Transgenic Arabidopsis plants expressing Escherichia coli pyrophosphatase display both altered carbon partitioning in their source leaves and reduced photosynthetic activity

The effects of the cytosolic expression of Escherichia coli pyrophosphatase (ppa) were investigated in the rosette leaves of transgenic Arabidopsis plants. During the daytime, glucose and fructose were found to accumulate at levels that were approximately two- to threefold higher in these plants than in the wild type. Interestingly, however, neither sucrose nor starch levels showed any distinctive build up in transgenic plants except under continuous white light growth conditions, during which they accumulated at high levels. Additionally, the leaves of transgenic Arabidopsis plants contain two- to threefold higher levels of inorganic phosphate (Pi) and two- to sixfold higher levels of uridine diphosphate-glucose than wild type plants during the diurnal cycle. In contrast, triose phosphate contents in the leaves of E. coli ppa transformants were either similar or slightly decreased when compared with wild type leaves. Furthermore, the photosynthetic activity of these transgenic plants was found to be reduced by 20–40% compared to normal levels. These results indicate that induction of ppa activity in the cytosol affects carbon partitioning between source and sink organs and also that the concomitant increase in Pi caused the accumulation of carbon metabolites and reduced photosynthetic activity.     

Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.)

 Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants. Received: 12 July 1999 / Accepted: 16 October 1999     

Improving freezing tolerance of transgenic tobacco expressing sucrose: sucrose 1-fructosyltransferase gene from Lactuca sativa

Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The 1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants.     

毛白杨蔗糖合酶基因PtSS2克隆与表达分析

为了解毛白杨蔗糖合酶(sucrose synthase,SS)基因功能和表达模式,以毛白杨茎段来源的cDNA为模板,根据毛果杨PtrSS2CDS序列信息设计特异引物,采用RT-PCR技术分离克隆了PtSS2基因。测序分析表明,PtSS2基因序列全长为2 412bp,编码803个氨基酸,蛋白大小为92.14kD,理论等电点6.00,属于酸性蛋白;氨基酸序列同源性分析表明,PtSS2与毛果杨PtrSS2和PtrSS1一致性分别高达100%和98.63%;结构域分析表明,PtSS2含有2个高度保守的功能域,即蔗糖合酶(5~552)和糖基化转移酶(554~744)功能域。qRT-PCR检测表明,PtSS2在毛白杨根、茎、叶、营养芽、雄雌花芽等各个组织中均有表达,但在营养芽和花芽中表达量较高,根部相对较低,总体呈组成型表达模式;在干旱胁迫下,PtSS2表达水平提升。研究结果推测,蔗糖合酶基因PtSS2在毛白杨各组织器官发育过程及干旱胁迫响应过程中具有重要功能。     

Intracellular site of sucrose synthesis in leaves

Improved conditions for extraction and assay increased rates of sucrose synthesis from uridine diphosphate glucose (UDPglucose) plus fructose 6-phosphate (F.6.P) catalysed by leaf extracts 20-fold. Rates of 17.9, 25·0, 9·2 and 27·7 μmol/hr/g fr. wt respectively were obtained from pea shoots, spinach, wheat and bean leaves. Chloroplasts isolated from pea shoots, in which half the plastids were intact, contained less than 4% of the total UDPglucose-fructosephosphate glucosyltransferase, more than 30% of the ribulose diphosphate (RuDP) carboxylase, and more than 40% of the total chlorophyll of the leaf. Although some of the UDPglucose-fructose-phosphate glucosyltransferase was associated with particles smaller than chloroplasts at least 85% of the enzyme was not precipitated at 38 000 g. UDPglucose pyrophosphorylase, also thought to be essential for sucrose synthesis, was distributed between the cell fractions in a similar manner to UDPglucose-fructosephosphate glucosyltransferase. It is concluded that sucrose synthesis in pea shoots and spinach leaves occurs mainly, in the cytoplasm.     

Pyrophosphate-dependent sucrose metabolism and its activation by fructose 2,6-bisphosphate in sucrose importing plant tissues

In the presence of pyrophosphate and uridine diphosphate, sucrose was cleaved to form glucose 1-phosphate and fructose with soluble extracts from sucrose importing plant tissues. The glucose 1-phosphate then was converted through glycolysis to triose phosphates in a pyrophosphate-dependent pathway which was activated by fructose 2,6-bisphosphate. Much less activity, less than 5%, was found in sucrose exporting tissue extracts from the same plants. These findings suggest that imported sucrose is metabolized in the cytoplasm of plant tissues by utilizing pyrophosphate and that sucrose metabolism is partially regulated by fructose 2,6-bisphosphate.     

