Synthesis Of Raffinose Family Oligosaccharides Research Articles

Overexpression of galactinol synthase 1 from Solanum commersonii (ScGolS1) confers freezing tolerance in transgenic potato

Potato (Solanum tuberosum L.) is the fourth largest food crop in the world. Low temperatures cause serious damage to potato plants every year, and freezing tolerance has become a hot spot in potato research. Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides (RFOs), and plays an important role in the response of plants to abiotic stress. In this study, the ScGolS1 gene from Solanum commersonii was cloned and introduced into the S. tuberosum cultivars ‘Atlantic’ and ‘Desiree’ via Agrobacterium-mediated transformation. Phenotyping assays showed that overexpression of ScGolS1 could significantly improve freezing tolerance in transgenic potato plants. Further physiological and biochemical experiments showed that the transgenic lines had lower relative conductivity, malondialdehyde content, and 3,3′-diaminobenzidine staining and a higher plant survival rate compared with wild type (WT) under cold stress. Moreover, the C-repeat binding factors (CBF1, CBF2 and CBF3), the downstream cold-responsive genes COR413 and COR47, and the ethylene-responsive factor (ERF) transcription factor genes ERF3, ERF4 and ERF6, which function in the ethylene signaling pathway, were all induced by freezing treatment and expressed at higher levels in the ScGolS1 overexpression lines compared with WT. Besides, the expression of some genes such as MIPS, STS and RS from the RFO metabolic pathway was up-regulated under cold stress, resulting in changes in the content of some soluble sugars. This indicated that ScGolS1 overexpression altered the sugar composition and enhanced freezing tolerance in transgenic potato by inducing the ethylene and CBF signaling pathways. These results provided theoretical support and genetic resources for freezing tolerance breeding in potato.

Characterization and Expression Profiles of Galaetinol Synthase and Raffinose Synthase in Rubber Tree

Galactinol synthase (GolS) and Raffinose synthase (RS) are key enzymes for the synthesis of Raffinose family oligosaccharides (RFOs) which are widely involved in the growth development and resistances to stresses. To explore the function of HbGolSs  and HbRSs  during leaf development and tapping period in Hevea brasiliensis, in this study, based on the established library data platform of rubber tree genome, we isolated and identified the family members of HbGolSs  and HbRSs , then we analyzed their expression profiles with emphasis through on-line molecular biological tools and PRISM software as well. The results demonstrated that HbGolS1  and HbRS1 were prime member in their separate family, HbGOlS1  was highly expressed in latex and leaf, and with leaf development, its level gradually increased till the ultimate in mature leave, especially, it was dramatically up-regulated by tapping but markedly inhibited by Ethrel treatment; Different from it, HbRS1  was little influenced by tapping or Ethrel treatment although it was specially expressed in latex. Together, this study demonstrated the expression characteristics of HbGolS1 and HbRS1  with leaf development and tapping, which has laid a foundation for further exploring the metabolism of RFOs and their roles in rubber trees.

A conserved NAG motif is critical to the catalytic activity of galactinol synthase, a key regulatory enzyme of RFO biosynthesis.

Galactinol synthase (GolS) catalyzes the key regulatory step in the biosynthesis of Raffinose Family Oligosaccharides (RFOs). Even though the physiological role and regulation of this enzyme has been well studied, little is known about active site amino acids and the structure-function relationship with substrates of this enzyme. In the present study, we investigate the active site amino acid and structure-function relationship for this enzyme. Using a combination of three-dimensional homology modeling, molecular docking along with a series of deletion, site-directed mutagenesis followed by in vitro biochemical and in vivo functional analysis; we have studied active site amino acids and their interaction with the substrate of chickpea and Arabidopsis GolS enzyme. Our study reveals that the GolS protein possesses GT8 family-specific several conserved motifs in which NAG motif plays a crucial role in substrate binding and catalytic activity of this enzyme. Deletion of entire NAG motif or deletion or the substitution (with alanine) of any residues of this motif results in complete loss of catalytic activity in in vitro condition. Furthermore, disruption of NAG motif of CaGolS1 enzyme disrupts it's in vivo cellular function in yeast as well as in planta. Together, our study offers a new insight into the active site amino acids and their substrate interaction for the catalytic activity of GolS enzyme. We demonstrate that NAG motif plays a vital role in substrate binding for the catalytic activity of galactinol synthase that affects overall RFO synthesis.

