The Mechanism Of O-Glycosylation By SlSEC And SlSPY In Modulating Cold Tolerance And Fruit Ripening In Tomato

Tomato(Solanum lycopersicum)is an important economic vegetable crop in China.However,the frequent occurrence of cold damage in winter and spring severely impairs plant growth and productivity.Plants undergo a range of changes to survive from cold stress.The physiological and biochemical methods to improve the cold resistance and shorten the growth cycle were used to minimizing damage and ensure quality of tomato.In this study,vegetable crop tomato was used to explore the role of O-glycosylation in defense responses and growth processes.The regulatory function of Oglycosyltransferase SlSEC and SlSPY in tomato cold tolerance and fruit ripening was identified.The main results are as follows:1.The transcript abundance of SlSEC1,SlSEC2 and SlSPY in different growth stages and during variant abiotic stresses response was identified.The transcript level of SlSEC and SlSPY in different tissues showed dynamic changes during growth and development.SlSEC and SlSPY were also involved in the response to various abiotic stresses.In addition,the slsec1 and slspy mutants exhibited different degrees of defects.Compared with the wild type,the slsec1 mutants only showed a slight semidwarf phenotype,while the slspy mutant showed more obvious slow-growing and had a dwarf phenotype.2.The O-GlcNAc transferase SlSEC1 was found to negatively regulates cold tolerance in tomato by promoting the stability of SlC3H39,which is a negative regulator of cold tolerance.The interaction between SlSEC1 and SlC3H39 was screened through yeast two-hybrid.Further study shows that SlSEC1 physically interacts with SlC3H39 proteins and promotes the stability of it by modulating O-GlcNAclation status of SlC3H39.SlC3H39 is rapidly induced by cold stress and negatively modulates plant cold tolerance.The slc3h39 mutants partially rescued the cold tolerance of SlSEC1 overexpressing lines.3.SlC3H39 negatively modulates plant cold tolerance through post-transcriptional regulation by binding to cold responding m RNAs 3’UTR and reducing those transcripts.The RNA-seq results shows that genes involved in defense response and cold response are up-regulated and genes involved in growth such as meristem development and photosynthesis are down-regulated in slc3h39 mutants compared with WT.Transient expression in vivo and nucleic acid binding assays in vitro confirmed that SlC3H39 had RNA binding ability and was involved in mediating RNA degradation.By associating the RNA-seq with RNA immunoprecipitation-seq,we identified that SlC3H39 binds to3’UTR of cold responding gene and decaying those transcripts.4.SlSPY positively regulates fruit ripening by changing the ethylene response in tomato.The accumulation of carotenoid content and fruit firmness in the WT,slspy mutants and SlSPY overexpressing lines indicate the positive role of SlSPY in fruit ripening.Further study indicates that SlSPY-regulated fruit ripening is closely related to ethylene response in tomatoes.5.SlEIN2 is essential for SlSPY in regulating fruit ripening in tomatoes.A central regulator of ethylene signaling SlEIN2 is a SlSPY interacting protein.Mass spectrometry analysis further identified that SlEIN2 has potential sites of O-glycans modifications.SlSPY promotes the stability and nuclear accumulation of SlEIN2,thus positive regulates the ethylene-promoted fruit ripening in tomato.In conclusion,this study reveals the role of plant O-glycosyltransferases SEC and SPY in defense responses and growth processes.we propose a SlSEC1-SlC3H39 module,which modulates tomato cold tolerance through post-transcriptional regulation.And a novel insight into the role of the SlSPY-SlEIN2 module in tomato fruit ripening was proposed.These findings provide positive guides for regulating the development and formatting the yield and quality of tomato.