Raffinose Synthase Gene Research Articles

Analysis of the Rice Raffinose Synthase (OsRS) Gene Family and Haplotype Diversity.

Based on the genome information of rice (Nipponbare), this study screened and identified six raffinose synthase (RS) genes and analyzed their physical and chemical properties, phylogenetic relationship, conserved domains, promoter cis-acting elements, and the function and genetic diversity of the gene-CDS-haplotype (gcHap). The results showed that these genes play key roles in abiotic stress response, such as OsRS5, whose expression in leaves changed significantly under high salt, drought, ABA, and MeJA treatments. In addition, the OsRS genes showed significant genetic variations in different rice populations. The main gcHaps of most OsRS loci had significant effects on key agronomic traits, and the frequency of these alleles varied significantly among different rice populations and subspecies. These findings provide direction for studying the RS gene family in other crops.

Redesigning soybean with improved oil and meal traits.

Soybean seed oil and meal composition traits can be combined without interference to provide additional value to the crop. Soybean [Glycine max (L.) Merr.] is an important crop worldwide; its overall value comes from seed oil and high protein meal. The development of soybean varieties with allele combinations for improved oil and meal quality is expected to provide a compositional value bundle for soybean. The high oleic and low linolenic acid seed oil trait (HOLL; > 70% oleic and < 3% linolenic acid) is targeted to optimize the health and functional properties of soybean oil. For soybean meal, metabolizable energy is improved by altering the carbohydrate profile with increased sucrose and decreased anti-nutritional factors, raffinose family of oligosaccharides (RFOs). Previous research identified four variant alleles of fatty acid desaturase (FAD) genes and two raffinose synthase (RS) genes necessary for the HOLL trait in soybean oil and Low or Ultra-Low (UL) RFO traits in soybean meal, respectively. We employed a molecular marker-assisted breeding approach to combine six alleles conferring the desired soybean oil and meal value traits. Eight environment field trials were conducted with twenty-four soybean lines to evaluate phenotypic interactions among the variant alleles of FAD and RS genes. The results indicated that the four FAD gene alleles conditioned the HOLL fatty acid profile of the seed oil regardless of the allele status of the RS genes. Independent of the allele combination of the FAD genes, soybean with two variant alleles of the RS genes had the desired RFO trait in the seeds. The results confirm the feasibility of soybean variety development with this unique combination of oil and meal traits.

Raffinose catabolism enhances maize waterlogging tolerance by stimulating adventitious root growth and development.

Raffinose mitigates plant heat, drought, and cold stresses; however, whether raffinose contributes to plant waterlogging tolerance is unknown. The maize raffinose synthase mutant zmrafs-1 had seedlings that lack raffinose, generated fewer and shorter adventitious roots, and were more sensitive to waterlogging stress, while overexpression of the raffinose synthase gene, ZmRAFS, increased raffinose content, stimulated adventitious root formation, and enhanced waterlogging tolerance of maize seedlings. Transcriptome analysis of null segregant seedlings compared with zmrafs-1, particularly when waterlogged, revealed that the expression of genes related to galactose metabolism and the auxin biosynthetic pathway were up-regulated by raffinose. Additionally, indole-3-acetic acid content was significantly decreased in zmrafs-1 seedlings and increased in ZmRAFS-overexpressing seedlings. Inhibition of the hydrolysis of raffinose by 1-deoxygalactonojirimycin decreased the waterlogging tolerance of maize seedlings, the expression of genes encoding proteins related to auxin transport-related genes, and the indole-3-acetic acid level in the seedlings, indicating that the hydrolysis of raffinose is necessary for maize waterlogging tolerance. These data demonstrate that raffinose catabolism stimulates adventitious root formation via the auxin signaling pathway to enhance maize waterlogging tolerance.

Analysis of Raffinose Synthase Gene Family in Bread Wheat and Identification of Drought Resistance and Salt Tolerance Function of TaRS15-3B.

