Functional Analysis Of A Chloroplast And Nucleus Dual-located WHIRLY1 Protein In Tomato Under Temperature Stress

Tomato(Solanum lycopersicum)is a typical temperature-sensitive vegetable crop,which is often damaged by chilling or heat stress in the process of protected cultivation in northern China,resulting in falling flowers and fruits as well as bringing huge economic losses to vegetable growers.More than 95% organics of crop yield come from photosynthesis.As an important arena for photosynthesis,chloroplast is particularly sensitive to temperature stress.In the chloroplast,the key enzyme(Rubisco)of carbon assimilation and the core protein of photosystem II are two sensitive targets for limiting the photosynthetic capacity and growth and development of crops under temperature stress.Under temperature stress,the activities of Rubisco in chloroplasts are inhibited,resulting in a large amount of excess light energy,which then leads to photoinhibition of photosystem II and reduces the photosynthetic capacity of tomato plants.Therefore,exploring the function of photoprotection related genes have potential benefits to provide effective strategies for enhancing temperature-sensitive crops resistance to temperature stress in future agricultural production.In our study,we found and identified a chloroplast and nucleus dual-localized SlWHIRLY1(SlWHY1)protein from tomato expression library induced by low temperature.qRT-PCR,Western blot and GUS staining assay confirmed that both the mRNA level and protein level of SlWHY1 could be induced by chilling stress.After chilling treatment(4°C),the SlWHY1 OE lines had obvious chilling tolerance phenotype,while its RNAi silencing line was not tolerant to chilling stress.Electron microscopic observation and analysis showed that the chloroplast of the OE plants retained more intact photosynthetic grana lamellae and less starch accumulation,while the grana lamellae of RNAi line were seriously damaged and the amount of starch was more.Transcriptome data showed that the PSBA gene encoding the photosystem II core protein(D1 protein)and two starch metabolism-related genes(ISA2 and AMY3-LIKE)had obvious different expression levels between SlWHY1 OE lines and the wild type.Then,we analyzed the regulatory mechanism of SlWHY1 in chloroplast and nucleus,respectively.Yeast one-hybrid,ChIP,and EMSA demonstrate that SlWHY1 can bind directly to the upstream region(A/GTTACCCT/A)of SlPSBA and enhance the de novo synthesis of D1 protein in chloroplasts,thus protect PSII.On the other hand,SlWHY1 in the nucleus can directly bind to the ERE elements in the promoters of two chloroplast-located starch metabolism-related genes ISA2 and AMY3-LIKE and regulate their expression,reducing starch accumulation in chloroplasts under low temperature stress.These results demonstrate that SlWHY1 enhances the resistance of tomato to chilling stress by maintaining the function of PSII and degrading starch.Thus,overexpression of WHY1 may be one of effective strategies for enhancing resistance to chilling stress of chilling-sensitive crops in agricultural production.Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco),which consists of eight large subunits(RBCLs)and eight small subunits(RBCSs),is a rate-limiting enzyme in the process of carbon assimilation and is particularly sensitive to chilling stress.However,it is not clear how plants maintain high Rubisco content under low temperature conditions.Here,we report that tomato WHIRLY1(SlWHY1)can maintain the Rubisco level under chilling stress by directly binding to the promoter region of SlRBCS1 and activating its expression.SlRBCS1 overexpressing lines had higher Rubisco contents and were more resistant to chilling stress compared with WT.qRT-PCR analyses showed that,among the five RBCS genes,only SlRBCS1 expression is up-regulated by chilling treatment.These results indicate that SlWHY1 specifically enhances the transcription level of SlRBCS1 and confers tomato plants tolerance to chilling stress.The amino acid sequence of SlRBCS1 is highly similar to that of other RBCS family members(92.67%),and only four specific amino acid residues(R57,R105,and NS113)are found.However,the substitution of these specific residues in SlRBCS1 did not affect its function during cold adaptation.Thus,we conclude that high level of Rubisco,rather than the specific amino acid residues in SlRBCS1 plays an important role in tomato tolerance to chilling stress.The regulation of heat shock factor(HSF)on heat shock protein(HSP)is one of the main defense responses of plants to heat stress.SlWHY1 can also be induced by high temperature.Phenotypic analysis after high temperature treatment showed that the wilting degree of SlWHY1 overexpression plant was lower than that of the wild type,which wasmanifested in better membrane stability,higher soluble sugar content and less active oxygen(ROS)accumulation.However,the phenotypic and physiological indexes of SlWHY1 RNAi line were opposite to those overexpressing plants under high temperature stress.Further analysis showed that the expression of SlHSP21.5A,encoding an endoplasmic reticulum-localized HSP,was up-regulated in SlWHY1 overexpressed plants and down-regulated in RNAi plants.The RNAi-mediated inhibition of SlHSP21.5A expression also resulted in reduced membrane stability and soluble sugar content,increased ROS accumulation compared to the WT under heat stress.In addition,SlWHY1 can bind to the elicitor response element-like(ERE-like)element in the promoter of SlHSP21.5A to activate its transcription.These findings suggest that SlWHY1 promotes thermotolerance of tomato by regulating SlHSP21.5A expression.