Genome-wide association mapping of lentil (Lens culinaris Medikus) prebiotic carbohydrates toward improved human health and crop stress tolerance

Lentil, a cool-season food legume, is rich in protein and micronutrients with a range of prebiotic carbohydrates, such as raffinose-family oligosaccharides (RFOs), fructooligosaccharides (FOSs), sugar alcohols (SAs), and resistant starch (RS), which contribute to lentil's health benefits. Beneficial microorganisms ferment prebiotic carbohydrates in the colon, which impart health benefits to the consumer. In addition, these carbohydrates are vital to lentil plant health associated with carbon transport, storage, and abiotic stress tolerance. Thus, lentil prebiotic carbohydrates are a potential nutritional breeding target for increasing crop resilience to climate change with increased global nutritional security. This study phenotyped a total of 143 accessions for prebiotic carbohydrates. A genome-wide association study (GWAS) was then performed to identify associated variants and neighboring candidate genes. All carbohydrates analyzed had broad-sense heritability estimates (H2) ranging from 0.22 to 0.44, comparable to those reported in the literature. Concentration ranges corresponded to percent recommended daily allowances of 2–9% SAs, 7–31% RFOs, 51–111% RS, and 57–116% total prebiotic carbohydrates. Significant SNPs and associated genes were identified for numerous traits, including a galactosyltransferase (Lcu.2RBY.1g019390) known to aid in RFO synthesis. Further studies in multiple field locations are necessary. Yet, these findings suggest the potential for molecular-assisted breeding for prebiotic carbohydrates in lentil to support human health and crop resilience to increase global food security.

Integrative transcriptomic and metabolomic analyses provide insight into the long-term submergence response mechanisms of young Salix variegata stems.

The mechanisms underlying long-term complete submergence tolerance in S. variegata involve enhanced oxidative stress responses, strengthened ethylene and ABA signaling, synthesis ofraffinose family oligosaccharides,unsaturated fatty acids, and specific stress-related amino acids. Salix variegata Franch. is a riparian shrub species that can tolerate long-term complete submergence; however, the molecular mechanisms underlying this trait remain to be elucidated. In this study, we subjected S. variegata plants to complete submergence for 60d and collected stems to perform transcriptomic and metabolomic analyses, as well as quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Results revealed that photosynthesis and the response to light stimulus were inhibited during submergence and recovered after desubmergence. Ethylene and abscisic acid (ABA) signaling could be important for the long-term submergence tolerance of S. variegata. Jasmonic acid (JA) signaling also participated in the response to submergence. Raffinose family oligosaccharides, highly unsaturated fatty acids, and specific stress-related amino acids accumulated in response to submergence, indicating that they may protect plants from submergence damage, as they do in response to other abiotic stressors. Several organic acids were produced in S. variegata plants after submergence, which may facilitate coping with the toxicity induced by submergence. After long-term submergence, cell wall reorganization and phenylpropanoid metabolic processes (the synthesis of specific phenolics and flavonoids) were activated, which may contribute to long-term S. variegata submergence tolerance; however, the detailed mechanisms require further investigation. Several transcription factors (TFs), such as MYB, continuously responded to submergence, indicating that they may play important roles in the responses and adaption to submergence. Genes related to oxidative stress tolerance were specifically expressed after desubmergence, potentially contributing to recovery of S. variegata plants within a short period of time.

Overexpression of GmGolS2-1, a soybean galactinol synthase gene, enhances transgenic tobacco drought tolerance

Plant adaptation to abiotic stress is significantly influenced by galactinol synthase (GolS), which is a regulatory enzyme that catalyzes the synthesis of raffinose family oligosaccharides (RFOs). Soybean GolS genes, GmGolS2-1 and GmGolS2-2 were isolated in this study. GmGolS2-1 was 993 bp and encoded a 330-amino acid polypeptide, and GmGolS2-2 was 975 bp and encoded a 324-amino acid polypeptide. The deduced amino acid sequences contained common features of plant GolS proteins: a DXD motif and a hydrophobic pentapeptide (APSAA). Significant increase of GmGolS2-1 and GmGolS2-2 transcripts under drought stress was observed under real-time fluorescence quantitative PCR (qPCR), while their transcripts only increased slightly under salt and cold stresses. The GmGolS2-1 promoter region contained four methyl jasmonate-responsive elements, two anaerobic-induced elements, a wound-responsive element, and two MYB binding sites. Overexpression of GmGolS2-1 enhanced the GolS activity along with expression of stress-associated genes in transgenic tobacco. Additionally, the overexpression of GmGolS2-1 resulted in lower relative electrolyte leakage and malondialdehyde (MDA) content and higher soluble carbohydrates and proline content in transgenic tobacco than wild-type (WT) tobacco under drought stress. These findings indicate that GmGolS2-1 improved transgenic tobacco drought resistance. Two galactinol synthase genes, GmGolS2-1 andGmGolS2-1, were isolated from soybean. They could be induced by abiotic stress. GmGolS2-1 enhances tolerance to drought in transgenic tobacco.

