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.     

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.     

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.     

Photochemical responses and oxidative stress in two clones of Coffea canephora under water deficit conditions

The effects of water deficit on photochemical parameters and activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as, cellular damages were investigated in two clones of Coffea canephora differing in drought tolerance. After 6 days without irrigation, predawn leaf water potential fell to −3.0 MPa that was accompanied by the suppression of net photosynthesis in both clones. The variable to maximum chlorophyll fluorescence ratio remained unchanged regardless of the imposed treatments. Both clones showed a similar decline (about 25%) in the photochemical quenching coefficient, but only the drought-sensitive clone exhibited an enhancement (31%) of thermal deactivation under water deficit conditions. The quantum yield of electron transport decreased similarly in both genotypes. Under drought conditions, activities of SOD, CAT and APX increased to a greater extent in the drought-tolerant clone than in the drought-sensitive one. This seemed to be matched with higher protection against oxidative stress, as judged from the lower levels of lipid peroxidation and electrolyte leakage in the drought-tolerant clone. Thus, the ability to increase the antioxidant system activity in order to limit cellular damages might be an important attribute linked to the drought tolerance in C. canephora.     

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.     

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.     

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。     

Low night temperatures inhibit galactinol synthase gene expression and phloem loading in melon leaves during fruit development

Low night temperatures seriously affect plant growth and fruit quality. To investigate the effect of low night temperatures on the expression of galactinol synthase genes (GOLS) and phloem loading of raffinose family oligosaccharides, particular stachyose and raffinose (RFO represents stachyose and raffinose in this paper) and to gain a better understanding of the relationship between the phloem loading of RFO and fruit development, melon (Cucumis melo L.) plants at the fruit development stage were treated with temperatures of 28/12°C or 28/9°C (day/night) with 28/15°C as the control. Both the CmGOLS1 and CmGOLS2 gene expression and the activity of galactinol synthase were clearly repressed after treatments with 9 and 12°C at night, and the effect of 9°C was more obvious. Furthermore, low night temperatures inhibited photosynthesis and caused the lower amounts of sucrose to supply the RFO synthesis. However, the total soluble sugar, RFO, and sucrose contents were increased in leaves subjected to low night temperatures. It is supposed that low night temperature blocked symplastic phloem loading, which led to the accumulation of RFO in the leaf cells. With increasing content of RFO in the leaves, the expression of GOLS genes was inhibited according to the principle of feedback, and therefore the decreased expression of GOLS limited RFO synthesis and was indirectly harmful to phloem loading, thereby affecting fruit development.     

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.     

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 .