Roles And Mechanisms Of SIGLRs-and SICaMs-Regulated Cold Tolerance In Tomato

Cold stress adversely affects the productivity and quality of tomato(Solanum lycopersicum)and other warm-season vegetables,subsequently restricting the year-round production and supply of vegetables.Therefore,it has become an urgent problem to overcome the cold stress obstacle for vegetable production in facility cultivation during winter and spring time.Researching on the response mechanisms of vegetable to cold stress and exploring the key factors inducing cold tolerance of vegetable plants by gene editing,plant growth regulators and environment regulation becomes very meaningful for the development of vegetable industry.Herein,the key factors involved in cold acclimation induced chilling tolerance in tomato were studied by using genetics,molecular biology,plant physiology,biochemistry,RNA sequencing and other means.We have also examined the potential roles of GLRs in cold acclimation induced chilling tolerance and explored the relationship between GLRs,H2O2 and GSH.In addition,we further screened CaMs in tomato towards cold response and analyzed the differentially expressed genes and their function by RNA sequencing.Major results are shown below:1.Cold acclimation can induce chilling tolerance in tomato plants.The time course of H2O2 accumulation and glutathione homeostasis during cold acclimation and subsequent chilling stress was further detailed.The results show that cold acclimation increased contents of H2O2 in tomato leaves.However,H2O2 content in the acclimated plants was not further increased by the chilling,while a continuous rise in the H2O2 content was observed in non-acclimated plants after being exposed to 4 ℃.Furthermore,cold acclimation induced a prolonged and consistent increase in the reduced glutathione(GSH)content and the ratio of GSH/GSSG in tomato plants.Meanwhile,the transcript levels of RESPIRATORY BURST OXIDASE HOMOLOG1(RBOH1),GSH1 and GSH2 were increased by cold acclimation.All these results suggest that cold acclimation-induced chilling tolerance is associated with accumulation of H2O2 and GSH.2.Time-course for the transcript levels of 13 GLRs in response to cold acclimation and subsequent chilling stress in tomato was examined by qRT-PCR.Among them,the transcript levels of SIGLR3.3 and SIGLR3.5 were significantly induced by the cold acclimation.We then demonstrated that GLR3.3 and GLR3.5 were involved in cold acclimation induced chilling tolerance by virus induced gene silencing(VIGS)and pharmacological approach.Both GLR3.3/GLR3.5 silencing and DNQX treatment compromised the acclimation-induced increases in the transcripts of RBOH1,activity of NADPH oxidase,the accumulation of apoplastic H2O2 and the ratio of reduced glutathione(GSH)to oxidized glutathione(GSSG),resulting in an attenuated chilling tolerance.These finding suggest that SlGLR3.3 and SIGLR3.5 are required for cold acclimation-induced cold tolerance and accumulation of H2O2 and GSH.3.The involvement of H2O2 and glutathione in the GLRs-induced chilling tolerance in cold acclimation was also investigated.We have applied either H2O2 or GSH to GLR3.3-or/and GLR3.5-silenced plants as well as pTRV control plants before exposure to 12 ℃ for cold acclimation.The results were shown below:First,exogenous applied H2O2 and GSH both enhanced chilling tolerance and increased H2O2 and GSH accumulation not only in pTRV plants but also in pTRV-GLR3.3 or/and 3.5 plants.Second,the application of GSH also significantly induced H2O2 accumulation in the leaves of GLR3.3-or/and 3.5-silenced plants.Importantly,the transcript levels of GLR3.3 and GLR3.5 were induced by application of GSH.We also used the RBOH1-RNAi(rbohl)plants and GSH1/2-cosilenced(pTRV-GSH1/2)VIGS plants to testify the dependency among H2O2 and GSH on GLR in cold acclimation-induced chilling tolerance.The results showed that silencing of RBOH1 or GSH1/GSH2,all compromised cold acclimation-induced chilling tolerance.Notably,the positive role of Glu and the negative role of DNQX on the chilling tolerance observed in WT or pTRV plants and were mostly abolished in rbohl and pTRV-GSH1/2 plants.These results not only unveil the critical role of RBOH1-derived H2O2 and GSH 1/2-derived GSH in GLRs-mediated cold tolerance,but also indicate the functional hierarchy of GSH that acts down-stream of H2O2 in cold acclimation-induced cold tolerance.4.The changes in the transcript levels of 6SlCaMs in response to cold acclimation and subsequent chilling stress in tomato were examined by qRT-PCR.These result showed that the transcript levels of SlCaM6 were significantly induced after getting exposure to 4 ℃ for 12h regardless of cold acclimation.Overexpression and CRISPR/Cas9 of SlCaM6 seedlings were performed by transgenic approaches.The results showed that cam6 showed increased cold tolerance,indicating that SlCaM6 negatively regulated cold tolerance in tomato plants.In addition,the transcript levels of COR47-like were decreased in SlCaM6-overexpressed plants exposed to cold stress.Furthermore,the subcellular localization of CaM6 and COR47-like showed that CaM6 was expressed in both nucleus and cytoplasm,while COR47-like was located within the membrane.BiFC,LCI and Split-ubiquitin membrane yeast two-hybrid assays further confirmed the interaction of SlCaM6 with COR47-like in vivo.A phenotype analysis also showed that COR47-like-silencing plants exhibited attenuated chilling tolerance.Thus,we conclude that SlCaM6 negatively regulates chilling tolerance in tomato by interacting with COR47-like.5.RNA-seq was employed to analyze gene expression profile of OE-CaM6,cam6 and WT for inquiring the genes that may be involved in regulation of cold tolerance.The results showed that 688 up-regulated and 677 down-regulated DEGs(differential expressed genes)were identified from OE-CaM6 plants compared with WT plants,when they were exposed to cold stress.Respectively,1068 up-regulated and 761 down-regulated DEGs were identified from cam6 plants compared with WT plants.All these DEGs divided into 5 groups which correspond to ethylene signaling,BR signaling,stress response,photosynthesis and metabolism.GO(Gene Ontology)analysis of DEGs suggest that these DEGs are enriched in response to stress,defense response and DNA binding.