Dissection of GA 20-oxidase members affecting tomato morphology by RNAi-mediated silencing

GA 20-oxidase is a key enzyme involved in gibberellin (GA) biosynthesis. In tomato, the GA 20-oxidase gene family consists of three members: GA20ox1, GA20ox2, and GA20ox3. To investigate the roles of these three genes in regulating plant growth and development, we used RNA interference technology to generate three kinds of transgenic tomato plants with suppressed expression of each three individual genes. Suppression of GA20ox1 or GA20ox2 resulted in shorter stems, a decreased length of internodes, and small dark green leaves while plants with decreased expression of GA20ox3 had no visible changes on stems and leaves. The plants of the three transgenic lines can flower and set fruits normally, but the seeds from these plants germinated slower than that from the normal plants. Decreased levels of endogenous GAs were detected in the apex of the three transgenic lines. These results demonstrate that the three GA 20-oxidase genes play different roles in the control of plan vegetative growth, but show no effects on flower and fruit development.Equal contribution authors: J. Xiao and H. Li.     

Antisense suppression of cucumber (Cucumis sativus L.) sucrose synthase 3 (CsSUS3) reduces hypoxic stress tolerance

Sucrose synthase (SUS; EC 2.4.1.13) plays important roles in sugar metabolism and abiotic stress response. But the genes encoding SUS in cucumber (Cucumis sativus L.) have not been well studied. Here, we isolated four cucumber sucrose synthase genes (CsSUS). Among them, CsSUS3, which highly expressed in the roots, was chosen for further study. Immunolocalization and subcellular localization analysis indicated that CsSUS3 localized in the cytosol and the plasma membrane, and mainly existed in the companion cells of phloem in the roots. When suffering hypoxia stress from flooding, CsSUS3 expression and SUS activity in roots increased, especially in the lateral roots; moreover, the soluble SUS activity increased clearly, but the membrane fraction hardly changed. Compared with the wild‐type cucumbers, the transgenic lines with antisense expression of CsSUS3 were more sensitive to flooding. After 6 d of flooding, the SUS activity, soluble sugar and uridine 5′‐diphosphate glucose (UDPG) content and the ratio of ATP/ADP in the roots of transgenic plants were significantly lower than that in wild‐type plants. Moreover, the transgenic lines grew more slowly with more yellow necrosis in the leaves. These findings suggested CsSUS3 participated in resisting hypoxic stress. Furthermore, the mechanism of CsSUS3 in resisting hypoxic stress was also discussed.     

Analysis of carbon metabolism in transgenic<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transformed with the cyanobacterial sucrose phosphate synthase gene

To investigate the effects of sucrose-phosphate synthase (SPS) on carbon partitioning, transgenicArabidopsis plants transformed withSynechocystis SPS were constructed. The integration, copy number and expression level were confirmed by Southern and Northern blot analyses. SPS activity in leaves from the transgenic and wild type plants was not significantly different. The level of sucrose and starch in the leaves of transgenic plant were slightly decreased compared to wild type. The glucose and fructose contents were increased up to two-fold compared to wild type during the light period. It is our speculation that the decreased sucrose level of the transgenic plant might be caused by the high acid invertase. These authors contributed equally to this work     

Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.     

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V _max SPS activity in leaf and fiber. Lines with the highest V _max SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of ¹⁴C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO₂ concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.     

Carbon-partitioning inArabidopsis is regulated by the fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase enzyme

To further elucidate the mechanisms underlying carbon-partitioning in plants, we established an experimental system by generating transgenicArabidopsis lines that overexpress both the fructose 6-phosphate, 2-kinase (F6P,2-K) and the fructose 2,6-bisphosphatase (F26BPase) domains. We also produced knockout transgenic plants for these domains via RNAi and T-DNA tagging. In F6P,2-K overexpressing transgenics, F6P,2-K activity increased slightly and Fru-2,6-P₂ levels were elevated by 80%, compared with the wild type (WT). F26BPase activity was similar between the WT and transgenic plants. However, when that domain was overexpressed, F26BPase activity was increased by 70% compared with the WT, whereas F6P,2-K activity was reduced to 85% of the WT level. In knockout and RNAi mutant lines that showed reduced F6P,2-K and F26BPase activities, levels of Fru-2,6-P₂ were only between 3 to 7% of those for the WT. In F6P,2-K overexpressing transgenic lines, the levels of starch, hexose, and triose phosphates slightly increased, while sucrose content was marginally reduced. In F26BPase overexpressing plants, however, the levels of soluble sugars and hexose phosphates were slightly increased, but starch and triose phosphate contents declined. Furthermore, compared with the WT, the levels of soluble sugars rose while starch and hexose phosphate quantities decreased in 2-kinase/fructose-2,6-bisphophatase knockout mutants. Therefore, our data reaffirms that Fru-2,6-P₂ contributes to the regulation of photosynthetic carbon-partitioning between starch and sucrose inArabidopsis leaves by limiting sucrose synthesis.     

Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning.

To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.     

