Functional Study Of SlHB2 And SlHB3 Genes In Tomato Abiotic Stress Response

As sessile organism,the growth and development of plant is affected by environmental conditions all the time.Adverse environmental conditions severely affect the growth and productivity of crop plants.Tomato?Solanum lycopersicum?is one of the most important vegetables and economic crop,and the model species for the study of genetic evolution and molecular biology.At the same time,the plant growth and development can be adversely affected by environmental stresses in most tomato cultivars.Many studies have been made to reveal the mechanism of plants response to stress conditions.HD-Zip I transcription factors,which are exclusively found in plants,have been described to be implicated in the signaling networks responsive to environmental stimuli.At present,functional studies of HD-Zip I genes from Arabidopsis thaliana,sunflower,rice,maize and tomato have been made.However,little information regarding stress-related HD-Zip I genes from tomato has been reported.The completion of the tomato genome-sequencing also provides great convenience for functional studies of tomato HD-Zip I genes.To find out the stress related transcription factors,a sequence search,using the conserved domain in HD-Zip I protein,has been made in the SGR database.Twenty-two tomato HD-Zip I proteins have been identified according to the result of conserved domain analysis by SMART and NCBI online software.Four tomato HD-Zip I genes,designated as SlHB2-SlHB5,have been screened out for the subsequent experiment on the basis of the phylogenetic analysis result and the analysis result of homologous genes in Arabidopsis.Structural analysis of promoter sequence,gene expression pattern under hormone and abiotic stress treatments were conducted in the present study.The result showed that the expression of SlHB2 was induced by SA,ABA,ACC and MeJA,ten hormones or stress-related cis-elements have also been found in the promoter region of this gene.Furthermore,a relatively high expression level of SlHB2 was observed in flowers and mature leaves,while the expression level in young leaves,B+4 and B+7 fruits was relatively low.Intermediate expression level was detected in the other samples.SlHB3,in the promoter region of which 8 cis-elements have been detected,was obviously up-regulated by exogenous hormone ABA and MeJA.The expression level of SlHB3 was relatively high in tomato fruit at B to B+7 stage,while relatively low expression levels were detected in the other tissues.Nine cis-elements were found in SlHB4 promoter region.The transcriptional level of SlHB4was remarkably induced by ACC and Me JA,and slightly induced by SA and IAA.A higher expression level of SlHB4 was detected in the sepal,root and flower than in the other tissues.Only 6 cis-elements have been found in SlHB5 promoter region.A rapidly up-regulation of SlHB5 has been found in the ACC and MeJA treatment,while a slight up-regulation was observed in the IAA treatment.It is worth noting that the expression levels of all the four HD-Zip I genes were found to be induced by abiotic stress,such as dehydration,salinity,low temperature and mechanical damage.According to the results of gene expression pattern and promoter prediction,Sl HB2and Sl HB3 were screened out for the subsequent gene functional study.To reveal the regulatory function of SlHB2 and SlHB3 in the growth and development of tomato,SlHB2-and SlHB3-RNAi vectors have been constructed in the study.Transgenic research indicated that down-regulation of SlHB2 transcriptional level resulted in the reduction of the tomato skin hardness and the up-regulation of the fruit cell wall metabolic related genes expression level,such as PG and PE,and finally reduced the storability of tomato fruits.Besides,a reduction in the size and weight of tomato seed has been found in the SlHB2-RNAi lines,which suggested that SlHB2 may play a role in the tomato seed development.The transcriptional level of SlHB2 was induced by various hormones and abiotic stress,which implied that SlHB2 may play important roles in the tomato environmental stress resistance.The result of drought stress treatment showed that the drought stress tolerance of SlHB2-RNAi lines was significantly enhanced.Meanwhile,the RWC and chlorophyll content were higher in the transgenic plants than that in WT plants.On the contrary,the RWL and the MDA content in the leaves of RNAi lines were lower.The higher expression levels of AREB,DREB,ERF1,TAS14,Cat2,P5CS,APX1 and APX2 in the SlHB2-RNAi lines than in the WT have also been detected after the dehydration treatment.In the plant salt stress treatment,in which the sprouted seed were used,a stronger inhibition of NaCl on WT seedling growth than SlHB2-RNAi has been observed.In the chlorophyll degradation experiment of detached leaf,an obvious degradation of chlorophyll was observed in the WT leaves,while the transgenic leaves displayed a slower degradation rate.At the same time,an enhanced salt stress tolerance of SlHB2-RNAi seedlings has also been observed in the plant salt stress treatment.In accordance with the enhanced tolerance to salt stress,transgenic plants contained higher chlorophyll content and lower MDA content than that in WT plants in salt tolerance experiment.The expression levels of TAS14,Cat1,Cat2,P5CS,APX1,APX2,PR1,PR5,ER5 and ERF1 in the root of transgenic lines were significantly higher than that in the WT.The results of H₂O₂ treatment showed that the inhibition of H₂O₂ on WT root growth was stronger than on the SlHB2-RNAi lines.Collectively,the silencing of SlHB2 resulted in the up-regulation of stress-related genes,and thus enhanced the drought and salt stress tolerance of transgenic lines,these results indicated that SlHB2 may play roles in the drought and salt stress response in tomato as a negative regulatory factor.According to the results of the phenotype investigation and qPCR test of ripening-associated genes,no obvious change has been observed in the SlHB3-RNAi tomato plants compared with WT.In the stress treatment of plants and seed germination experiment,no obvious difference in drought and salt stress tolerance between transgenic lines and WT plant has been observed.These results suggested that down-regulation of SlHB3 cannot significantly affect the process of tomato growth and development and stress tolerance that described in our study.According to these results,we speculated that SlHB3 and its paralogs are functional redundancy.The silence of SlHB3 alone can not reveal its regulatory function in the tomato growth and development for the functional compensation of homologs.In summary,four HD-Zip I genes were identified in our study,and the gene expression pattern analysis and bioinformatics analysis of these genes have also been conducted.Then,we focused on the gene function analysis of SlHB2 and SlHB3 in tomato abiotic stress response.The results of our study laid a foundation for the elucidation of functions of SlHB2 and SlHB3 and tomato breeding for stress tolerance,and indicated the direction of the future study on these two genes.