectsof cell function, but their role in the regulation of biosyntheticprocesses is still unclear. It was previously reported thatdecreased expression of plastidial adenylate kinase, catalysingthe interconversion of ATP and AMP to ADP, leads to increasedadenylate pools and starch content in transgenic potato tubers.However, the underlying mechanisms were not elucidated. Here,it is shown that decreased expression of plastidial adenylatekinase in growing tubers leads to increased rates of respiratoryoxygen consumption and increased carbon fluxes into starch.Increased rates of starch synthesis were accompanied by post-translationalredox-activation of ADP-glucose pyrophosphorylase (AGPase),catalysing the key regulatory step of starch synthesis in theplastid, while there were no substantial changes in metabolicintermediates or sugar levels. A similar increase in post-translationalredox-activation of AGPase was found after supplying adenineto wild-type potato tuber discs to increase adenine nucleotidelevels. Results provide first evidence for a link between redox-activationof AGPase and adenine nucleotide levels in plants. Key words: Adenylate kinase, ADPglucose pyrophosphorylase, plastid, redox-regulation, potato, respiration, starch Received 18 September 2007; Revised 12 November 2007 Accepted 13 November 2007     

反义酸性转化酶基因低温贮藏马铃薯块茎还原糖和淀粉含量的影响

对2个含有酸性转化酶(AcInv)反义基因的转基因马铃薯品系及对照品种进行低温贮藏(4℃)及室温还暖处理.随低温贮藏时间的延长,供试材料均表现出还原糖含量升高,总淀粉含量下降的趋势.低温处理40 d时,"Ac转Atlantic"和"Ac转甘农薯2号"的还原糖含量比未转基因品种低23%和18%.总淀粉含量分别比未处理前下降约1.0%和1.3%,支链淀粉含量分别下降约1.4%和1.7%,淀粉直/支比明显低于对照,分别为0.29和0.38.块茎的石蜡切片显示,转基因块茎中深蓝色淀粉颗粒明显少于未转基因对照.另外,对低温贮藏的块茎室温还暖后,2个转基因品系的还原糖含量仍低于对照品种.实验结果证明反义AcInv基因对低温贮藏下块茎还原糖和淀粉含量具有下向调节作用.     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.     

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.     

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     

Embryo-specific reduction of ADP-Glc pyrophosphorylase leads to an inhibition of starch synthesis and a delay in oil accumulation in developing seeds of oilseed rape

In oil-storing Brassica napus (rape) seeds, starch deposition occurs only transiently in the early stages of development, and starch is absent from mature seeds. This work investigates the influence of a reduction of ADP-Glc pyrophosphorylase (AGPase) on storage metabolism in these seeds. To manipulate the activity of AGPase in a seed-specific manner, a cDNA encoding the small subunit of AGPase was expressed in the sense or antisense orientation under the control of an embryo-specific thioesterase promoter. Lines were selected showing an embryo-specific decrease in AGPase due to antisense and cosuppression at different stages of development. At early developmental stages (25 days after flowering), a 50% decrease in AGPase activity was accompanied by similar decreases in starch content and the rate of starch synthesis measured by injecting (14)C-Suc into seeds in planta. In parallel to inhibition of starch synthesis, the level of ADP-Glc decreased, whereas Glc 1-phosphate levels increased, providing biochemical evidence that inhibition of starch synthesis was due to repression of AGPase. At 25 days after flowering, repression of starch synthesis also led to a decrease in the rate of (14)C-Suc degradation and its further metabolism via other metabolic pathways. This was not accompanied by an increase in the levels of soluble sugars, indicating that Suc import was inhibited in parallel. Flux through glycolysis, the activities of hexokinase, and inorganic pyrophosphate-dependent phosphofructokinase, and the adenylate energy state (ATP to ADP ratio) of the transgenic seeds decreased, indicating inhibition of glycolysis and respiration compared to wild type. This was accompanied by a marked decrease in the rate of storage lipid (triacylglycerol) synthesis and in the fatty acid content of seeds. In mature seeds, glycolytic enzyme activities, metabolite levels, and ATP levels remained unchanged, and the fatty acid content was only marginally lower compared to wild type, indicating that the influence of AGPase on carbon metabolism and oil accumulation was largely compensated for in the later stages of seed development. Results indicate that AGPase exerts high control over starch synthesis at early stages of seed development where it is involved in establishing the sink activity of the embryo and the onset of oil accumulation.     