On the Inverse Correlation of Protein and Oil: Examining the Effects of Altered Central Carbon Metabolism on Seed Composition Using Soybean Fast Neutron Mutants.

Protein and oil levels measured at maturity are inversely correlated across soybean lines; however, carbon is in limited supply during maturation resulting in tradeoffs for the production of other reserves including oligosaccharides. During the late stages of seed development, the allocation of carbon for storage reserves changes. Lipid and protein levels decline while concentrations of indigestible raffinose family oligosaccharides (RFOs) increase, leading to a decreased crop value. Since the maternal source of carbon is diminished during seed maturation stages of development, carbon supplied to RFO synthesis likely comes from an internal, turned-over source and may contribute to the reduction in protein and lipid content in mature seeds. In this study, fast neutron (FN) mutagenized soybean populations with deletions in central carbon metabolic genes were examined for trends in oil, protein, sugar, and RFO accumulation leading to an altered final composition. Two lines with concurrent increases in oil and protein, by combined 10%, were identified. A delayed switch in carbon allocation towards RFO biosynthesis resulted in extended lipid accumulation and without compromising protein. Strategies for future soybean improvement using FN resources are described.

Getting that sugar, one way or another.

Getting that sugar, one way or another.

Arabidopsis galactinol synthases 1 (AtGOLS1) negatively regulates seed germination

Seed germination begins the growth phases of plants and its rate is affected not only by plant hormones, including abscisic acid (ABA), gibberellin (GA) and brassinosteroids (BRs), but also by environmental factors. In this study, we searched for additional chemical reagents that affect seed germination, using the det2-1 and ga1-3 mutants that showed reduced seed germination due to defective BR- or GA- biosynthesis, respectively. We found that the reducing reagent dithiothreitol (DTT) specifically enhanced seed germination of det2-1 compared with that of ga1-3. To further investigate the underlying molecular mechanism for this phenomenon, we identified AtGOLS1 as a differentially expressed gene in germinating seeds treated with DTT by GeneFishing analysis. AtGOLS1 encodes a galactinol synthase, critical for the first step in raffinose family oligosaccharides synthesis during seed maturation. We observed that expression of AtGOLS1 decreased when conditions were favorable for seed germination. We also determined that the seed germination rate was faster in T-DNA knockout atgols1 mutant and transgenic plants transformed with an RNA interference construct targeting AtGOLS1 compared with wild type plants. The double mutant of det2-1 and atgols1 also suppressed the reduced seed germination of the det2-1. Taken together, our results suggest that AtGOLS1 acts as a negative regulator in seed germination.

Drought and salt stress in Macrotyloma uniflorum leads to common and specific transcriptomic responses and reveals importance of raffinose family oligosaccharides in stress tolerance

Abiotic stresses affect crop productivity worldwide. Understanding molecular mechanisms of plant abiotic stress tolerance is important for developing stress tolerant crop plants for sustaining crop productivity in future. Horse gram (Macrotyloma uniflorum Lam.) is a grain legume largely cultivated by marginal farmers and is known for excellent nutritional and remedial properties and better adaptability to harsh environmental conditions. In this study, we subjected M. uniflorum seedlings to salt and drought stress and carried out whole transcriptome profiling of leaf and root tissues. Assessing the global transcriptome changes under drought and salt stress resulted in identification of several genes contributing to stress response in this species. Overall, more transcriptomic changes were observed in root tissue compared to leaf tissue. The study identified stress responsive genes commonly and differently regulated under stress/tissue conditions. Several Differentially Expressing Genes (DEGs) in our data were identified as enzymes involved in the synthesis of raffinose family oligosaccharides (RFOs), pointing towards major role for RFOs such as galactinol and raffinose in tolerance to drought/salt stress in M. uniflorum, possibly through osmoprotection and/or ROS scavenging. The upregulation of a few genes reported to result in ABA insensitivity point towards reduced abscisic acid sensitivity and increased water loss rate in M. uniflorum under the experimental conditions. Our data will facilitate further molecular studies on stress tolerance of M. uniflorum. The DEGs from our results are potential candidates for understanding and engineering abiotic stress tolerance in plants.