Raffinose synthase (RS) plays a crucial role in plant growth and development, as well as in responses to biotic stresses and abiotic stresses, yet few studies have been conducted on its role in bread wheat. Therefore, in this study we screened and identified a family of bread wheat raffinose synthase genes based on bread wheat genome information and analyzed their physicochemical properties, phylogenetic evolutionary relationships, conserved structural domains, promoter cis-acting elements, and expression patterns. The BSMV-induced silencing of TaRS15-3B resulted in the bread wheat seedlings being susceptible to drought and salt stress and reduced the expression levels of stress-related and ROS-scavenging genes in bread wheat plants. This further affected the ability of bread wheat to cope with drought and salt stress. In conclusion, this study revealed that the RS gene family in bread wheat plays an important role in plant response to abiotic stresses and that the TaRS15-3B gene can improve the tolerance of transgenic bread wheat to drought and salt stresses, provide directions for the study of other RS gene families in bread wheat, and supply candidate genes for use in molecular breeding of bread wheat for stress resistance.

Identification and Alternative Splicing Profile of the Raffinose synthase Gene in Grass Species.

Raffinose synthase (Rafs) is an important enzyme in the synthesis pathway of raffinose from sucrose and galactinol in higher plants and is involved in the regulation of seed development and plant responses to abiotic stresses. In this study, we analyzed the Rafs families and profiled their alternative splicing patterns at the genome-wide scale from 10 grass species representing crops and grasses. A total of 73 Rafs genes were identified from grass species such as rice, maize, foxtail millet, and switchgrass. These Rafs genes were assigned to six groups based the phylogenetic analysis. We compared the gene structures, protein domains, and expression patterns of Rafs genes, and also unraveled the alternative transcripts of them. In addition, different conserved sequences were observed at these putative splice sites among grass species. The subcellular localization of PvRafs5 suggested that the Rafs gene was expressed in the cytoplasm or cell membrane. Our findings provide comprehensive knowledge of the Rafs families in terms of genes and proteins, which will facilitate further functional characterization in grass species in response to abiotic stress.

Systematic Analysis of Galactinol Synthase and Raffinose Synthase Gene Families in Potato and Their Expression Patterns in Development and Abiotic Stress Responses.

Raffinose family oligosaccharides (RFOs) are very important for plant growth, development, and abiotic stress tolerance. Galactinol synthase (GolS) and raffinose synthase (RFS) are critical enzymes involved in RFO biosynthesis. However, the whole-genome identification and stress responses of their coding genes in potato remain unexplored. In this study, four StGolS and nine StRFS genes were identified and classified into three and five subgroups, respectively. Remarkably, a total of two StGolS and four StRFS genes in potato were identified to form collinear pairs with those in both Arabidopsis and tomato, respectively. Subsequent analysis revealed that StGolS4 exhibited significantly high expression levels in transport-related tissues, PEG-6000, and ABA treatments, with remarkable upregulation under salt stress. Additionally, StRFS5 showed similar responses to StGolS4, but StRFS4 and StRFS8 gene expression increased significantly under salt treatment and decreased in PEG-6000 and ABA treatments. Overall, these results lay a foundation for further research on the functional characteristics and molecular mechanisms of these two gene families in response to ABA, salt, and drought stresses, and provide a theoretical foundation and new gene resources for the abiotic-stress-tolerant breeding of potato.

Spatio-temporal expression pattern of Raffinose Synthase genes determine the levels of Raffinose Family Oligosaccharides in peanut (Arachis hypogaea L.) seed

Raffinose family oligosaccharides (RFOs) are known to have important physiological functions in plants. However, the presence of RFOs in legumes causes flatulence, hence are considered antinutrients. To reduce the RFOs content to a desirable limit without compromising normal plant development and functioning, the identification of important regulatory genes associated with the biosynthetic pathway is a prerequisite. In the present study, through comparative RNA sequencing in contrasting genotypes for seed RFOs content at different seed maturity stages, differentially expressed genes (DEGs) associated with the pathway were identified. The DEGs exhibited spatio-temporal expression patterns with high RFOs variety showing early induction of RFOs biosynthetic genes and low RFOs variety showing a late expression at seed maturity. Selective and seed-specific differential expression of raffinose synthase genes (AhRS14 and AhRS6) suggested their regulatory role in RFOs accumulation in peanut seeds, thereby serving as promising targets in low RFOs peanut breeding programs. Despite stachyose being the major seed RFOs fraction, differential expression of raffinose synthase genes indicated the complex metabolic regulation of this pathway. The transcriptomic resource and the genes identified in this study could be studied further to develop low RFOs varieties, thus improving the overall nutritional quality of peanuts.

