| High temperature was one of the major abiotic stresses and tomato(Solanum lycopersicum L.)was often suffered from high-temperature stress when it was cultivated.This resulted in decrease of tomato yield and quality together with decline of product offering and economic benefits.Therefore,identifying heat stress-responsive genes and breeding heattolerant varieties were effective way and urgent need to address this issue.Tomato heat tolerance was a quantitative trait,and QTL mapping combined with RNA-seq were effective way to identify genes responsible for heat tolerance.Here,the heat-susceptible cultivated tomato line ‘LA1698’(Solanum lycopersicum L.)and the heat-tolerant currant tomato line‘LA2093’(Solanum pimpinellifolium L.)were used as materials,and conventional QTL analysis combined with QTL-seq technology were applied to comprehensively detect heattolerance QTLs.In addition,we integrated the QTL mapping results with RNA-seq to identify heat-related genes within the major QTLs.And after screening,Cathepsin B-like protease 2(CathB2)gene was selected as key heat stress-responsive gene.Then,the study identified the two SlCathB2 genes named SlCathB2-1 and SlCathB2-2,and analyzed their expression profile.Furthermore,the study analyzed the involvement and molecular mechanism of SlCathB2-1and SlCathB2-2 in high-temperature stress response.The main results were as follows:1.A total of five major heat-tolerant QTLs were detected combined with conventional QTL mapping and QTL-seq: qHII-1-1,qHII-1-2,qHII-1-3,qHII-2-1 and qCC-1-5(qREC-1-3).qHII-1-1,qHII-1-2 and qHII-1-3 were located,respectively,in the intervals of 1.43,1.17 and 1.19 Mb on chromosome 1,while the interval of qHII-2-1 was located in the intervals of1.87 Mb on chromosome 2.The locations observed with conventional QTL mapping and QTL-seq were consistent.qCC-1-5 and qREC-1-3 for CC and REC,respectively,were located at the same position by conventional QTL mapping.Although qCC-1-5 was not detected in QTL-seq analysis,its phenotypic variation(16.48%)and positive additive effect(0.22)were the highest among all heat tolerance QTLs,showing that the QTL was closely related to heat tolerance and was a major heat-tolerant region.This study demonstrated that the combination of conventional QTL mapping and QTL-seq analysis using different physiological and phenotypic indexes can quickly,efficiently and accurately detect the major QTLs for target traits.2.The heat-susceptible genotype ‘LA1698’ and the heat-tolerant genotype ‘LA2093’were employed for RNA-seq analysis.After sequencing and analyzing,3686 and 3781 differentially expressed genes(DEGs)were identified in ‘LA1698’ and ‘LA2093’,respectively.The functional annotation showed that DEGs were mainly involved in DNA integration,DNA metabolic process,nucleic acid metabolic process,zinc ion binding and transition metal ion binding under high-temperature stress.The results verified by qRT-PCR were consistent with the up-and downregulation trends of the RNA-seq results,indicating that the results from RNA-seq were reliable.A total of 91 genes were found in the major QTLs by combining with QTL mapping and RNA-seq analysis.The largest number of DEGs,61,was found in qCC-1-5.Then,25 genes with high expression level were selected within the 91 DEGs for candidate-gene screening by qRT-PCR analysis.According to the expression profile of 25 genes in two parents under high-temperature stress and biological information analysis,four genes were finally screened,namely,cathepsin B-like protease 2(SlCathB2),glutathione S-transferase zeta class-like isoform X1(SlGST),ubiquitin-conjugating enzyme 5 E2-23 k Da(SlUBC5)and arginase 1(SlARG1).Among them,SlCathB2 was contained in qHII-2-1,while the rest of three genes were contained in qCC-1-5(qREC-1-3).Once functionally validated,the candidate genes could be utilized as potential candidates to enhance heat tolerance as well as yield.3.We reported the analysis of two SlCathB2 genes named SlCathB2-1 and SlCathB2-2.Bioinformatics analysis showed that some elements related to abiotic stress-responses were found in promoter regions of two SlCathB2 genes.SlCathB2-1 and SlCathB2-2 had the same protein domain,and their protein sequence shared close evolutionary relationship with the CathB2 of Solanum tuberosum.Tissue expression analysis of SlCathB2-1 and SlCathB2-2 and their responses to various abiotic stresses and hormone treatments revealed that SlCathB2-1and SlCathB2-2 differentially expressed in various tissues.Meanwhile,the genes positively responded to high temperature,drought,salt,ABA,and SA treatments with higher expression under high-temperature stress.Then,the expression of SlCathB2 in the leaves of tomato genotypes with different thermotolerance under moderately elevated temperature(MET,33 °C)and acutely elevated temperature(AET,40 °C)stresses were further analyzed.The results showed that the expression levels of SlCathB2-1 and SlCathB2-2 in heat-sensitive tomato genotypes(S1 and S2)were higher than that in heat-tolerant genotypes(T1 and T2)under MET and AET stresses.Moreover,the expression in the moderate thermotolerant genotypes(M1 and M2)showed no significant changes as compared with the control.Furthermore,SlCathB2-1 and SlCathB2-2 genes showed similar expression patterns in tomato genotypes with same behavior for thermotolerance under MET and AET stresses,but exhibited higher expression level under AET stress.Overall,the results confirmed that SlCathB2-1 and SlCathB2-2 participated in high-temperature stress response and they may be negative regulators of high-temperature stress response.This study lays foundation for enriching the regulation mechanism of tomato high temperature response and functional studies of SlCathB2-1 and SlCathB2-2 in tomato.4.Overexpression of SlCathB2-1 and SlCathB2-2 resulted in reduced tolerance to hightemperature stress,while silencing the genes resulted in enhanced thermotolerance.Furthermore,the possible molecular regulation mechanisms underlying SlCathB2-1 and SlCathB2-2-mediated responses to high-temperature stress were investigated.We found that SlCathB2-1 and SlCathB2-2 negatively regulated antioxidant capacity by regulating a set of genes involved in antioxidant defense and ROS(Reactive oxygen species)signal transduction.The study also demonstrated that SlCathB2-1 and SlCathB2-2 positively regulated ER-stressinduced PCD(ERSID)by regulating unfolded protein response(UPR)gene expression.Furthermore,using yeast two-hybrid(Y2H)analysis,SlCathB2-1 and SlCathB2-2 interacting with proteasome subunit beta type-4(PBA4)was identified in ERSID regulation pathway,and only SlCathB2-1 interacted with PBA4 was detected by bimolecular fluorescence complementation(Bi FC).In addition,the study found that silencing of SlCathB2-1 did not change the expression of SlCathB2-2,while the expression of SlCathB2-1 was triggered in SlCathB2-2 silencing plants,which suggesting that SlCathB2-2 was an upstream regulator of SlCathB2-1.We found that there was no interaction between SlCathB2-1 and SlCathB2-2 by Y2 H analysis.Overall,the study identified the SlCathB2-1 and SlCathB2-2 as new negative regulators for high-temperature stress and presented a new high temperature-responsive pathway.This provided the foundation for the construction of heat-tolerant molecular mechanisms and breeding strategies aiming to improve thermotolerance. |
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