Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana

Raffinose family oligosaccharides (RFO) accumulating during seed development are thought to play a role in the desiccation tolerance of seeds. However, the functions of RFO in desiccation tolerance have not been elucidated. Here we examine the functions of RFO in Arabidopsis thaliana plants under drought- and cold-stress conditions, based on the analyses of function and expression of genes involved in RFO biosynthesis. Sugar analysis showed that drought-, high salinity- and cold-treated Arabidopsis plants accumulate a large amount of raffinose and galactinol, but not stachyose. Raffinose and galactinol were not detected in unstressed plants. This suggests that raffinose and galactinol are involved in tolerance to drought, high salinity and cold stresses. Galactinol synthase (GolS) catalyses the first step in the biosynthesis of RFO from UDP-galactose. We identified three stress-responsive GolS genes (AtGolS1, 2 and 3) among seven Arabidopsis GolS genes. AtGolS1 and 2 were induced by drought and high-salinity stresses, but not by cold stress. By contrast, AtGolS3 was induced by cold stress but not by drought or salt stress. All the GST fusion proteins of GST-AtGolS1, 2 and 3 expressed in Escherichia coli had galactinol synthase activities. Overexpression of AtGolS2 in transgenic Arabidopsis caused an increase in endogenous galactinol and raffinose, and showed reduced transpiration from leaves to improve drought tolerance. These results show that stress-inducible galactinol synthase plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions, and that galactinol and raffinose may function as osmoprotectants in drought-stress tolerance of plants.     

植物中棉子糖系列寡糖代谢及其调控关键酶研究进展

棉子糖系列寡糖代谢与植物生长发育、逆境胁迫、种子耐贮性及脱水耐性等关系密切.棉子糖系列寡糖的合成从棉子糖的合成开始,由半乳糖苷肌醇上的半乳糖基的转移依次生成棉子糖、水苏糖、毛蕊花糖等.寡糖代谢是一个复杂的调控体系,其中肌醇-1-磷酸合成酶、肌醇半乳糖苷合成酶、蔗糖合成酶、棉子糖合成酶、水苏糖合成酶和毛蕊花糖合成酶等参与了棉子糖系列寡糖的生物合成过程.本文对植物中棉子糖系列寡糖的代谢及其重要调控酶的特性、功能及分子生物学研究进展进行综述.     

Physiological aspects of raffinose family oligosaccharides in plants: protection against abiotic stress

Abiotic stresses resulting from water deficit, high salinity or periods of drought adversely affect plant growth and development and represent major selective forces during plant evolution. The raffinose family oligosaccharides (RFOs) are synthesised from sucrose by the subsequent addition of activated galactinol moieties donated by galactinol. RFOs are characterised as compatible solutes involved in stress tolerance defence mechanisms, although evidence also suggests that they act as antioxidants, are part of carbon partitioning strategies and may serve as signals in response to stress. The key enzyme and regulatory point in RFO biosynthesis is galactinol synthase (GolS), and an increase of GolS in expression and activity is often associated with abiotic stress. It has also been shown that different GolS isoforms are expressed in response to different types of abiotic stress, suggesting that the timing and accumulation of RFOs are controlled for each abiotic stress. However, the accumulation of RFOs in response to stress is not universal and other functional roles have been suggested for RFOs, such as being part of a carbon storage mechanism. Transgenic Arabidopsis plants with increased galactinol and raffinose concentrations had better ROS scavenging capacity, while many sugars have been shown in vitro to have antioxidant activity, suggesting that RFOs may also act as antioxidants. The RFO pathway also interacts with other carbohydrate pathways, such as that of O‐methyl inositol (OMI), which shows that the functional relevance of RFOs must not be seen in isolation to overall carbon re‐allocation during stress responses.     

Expression of a GALACTINOL SYNTHASE gene is positively associated with desiccation tolerance of Brassica napus seeds during development

Desiccation tolerance of seeds is positively correlated with raffinose family oligosaccharides (RFOs). However, RFOs’ role in desiccation tolerance is still a matter of controversy. The aim of this work was to monitor the accumulation of RFO during acquisition of desiccation tolerance in rapeseed (Brassica napus L.). Rapeseeds become desiccation tolerant at 21-24 d after flowering (DAF), and the time was coincident with an accumulation of raffinose and stachyose. A gene encoding galactinol synthase (GolS; EC2.4.1.123), involved in RFO biosynthesis, was cloned and functionally characterized. Enzymatic properties of recombinant galactinol synthase were also determined. Accumulation of BnGOLS-1 mRNA in developing rapeseeds was concomitant with dry weight deposition and the acquisition of desiccation tolerance, and was concurrent with the formation of raffinose and stachyose. The physiological implications of BnGOLS-1 expression patterns in developing seeds are discussed in light of the hypothesized role of RFOs in seed desiccation tolerance.     