Multiplex CRISPR/Cas9-mediated raffinose synthase gene editing reduces raffinose family oligosaccharides in soybean.

Soybean [Glycine max (L.) Merr.] is an important world economic crop. It is rich in oil, protein, and starch, and soluble carbohydrates in soybean seeds are also important for human and livestock consumption. The predominant soluble carbohydrate in soybean seed is composed of sucrose and raffinose family oligosaccharides (RFOs). Among these carbohydrates, only sucrose can be digested by humans and monogastric animals and is beneficial for metabolizable energy, while RFOs are anti-nutritional factors in diets, usually leading to flatulence and indigestion, ultimately reducing energy efficiency. Hence, breeding efforts to remove RFOs from soybean seeds can increase metabolizable energy and improve nutritional quality. The objective of this research is to use the multiplex Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing system to induce the knockout of soybean raffinose synthase (RS) genes RS2 and RS3 simultaneously to reduce RFOs in mature seeds. First, we constructed five types of multiplex gene editing systems and compared their editing efficiency in soybean hairy roots. We confirmed that the two-component transcriptional unit (TCTU) and single transcriptional unit (STU) systems with transfer RNA (tRNA) as the cleavage site performed better than other systems. The average editing efficiency at the four targets with TCTU-tRNA and STU-tRNA was 50.5% and 46.7%, respectively. Then, we designed four single-guide RNA (sgRNA) targets to induce mutations at RS2 and RS3 by using the TCTU-tRNA system. After the soybean transformation, we obtained several RS2 and RS3 mutation plants, and a subset of alleles was successfully transferred to the progeny. We identified null single and double mutants at the T2 generation and analyzed the seed carbohydrate content of their progeny. The RS2 and RS3 double mutants and the RS2 single mutant exhibited dramatically reduced levels of raffinose and stachyose in mature seeds. Further analysis of the growth and development of these mutants showed that there were no penalties on these phenotypes. Our results indicate that knocking out RS genes by multiplex CRISPR/Cas9-mediated gene editing is an efficient way to reduce RFOs in soybean. This research demonstrates the potential of using elite soybean cultivars to improve the soybean meal trait by multiplex CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-mediated gene editing.

Identification and Expression Analysis of the Genes Involved in the Raffinose Family Oligosaccharides Pathway of Phaseolus vulgaris and Glycine max.

Raffinose family oligosaccharides (RFO) play an important role in plants but are also considered to be antinutritional factors. A profound understanding of the galactinol and RFO biosynthetic gene families and the expression patterns of the individual genes is a prerequisite for the sustainable reduction of the RFO content in the seeds, without compromising normal plant development and functioning. In this paper, an overview of the annotation and genetic structure of all galactinol- and RFO biosynthesis genes is given for soybean and common bean. In common bean, three galactinol synthase genes, two raffinose synthase genes and one stachyose synthase gene were identified for the first time. To discover the expression patterns of these genes in different tissues, two expression atlases have been created through re-analysis of publicly available RNA-seq data. De novo expression analysis through an RNA-seq study during seed development of three varieties of common bean gave more insight into the expression patterns of these genes during the seed development. The results of the expression analysis suggest that different classes of galactinol- and RFO synthase genes have tissue-specific expression patterns in soybean and common bean. With the obtained knowledge, important galactinol- and RFO synthase genes that specifically play a key role in the accumulation of RFOs in the seeds are identified. These candidate genes may play a pivotal role in reducing the RFO content in the seeds of important legumes which could improve the nutritional quality of these beans and would solve the discomforts associated with their consumption.

