The Mechanism Of Raffinose Family Oligosaccharide Metabolism Regulating Seed Vigor,Low-temperature Adaptation And Source-Sink Allocation In Cucumber

Raffinose family oligosaccharides(RFOs)are ubiquitous in high plants,which have various physiological functions including nutrient storage,stress adaptation and assimilate transport.RFOs also play important physiological roles in seed nutrient accumulation,stress adaptation and assimilate tanslocation in cucumber(Cucumis sativus),a typical RFOs-translocating species.However,to date how RFOs metabolism coordinating these functions in cucumber plants is still unknown.On the other hand,cucumber is an important vegetable cultivated in the world.At present,the scale of cucumber seed industry is large in China,and there is still a big gap of cucumber yield between China and other developed countries.In addition,cucumber originated from the subtropical rainforest region and is sensitive to low temperature.Therefore,we investigated the mechanism of RFOs metabolism in regulating cucumber seed vigor,cold adaptation and source-sink allocation in this study.The results should lead to new findings which is different from that in sucrose-transporting plants,and provide beneficial information for cucumber seed production and high-yield and stress-resistance breeding and cultivation,which is of great theoretical and practical significance.In this paper,based on different ecotypes and current main cucumber varieties,the functions of RFOs metabolism in cucumber seed vigor,low-temperature adaptation and source-sink carbohydrate partitioning were demonstrated.Multiple technologies including morphological observation,physiological measurements,omics analysis,in situ hybridization,dual luciferase assay and plant transformation were used.The main results obtained are as follows:1.The content of raffinose in seeds is a key factor determining seed vigor.During cucumber seed development,monosaccharides and disaccharides were dominant soluble sugars in seeds before 30 day post-anthesis(DPA),while the proportion of RFOs in total sugars increased significantly after 30 DPA.The accumulation of starch,protein and lipid significantly increased in seeds after 25 DPA.The correlation analysis between concentrations of materials mentioned above and seed vigor among 10 cucumber varieties showed that raffinose content at 35 DPA was significantly positively correlated with seed vigor(r=0.823),which could be used as a credible indicator for predicting seeds and screening cucumber varieties with high seed vigor.This conclusion was further verified by seed experiments of other 5 cucumber varieties.2.CsGolS1 improves cucumber cold tolerance by accelerating assimilate translocation.Expression of all four isoforms of CsGolSs in the cucumber genome were induced by cold,heat,salt and drought stresses.β-Glucuronidase staining and tissue enrichment experiments suggested that CsGolS1 was specifically expressed in vascular tissues,whereas the other three CsGolSs were only identified in mesophyll cells.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.Further investigation indicated that CsGolS1 exerted double roles in both assimilate loading and stress response in minor veins,which improved assimilate transport and the stress resistance of cucumber under adverse conditions.3.Galactinol and RFOs may act as signaling molecules in cucumber low-temperature adaptation.Compared with the wild-type plants,the contents of galactinol,raffinose,stachyose,glucose and sucrose were significantly higher in the leaves of CsGolSl overexpression plants at 1 h after cold treatment,indicating that overexpressing CsGolS1 significantly enhanced the cold tolerance of cucumber plants.Transcriptome analysis of two types of plants under cold stress indicated that the CsGolSl overexpression triggered the C-repeat binding transcription factors(CBF s)-mediated signal through an ABA-independent pathway.On the other hand,the expression difference of two key factors in the hexose and sucrose signaling pathways,hexokinase and sucrose non-fermentation-related kinase 1,were not significant between WT and CsGolSl overexpression plants,indicating that a unique low temperature signaling pathway mediated by galactinol or RFOs may exist in cucumber.4.asCsSTS,a cis-natural antisense noncoding transcript of CsSTS,involved in the regulation of source-sink allocation in cucumber.Comparing to leaves without fruit at the node,CsSTS expression and CsSTS enzyme activity were upregulated in the vasculature and downregulated in mesophyll tissues of cucumber fruiting node leaves.In situ hybridization and tissue enrichment experiments revealed that asCsSTS mainly expressed in mesophyll tissues.In vitro overexpression,RNA interference,and dual luciferase reporter experiments indicated that CsSTSs are negatively regulated by asCsSTS.Fluorescence in situ hybridization assays revealed that asCsSTS transcript localized in leaf cytoplasm,indicating that the regulation of CsSTS by asCsSTS was a posttranscriptional process.Further investigation suggested that this regulation occurred by reducing CsSTS transcript stability through a DICER-like protein-mediated pathway.Chemically induced overexpression and RNA interference of asCsSTS led to promotion or inhibition,respectively,of assimilate export from leaves,and altered fruit growth rates.In conclusion,our results identified a key indicator of cucumber seed vigor,demonstrated a new mechanism of stress adaptation of RFOs-translocating species cucumber,and found a new regulatory mechanism that noncoding RNA regulating cucumber source-sink allocation.These results enriched the regulatory network of cold adaptation and source-sink allocation in cucumber,and provided candidate genes and technological methods for cucumber seed production and high-yield and stress-resistance cultivation and breeding.