Frost tolerance in excised leaves of the common bugle (Ajuga reptans L.) correlates positively with the concentrations of raffinose family oligosaccharides (RFOs)

Mass increases in raffinose family oligosaccharides (RFOs, α 1,6-galactosyl extensions of sucrose) are well documented in the generative tissues of many plants upon cold acclimation, and they (i.e. mainly the two shortest RFO members, raffinose and stachyose) have been suggested as frost stress protectants. Our focus here was on the longer RFO members as they commonly occur in the frost-hardy evergreen labiate Ajuga reptans in its natural habitat, and accumulate to their highest concentrations in winter when the plant is faced with sub-zero temperatures. We examined the effects of RFO concentration and chain length on frost tolerance using excised leaves which accumulate long-chain RFOs under both cold and warm conditions, thereby uncoupling the acclimation temperature from RFO production. We demonstrated that frost tolerance in excised A. reptans leaves correlates positively with long-chain RFO accumulation under both acclimation temperatures. After 24 d post-excision in the warm, the leaves had increased their RFO concentrations (mainly long-chain RFOs) 22-fold to 78 mg g⁻¹ fresh weight, and decreased their EL₅₀ values (temperature at which 50% leakage occurred) from −10.5 to −24.5 °C, suggesting a protective role for these oligosaccharides in the natural frost tolerance of A. reptans .     

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.     

Analysis of the raffinose family oligosaccharide pathway in pea seeds with contrasting carbohydrate composition.

Raffinose family oligosaccharides (RFOs) are synthesized by a set of galactosyltransferases, which sequentially add galactose units from galactinol to sucrose. The accumulation of RFOs was studied in maturing seeds of two pea (Pisum sativum) lines with contrasting RFO composition. Seeds of the line SD1 accumulated stachyose as the predominant RFO, whereas verbascose, the next higher homolog of stachyose, was almost absent. In seeds of the line RRRbRb, a high level of verbascose was accumulated alongside with stachyose. The increase in verbascose in developing RRRbRb seeds was associated with galactinol-dependent verbascose synthase activity. In addition, a galactinol-independent enzyme activity was detected, which catalyzed transfer of a galactose residue from one stachyose molecule to another. The two enzyme activities synthesizing verbascose showed an optimum at pH 7.0. Both activities were almost undetectable in SD1. Maximum activity of stachyose synthase was about 4-fold higher in RRRbRb compared with SD1, whereas the activities of galactinol synthase and raffinose synthase were only about 1.5-fold higher in RRRbRb. The levels of galactinol synthase and stachyose synthase activity were reflected by steady-state levels of corresponding mRNAs. We suggest that the accumulation of verbascose in RRRbRb was controlled by a coordinated up-regulation of the last steps of verbascose biosynthesis.     

Transport and metabolism of raffinose family oligosaccharides in transgenic potato

Raffinose family oligosaccharides (RFOs) are involved in the storage and transport of carbon and serve as compatible solutes for protection against abiotic stresses like drought or cold. RFOs are usually transported in plant species that load sugars symplastically into the phloem. Loading probably occurs by a polymer trapping mechanism which establishes a concentration gradient of assimilates between the mesophyll and the vasculature. Transgenic approaches have demonstrated phloem transport of small molecules produced in the companion cells of apoplastic loading species, but these molecules have been non-native transport substances to plants. In this study, transgenic potato plants with constitutive or companion cell specific overexpression of galactinol synthase (GS) or GS plus raffinose synthase (RS) are characterized, which together provide new insights into the metabolism and transport of RFOs in plants. It is demonstrated that raffinose and galactinol are both transported in the phloem and that, whilst the effect of GS overexpression is promoter-independent, that of RS is dependent on the promoter used. The presence of significant amounts of galactinol in the phloem is shown and also that transgenic potato is unable to transport large amounts of raffinose despite high RS expression and substrate concentrations. These data indicate that there may be additional features of intermediary cells, the specialized companion cells of RFO transporting plants, required for significant RFO synthesis and transport that are currently not well-understood.     

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.     

Galactinol synthase across evolutionary diverse taxa: Functional preference for higher plants?

Galactinol synthase (GolS), a GT8 family glycosyltransferase, synthesizes galactinol and raffinose series of oligosaccharides (RFOs). Identification and analysis of conserved domains in GTs among evolutionarily diverse taxa, structure prediction by homology modeling and determination of substrate binding pocket followed by phylogenetic analysis of GolS sequences establish presence of functional GolS predominantly in higher plants, fungi having the closest possible ancestral sequences. Evolutionary preference for a functional GolS expression in higher plants might have arisen in response to the need for galactinol and RFO synthesis to combat abiotic stress, in contrast to other organisms lacking functional GolS for such functions.     