Transcriptomic analysis of grapevine Dof transcription factor gene family in response to cold stress and functional analyses of the VaDof17d gene

Dof genes enhance cold tolerance in grapevine and VaDof17d is tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides. DNA-binding with one finger (Dof) proteins comprise a large family that plays important roles in the regulation of abiotic stresses. No in-depth analysis of Dof genes has been performed in the grapevine. In this study, we analyzed a total of 25 putative Dof genes in grapevine at genomic and transcriptomic levels, compiled expression profiles of 11 selected VaDof genes under cold stress and studied the potential function of the VaDof17d gene in grapevine calli. The 25 Dof proteins can be classified into four phylogenetic groups. RNA-seq and qRT-PCR results demonstrated that a total of 11 VaDof genes responded to cold stress. Comparative mRNA sequencing of 35S::VaDof17d grape calli showed that VaDof17d was tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides (RFOs), as observed by the up-regulation of galactinol synthase (GolS) and raffinose synthase genes. We found that the Dof17d-ED (CRISPR/Cas9-mediated mutagenesis of Dof17d-ED) mutant had low cold tolerance with a decreased RFOs level during cold stress. These results formed the fundamental knowledge for further analysis of the biological roles of Dof genes in the grapevine's adaption to cold stresses.

The Mitochondrial Iron-Regulated (MIR) gene is Oryza genus specific and evolved before speciation within the Oryza sativa complex.

The MIR gene is not an Oryza sativa orphan gene, but an Oryza genus-specific gene that evolved before AA lineage speciation by a complex origination process. Rice (Oryza sativa L.) is a model species and an economically relevant crop. TheOryzagenus comprises 25 species, with genomic data available for several Oryzaspecies, making it a model for genetics and evolution. TheMitochondrial Iron-Regulated(MIR) gene was previously implicated inthe O. sativaFe deficiency response, and was considered an orphan gene present only in rice. Here we show that MIR is also found in otherOryzaspecies that belong to the Oryza sativa complex, which have AA genome type and constitute the primary gene pool for O. sativa breeding.Our data suggest thatMIRoriginated in a stepwise process, in which sequences derived from an exon fragment of the raffinose synthase gene were pseudogenized into non-coding, which in turn originated the MIR gene de novo. All species with a putative functionalMIRgene conserve their regulation by Fe deficiency, with the exception ofOryza barthii. InO. barthii, the MIR coding sequence was translocated to a different chromosomal position and separated from its regulatory region, leading to a lack of Fe deficiency responsiveness. Moreover, the MIR co-expression subnetwork cluster inO. sativais responsive to Fe deficiency, evidencing the importance of the newly originated gene in Fe uptake. This work establishes thatMIRis not an orphan gene as previously proposed, but a de novo originated gene within thegenus Oryza. We also showed that MIR is undergoing genomic changes in one species (O. barthii), with an impact onFe deficiency response.

The GARP/MYB-related grape transcription factor AQUILO improves cold tolerance and promotes the accumulation of raffinose family oligosaccharides

Grapevine (Vitis vinifera L.) is a widely cultivated fruit crop whose growth and productivity are greatly affected by low temperatures. On the other hand, wild Vitis species represent valuable genetic resources of natural stress tolerance. We have isolated and characterized a MYB-like gene encoding a putative GARP-type transcription factor from Amur grape (V. amurensis) designated as VaAQUILO. AQUILO (AQ) is induced by cold in both V. amurensis and V. vinifera, and its overexpression results in significantly improved tolerance to cold both in transgenic Arabidopsis and in Amur grape calli. In Arabidopsis, the ectopic expression of VaAQ increased antioxidant enzyme activities and up-regulated reactive oxygen species- (ROS) scavenging-related genes. Comparative mRNA sequencing profiling of 35S:VaAQ Arabidopsis plants suggests that this transcription factor is related to phosphate homeostasis like their Arabidopsis closest homologues: AtHRS1 and AtHHO2. However, when a cold stress is imposed, AQ is tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides (RFOs), as observed by the up-regulation of galactinol synthase (GoLS) and raffinose synthase genes. Gene co-expression network (GCN) and cis-regulatory element (CRE) analyses in grapevine indicated AQ as potentially regulating VvGoLS genes. Increased RFO content was confirmed in both transgenic Arabidopsis and Amur grape calli overexpressing VaAQ. Taken together, our results imply that AQ improves cold tolerance through promoting the accumulation of osmoprotectants.