Purification and properties of sucrose synthetase from morning-glory callus cells

Sucrose synthetase was purified about 130-fold from morning-glory (Pharbitis nil Choisy cv. Murasaki) callus cells, and the properties of sucrose synthesis and cleavage activities of the enzyme were compared. The enzyme preparation gave a single band by disc electrophoresis. The molecular mass of the enzyme was estimated to be 4.2 × 10⁵ by gel filtration. The enzyme preparation gave two bands by SDS disc electrophoresis, suggesting the molecular mass of about 3.8 ×10⁴ and 7.0 × 10⁴. The pH optima of sucrose synthesis and cleavage activities of the enzyme were different from each other, giving pH 9.0 and pH 6.5 respectively. MgCl², MnCl² and CaCl² activated the sucrose synthesis activity about two times the normal rate and conversely inhibited the sucrose cleavage activity. F-6-P was not replaced by fructose. UDP was the only valuable substrate as a nucleotide diphosphate. The enzyme showed the negative ecoperativity effect of UDPG suggesting to be an allosteric enzyme. The Km values of sucrose and fructose were calculated to be 167 mM and 5 mM, respectively. UDP suggested substrate inhibition. The apparent equilibrium constant varied between 1 to 3. Based on these results, the role of the enzyme in the sucrose metabolism of morning-glory callus cells will be discussed.     

Inhibition of sucrose phosphatase by sucrose

1. Partially purified sucrose phosphatase from immature stem tissue of sugarcane is inhibited by sucrose. The enzyme was also inhibited by maltose, melezitose and 6-kestose but not by eight other sugars, including glucose and fructose. 2. The relative effectiveness of sucrose, maltose and melezitose as inhibitors is different for sucrose phosphatase from different plants. 3. The inhibition of the sugar-cane enzyme by sucrose was shown to be partially competitive. The K(i) for sucrose is about 10mm. 4. Melezitose is also a partially competitive inhibitor of the enzyme but the inhibition by maltose is probably mixed. 5. The possibility that sucrose controls both the rate of accumulation of sucrose in stems of sugar-cane and sucrose synthesis in leaves by inhibiting sucrose phosphatase is discussed.     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size,but only small changes in carbohydrate metabolism

A potato (Solanum tuberosum L. ) cDNA coding for the chloroplastic isoform of fructose 1,6-bisphosphatase (cp-FBPase) was utilized to repress its activity in tomatoes (Lycopersicon esculentum Mill.) using antisense techniques. The patatin B33 promoter was used to ensure fruit specificity of the antisense effect. Transgenic plants were isolated in which fructose 1,6-bisphosphatase activity was reduced by more than 50% of the control in green fruits. Immunoblots indicated that the plastidial isoform was almost completely eliminated in the most strongly inhibited lines. Fruits of the transgenic plants were analyzed for levels of metabolites during fruit development. Glucose and fructose concentrations were increased in green fruits in the transgenic lines, but unchanged at later stages of development. The sucrose concentration was low, and was not significantly altered in the transgenic lines. There was net degradation of starch over the developmental period, but the starch content was not decreased. In green fruit the levels of hexose phosphates were unchanged, whilst the level of 3-phosphoglyceric acid was significantly increased in one line. Most importantly the deduced ratio of hexose phosphate to 3-phosphoglyceric acid decreased, consistent with an in vivo inhibition of fructose 1,6-bisphosphatase activity. One consequence of this reduction of in vivo activity of cp-FBPase was that the average weight of fully ripe fruits was significantly decreased by up to 20% in all transgenic lines in comparison with the control.Abbreviations AGPase ADP-glucose pyrophosphorylase - cp-FBPase Chloroplastidic fructose 1,6-bisphosphatase - cy-FBPase Cytoplasmic fructose 1,6-bisphosphatase - DAF Days after flowering     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate     

Expression of a yeast-derived invertase in companion cells results in long-distance transport of a trisaccharide in an apoplastic loader and influences sucrose transport

Companion cell-specific expression of a cytosolic invertase from yeast (Saccharomyces cerevisiae) was used as a tool to synthesise oligosaccharides in the sieve element/companion cell complex and study whether oligosaccharides could be transported in the phloem of an apoplastically loading species. Potato (Solanum tuberosum L.) plants expressing the invertase under the control of the Agrobacterium tumefaciens rolC promoter produced the trisaccharide 6-kestose in leaves, which was transported via the phloem and accumulated in tubers of transgenic plants. In graft experiments with rolC invertase plants as scion and wild-type rootstocks, 6-kestose accumulated in tubers to levels comparable to sucrose. This shows that long-distance transport of oligosaccharides is possible in apoplastically loading plants, which normally transport only sucrose. The additional transport route for assimilates neither led to elevated photosynthetic activity nor to increased tuber yield. Enhanced sucrose turnover in companion cells caused large amounts of glucose and fructose to be exuded from leaf petioles, and elevated levels of sucrose were detected in phloem exudates. While the latter indicates a higher capacity for sucrose loading into the phloem due to increased metabolic activity of companion cells, the massive release of hexoses catalysed by the invertase seemed to interfere with assimilate delivery to sink organs.Abbreviations HPAEC High-performance liquid anion-exchange chromatography - SE–CCC Sieve element/companion cell complex - WT Wild type