Functional Characterization Of CaATG8c In Plant Tolerance To Abiotic Stresses And Genome-wide Identification And Expression Analysis Of Autophagy Related Genes In Capsicum Annuum L.

Pepper(Capsicum annuum L.),an important vegetable crop and spice,has been widely planted around the world.In recent years,due to climate changing,pepper is often exposed to environmental stresses,leading to decreased yields and low quality of pepper fruits.Abiotic stresses negatively affect plants growth and development by inducing protein misfolding,denaturation,oxidation and aggregation.The removal of damaged proteins can be conducted by Autophagy is one of the ubiquitous and highly conservative protein degradation systems in eukaryotic cells,which can remove the damaged proteins.However,the study on the relationship between pepper autophagy and tolerance to abiotic stresses is limited.By cDNA-AFLP in previous research work,we obtained a specific gene segment of CaATG8 c induced by heat stress.On this basis,pepper thermotolerant line R9 and thermosensitive line B6 were selected as materials to study the effects of silencing or ectopic expression of CaATG8 c gene on plant tolerance to environmental stresses.Besides,the genome-wide identification of CaATG genes was performed based on the published pepper genome database,and the expression of CaATG genes under abiotic stress were analyzed by qRT-PCR.The main results of the study are as follows:1.The sequence structure of pepper CaATG8 c gene was analyzed.The open reading frame was 366 bp,and molecular weight 14.07 kDa,isoelectric point 8.3.This gene contains conservative amino acid sequence,and several cis-acting elements related to growth and stress response were found in its promoter regions.The expression of CaATG8 c was higher in roots and leaf,and by subcellular location study,the CaATG8 c protein was mostly distributed in cell cytoplasm.2.By the tobacco rattle virus(TRV)-based VIGS technique,the CaATG8 c gene in pepper line R9 was silenced.Under heat stress,gene-silenced pepper plants contained higher malondialdehyde content,lower chlorophyll content than the control plants,and the SOD activity of the control plants was increased,while decreased in gene-silenced plants.these results suggest that the silencing of CaATG8 c gene reduces the heat-induced active oxygen removal ability,which results in the decrease of pepper tolerance to heat stress.Under salt stress,compared with the control plants,the chlorosis in leaf discs of gene-silenced plants wasmore serious,showing the association of CaATG8 c gene with plant salt tolerance.3.Agrobacterium-mediated transformation had been used to introduce the CaATG8 c gene into Arabidopsis.Under heat stress,transgenic plants had a lower survival rate compared with the control(the transgenic plants with empty vector load and wild type)plants,suggesting that the overexpression of CaATG8 c gene reduced Arabidopsis thaliana tolerance to heat stress.Under salt stress,in CaATG8 c overexpression Arabidopsis plants,leaf chlorosis was more serious and the survival rate is lower,and the root growth has also been greatly inhibited.these data indicate CaATG8 c overexpression plants is more sensitive to salt stress.4.The pepper CaATG genes were composed of 15 core ATG members,including 29 ATG proteins with their respective conserved functional domains,which involved in the whole process of autophagy.To investigate the spatial and temporal expression patterns of CaATG genes,we retrieved the data of 26 CaATG genes from the published genomic RNA-seq databases.As a whole,the expression of CaATG genes showed tissue-and organ-specific pattern,in which most of the CaATG genes were abundantly transcribed in root and stem,while CaATG8 a and CaATG8 c remained at higher expressions levels in each tested tissue.In addition,with the increment in days after anthesis or breaker,a majority of the CaATG genes displayed up-regulated expression in pepper pericarp and placenta,suggesting the linkage of autophagy to the development and maturity of pepper fruit.Under abiotic stresses of salt,drought,heat,cold and carbohydrate starvation,the accumulation of autophagosome punctate increased and the expression level of CaATG genes changed with stress type-dependent pattern,which indicates the linkage of autophagy to pepper response to abiotic stresses.5.After treated with heat stress,both the number of up-regulated CaATG genes and the increment of autophagosome punctate were higher in pepper thermotolerant line R9 than those in thermosensitive line B6,implying an association of autophagy with plant heat tolerance.