Regulation of Seed Vigor by Manipulation of Raffinose Family Oligosaccharides in Maize and Arabidopsis thaliana

Raffinose family oligosaccharides (RFOs) accumulate in seeds during maturation desiccation in many plant species. However, it remains unclear whether RFOs have a role in establishing seed vigor. GALACTINOL SYNTHASE (GOLS), RAFFINOSE SYNTHASE (RS), and STACHYOSE SYNTHASE (STS) are the enzymes responsible for RFO biosynthesis in plants. Interestingly, only raffinose is detected in maize seeds, and a unique maize RS gene (ZmRS) was identified. In this study, we found that two independent mutator (Mu)-interrupted zmrs lines, containing no raffinose but hyperaccumulating galactinol, have significantly reduced seed vigor, compared with null segregant controls. Unlike maize, Arabidopsis thaliana seeds contain several RFOs (raffinose, stachyose, and verbascose). Manipulation of A. thaliana RFO content by overexpressing ZmGOLS2, ZmRS, or AtSTS demonstrated that co-overexpression of ZmGOLS2 and ZmRS, or overexpression of ZmGOLS2 alone, significantly increased the total content of RFOs and enhanced Arabidopsis seed vigor. Surprisingly, while overexpression of ZmRS increased seed raffinose content, its overexpression dramatically decreased seed vigor and reduced the seed amounts of galactinol, stachyose, and verbascose. In contrast, the atrs5 mutant seeds are similar to those of the wild type with regard to seed vigor and RFO content, except for stachyose, which accumulated in atrs5 seeds. Total RFOs, RFO/sucrose ratio, but not absolute individual RFO amounts, positively correlated with A. thaliana seed vigor, to which stachyose and verbascose contribute more than raffinose. Taken together, these results provide new insights into regulatory mechanisms of seed vigor and reveal distinct requirement for RFOs in modulating seed vigor in a monocot and a dicot.

Isolation, functional characterization and stress responses of raffinose synthase genes in sugar beet

Raffinose (sucrosylgalactoside oligosaccharide) is a water soluble carbohydrate and accumulates in response to abiotic stresses in plants. Plant raffinose synthases are poorly characterized, and the genes involved in raffinose biosynthesis are unknown in sugar beet. Here, we report the isolation of two genes encoding raffinose synthase (BvRS1 and BvRS2) as well as a gene encoding galactinol synthase (BvGolS1) from sugar beet. BvRS1 and BvRS2 show high homologies to Arabidopsis raffinose synthase AtRS5. BvRS1 and BvGolS1 were expressed in Escherichia coli. Crude extracts showed the activities of raffinose synthase and galactinol synthase. The K m values of BvRS1 for galactinol and sucrose and the K m values of BvGolS1 for UDP-galactose and myo-inositol were determined. The expression levels of BvRS1 were significantly higher than that of BvRS2. The mRNA for BvRS1 was rapidly induced by cold stress whereas the mRNA for BvRS2 was slowly induced by cold and salt stresses. These data suggest that BvRS1 and BvRS2 encode raffinose synthase genes responsible to cold and salt stress, respectively.

Silencing of Soybean Raffinose Synthase Gene Reduced Raffinose Family Oligosaccharides and Increased True Metabolizable Energy of Poultry Feed.

Soybean [Glycine max (L.) Merr.] is the number one oil and protein crop in the United States, but the seed contains several anti-nutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2), to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobacterium-mediated transformation. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and Southern blot. The efficiency of mRNA silencing was confirmed by real-time quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines were further analyzed for other phenotypes such as development and yield. Additionally, a precision-fed rooster assay was conducted to measure the true metabolizable energy (TME) in full-fat soybean meal made from the wild-type or transgenic low-raffinose soybean lines. Transgenic low-raffinose soy had a measured TME of 2,703 kcal/kg, an increase as compared with 2,411 kcal/kg for wild-type. As low digestible energy is a major limiting factor in the percent of soybean meal that can be used in poultry diets, these results may substantiate the use of higher concentrations of low-raffinose, full-fat soy in formulated livestock diets.

Molecular cloning of AtRS4, a seed specific multifunctional RFO synthase/galactosylhydrolase in Arabidopsis thaliana.

Stachyose is among the raffinose family oligosaccharides (RFOs) one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, RFOs are indigestible for humans and can contribute to diverse abdominal disorders. In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the RFO physiology in A. thaliana. In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970) as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 μM) as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only semi-quantitative PCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the RFO physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf accumulation under abiotic stress.

Effects of cold acclimation on sugar metabolism and sugar-related gene expression in tea plant during the winter season.

