| Pepper(Capsicum annuum L.),which is native to the tropics in Central and South America,is suitable for growing in warm place but sensitive to high temperature.The optimum temperature for pepper growth ranges from 20℃ to30℃.Pepper is subject to heat stress when the ambient temperature is above 32℃.Heat stress affects pollen development and pollen viability,leading to pollen abortion and causing yield lost about 70%.Due to global warming,heat stress has become an important factor limiting the yield and quality of pepper in summer,especially under the facilities culture conditions.Indeed,heat stress restrict the development of pepper industry seriously.Therefore,it is meaningful for the development of pepper industry to screen high-quality heat-tolerant cultivars,explore the molecular mechanism of heat stress response(HSR),and investigate the heat-resistant mechanism of pepper.In the present study,the resistance to heat stress of pepper was evaluated and identified.In addition,we investigated the changes of physiology,transcriptome and metabolome in heat-tolerant(17CL30)and heat-sensitive(05S180)pepper seedlings under heat stress.The main contents and research results are as follows:(1)Evaluation and identification of resistance to heat stress in germplasm resources of pepper.Twenty pepper materials(numbered from A1 to A20)with different heat-tolerance were used in this study and 13 physiological indicators of the pepper seedlings under heat stress(40℃ for 16 h at day and 30 ℃ for 8 h at night)were taken to analyze the heat-tolerance of pepper by principal component analysis,subordinative function analysis,cluster analysis and regression analysis.The results showed that 20 pepper materials were divided into four categories,strongly heat-tolerance(D≥0.6),mildly heat-tolerance(0.55≤D<0.6),heat-sensitive(0.45≤D<0.55),strongly heat-sensitive(0.35≤D<0.45),and the D value represents the average of membership function of comprehensive index.Among them,A13(17CL30)was identified as heat-tolerance material and A17(05S180)was identified as heat-sensitive material.(2)The physiology response of pepper under heat stress.The physiological mechanism of heat resistance in pepper(17CL30 and 05S180)was studied based on morphology,ultrastructure,biomembrane system,osmotic regulation system,antioxidant system,systematically.The results showed that heat stress(40℃)can cause leaf wilting and cause damage to its ultrastructure.17CL30 suffering less heat damage than 05S180 can maintain relatively complete cell structures to maintain metabolism,growth and development in pepper.Compared with the heat-sensitive pepper material,the heat-tolerance pepper material can reduce electrolyte leaking by increasing the content of osmosis-regulating substance after heat stress,thereby maintaining cellular homeostasis.In addition,the accumulation of enzymatic and non-enzymatic antioxidants contributed to alleviate oxidation damage caused by heat stress.And the content of antioxidants in 17CL30 was significantly higher than that of 05S180.(3)Transcriptome analysis of pepper under heat stress.17CL30 and 05S180 were used to perform RNA-Seq of pepper leaves at seedling stage under heat stress(40℃).The results showed that there were 5754 differentially expressed genes(DEGs)(2954 up-regulated genes and 2800 down regulated genes)in 17CL30 and5756 DEGs(3043 up-regulated genes and 2713 up-regulated genes)in 05S180 under heat stress.Among them,2455 DEGs were only identified in 17CL30.The differential expression of these genes and some common DEGs might help explain why 17CL30 was more heat-tolerant.Go and KEGG annotation of DEGs showed that 17CL30 had stronger heat tolerance owing to stronger signal transduction,carbohydrate metabolism and ROS scavenging ability than 05S180.(4)Metabolome analysis of pepper under heat stress.Metabolite profiling of pepper leaves(17CL30 and 05S180)under heat stress(40℃)was carried out using a widely targeted metabolomic method based on LC-ESI-MS/MS system.Briefly,we identified 94 differentially accumulated metabolites(DAMs)in 17CL30 and108 DAMs in 05S180.According to the composition of DAMs,soluble sugars,amino acids and its derivatives,flavone and organic acids were related to the heat resistance of pepper.Among them,GSH(amino acids derivative)might play a key role in the HSR process of pepper.(5)Muti-omics analysis of pepper under heat stress.The correlation analysis of identified DEGs and DAMs was carried out by integrating transcriptomics and metabolomics to reveal the response mechanism of pepper to heat stress and heat resistance mechanism systematically.Transcriptome and metabolome were reprogrammed to active the response mechanism to heat stress.According to KEGG annotation,glutathione metabolic pathway played a critical role in pepper response to heat stress and the changes of related genes and metabolites might contribute to ROS scavenging.(6)Identification and expression analysis of GST gene family in pepper.In the present study,the pepper GST gene family was identified and analyzed using bioinformatics tools and molecular techniques.The results showed that a total of80 GSTs were identified.All the identified GST members belong to nine different classes according to the classification methods of other species.In addition,a set of analysis including chromosome location,subcellular localization,conserved domain,conserved motif,intron/exon composition,phylogenetic classification and putative promoter were performed to lay a foundation for further understanding of the structure and function of GSTs.At the same time,the RNA-Seq data was used to analyze the expression profiling of GSTs in different tissue and different stress conditions for exploring the GSTs involved in pepper growth and development and stress response.CaGSTU23 involved in the HSR process of pepper.Overexpression of CaGSTU23 can improve the heat resistance of Arabidopsis,while CaGSTU23 silencing pepper showed heat sensitive.This laid a foundation for further exploration of the function of CaGSTs. |