Molecular Cloning and Characterization of Galactinol Synthases in Camellia sinensis with Different Responses to Biotic and Abiotic Stressors.

Galactinol synthase (GolS) is a key biocatalyst for the synthesis of raffinose family oligosaccharides (RFOs). RFOs accumulation plays a critical role in abiotic stress adaptation, but the relationship between expression of GolS genes and biotic stress adaptation remains unclear. In this study, two CsGolS genes were found to be highly up-regulated in a transcriptome library of Ectropic oblique-attacked Camellia sinensis. Three complete CsGolS genes were then cloned and characterized. Gene transcriptional analyses under biotic and abiotic stress conditions indicated that the CsGolS1 gene was sensitive to water deficit, low temperature, and abscisic acid, while CsGolS2 and CsGolS3 genes were sensitive to pest attack and phytohormones. The gene regulation and RFOs determination results indicated that CsGolS1 was primarily related to abiotic stress and CsGolS2 and CsGolS3 were related to biotic stress. GolS-mediated biotic stress adaptations have not been studied in depth, so further analysis of this new biological function is required.

Molecular, physiological, and agronomical characterization, in greenhouse and in field conditions, of soybean plants genetically modified with AtGolS2 gene for drought tolerance

Water deficit may occur at any stage of crop development, affecting productivity and causing economic losses. In response to drought, raffinose family oligosaccharides (RFOs) are accumulated in plant tissues stabilizing and protecting cell membranes and keeping the vital functions. The enzyme galactinol synthase (GolS, EC 2.4.1.123) catalyzes the first step in the biosynthesis of RFOs. In our study, soybean events overexpressing 35S:AtGolS2 were molecularly, physiological, and agronomical characterized, under drought simulated in greenhouse and in field conditions during the crop season 2014/2015. The conventional soybean cultivar BRS 184 was transformed and five positive events were obtained. Four events transmitted the transgene to further generations and in the events 2Ia1 and 2Ia4, two to four copies of AtGols2 gene were observed. Results in greenhouse showed that the overexpression of AtGolS2 in genetically modified (GM) plants led to increased galactinol transcripts, probably resulting in changes in carbohydrate metabolism. Accumulation of these transcripts that may have acted as osmoprotectors, lead to higher drought tolerance and survival rate of 2Ia4 plants. In addition, in field conditions, higher yield was observed for 2Ia4 plants under irrigated (IRR) and non-irrigated (NIRR) treatments. This result can be due to the increased synthesis of RFOs even under well-watered conditions. This field screening showed promising results for drought tolerance, suggesting that 2Ia4 plants may be useful in a breeding program for the development of drought-tolerant plants. However, additional studies are needed in further crop seasons and other sites to better characterize how these plants may outperform the WT plants under water deficit.

Overexpression of TsGOLS2, a galactinol synthase, in Arabidopsis thaliana enhances tolerance to high salinity and osmotic stresses

Galactinol synthase (GOLS, EC 2.4.1.123), a key enzyme in the synthesis of raffinose family oligosaccharides (RFOs), catalyzes the condensation of UDP-galactose with myo-inositol to produce galactinol as the sole donor for the synthesis of RFOs. RFOs have been implicated in mitigating effects of environmental stresses on plants. TsGOLS2, was cloned from Thellungiella salsuginea with high homology to AtGOLS2. TsGOLS2 was up-regulated by several abiotic stresses. We overexpressed TsGOLS2 in Arabidopsis thaliana. The contents of galactinol, raffinose, and α-ketoglutaric acid were significantly increased in transgenic plants. Compared to wild type plants, salt-stressed transgenic A.thaliana exhibited higher germination rate, photosynthesis ability, and seedling growth. After being treated with osmotic stress by high concentration of sorbitol, transgenic plants retained high germination rates and grew well during early development. These results indicated that overexpression of TsGOLS2 in A.thaliana improved the tolerance of transgenic plants to high salinity and osmotic stress.

