Raffinose Family Oligosaccharides Biosynthesis Research Articles

SlGolS2 influences the metabolism of chlorophyll, carotenoid, and ethylene in tomato fruits.

Galactinol synthase (GolS), which catalyzes the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR-Cas9 gene editing technology to create the slgols2 mutation showing uniformly green fruits without the dark green shoulder, and promoting fruit ripening. Further analysis revealed that the galactinol was undetected in slgols2 ovaries and fruits. Additionally, the mutant suppressed the accumulation of chlorophyll (Chl) and chloroplast development in tomato fruits. RNA sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development, such as SlPORB, SlGLK2, and SlCABs were downregulated in slgols2 fruits. Moreover, slgols2 lines prompted early color transformation and ethylene release by regulating the expression of genes such as SlPSY1, SlCRTISO, SlACS2, SlACS4, SlE4, SlE8, SlRIN, SlNOR and SlAP2a. Overall, our study provides evidence for the involvement of SlGolS2 in the pigment and ethylene metabolism of tomato fruits.

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.

Transcriptional regulation of the raffinose family oligosaccharides pathway in Sorghum bicolor reveals potential roles in leaf sucrose transport and stem sucrose accumulation.

Bioenergy sorghum hybrids are being developed with enhanced drought tolerance and high levels of stem sugars. Raffinose family oligosaccharides (RFOs) contribute to plant environmental stress tolerance, sugar storage, transport, and signaling. To better understand the role of RFOs in sorghum, genes involved in myo-inositol and RFO metabolism were identified and relative transcript abundance analyzed during development. Genes involved in RFO biosynthesis (SbMIPS1, SbInsPase, SbGolS1, SbRS) were more highly expressed in leaves compared to stems and roots, with peak expression early in the morning in leaves. SbGolS, SbRS, SbAGA1 and SbAGA2 were also expressed at high levels in the leaf collar and leaf sheath. In leaf blades, genes involved in myo-inositol biosynthesis (SbMIPS1, SbInsPase) were expressed in bundle sheath cells, whereas genes involved in galactinol and raffinose synthesis (SbGolS1, SbRS) were expressed in mesophyll cells. Furthermore, SbAGA1 and SbAGA2, genes that encode neutral-alkaline alpha-galactosidases that hydrolyze raffinose, were differentially expressed in minor vein bundle sheath cells and major vein and mid-rib vascular and xylem parenchyma. This suggests that raffinose synthesized from sucrose and galactinol in mesophyll cells diffuses into vascular bundles where hydrolysis releases sucrose for long distance phloem transport. Increased expression (>20-fold) of SbAGA1 and SbAGA2 in stem storage pith parenchyma of sweet sorghum between floral initiation and grain maturity, and higher expression in sweet sorghum compared to grain sorghum, indicates these genes may play a key role in non-structural carbohydrate accumulation in stems.

Galactinol synthase 1 improves cucumber performance under cold stress by enhancing assimilate translocation.

Cucumber (Cucumis sativus L.) predominantly translocates raffinose family oligosaccharides (RFOs) in the phloem and accumulates RFOs in leaves. Galactinol synthase (GolS) catalyzes the critical step of RFO biosynthesis, and determining the functional diversity of multiple GolS isoforms in cucumber is of scientific significance. In this study, we found that all four isoforms of CsGolS in the cucumber genome were upregulated by different abiotic stresses. β-Glucuronidase staining and tissue separation experiments suggested that CsGolS1 is expressed in vascular tissues, whereas the other three CsGolSs are located in mesophyll cells. Further investigation indicates that CsGolS1 plays double roles in both assimilate loading and stress response in minor veins, which could increase the RFO concentration in the phloem sap and then improve assimilate transport under adverse conditions. Cold-induced minor vein-specific overexpression of CsGolS1 enhanced the assimilate translocation efficiency and accelerated the growth rates of sink leaves, fruits, and whole plants under cold stress. Finally, our results demonstrate a previously unknown response to adverse environments and provide a potential biotechnological strategy to improve the stress resistance of cucumber.

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.

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.

CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds.

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

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.

Preparation and identification of oligosaccharides in lotus seeds and determination of their distribution in different parts of lotus.

Three fractions (I-III) were separated from crude oligosaccharides of lotus seeds by fast protein liquid chromatography with final purity of 97.6, 96.3, and 96.8%, respectively. The fractions were identified as sucrose, raffinose, and stachyose by using TLC, HPLC with charged aerosol detector (CAD), LC-MS, and methylation analysis. Subsequently sucrose and raffinose family oligosaccharides (RFOs) with degree of polymerization (DP) 3-5 (raffinose, stachyose, and verbascose) have been quantified by HPLC-CAD for the first time. All calibration curves for investigated analytes showed good linear regression (R2 >0.9952). Their limit of detection and limit of quantity were in the ranges 0.14-0.28 and 0.36-0.48μg/mL, respectively. The recoveries ranged from 96.6 to 103.4%. The contents of sucrose and RFOs DP3-DP5 were different in lotus seeds and other parts of lotus samples, but similar in their own variety. Additionally, the distribution of RFOs in different parts of lotus were also compared and the results indicated that RFOs might be mainly synthesized in lotus seeds. This work is helpful for understanding the way of biosynthesis of RFOs in lotus as well as quality control of plants containing RFOs.