Sugar plays an essential role in plant cold acclimation (CA), but the interaction between CA and sugar remains unclear in tea plants. In this study, during the whole winter season, we investigated the variations of sugar contents and the expression of a large number of sugar-related genes in tea leaves. Results indicated that cold tolerance of tea plant was improved with the development of CA during early winter season. At this stage, starch was dramatically degraded, whereas the content of total sugars and several specific sugars including sucrose, glucose and fructose were constantly elevated. Beyond the CA stage, the content of starch was maintained at a low level during winter hardiness (WH) period and then was elevated during de-acclimation (DC) period. Conversely, the content of sugar reached a peak at WH stage followed by a decrease during DC stage. Moreover, gene expression results showed that, during CA period, sugar metabolism-related genes exhibited different expression pattern, in which beta-amylase gene (CsBAM), invertase gene (CsINV5) and raffinose synthase gene (CsRS2) engaged in starch, sucrose and raffinose metabolism respectively were solidly up-regulated; the expressions of sugar transporters were stimulated in general except the down-regulations of CsSWEET2, 3, 16, CsERD6.7 and CsINT2; interestingly, the sugar-signaling related CsHXK3 and CsHXK2 had opposite expression patterns at the early stage of CA. These provided comprehensive insight into the effects of CA on carbohydrates indicating that sugar accumulation contributes to tea plant cold tolerance during winter season, and a simply model of sugar regulation in response to cold stimuli is proposed.

Development of Molecular Markers for Low Raffinose and Stachyose in Korean Soybean Cultivars

A novel allele of the putative soybean raffinose synthase gene, RS2, was discovered in PI200508 that is associated with the low raffinose and stachyose content. Soybean line PI200508 was identified as expressing reduced levels of raffinose and stachyose as well as elevated levels of sucrose. The RS2 mutant gene shows three base pairs InDel with the normal gene. Based on InDel region we developed novel co-dominant and dominant marker. The aim of this study was to develop Korean soybean cultivars, Daewon, Cheongja, and Danmiput, containing low levels of raffinose and stachyose. A specific markers assay for the PI200508 RS2 allele was developed to allow direct selection of the low raffinose and stachyose phenotype. Our findings highlight the efficiency of allele-specific markers in selection, which is evident in the matching genotype and results of the HPLC in the F2 generations of Daewon×PI200508 population.

Molecular cloning, characteristics and low temperature response of raffinose synthase gene in Cucumis sativus L.

Raffinose synthase (RS, EC2.4.1.82) is one of the key enzymes that channels sucrose into the raffinose family oligosaccharides (RFOs) biosynthetic pathway. However, the gene encoding RS is poorly characterized in cucumber (Cucumis sativus L.), which is a typical RFOs-translocating plant species. Here we isolated the gene encoding RS (CsRS) from the leaves of cucumber plants. The complete cDNA of CsRS consisted of 2552 nucleotides with an open reading frame encoding a polypeptide of 784 amino acid residues. Reverse transcription-polymerase chain reaction and RNA hybridization analysis revealed that expression of CsRS was the highest in leaves followed by roots, fruits, and stems. The RS activity was up-regulated and the raffinose content was high in the leaves of transgenic tobacco with over-expression of CsRS, while both the RS activity and the raffinose content decreased in the transgenic cucumber plants with anti-sense expression of CsRS. The expression of CsRS could be induced by low temperature and exogenous phytohormone abscisic acid (ABA). In cucumber growing under low temperature stress, CsRS expression, RS activity and raffinose content increased gradually in the leaves, the fruits, the stems and the roots. The most notable increase was observed in the leaves. Similarly, the expression of CsRS was induced in cucumber leaves and fruits with 200μM and 150μM ABA treatments, respectively.

Expression analysis of the gene family associated with raffinose accumulation in rice seedlings under cold stress

A considerable increase in the concentration of raffinose was detected in rice seedlings exposed to chilling for more than 4 days. The content of raffinose in leaf blades increased from hardly detectable levels (0–1 days) to approximately 9 mg/g FW after 11 days of chilling treatment. Accumulation occurred in leaf blades but not in sheaths. Analysis of the expression of candidate genes related to galactinol and raffinose synthesis revealed that transcript levels of two galactinol synthase and four raffinose synthase genes increased in leaf blades before the accumulation of raffinose became detectable.