Winter induction of the galactinol synthase gene is associated with endodormancy in chestnut trees

From the start of autumn and throughout winter dormancy, starch stored in the stems and buds of woody plants is broken down into oligosaccharides, mainly raffinose family oligosaccharides (RFOs). Given that RFO synthesis protects plants against winter cold, the enzyme galactinol synthase (catalyzing the first step in RFO synthesis) could play an important role in this process. Here, we characterized a galactinol synthase gene (CsGolS1) in chestnut and examined its expression in relation to tree dormancy. In chestnut branches, CsGolS1 mRNA levels paralleled the induction and course of winter dormancy, starting to rise at the beginning of autumn and elevated levels persisting until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsGolS1 gene in the stems. However, the permanent induction of stem CsGolS1 during winter months, rather than being a direct response to the cold, was found to be associated with the endodormancy state. This observation points to transcriptional mechanisms controlling the maintenance of endodormancy.

Synthesis of raffinose family oligosaccharides by regioselective de-O-benzylation with Co2(CO)8/Et3SiH/CO system

A convenient approach for synthesis of raffinose, stachyose, and verbascose using sucrose as the starting material is presented. The key step is the regioselective de-O-benzylation with Co2(CO)8/Et3SiH/CO system, followed by a high α-selective glycosylation. The newly developed de-O-benzylation system is efficient in removing the primary benzyl groups of sucrose and raffinose under mild condition and with high selectivity. Using thioglycoside as donor, NIS/AgOTf as promoter and DTBMP as additive, glycosylation of acid labile sucrose substrate is achieved in high yield.

A cold responsive galactinol synthase gene from Medicago falcata (MfGolS1) is induced by myo‐inositol and confers multiple tolerances to abiotic stresses

Galactinol synthase (GolS, EC 2.4.1.123) catalyzes formation of galactinol and the subsequent synthesis of raffinose family oligosaccharides. The relationship of GolS to drought and salt tolerance has been well documented, however, little information is available about the role of GolS gene in cold tolerance. A coding sequence of MfGolS1 cDNA was cloned from Medicago sativa spp falcata (i.e. M. falcata), a species that exhibits greater cold tolerance than alfalfa (M. sativa). MfGolS1 transcript was not detected in untreated vegetative tissues using RNA blot hybridization; however, it was greatly induced in leaves, but not in stem and petiole, after cold treatment. Higher levels of MfGolS1 transcript were induced and maintained in M. falcata than in M. sativa during cold acclimation. Accordingly, more sugars including sucrose, galactinol, raffinose and stachyose were accumulated in M. falcata than in M. sativa. The data indicated that MfGolS1 transcript and its resultant sugar accumulation were associated with the differential cold tolerance between M. falcata and M. sativa. MfGolS1 transcript was weakly induced by dehydration and salt stresses, but not responsive to abscisic acid. MfGolS1 could be induced by myo-inositol, which is proposed to participate in cold-induced MfGolS1 expression. Overexpression of MfGolS1 in tobacco resulted in elevated tolerance to freezing and chilling in transgenic plants as a result of enhanced levels of galactinol, raffinose and stachyose. Tolerance to drought and salt stresses was also increased in the transgenic tobacco plants. It is suggested that MfGolS1 plays an important role in plant tolerance to abiotic stresses.

Molecular Cloning and Characterization of GhGolS1, a Novel Gene Encoding Galactinol Synthase from Cotton (Gossypium hirsutum)

Galactinol synthase (GolS) is a key initial regulatory enzyme in the synthesis of raffinose family oligosaccharides (RFOs), sugars that play essential roles in plant growth and development. We have cloned the GhGolS1 gene (GenBank accession number: JF813792), which encodes the cotton GolS1 protein by rapid amplification of cDNA ends–polymerase chain reaction (RACE–PCR). The full-length cDNA is 1,729-bp long and encoded an open reading frame (ORF) of 343 amino acids with a deduced molecular weight of 39.2 kDa. The GhGolS1 protein shared 74–78% identity at the amino acid level with GolS isolated from Ricinus communis, Populus trichocarpa, Solanum lycopersicum, Arabidopsis thaliana and Capsicum annuum. The corresponding genomic DNA, which contained three exons and two introns, was isolated and analyzed. The 5′ flanking region was also analyzed to identify a group of putative cis-acting elements. DNA gel blot analysis showed that in the cotton genome, the GhGolS gene family contains at least three members. Real-time PCR (RT-PCR) analysis revealed that GhGolS1 is expressed in cotton leaves, anthers and 35 DPA fibers, and its expression level in anthers is much higher than that in leaves and 35 DPA fibers. It was minimally expressed in other tissues. Additionally, the GhGolS1 protein was localized to the cell membrane. On the basis of these results, we propose that GhGolS1 has critical roles in cotton male fertility, fiber quality and seed development.