Genome-wide identification and expression analyses of genes involved in raffinose accumulation in sesame

Sesame (Sesamum indicum L.) is an important oilseed crop. However, multiple abiotic stresses severely affect sesame growth and production. Raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, play an important role in desiccation tolerance of plants and developing seeds. In the present study, three types of key enzymes, galactinol synthase (GolS), raffinose synthase (RafS) and stachyose synthase (StaS), responsible for the biosynthesis of RFOs were identified at the genome-wide scale in sesame. A total of 7 SiGolS and 15 SiRS genes were identified in the sesame genome. Transcriptome analyses showed that SiGolS and SiRS genes exhibited distinct expression profiles in different tissues and seed developmental stages. Comparative expression analyses under various abiotic stresses indicated that most of SiGolS and SiRS genes were significantly regulated by drought, osmotic, salt, and waterlogging stresses, but slightly affected by cold stress. The up-regulation of several SiGolS and SiRS genes by multiple abiotic stresses suggested their active implication in sesame abiotic stress responses. Taken together, these results shed light on the RFOs-mediated abiotic stress resistance in sesame and provide a useful framework for improving abiotic stress resistance of sesame through genetic engineering.

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.

Stress-Inducible Galactinol Synthase of Chickpea (CaGolS) is Implicated in Heat and Oxidative Stress Tolerance Through Reducing Stress-Induced Excessive Reactive Oxygen Species Accumulation.

Raffinose family oligosaccharides (RFOs) participate in various aspects of plant physiology, and galactinol synthase (GolS; EC 2.4.1.123) catalyzes the key step of RFO biosynthesis. Stress-induced accumulation of RFOs, in particular galactinol and raffinose, has been reported in a few plants; however, their precise role and mechanistic insight in stress adaptation remain elusive. In the present study, we have shown that the GolS activity as well as galactinol and raffinose content are significantly increased in response to various abiotic stresses in chickpea. Transcriptional analysis indicated that the CaGolS1 and CaGolS2 genes are induced in response to different abiotic stresses. Interestingly, heat and oxidative stress preferentially induce CaGolS1 over CaGolS2. In silco analysis revealed several common yet distinct cis-acting regulatory elements in their 5'-upstream regulatory sequences. Further, in vitro biochemical analysis revealed that the CaGolS1 enzyme functions better in stressful conditions than the CaGolS2 enzyme. Finally, Arabidopsis transgenic plants constitutively overexpressing CaGolS1 or CaGolS2 exhibit not only significantly increased galactinol but also raffinose content, and display better growth responses than wild-type or vector control plants when exposed to heat and oxidative stress. Further, improved tolerance of transgenic lines is associated with reduced accumulation of reactive oxygen species (ROS) and consequent lipid peroxidation as compared with control plants. Collectively, our data imply that GolS enzyme activity and consequent galactinol and raffinose content are significantly increased in response to stresses to mitigate stress-induced growth inhibition by restricting excessive ROS accumulation and consequent lipid peroxidation in plants.

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 Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis.

Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis.

Overexpression of a Common Wheat Gene GALACTINOL SYNTHASE3 Enhances Tolerance to Zinc in Arabidopsis and Rice Through the Modulation of Reactive Oxygen Species Production

Galactinol synthase (GolS, EC 2.4.1.123), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), plays roles in plant growth and developmental processes. The in vitro roles of GolS in plant responses against heavy metal stress are not well clarified. In the present study, a suppression-subtractive hybridization (SSH) cDNA library has been constructed using RNA extracted from wheat cultivar Jinan 18 treated with ZnCl2 as the tester and RNA from untreated seedlings as the driver. Sixteen expressed sequence tags (ESTs) highly homologous with known proteins associated with stress tolerance have been obtained. Among these, a 1000-bp cDNA sequence encoding GolS protein has been isolated and designated as TaGolS3. Real-time quantitative PCR (qPCR) analysis revealed that TaGolS3 was mainly expressed in young roots and upregulated by exogenous ABA treatment and several abiotic stresses, such as ZnCl2, CuCl2, low temperature, and NaCl. Subcellular localization analysis showed that TaGolS3 protein is a nuclear-localized protein. A detailed analysis of Arabidopsis and rice transgenic plants overexpressing TaGolS3 gene displayed that transgenic plants exhibited increased lateral root number, primary root length, plant survival rate, and plant height. Moreover, in comparison with the wild-type (WT) plants, the TaGolS3-overexpressing lines showed a higher expression of ROS-scavenging genes, activities of antioxidative enzymes, proline contents, and a lower level of malondialdehyde (MDA) contents and electrolyte leakage under zinc stress. These results confirmed the positive roles of TaGolS3 in improving plant tolerance to heavy metal stress, indicating a potential resource in the transgenic breeding to enhance heavy metal stress tolerance in crop plants.

