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.     

Enzymatic control of the accumulation of verbascose in pea seeds

Verbascose, the pentasaccharide of the raffinose family of oligosaccharides, consists of galactose units joined to sucrose. In pea (Pisum sativum) seeds, the content of verbascose is highly variable. In a previous study on a high‐verbascose pea cultivar, the present authors have demonstrated that verbascose is synthesized by a multifunctional stachyose synthase (EC 2.4.1.67), which utilizes raffinose as well as stachyose as a galactosyl acceptor. Herein the results of a study of the cloning and functional expression of stachyose synthase from the low‐verbascose genotype SD1 are reported and it is demonstrated that this line contains a protein with a reduced ability to synthesize verbascose. Analysis of seeds from seven pea lines revealed a positive correlation between verbascose synthase activity and verbascose content. Among these genotypes, only the SD1 line showed low verbascose synthase activity when the data were normalized to stachyose synthase activity. These results suggest that differences in the level of verbascose synthase activity could be caused by mutations in the stachyose synthase gene as well as by variation in the amount of the protein. The lines were also analysed for activity of α‐galactosidase, a catabolic enzyme that could limit the extent of verbascose accumulation. No relationship was found between α‐galactosidase activity and the amount of raffinose family oligosaccharides.     

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.     

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.     

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。     

Purification and characterization of the raffinose oligosaccharide chain elongation enzyme,galactan : galactan galactosyltransferase (GGT), from Ajuga reptans leaves

Galactan: galactan galactosyltransferase (GGT), an enzyme involved in the biosynthesis of the long-chain raffinose family of oligosaccharides (RFOs) in Ajuga reptans, catalyses the transfer of an alpha-galactosyl residue from one molecule of RFO to another one resulting in the next higher RFO oligomer. This novel galactinol (alpha-galactosyl-myo-inositol)-independent alpha-galactosyltransferase is responsible for the accumulation of long-chain RFOs in vivo. Warm treatment (20 degrees C) of excised leaves resulted in a 34-fold increase of RFO concentration and a 200-fold increase of GGT activity after 28 days. Cold treatment (10 degrees C/3 degrees C day/night) resulted in a 26- and 130-fold increase, respectively. These data support the role of GGT as a key enzyme in the synthesis and accumulation of long-chain RFOs. GGT was purified from leaves in a 4-step procedure which involved fractionated precipitation with ammonium sulphate as well as lectin affinity, anion exchange, and size-exclusion chromatography and resulted in a 200-fold purification. Purified GGT had an isoelectric point of 4.7, a pH optimum around 5, and its transferase reaction displayed saturable concentration dependence for both raffinose (Km = 42 mM) and stachyose (Km = 58 mM). GGT is a glycoprotein with a 10% glycan portion. The native molecular mass was 212 kDa as determined by size-exclusion chromatography. Purified GGT showed one single active band after native PAGE or IEF separation, respectively, which separated into three bands on SDS-PAGE at 48 kDa, 66 kDa, and 60 kDa. The amino acid sequence of four tryptic peptides obtained from the major 48-kDa band showed a high homology to plant alpha-galactosidase (EC 3.2.1.22) sequences. GGT differed, however, in its substrate specificity from alpha-galactosidases; it neither hydrolysed nor transferred alpha-galactosyl-groups from melibiose, galactinol, UDP-galactose, manninotriose, and manninotetrose. Galactinol, sucrose, and galactose inhibited the GGT reaction considerably at 10-50 mM.     

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.     

Soluble sugar content of white spruce (Picea glauca) seeds during and after germination

In white spruce ( Picea glauca [Moench.] Voss.) seeds, the raffinose family oligosaccharides (RFOs) provide carbon reserves for the early stages of germination prior to radicle protrusion. Some seedlots contain seeds that are dormant, failing to complete germination under optimal conditions. Since dormancy may be imposed through a metabolic block in reserve mobilization, the goal of this project was to identify any impediment to RFO mobilization in dormant relative to nondormant seeds. Desiccated seeds contain primarily, and in order of abundance on a molar basis, sucrose and the first 3 members of the RFOs, raffinose, stachyose and verbascose. Upon radicle protrusion at 25°C, the contents of RFOs decreased to low amounts in all seed parts, regardless of prior dormancy status and sucrose was metabolized to glucose and fructose, which increased in seed parts. During moist chilling at 4°C, RFO content initially decreased before stabilizing and then increasing. In seeds that did not complete germination, the synthesis of RFOs at 4°C favored verbascose, so that at the end of 14 (nondormant) or 35 (dormant) weeks, verbascose contents in megagametophytes exceeded the amount initially present in the desiccated seed. This was also true in the embryos of the dormant seedlot. In seed parts from both seedlots after months of moist chilling, stachyose amounts exceeded raffinose amounts. Upon radicle protrusion at 4°C, RFO contents decreased to amounts most similar to those present in seeds that completed germination at 25°C. Hence, the RFOs are utilized as a source of energy, regardless of the temperature at which white spruce seeds complete germination. Based on the similarity of sugar contents in seed parts between dormant and nondormant seeds that did not complete germination, differences in sugar metabolism are probably not the basis of dormancy in white spruce seeds.     

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.     

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.     

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.     

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.     

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.     

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.     

Metabolism of the Raffinose Family Oligosaccharides in Leaves of Ajuga reptans L. (Inter- and Intracellular Compartmentation)

We recently suggested that leaves of the frost-hardy species Ajuga reptans L. (Lamiaceace) contain two pools of raffinose family oligosaccharides (RFO): a large long-term storage pool in the mesophyll, possibly also involved in frost resistance, and a transport pool in the phloem (M. Bachmann, P. Matile, F. Keller [1994] Plant Physiol 105: 1335-1345). In the present study, the inter- and intracellular compartmentation of anabolic RFO metabolism was investigated by comparing whole-leaf tissue with mesophyll protoplasts and vacuoles. The studies showed the mesophyll to be the primary site of RFO synthesis in A. reptans. Mesophyll protoplasts were capable of RFO formation upon in vitro 14CO2 photosynthesis. Sucrose-phosphate synthase, galactinol synthase, and the galactinol-independent galactosyltransferase, which is responsible for RFO chain elongation, were located predominantly in the mesophyll protoplasts. The percentage of stachyose synthase in the mesophyll changed greatly during the cold-acclimation period (from 26% at the beginning to 88% after 20 d). The remainder was most probably in the intermediary cells of the phloem. Compartmentation studies in which mesophyll protoplasts were compared with vacuoles isolated from them showed that, of the components of the RFO storage pool, galactinol synthase, stachyose synthase, myo-inositol, galactinol, and sucrose were extravacuolar (most probably cytosolic), whereas galactinol-independent galactosyltransferase and higher RFO oligomers (with degree of polymerization 4) were vacuolar. Raffinose was found in both locations and might serve as a cryoprotectant.     

Expression of a GALACTINOL SYNTHASE gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.     

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。