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.     

Allocation of raffinose family oligosaccharides to transport and storage pools in Ajuga reptans: the roles of two distinct galactinol synthases

Raffinose family oligosaccharides (RFOs) are important phloem transport and storage carbohydrates for many plants. Ajuga reptans, a frost-hardy evergreen labiate, ideally combines these two physiological roles and served as our model plant to study the regulation and importance of RFO metabolism. Galactinol is the galactosyl donor for the synthesis of raffinose (RFO-trisaccharide) and stachyose (RFO-tetrasaccharide), and its synthesis by galactinol synthase (GolS) is the first committed step of the RFO biosynthetic pathway. Two cDNAs encoding two distinct GolS were isolated from A. reptans source and sink leaves, designated GolS-1 and GolS-2, respectively. Warm- and cold-grown sink and source leaves were compared, revealing both isoforms to be cold-inducible and GolS-1 to be source leaf-specific; GolS-1 expression correlated positively with GolS activity. Conversely, GolS-2 expression was comparatively much lower and its contribution to the total extractable GolS activity is most probably only minor. These observations, together with results from phloem exudation and leaf shading experiments suggest that GolS-1 is mainly involved in the synthesis of storage RFOs and GolS-2 in the synthesis of transport RFOs. Furthermore, in situ hybridization studies showed GolS-1 to be primarily expressed in the mesophyll, the site of RFO storage, and GolS-2 in the phloem-associated intermediary cells known for their role in RFO phloem loading. A model depicting the spatial compartmentation of the two GolS isoforms is proposed.     

myo-Inositol and sucrose concentrations affect the accumulation of raffinose family oligosaccharides in seeds

Raffinose family oligosaccharides (RFOs) fulfil multiple functions in plants. In seeds, they possibly protect cellular structures during desiccation and constitute carbon reserves for early germination. Their biosynthesis proceeds by the transfer of galactose units from galactinol to sucrose. Galactinol synthase (GolS), which mediates the synthesis of galactinol from myo-inositol and UDP-galactose, has been proposed to be the key enzyme of the pathway. However, no significant relationship was detected between the extractable GolS activity and the amount of RFOs in seeds from seven pea (Pisum sativum L.) genotypes selected for high variation in RFO content. Instead, a highly significant correlation was found between the levels of myo-inositol and RFOs. Moderately strong relationships were also found between sucrose and RFO content as well as between myo-inositol and galactinol. Further evidence for a key role of myo-inositol for the synthesis of galactinol was obtained by feeding exogenous myo-inositol to intact pea seeds and by the analysis of four barley (Hordeum vulgare L.) low phytic acid mutants. In seeds of three of these mutants, the reduced demand for myo-inositol for the synthesis of phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate) was associated with an increased level in myo-inositol. The mutants seeds also contained more galactinol than wild-type seeds. The results suggest that the extent of RFO accumulation is controlled by the levels of the initial substrates, myo-inositol and sucrose, rather than by GolS activity alone.     

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.     

Stachyose synthesis in mature leaves of Cucumis melo. Purification and characterization of stachyose synthase (EC 2.4.1.67)

Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH₄)₂SO₄ precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg^-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K _m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K _i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K _m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE diethylaminoethyl - DTT dithiothreitol - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged.     

Cold-induced accumulation of raffinose family oligosaccharides in somatic embryos of Norway spruce (<Emphasis Type="Italic">Picea abies</Emphasis>)

Summary Exposure of mature cotyledonary somatic embryos of Picea abies to low temperature (4°C) resulted in the accumulation of raffinose family oligosaccharides (RFOs)—raffinose and stachyose. The RFO content represented approximately 20% of the total soluble saccharides with the RFO: sucrose ratio being almost 1∶3 (molar basis) after 3 wk of cold exposure. This treatment, like desiccation, brings the endogenous saccharide spectrum nearer to that of mature zygotic embryos of the same species (zygotic embryos, RFO: sucrose ratio 1∶1.5 on a molar basis). Based on indications that RFOs are at least partly responsible for the positive effects of desiccation, we propose cold treatment as an alternative to slow desiccation for conifer somatic embryogenesis protocols.     

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.     

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。