Osmotic stress induces genes, enzymes and accumulation of galactinol, raffinose and stachyose in seedlings of pea (Pisum sativum L.)

The objective of the present study was to recognize the molecular background of the accumulation of raffinose family oligosaccharides (RFOs) in pea (Pisum sativum L.) seedlings under osmotic stress conditions. The exposure of 5-day-old pea seedlings to osmotic stress for 48 h created by immersing roots in PEG8000 solution (−1.5 MPa) induced synthesis of galactinol and RFOs (raffinose and stachyose) in the epicotyl and root tissues, but not in cotyledons. After 24 h of recovery, galactinol completely disappeared, raffinose decreased fourfold and stachyose decreased twofold in roots, but increased in epicotyls. The temporary accumulation of RFOs resulted from a dramatic increase in the enzymatic activity and changes in expression of galactinol synthase (PsGolS), raffinose synthase (PsRS) and stachyose synthase (PsSTS) genes. PsGolS was induced by osmotic stress in both epicotyls as well as in roots. PsRS and PsSTS were induced only in epicotyls, but repressed or remained unaffected in roots, respectively. During recovery, the expression and activity of PsGolS, PsRS and PsSTS dramatically decreased. The expression of PsGolS gene, that level of mRNA transcript significantly decreased during recovery and whose promoter region was identified to contain some stress-related regulating elements, seems to play a crucial role in the biosynthesis of RFOs under osmotic stress. Possible signals that may trigger the induction of expression of PsGolS, PsRS and PsSTS genes and accumulation of RFOs in pea seedlings are discussed.

Overexpression of cold-inducible wheat galactinol synthase confers tolerance to chilling stress in transgenic rice.

Galactinol synthase (GolS) is considered to be a key regulator of the biosynthesis of Raffinose family oligosaccharides (RFOs). Accumulation of RFOs has been reported to play a role in protection against abiotic stresses. We identified two cDNAs encoding galactinol synthase from wheat (Triticum aestivum L.), which we designated as TaGolS1 and TaGolS2. Expression of the two TaGolS genes was induced by cold stress but not by drought, heat stress or ABA treatment in wheat. We generated transgenic lines of rice (Oryza sativa L.) constitutively overexpressing TaGolS1 or TaGolS2. These transgenic plants accumulated significantly higher levels of galactinol and raffinose than did wild-type plants and exhibited enhanced cold-stress tolerance. The results demonstrate the involvement of galactinol and raffinose in the development of chilling stress in rice and indicate that the genetic modification of the biosynthesis of RFOs by transformation with GolS genes could be an effective method for enhancing chilling-stress tolerance in rice.

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.

Genotype and Growing Environment Interaction Shows a Positive Correlation between Substrates of Raffinose Family Oligosaccharides (RFO) Biosynthesis and Their Accumulation in Chickpea (Cicer arietinum L.) Seeds

To develop genetic improvement strategies to modulate raffinose family oligosaccharides (RFO) concentration in chickpea ( Cicer arietinum L.) seeds, RFO and their precursor concentrations were analyzed in 171 chickpea genotypes from diverse geographical origins. The genotypes were grown in replicated trials over two years in the field (Patancheru, India) and in the greenhouse (Saskatoon, Canada). Analysis of variance revealed a significant impact of genotype, environment, and their interaction on RFO concentration in chickpea seeds. Total RFO concentration ranged from 1.58 to 5.31 mmol/100 g and from 2.11 to 5.83 mmol/100 g in desi and kabuli genotypes, respectively. Sucrose (0.60-3.59 g/100 g) and stachyose (0.18-2.38 g/100 g) were distinguished as the major soluble sugar and RFO, respectively. Correlation analysis revealed a significant positive correlation between substrate and product concentration in RFO biosynthesis. In chickpea seeds, raffinose, stachyose, and verbascose showed a moderate broad sense heritability (0.25-0.56), suggesting the use of a multilocation trials based approach in chickpea seed quality improvement programs.

Effect of CMV infection and high temperatures on the enzymes involved in raffinose family oligosaccharide biosynthesis in melon plants

Ultrastructural and molecular studies have provided experimental evidence for the classification of cucurbits as symplastic loaders, mainly translocating the raffinose family oligosaccharides (RFOs) raffinose and stachyose. Earlier studies established that cucumber mosaic virus (CMV) infection causes a significant increase in the sucrose-to-RFO ratio in the phloem sap of melon plants. The alteration in phloem sap sugar composition was associated with upregulation of CmSUT1 transcript within the vascular bundles.The current research aimed to explore the effect of CMV infection on the enzymes involved in symplastic phloem loading and RFO biosynthesis. Viral infection did not affect the activity of either raffinose or stachyose synthases in source leaves, but caused upregulation of the respective transcripts. Interestingly, activity of galactinol synthase was higher in CMV-infected leaves, associated with upregulation of CmGAS2. A significant increase in CmGAS2 expression in source leaves of melon plants exposed to high temperatures indicated that this response is common for both biotic and abiotic stresses. However, the effect of CMV or heat stress on phloem sap sugar composition is not due to alteration in RFO biosynthesis.