Functional Analysis Of Low-temperature Tolerance Conferred By The GPA1-encoded G Protein Subunit In Cucumis Sativus

Cucumber(Cucumis sativus L.)is an important vegetable crop cultivated in China.Cold stress conditions are an important factor limiting yield during winter in the northern regions where cucumber plants are grown.Plant G-proteins are membrane proteins comprised of three different subunits involved in signal transduction and response to stresses.The physiological and molecular characteristics of the Ga subunit have been reported in model plant species such as Arabidopsis thaliana and rice,however,to date there are no reports characterizing the biological function of the Ga subunit in cucumber.This study reports the cloning of the CsGPAl gene,encoding the G-protein a subunit in cucumber,and characterization of its biological function through analysis of expression patterns and subcellular localization.In particular this research explores the physiological and molecular mechanisms of CsGPA1 in response to low temperature stress.The main findings of this work are as follows:Ⅰ.In C.sativus,CsGPAl,encoding the G-protein a subunit,is only a single copy gene.Experiments using qRT-PCR showed that the expression of CsGPA1 was detected in all organs and tissues,and that gene expression in roots was relatively higher than in young leaves.CsGPA1 has lower transcriptional levels in the tissue of other organs such as mature leaves,stems,female flowers,male flowers,and fruits.Ⅱ.Immunohistochemical localization methods found that CsGPAl was expressed in the petiole,stem,and root tip of cucumber.Among these organs,it is mainly present in the mesenchymal tissues of the inner and outer phloem,and especially in the meristematic epidermal cells of the root tip.Subcellular localization experiments also showed that CsGPAl is a transmembrane protein localized to the plasma membrane of cells.Ⅲ.In comparison with wild-type seedlings,transgenic CsPA1-overexpressing lines exhibited enhanced seed germination and earlier seedling development,as measured by hypocotyl length,stem diameter,surface area,projected area,root volume,and root length.In contrast,RNAi silencing of the CsGPA1 gene inhibited seedling growth and development.Our findings suggested that CsGPAl may promote cell division in cucumber seedlings.To further explore this finding,endogenous hormone levels were measured in CSGPA1-overexpressing,RNAi,and wild-type seedlings.Quantitative RT-PCR and salicylic acid supplement analysis suggested that CsGPA1 may regulate endogenous salicylic acid content in young cucumber seedlings grown in darkness by modulating the expression levels of the key salicylic acid synthesis genes CsCBP60g,CsEDS1,and CsSID2 in the cotyledons,and CsSRl in the hypocotyl and roots.We concluded that CsGPAl positively regulates seed germination,early seedling development in darkness,and is part of the salicylic acid signaling pathway.IV.After 60 h of treatment at low temperature(6 ℃),CsGPA1-RNAi-interfering transgenic cucumber plants showed a more severe drooping and wilting phenotype in the leaves of seedlings than in wild-type plants.This indicated that decreased expression of CsGPAl in the RNAi transient suppression strain can make the plant more susceptible to low temperature.Compared with the wild type,the gene expression levels of CsWCOR413PM,CsICE1,CsCBF1,CsCBF3,and CsRD-29 in the leaves of the plants were relatively low during the treatment,whereas the relative expression of the CsHOS1 gene was high.Similarly,the relative conductivity of REC and MDA content were found to be at high levels,while the activities of Proline(Pro),soluble proteins,and antioxidant enzymes(SOD,POD and CAT)were all low in comparison with the wild type.These results indicated that RNA interference targeting CsGPA1 was not conducive to low temperature tolerance.Moreover,a cucumber leaf cDNA expression library was established using split ubiquitin yeast two-hybrid,and 11 candidate proteins were screened that potentially interact with CsGPA1.GST pull-down experiments confirmed that interaction between CsWCOR413PM and CsGPAl proteins may be involved in the response to low temperature for cucumber seedlings.In brief,the alpha subunit(CsGPA1)of cucumber G-protein is localized to the plasma membrane,phloem,and especially the epidermal cells of the root apical meristem.The interaction between CsWCOR413PM and CsGPA1 was demonstrated by mitotic ubiquitin yeast heterozygosity and GST pull-down tests.CsGPAl positively regulates cucumber seed germination and participates in early seedling growth under dark conditions.CsGPA1 participates in the response of cucumber seedlings to low temperature stress.