复苏植物旋蒴苣苔棉子糖合酶基因的克隆和表达

复苏植物是研究植物耐脱水机制的特殊模式植物和宝贵的耐旱基因资源植物。以复苏植物旋蒴苣苔(Boea hygrometrica) 为材料研究其在脱水和复水过程中棉子糖系列寡糖含量的变化, 并克隆了旋蒴苣苔棉子糖合酶基因BhRFS。荧光定量PCR检测表明, BhRFS受干旱、低温(4°C)、高盐(200 mmol·L^–1NaCl)和ABA(100 μmol·L^–1)诱导表达上调, 而高温(37°C)抑制其表达, H₂O₂(200 μmol·L^–1)处理对其没有影响。研究结果表明, BhRFS可能参与了多种非生物逆境胁迫抗性反应, 并受到ABA依赖的信号通路调控。     

油菜（Brassica napus L.）种子脱水耐性获得过程中肌醇半乳糖苷合成酶活性与可溶性糖含量的变化

在这个研究中测量不同发育时期的油菜种子中可溶性糖含量与肌醇半乳糖苷合成酶（galactinol synthase,GOLS）活性,将二者的变化趋势与种子脱水耐性获得的过程相比较并对结果进行相关性分析。结果显示油菜种子脱水耐性获得过程中,葡萄糖和果糖含量均随着发育期的延长而下降,蔗糖则保持较高水平;肌醇含量下降而肌醇半乳糖苷含量上升;棉子糖系列寡糖（raffinose familyolig osaccharides,RFO）含量随着种子发育而上升,特别是水苏糖,在成熟种子中可以达到相当高的浓度。油菜种子发育中期,细胞内GOLS活性开始上升,至贮藏物积累完成时达到最大。GOLS活性变化与种子肌醇半乳糖苷积累速度、RFO含量及种子的脱水耐性呈一定的正相关关系。我们认为GOLS促使RFO积累,从而对种子脱水耐性的获得产生重要影响。     

Galactinol synthase activity and soluble sugars in developing seeds of four soybean genotypes

Galactinol synthase (UDP-galactose:inositol galactosyltransferase) is the first unique enzyme in the biosynthetic pathway of raffinose saccharides. Its role as a regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean (Glycine max L. Merrill) seeds was examined. Galactinol synthase activity and concentrations of sucrose, stachyose, and raffinose were compared during seed development between two genotypes that were high and two genotypes that were low in mature seed raffinose saccharide concentration. In all genotypes, sucrose concentration increased as seed development progressed, but in both low raffinose saccharide genotypes, greater increases in sucrose concentration were observed late in seed development. Sucrose to stachyose ratios in mature seeds were 2.3-fold greater in low raffinose saccharide genotypes than in the high raffinose saccharide genotypes. During seed development, higher levels of galactinol synthase activity were observed in the high raffinose saccharide genotypes than in the low raffinose saccharide genotypes. A common linear relationship for all four soybean genotypes was shown to exist between galactinol formed estimated from galactinol synthase activity data and the concentration of galactose present in raffinose saccharides. Results of this study implied that galactinol synthase is an important regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean seeds.     

Raffinose Synthesis in Chlorella vulgaris Cultures after a Cold Shock

Chlorella vulgaris cultures have been submitted to a chilling shock, bringing down the growing temperature from to 24°C to 4°C. Growth was stopped immediately, and concomitantly there was an accumulation of sucrose and a decrease in the starch content. The enzymes involved in sucrose metabolism were differentially affected by the chilling shock. Sucrose phosphate synthase activity increased while sucrose synthase was not affected. Simultaneously with the chilling shock, raffinose began to accumulate. When algal cultures were returned at 24°C, raffinose disappeared. The presence of raffinose in algal cells has not been reported before.     

Fructans,but not the sucrosyl-galactosides,raffinose and loliose,are affected by drought stress in perennial ryegrass

The aim of this study was to evaluate the putative role of the sucrosyl-galactosides, loliose [alpha-D-Gal (1,3) alpha-D-Glc (1,2) beta-D-Fru] and raffinose [alpha-D-Gal (1,6) alpha-D-Glc (1,2) beta-D-Fru], in drought tolerance of perennial ryegrass and to compare it with that of fructans. To that end, the loliose biosynthetic pathway was first established and shown to operate by a UDP-Gal: sucrose (Suc) 3-galactosyltransferase, tentatively termed loliose synthase. Drought stress increased neither the concentrations of loliose and raffinose nor the activities of loliose synthase and raffinose synthase (EC 2.4.1.82). Moreover, the concentrations of the raffinose precursors, myoinositol and galactinol, as well as the gene expressions of myoinositol 1-phosphate synthase (EC 5.5.1.4) and galactinol synthase (EC 2.4.1.123) were either decreased or unaffected by drought stress. Taken together, these data are not in favor of an obvious role of sucrosyl-galactosides in drought tolerance of perennial ryegrass at the vegetative stage. By contrast, drought stress caused fructans to accumulate in leaf tissues, mainly in leaf sheaths and elongating leaf bases. This increase was mainly due to the accumulation of long-chain fructans (degree of polymerization > 8) and was not accompanied by a Suc increase. Interestingly, Suc but not fructan concentrations greatly increased in drought-stressed roots. Putative roles of fructans and sucrosyl-galactosides are discussed in relation to the acquisition of stress tolerance.     

