Purification and characterization of stachyose synthase from lentil (Lens culinaris) seeds: galactopinitol and stachyose synthesis.

Stachyose synthase (STS) (EC 2.4.1.67) was purified 313-fold from mature seeds of lentil. The final preparation had a specific activity of 9.09 nkat stachyose formed per milligram of protein. The enzyme was a monomeric protein with a molecular mass of 88.6 kDa (SDS-PAGE) and an isoelectric point of 4.8 (chromatofocusing). Western analysis revealed cross-reactivity of polyclonal antibodies raised against STS from adzuki bean with the lentil enzyme. The purified enzyme catalyzed a range of different galactosyl transfer reactions. In addition to the genuine STS reaction (raffinose + galactinol --> stachyose + myo-inositol), the enzyme catalyzed the reversible galactosyl transfer from galactinol to d-pinitol (1d-3-O-methyl-chiro-inositol), yielding galactopinitol A (O-alpha-d-galactopyranosyl-(1 --> 2)-4-O-methyl-d-chiro-inositol) and myo-inositol. Galactopinitol A could be further galactosylated by STS to give ciceritol (O-alpha-d-galactopyranosyl-(1 --> 6)-O-alpha-d-galactopyranosyl-(1 --> 2)-4-O-methyl-d-chiro-inositol). Enzymatic synthesis of galactopinitol A and ciceritol is a new observation. However, STS was not only able to utilize galactopinitol A as galactosyl acceptor, but also as galactosyl donor to form stachyose from raffinose. The role of STS in the metabolism of galactosyl cyclitols and oligosaccharides in plant seeds is discussed.     

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.     

Stachyose synthesis in leaves of Cucurbita pepo

An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (K_m 7.7 mM) and the acceptor raffinose (K_m 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co²⁺, Hg²⁺, Mn²⁺ and Ni²⁺ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (K_i 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase.     

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.     

Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase

Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT-PCR, followed by rapid amplification of cDNA ends (RACE-PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.     

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.     

Isolation of galactinol from leaves of Cucumis sativus

A simple procedure for the isolation of galactinol from leaves of Cucumimis sativus L. has been developed. The procedure yielded approx. 60% of the galactinol originally present in leaves with an apparent purity of 97%. Gas chromatographic and mass spectral analysis indicated that the compound was identical to galactinol isolated from sugar beet. The cucumber leaf galactinol was found to be suitable as a substrate for a galactosyl transferase enzyme which catalyses the formation of stachyose.     

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.     

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.     

Distribution and immunolocalization of stachyose synthase in Cucumis melo L.

Indirect evidence for the site of stachyose biosynthesis has been provided by determining the occurrence and distribution of stachyose, raffinose and galactinol, the donor of the galactosyl moiety for stachyose synthesis, in Cucumis melo L. cv. Ranjadew. Studies of enzyme activities for the synthesis of these sugars and their distribution in different plant organs and isolates has led to the conclusion that stachyose is synthesized mainly in mature leaves and seeds. Nevertheless, stachyose-synthase activity varied with leaf age, the developmental stage of a plant, the growing season and the plant cultivar used. No stachyose or stachyose-synthase activity could be detected in isolated mesophyll protoplasts and chloroplasts, whereas both were found in a minor-vein-enriched fraction isolated from mature leaves. The conclusion that stachyose biosynthesis is associated with minor veins was confirmed by immunolocalization of the enzyme. Positive specific immunoreactivity of stachyose synthase with polyclonal anti-stachyose-synthase antibodies, labeled with protein A-gold, was detected in intermediary cells of leaf minor veins. The implication of this local synthesis of the main transport sugar for phloem loading in mature leaves of Cucumis melo is discussed.Abbreviation RUBPCase ribulose-1,5-bisphosphate carboxylase This work was supported by Deutsche Forschungsgemeinschaft. The excellent assistance of Ms. B. Müller in preparing the samples for electron microscopy is gratefully acknowledged. The authors thank Professor H.J. Schneider-Poetsch for anti-RuBPCase antibodies.     

Stachyose Synthesis in Source Leaf Tissues of the CAM Plant Xerosicyos danguyi H. Humb

Leaf tissues from Xerosicyos danguyi H. Humb., a succulent member of the Cucurbitaceae, were found to possess both galactinol synthase activity and the capacity for photosynthetic production of stachyose, the phloem transport oligosaccharide common to other nonsucculent cucurbits. The amounts of stachyose isolated from leaf tissues, and the extractable activity of galactinol synthase, were somewhat higher in leaf tissues obtained from plants operating in the Crassulacean acid metabolism (CAM) mode (well watered plants) compared to leaf tissues from plants operating in the CAM-idling mode (water-stressed plants). In contrast, in leaf discs, the photosynthetic incorporation of label into stachyose following pulse labeling with ¹⁴CO₂ was similar for stressed and for nonstressed tissues. Stachyose could be extracted from, and was synthesized photosynthetically by, leaf discs which contained no vascular tissues, indicating that synthesis of stachyose can occur in photosynthetic mesophyll cells of Xerosicyos.     

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.     

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。     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Carbohydrate Changes during Maturation of Cucumber Fruit : Implications for Sugar Metabolism and Transport

Changes in the carbohydrate profiles in the mesocarp, endocarp, and seeds of maturing cucumber (Cucumis sativus, L.) fruit were analyzed. Fruit maturity was measured by a decrease in endocarp pH, which was found to correlate with a loss in peel chlorophyll and an increase in citric acid content. Concentrations of glucose and fructose (8.6-10.3 milligrams per gram fresh weight, respectively) were found to be higher than the concentration of sucrose (0.3 milligrams per gram fresh weight) in both mesocarp and endocarp tissue. Neither raffinose nor stachyose were found in these tissues. The levels of glucose and fructose in seeds decreased during development, but sucrose, raffinose, and stachyose accumulated during the late stages of maturation. Both raffinose and stachyose were found in the seeds of six lines of Cucumis sativus L. This accumulation of raffinose saccharides coincided with an increase in galactinol synthase activity in the seeds. Funiculi from maturing fruit were found to be high in sucrose concentration (4.8 milligrams per gram fresh weight) but devoid of both raffinose and stachyose. The results indicated that sucrose is the transport sugar from the peduncle to seed, and that raffinose saccharide accumulation in the seed is the result of in situ biosynthesis and not from direct vascular transport of these oligosaccharides into the seeds.     

Sugar synthesis and phloem loading in Coleus blumei leaves

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of ¹⁴CO₂ was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either ¹⁴CO₂ or [¹⁴C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [¹⁴C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to ¹⁴CO₂ for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ¹⁴CO₂, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244–1249) indicate that the phloem loading pathway in Coleus is probably symplastic.Abbreviations 3-OMG 3-O-methyl glucose - PCMBS p-chloromercuribenzenesulfonic acid - SE-CCC sieve-element-companion-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

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.     

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.     

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.     

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.