种子中的棉子糖半乳糖苷系列寡糖研究进展

棉子糖半乳糖苷系列寡糖广泛分布在许多种植物种子中,并存在于干燥后仍能保持活力的组织内,如禾谷类种子的胚及糊粉层,豆类及其他双子叶植物的子叶和胚轴组织等。棉子糖半乳糖苷系列寡糖在禾谷类种子的非自溶性中央胚乳中不合成,但存在于蓖麻种子的自溶性胚乳细胞中。棉子糖半乳糖苷系列寡糖在种子发育后期累积,并持续到种子大量成熟直到脱水阶段。棉子糖半乳糖苷系列寡糖主要包括棉子糖、水苏糖和毛蕊花糖,是种子中最广泛的低分子量α_半乳糖苷。许多植物正常性种子的发育伴随着棉子糖半乳糖苷系列寡糖的累积,这些糖的累积已被认为在种子脱水耐性获得、种子活力、糖的运输及植物的抗冷驯化等过程 中起重要作用。本文从种子的脱水耐性获得、植物的冷驯化、细胞内定位及生物合成等方面综述了棉子糖半乳糖苷系列寡糖的研究进展。     

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.     

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。     

种子脱水耐性与糖的关系

糖类在植物种子中的累积随种子的发育阶段和种子类型不同而不同,并与种子脱水耐性的变化相联系。许多正常性植物种子的发育伴随着某些糖的累积,这些糖的累积已被认为在种子脱水耐性获得中起重要作用。但糖对种子脱水耐性的影响不是单独的,而是与ABA和蛋白质等物质协同作用。种子脱水耐性不仅与糖的种类和含量有关,而且与种子所处的生理状态和发育进程有关。本文综述了种子脱水耐性与糖关系的研究进展。     

种子的脱水行为及其分子机制

成熟脱水是种子发育的末端事件。根据种子的脱水行为,可以把种子分为正常性、顽拗性和中间性种子。许多过程或者机制授予或者提高种子的脱水耐性,不同的过程可能在不同的水合水平上对水分丧失起保护作用,这些过程的缺乏或者无效表达可能决定个别物种的种子的脱水敏感性程度。到目前为止,涉及种子脱水耐性的过程或者机制有：细胞内脱分化;代谢的‘关闭’;抗氧化系统的存在和有效运转;保护性分子(包括胚胎发育后期高丰度表达蛋白,蔗糖、寡糖或者半乳糖苷环多醇,亲水脂分子,油素)的存在;以及在重新水合过程中修复机制的存在和运转。     

种子发育与萌发过程中的程序性细胞死亡

禾谷类种子胚乳发育过程中的程序性细胞死亡(PCD)主要发生在种子成熟期的后期,并伴随着生物合成的停止和自然脱水;乙烯和活性氧促进胚乳发育中的PCD,而ABA起负调节作用。种子萌发过程中糊粉层降解的PCD被GA、Ca^2 和活性氧促进,被ABA和抗氧化剂抑制。种子人工老化和劣变种子萌发过程中可能存在PCD事件,其研究对延长种子的贮藏寿命和提高播种品质具有重要的意义。     

种子脱水耐性及其与种子类型和发育阶段的相关性

种子脱水耐性是种子发育过程中获得的综合特性,是判断种子贮藏持性的一个重要依据。当种子获得脱水耐性时,生理,形态和结构会发生相应的变化,包括糖,蛋白质,脂类和抗氧化系统等保护性物质的合成,各种保护性物质不是单独作用的,而是协同调节种子的脱水耐性。不同的植物种子,其脱水耐性不同,并且随着种子的发育而变化。关于种子脱水耐性的获得,主要有2种观点,一种认为是数量性状,另一种认为是突变性状。种子库收集种子保存时,适时采集和适度脱水才能有效地延长种子的贮藏寿命。     

成熟脱水对种子发育和萌发的作用

成熟脱水是正常性种子发育的末端事件。种子在成熟时胚的脱水耐性增加;当种子萌发时胚变得不耐脱水。当种子获得脱水耐性时,糖、蛋白质和抗氧化防御系统等保护性物质积累;当脱水耐性丧失时,这些物质被降解。成熟脱水是种子从发育过程向萌发过程转变的“开关”,它降低发育的蛋白质和ｍＲＮＡ的合成,终止发育事件和促进萌发事件。顽拗性种子不经历成熟脱水的发育阶段,对脱水高度敏感。     

成熟脱水对种子发育和萌发的作用

成熟脱水是正常性种子发育的末端事件。种子在成熟时胚的脱水耐性增加;当种子萌发时胚变得不耐脱水。当种子获得脱水耐性时,糖、蛋白质和抗氧化防御系统等保护性物质积累;当脱水耐性丧失时,这些物质被降解。成熟脱水是种子从发育过程向萌发过程转变的“开关”,它降低发育的蛋白质和mRNA的合成,终止发育事件和促进萌发事件。顽拗性种子不经历成熟脱水的发育阶段,对脱水高度敏感。     