Water Deficit Effects on Raffinose Family Oligosaccharide Metabolism in Coleus

Variegated coleus (Coleus blumei Benth.) plants were exposed to a restricted water supply for 21 d. The relative water content in leaf tissues was reduced from 80% (control) to 60% (drought-stressed). Under drought conditions, the stomatal conductance and leaf photosynthetic rate were reduced. In green leaf tissues, drought stress also greatly decreased the diurnal light-period levels of the raffinose family oligosaccharides (RFOs) stachyose and raffinose, as well as those of other non-structural carbohydrates (galactinol, sucrose, hexoses, and starch). However, drought had little effect on soluble carbohydrate content of white, non-photosynthetic leaf tissues. In green tissues, galactinol synthase activity was depressed by drought stress. An accumulation of O-methyl-inositol was also observed, which is consistent with the induction of myoinositol-6-O-methyltransferase activity seen in the stressed green tissues. In source tissues, RFO metabolism is apparently reduced by drought stress through a combined effect of decreased photosynthesis and reduced galactinol synthase activity. Moreover, a further reduction in RFO biosynthesis may have been due to a switch in carbon partitioning to O-methyl-inositol biosynthesis, creating competition for myoinositol, a metabolite shared by both biochemical pathways.     

植物肌醇半乳糖苷合酶的生理功能和调控机制

植物肌醇半乳糖苷合酶（galactinol synthase, GolS）是高等植物棉子糖类寡糖合成途径中的关键酶,为棉子糖系列寡糖提供活化的半乳糖基,调控植物体内棉子糖（raffinose, RFO）系列寡糖的生物合成与积累。编码该酶的基因属于糖基转移酶（glycosyltransferases, GTs）GT8基因家族的亚家族。GolS参与合成的最终产物棉子糖家族低聚糖（raffinose family oligosaccharides,RFOs）是植物中重要的碳水化合物存在形式,在细胞内可溶性强,可作为脱水保护剂;还能发挥稳定膜结构的作用。同时,GolS催化合成的直接产物肌醇半乳糖苷（galactinol）和RFOs都能作为羟基自由基捕获分子参与活性氧的清除。因此,GolS参与的代谢途径在植物碳同化物的贮存与运输、生物和非生物逆境响应、种子的脱水效应等生命过程中均发挥了重要作用。GolS基因结构差异与表达模式不同,导致不同GolS基因参与的生物学功能具有很大的差异。研究植物中不同GolS基因的结构特征,组织特异性表达特性及它们响应不同生长发育阶段、环境变化的表达特性,对了解GolS参与的生物学功能具有重要意义。同时,在分子生物学水平上,深入了解调控植物GolS基因的分子调控机制,为通过遗传工程或分子辅助育种等手段,利用GolS改良农林作物的经济性状提供理论支持。本文针对近年来植物中GolS基因的生理功能和调控机制的研究进行了综述。     

植物肌醇半乳糖苷合酶的生理功能和调控机制

植物肌醇半乳糖苷合酶（galactinol synthase, GolS）是高等植物棉子糖类寡糖合成途径中的关键酶,为棉子糖系列寡糖提供活化的半乳糖基,调控植物体内棉子糖（raffinose, RFO）系列寡糖的生物合成与积累。编码该酶的基因属于糖基转移酶（glycosyltransferases, GTs）GT8基因家族的亚家族。GolS参与合成的最终产物棉子糖家族低聚糖（raffinose family oligosaccharides,RFOs）是植物中重要的碳水化合物存在形式,在细胞内可溶性强,可作为脱水保护剂;还能发挥稳定膜结构的作用。同时,GolS催化合成的直接产物肌醇半乳糖苷（galactinol）和RFOs都能作为羟基自由基捕获分子参与活性氧的清除。因此,GolS参与的代谢途径在植物碳同化物的贮存与运输、生物和非生物逆境响应、种子的脱水效应等生命过程中均发挥了重要作用。GolS基因结构差异与表达模式不同,导致不同GolS基因参与的生物学功能具有很大的差异。研究植物中不同GolS基因的结构特征,组织特异性表达特性及它们响应不同生长发育阶段、环境变化的表达特性,对了解GolS参与的生物学功能具有重要意义。同时,在分子生物学水平上,深入了解调控植物GolS基因的分子调控机制,为通过遗传工程或分子辅助育种等手段,利用GolS改良农林作物的经济性状提供理论支持。本文针对近年来植物中GolS基因的生理功能和调控机制的研究进行了综述。     

