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.     

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.     

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.     

The role of raffinose in the cold acclimation response of Arabidopsis thaliana

In many plants raffinose family oligosaccharides are accumulated during cold acclimation. The contribution of raffinose accumulation to freezing tolerance is not clear. Here, we investigated whether synthesis of raffinose is an essential component for acquiring frost tolerance. We created transgenic lines of Arabidopsis thaliana accessions Columbia-0 and Cape Verde Islands constitutively overexpressing a galactinol synthase (GS) gene from cucumber. GS overexpressing lines contained up to 20 times as much raffinose as the respective wild-type under non-acclimated conditions and up to 2.3 times more after 14 days of cold acclimation at 4 degrees C. Furthermore, we used a mutant carrying a knockout of the endogenous raffinose synthase (RS) gene. Raffinose was completely absent in this mutant. However, neither the freezing tolerance of non-acclimated leaves, nor their ability to cold acclimate were influenced in the RS mutant or in the GS overexpressing lines. We conclude that raffinose is not essential for basic freezing tolerance or for cold acclimation of A. thaliana.     

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 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.     

Metabolism of the Raffinose Family Oligosaccharides in Leaves of Ajuga reptans L. (Cold Acclimation,Translocation, and Sink to Source Transition: Discovery of Chain Elongation Enzyme)

Ajuga reptans is a frost-hardy, perennial labiate that is known for its high content of raffinose family oligosaccharide(s) (RFO). Seasonal variations in soluble nonstructural carbohydrate levels in above-ground parts of Ajuga showed that the RFO were by far the most predominant components throughout the whole year. RFO were lowest in summer (75 mg/g fresh weight) and highest in fall/winter (200 mg/g fresh weight), whereas sucrose and starch were only minor components. Cold treatment (14 d at 10/3[deg]C, day/night) of plants that were precultivated under warm conditions (25[deg]C) lowered the temperature optimum of net photosynthesis from 16[deg] to 8[deg]C, decreased the maximum rate, and increased the total nonstructural carbohydrate content of leaves by a factor of about 10, mainly because of an increase of RFO. The degree of polymerization of the RFO increased sequentially up to at least 15. A novel, galactinol-independent galactosyltransferase enzyme was found, forming from two molecules of RFO, the next higher and lower degree of polymerization of RFO. The enzyme had a pH optimum of 4.5 to 5.0 and may be responsible for RFO chain elongation. RFO were the main carbohydrates translocated in the phloem, with stachyose being by far the most dominant form. Studies of carbon balance during leaf development revealed a transition point between import and export at approximately 25% maximal leaf area. RFO synthesis could be detected even before the commencement of export, suggesting the existence of a nonphloem-linked RFO pool even in very young leaves. Taken together, it seems that Ajuga leaves contain two pools of RFO metabolism, a pronounced long-term storage pool in the mesophyll, possibly also involved in frost resistance, and a transport pool in the phloem.     

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.     

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.     

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.     

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.     

The influence of thermal parameters on the acclimation responses of pinfish Lagodon rhomboides exposed to static and decreasing low temperatures

Pinfish Lagodon rhomboides acclimation rates were determined by modelling changes in critical thermal minimum ( T _{crit min}, ° C) estimates at set intervals following a temperature decrease of 3–4° C. The results showed that pinfish gained a total of 3·7° C of cold tolerance over a range of acclimation temperatures ( T _acc, ° C) from (23–12° C), that cold tolerance increased with exposure time to the reduced temperature at all T _acc, but that the rate of cold tolerance accruement (mean 0·14° C day⁻¹) was independent of T _acc. A highly significant ( P < 0·001) multivariate predictive model was generated that described the acclimation rates and thermal tolerance of pinfish exposed to reduction in water temperature: log₁₀ T _{crit min}= 0·41597 − 0·01704 T _acc+ 0·04320 T _plunge− 0·08376[log₁₀ ( t + 1)], where T _plunge is plunge temperature (° C) and t is the time (days). A comparison of the present data, with acclimation rate data for other species, suggests that factors such as latitude or geographic range may play a more important role than ambient temperature in determining cold acclimation rates in fishes.     

Carbohydrate content of Eucalyptus gunnii leaves along an annual cycle in the field and during induced frost-hardening in controlled conditions

The annual changes in frost hardiness were studied for three Eucalyptus gunnii genotypes. Frost resistance evaluated on leaf discs by the electrolyte leakage method reached a maximum in the coldest period and a minimum in summer demonstrating winter frost hardening. Genotype 634 exhibited a higher intrinsic resistance than the other genotypes both in the hardened and in the non-hardened stages. Plants of this genotype were also frost acclimated in controlled conditions by a progressive decrease of culture temperature (25 to 0 °C) but the degree of hardening appeared to be lower in these conditions. The carbohydrate patterns in leaves varied with acclimation. In controlled conditions the leaves of genotype 634 exhibited a rise in sucrose, fructose and raffinose concentration up to a temperature of 10 to 7 °C which subsequently decreased. In natural conditions a comparison of the three genotypes allowed us to correlate the higher intrinsic resistance of genotype 634 to a higher soluble sugar content. During acclimation fructose and raffinose changes were also correlated to an increase in cold resistance even though the kinetics of these changes differed in controlled and natural conditions. The starch content was very low in the various genotypes in the different conditions but oligosaccharides such as stachyose and possibly verbascose were detected. The results point out the relationships occurring between increased frost resistance and changes in fructose and raffinose concentration in E. gunnii leaves.     

种子中的棉子糖半乳糖苷系列寡糖研究进展

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

种子中的棉子糖半乳糖苷系列寡糖研究进展

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