与种子耐脱水性有关的基础物质研究进展

耐脱水的获得和维持与种子的类型有关,正常型种子耐脱水,而顽拗形种子对脱水高度敏感。正常型种子的脱水耐性随发育过程而变化,种子成熟时胚的脱水耐性增强,而萌发时胚变为不耐脱水。当种子获得脱水耐性时,糖、LEA蛋白质和抗氧化防御系统等保护性物质积累。但脱水耐性是一种复杂的数量的特性,任何一种单一的机制都不能充分地解释脱水耐性,各种保护性物质协同调节脱水耐性。本文综述了近几年来关于种子耐脱水性与保护性物质相关性的研究进展。     

Chain Elongation of raffinose in pea seeds. Isolation, characterization, and molecular cloning of mutifunctional enzyme catalyzing the synthesis of stachyose and verbascose.

Raffinose oligosaccharides are major soluble carbohydrates in seeds and other tissues of plants. Their biosynthesis proceeds by stepwise addition of galactose units to sucrose, which are provided by the unusual donor galactinol (O-alpha-d-galactopyranosyl-(1-->1)-l-myo-inositol). Chain elongation may also proceed by transfer of galactose units between raffinose oligosaccharides. We here report on the purification, characterization, and heterologous expression of a multifunctional stachyose synthase (EC ) from developing pea (Pisum sativum L.) seeds. The protein, a member of family 36 of glycoside hydrolases, catalyzes the synthesis of stachyose, the tetrasaccharide of the raffinose series, by galactosyl transfer from galactinol to raffinose. It also mediates the synthesis of the pentasaccharide verbascose by galactosyl transfer from galactinol to stachyose as well as by self-transfer of the terminal galactose residue from one stachyose molecule to another. These activities show optima at pH 7.0. The enzyme also catalyzes hydrolysis of the terminal galactose residue of its substrates, but is unable to initiate the synthesis of raffinose oligosaccharides by galactosyl transfer from galactinol to sucrose. A minimum reaction mechanism which accounts for the broad substrate specificity and the steady-state kinetic properties of the protein is presented.     

Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase,partial purification of the enzyme from maturing seeds,and steady-state kinetic analysis of raffinose synthesis

Raffinose (O-alpha- D-galactopyranosyl-(1-->6)- O-alpha- D-glucopyranosyl-(1<-->2)- O-beta- D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea ( Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol ( O-alpha- D-galactopyranosyl-(1-->1)- L- myo-inositol), p-nitrophenyl alpha- D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for alpha-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.     

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.     

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.     

Differences in accumulation of soluble α-galactosides during seed maturation of several <Emphasis Type="Italic">Vicia</Emphasis> species

Composition and levels of soluble α-galactosides: raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C) in developing seeds were measured by high resolution gas chromatography (HRGC) method. The studies were performed on maturing seeds of several wild and cultivated Vicia species: Vicia angustifolia L. (common vetch), Vicia cracca L. (bird vetch), Vicia grandiflora Scop. (large yellow vetch), Vicia hirsuta (L.) S.F.Gray (tiny vetch), Vicia sativa L. (garden vetch, spring-growing cultivar Kwarta), and Vicia villosa Roth (winter vetch). In all Vicia species similar patterns in the accumulation of RFOs were observed. Galactinol — the donor of galactosyl moieties in α-galactosides biosynthesis was present in the middle stage of seed development, before appearing measurable levels of RFOs. Accumulation of RFOs started parallel with seed desiccation process. At first accumulation of the raffinose, then few days later stachyose and finally verbascose was noticed. In the final stage of seed maturation the verbascose was the main soluble α-galactoside (up to 3% of dry weight, V. sativa). Besides the RFOs seeds of three Vicia species (V. cracca, V. hirsuta, and V. villosa) accumulated d-pinitol and its α-galactosides (Gal-C). Mono-galactosylpinitols (similar to raffinose) appeared in these species 2–4 days after galactinol, di-galactosyl pinitol A (common name: ciceritol) and di-galactosyl myo-inositol were present several days later than raffinose, and accumulation of tri-galactosyl pinitol A (TGPA) began after accumulation of stachyose. Matured seeds of V. hirsuta contained much more RFOs than Gal-C, opposite to seeds of V. villosa, and V. cracca where concentration of Gal-C was 4–8-fold higher than RFOs. In V. cracca seeds RFOs were almost replaced by Gal-C. In seeds of V. cracca and V. villosa the level of d-pinitol was significantly higher, than the level of myo-inositol. Contents of both cyclitols declined rapidly at the beginning of seed desiccation, when accumulation of RFOs and Gal-C quickly increased. We suggest that α-galactosides of d-pinitol can substitute raffinose family oligosaccharides and play similar role during seed maturation and storage.     

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。