Raffinose in seedlings of winter vetch (<Emphasis Type="Italic">Vicia villosa</Emphasis> Roth.) under osmotic stress and followed by recovery

During germination of winter vetch (Vicia villosa Roth.) seeds, the degradation of raffinose family oligosaccharides and galactosyl pinitols occurred faster in axis than in cotyledons. After 7 days of germination, all α-d-galactosides disappeared and the soluble carbohydrates in seedling tissues consisted of d-pinitol, sucrose, fructose, glucose and myo-inositol. Osmotic stress caused by incubation of seedlings in PEG 8000 solution (−0.5, −1.0, and −1.5 MPa) for 48 h induced the activity of crucial enzymes of the RFOs pathway, i.e. galactinol synthase and raffinose synthase, in both the root and epicotyl but not in cotyledons. The root and epicotyl accumulated elevated amounts of galactinol and raffinose as the osmotic potential was lowered. This process was transient because when PEG solution was replaced with water, galactinol and raffinose were degraded, thus confirming their direct involvement in the response of tissues to osmotic stress. Among other soluble carbohydrates, only sucrose accumulated in response to stress. The results did not show potential role of d-pinitol in the adjustment of winter vetch seedlings to osmotic stress.     

Isolation,functional characterization and stress responses of raffinose synthase genes in sugar beet

Raffinose (sucrosylgalactoside oligosaccharide) is a water soluble carbohydrate and accumulates in response to abiotic stresses in plants. Plant raffinose synthases are poorly characterized, and the genes involved in raffinose biosynthesis are unknown in sugar beet. Here, we report the isolation of two genes encoding raffinose synthase (BvRS1 and BvRS2) as well as a gene encoding galactinol synthase (BvGolS1) from sugar beet. BvRS1 and BvRS2 show high homologies to Arabidopsis raffinose synthase AtRS5. BvRS1 and BvGolS1 were expressed in Escherichia coli. Crude extracts showed the activities of raffinose synthase and galactinol synthase. The K _m values of BvRS1 for galactinol and sucrose and the K _m values of BvGolS1 for UDP-galactose and myo-inositol were determined. The expression levels of BvRS1 were significantly higher than that of BvRS2. The mRNA for BvRS1 was rapidly induced by cold stress whereas the mRNA for BvRS2 was slowly induced by cold and salt stresses. These data suggest that BvRS1 and BvRS2 encode raffinose synthase genes responsible to cold and salt stress, respectively.     

摘除雌花对甜瓜成熟叶片中糖及相关酶活性的影响

甜瓜有果株的成熟叶片中蔗糖、葡萄糖、果糖含量与无果株的无显著差异,水苏糖与棉子糖含量略低于无果株,肌醇半乳糖苷(合成水苏糖的前体)含量显著低于无果株,蔗糖磷酸合成酶(SPS)和肌醇半乳糖苷合成酶活性与无果株的无显著差异,水苏糖合成酶活性显著高于无果株。     

Localization of galactinol,raffinose, and stachyose synthesis in Cucurbita pepo leaves

The biochemical pathway of stachyose synthesis was localized by immunocytochemical and ¹⁴C-labeling techniques in mature Cucurbita pepo L. leaves. Galactinol synthase (GaS; EC 2.4.1.123), the first unique enzyme in this pathway, was immunolocalized within the intermediary cells of minor veins in conventionally fixed and cryo-fixed, resin-embedded sections using polyclonal anti-GaS antibodies and protein A-gold. Intermediary cells are specialized companion cells with extensive symplastic connections to the bundle sheath. Gold particles were not seen over the non-specialized companion cells of larger veins or over intermediary cells in young leaves prior to the sink-source transition. In another approach to localization, radiolabel was measured in isolated mesophyll tissue and whole tissue of leaves that were lyophilized following a 90-s exposure to ¹⁴CO₂. Mesophyll, obtained by abrasion of the leaf surface, contained labeled sucrose, galactinol, raffinose and stachyose. However, the latter three labeled compounds constituted a smaller proportion of the neutral fraction than in whole-tissue samples, which also contained minor veins. We conclude that synthesis of galactinol, raffinose, and stachyose occurs in both mesophyll and intermediary cells, predominantly the latter.Abbreviations GaS galactinol synthase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank John Pierce, Phillip Kerr, and Brace Schweiger for the gift of anti-GaS antibody and M.K. Kandasamy for helpful discussions. This research was supported by National Science Foundation grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.