Expression of three galactinol synthase isoforms in Coffea arabica L. and accumulation of raffinose and stachyose in response to abiotic stresses

Galactinol synthase (EC 2.4.1.123; GolS) catalyzes the first step in the synthesis of raffinose family oligosaccharides (RFOs). Their accumulation in response to abiotic stresses implies a role for RFOs in stress adaptation. In this study, the expression patterns of three isoforms of galactinol synthase (CaGolS1-2-3) from Coffea arabica were evaluated in response to water deficit, salinity and heat stress. All CaGolS isoforms were highly expressed in leaves while little to no expression were detected in flower buds, flowers, plagiotropic shoots, roots, endosperm and pericarp of mature fruits. Transcriptional analysis indicated that the genes were differentially regulated under water deficit, high salt and heat stress. CaGolS1 isoform is constitutively expressed in plants under normal growth conditions and was the most responsive during all stress treatments. CaGolS2 is unique among the three isoforms in that it was detected only under severe water deficit and salt stresses. CaGolS3 was primarily expressed under moderate and severe drought. This isoform was induced only at the third day of heat and under high salt stress. The increase in GolS transcription was not reflected into the amount of galactinol in coffee leaves, as specific glycosyltransferases most likely used galactinol to transfer galactose units to higher homologous oligosaccharides, as suggested by the increase of raffinose and stachyose during the stresses.

Phloem loading, plant growth form, and climate

Plasmodesmatal frequencies in the phloem of leaf minor veins vary considerably, suggesting that photoassimilate is loaded into the phloem by different strategies. The ecophysiological basis for multiple loading types is unknown. We updated the analysis of van Bel and Gamalei (Plant Cell Environ 15: 265-270, 1992) with more current phylogenetic data and by treating separately two symplastic loading types, those that load actively by polymer trapping (synthesis of raffinose family oligosaccharides--RFOs), and those that load passively, by diffusion. The results indicate a stronger association between passive, symplastic loading and the tree growth form than previously recognized. Apoplastic loading is highly correlated with the herbaceous habit. There is no correlation between RFO families and growth form. At the family level, there are no correlations between minor vein types and climate that cannot be explained by the dearth of woody plants in the arctic for reasons unassociated with phloem loading. However, at the species level, a floristic analysis uncovered a correlation between the RFO trait and species frequency in tropical and subtropical regions of the world. The correlations between loading types and both growth form and climate are subtle, probably indirect, and poorly understood.

Low night-temperatures affect the metabolism of raffinose-family oligosaccharides in melon (Cucumis melo L.) leaves during fruit expansion

SummaryThis study served to reveal the effects and mechanism of low night-temperatures on the metabolism of raffinose-family oligosaccharides in melon (Cucumis melo L.) leaves. We investigated the carbohydrate contents and sugar-metabolising enzyme activities in leaves under normal (15°C; control) and two low night-temperature (12°C or 9°C) conditions during fruit expansion. The results showed that the greatest difference in carbohydrate metabolism occurred between 9°C at night and the 15°C control. Compared to the normal night temperature, total carbohydrate accumulation, in particular raffinose, increased and the ratio of raffinose to total carbohydrates exhibited the greatest increase in melon leaves treated at 9°C at night, while the ratio of sucrose to total carbohydrates declined at the end of the same treatment. These data suggested that 9°C at night played an important role in partitioning carbon from sucrose to raffinose-family oligosaccharides (RFO), and that raffinose may serve dual purposes in stress protection and carbon storage. Meanwhile, the activities of sugar-metabolising enzymes were notably affected by 9°C at night. Sucrose synthase (SS), sucrose phosphate synthase (SPS) and, in particular, galactinol and stachyose synthase activities decreased markedly, while acid invertase (AI) and neutral invertase (NI) activities increased sharply, which restricted sucrose and RFO synthesis. Overall, these data suggest that the decline in stachyose and galactinol synthase activities were the main limiting factor for RFO synthesis in melon leaves at 9°C at night. They also indicated that the reduction in stachyose metabolism may be an important factor during the development of unmarketable fruit.