Response of potato tuber cell division and growth to shade and elevated CO2

Plants adjust their sink-organ growth rates, development and distribution of dry matter in response to whole-plant photosynthate status. To advance understanding of these processes, potato (Solanum tuberosum L.) plants were subjected to CO(2) and light flux treatments, and early tuber growth was assessed. Atmospheric CO(2) (700 or 350 micro mol mol(-1)) and light flux (shade and control illumination) treatments were imposed at two growth stages: tuber initiation (TI) and tuber bulking (TB). Elevated CO(2) increased accumulation of total net biomass when imposed at both stages, and increased tuber growth rate by about 36 %, but did not increase the number of tubers. Elevated CO(2) increased the number of cells in tubers at both TI and TB stages, whereas shade substantially decreased the number of cells at both stages. Generally, treatments did not affect cell volume or the proportion of nuclei endoreduplicating (repeated nuclear DNA replication in the absence of cell division), but the shade treatment led to a decrease in cell volume at TB and a decrease in endoreduplication at TI. Elevated CO(2) increased, and shade decreased, glucose concentration and soluble invertase activity in the cambial zones at both TI and TB, whereas sucrose concentration and activities of glucokinase, fructokinase, cell-wall-bound invertase and thymidine kinase were unaffected. Modulation of tuber cell division was responsible for much of the growth response to whole-plant photosynthate status, and treatments affected cambial-zone glucose and soluble invertase in a pattern suggesting involvement of a glucose signalling pathway.     

Transgenic 14-3-3 isoforms in plants: the metabolite profiling of repressed 14-3-3 protein synthesis in transgenic potato plants

14-3-3 proteins are abundant eukaryotic proteins that interact with many other proteins, thereby modulating their function and thus cell metabolism. The data from mRNA analysis confirm the developmental regulation of 14-3-3 isoform expression in potato plants. In order to test whether or not 14-3-3 protein expression affects plant phenotype and metabolism, transgenic potato plants either overexpressing Cucurbita pepo 14-3-3 or underexpressing endogenous 14-3-3 isoforms were analysed. An increase in tuber number and a decrease in tuber size in the overexpressed transformant was observed; the transgenic plants contain more chlorophyll than the control and they lose it more slowly than the control when transferred to the dark. The 14-3-3-repressed transgenic plants showed a decrease in tuber number and an increase in tuber size; an increase in the fresh weight of the transgenic tubers was also detected. The increased catecholamine level was accompanied by an increased ratio of soluble sugars to starch in overexpressed transformant. The opposite effect was detected in 14-3-3-repressed transgenic plants. All the repressed plants showed significant increases in nitrate reductase (NR) activity, suggesting that the regulation of NR occurs in vivo, and is not isoform-dependent. The increase in NR activity resulted in a significant decrease in nitrate level. The level of sucrose phosphate synthase activity was also significantly increased in all 14-3-3-underexpressed transgenes, and remarkably the increase in enzyme activity was accompanied by respective changes in sucrose levels in the tubers. The most intriguing finding was the significant (2-3-fold) increase in ethylene content in all the 14-3-3-repressed transgenic lines, which probably resulted from a methionine level increase. The substantial increase of ethylene level in the repressed forms might explain the significant shortening of the vegetation period of the analysed transgenic plants.     

A transgenic study on affecting potato tuber yield by expressing the rice sucrose transporter genes OsSUT5Z and OsSUT2M

In many plants, sucrose transporters are essential for both sucrose exports from sources and imports into sinks, indicating a function in assimilate partitioning. To investigate whether sucrose transporters can improve the yield of starch plant, potato plants (Solanum tuberosum L. cv. Désirée) were transformed with cDNAs of the rice sucrose transporter genes OsSUT5Z and OsSUT2M under the control of a tuber-specific, class-I patatin promoter. Compared to the controls, the average fructose content of OsSUT5Z transgenic tubers significantly increased. However, the content of the sugars and starch in the OsSUT2M transgenic potato tubers showed no obvious difference. Correspondingly, the average tuber yield, average number of tubers per plant and average weight of single tuber showed no significant difference in OsSUT2M transgenic tubers with controls. In the OsSUT5Z transgenic lines, the average tuber yield per plant was 1.9-fold higher than the controls, and the average number of tubers per plant increased by more than 10 tubers on average, whereas the average weight of a single tuber did not increase significantly. These results suggested that the average number of tubers per plant showed more contribution than the average weight of a single tuber to the tuber yield per plant.