The Roles Of BAG8 And BAG9 In Response To Temperature Stress In Tomato

Tomato(Solanum lycopersicum)is a thermophilic horticultural crop with multiple values such as edible and medicinal use.At present,facility tomato industry has developed rapidly in China,but there are also series problems,especially in recent years,the occurrence of extreme temperature weather,which seriously affects the improvement of tomato yield and quality.BAG(Bcl-2 associated athanogene)is a multifunctional chaperone regulatory protein,which has been widely reported to be involved in regulating growth,development and stress response pathways such as salt and drought in Arabidopsis,but the mechanism of BAG in response to temperature stress in tomato is still unknown.In this study,we identified 10 BAG genes in tomato and analyzed their phylogenetic evolution,protein functions and transcriptional regulation by bioinformatics.We screened out two key genes BAG8 and BAG9,and explored their functions in response to stress by physiological and molecular techniques.We identified the localization,interaction proteins and preliminarily explored the biological functions of BAG8 and BAG9.The main findings are as follows:1.Ten BAG genes were identified,and it was cleared that BAG family was widely involved in response to abiotic stress in tomato.According to homology and evolution with Arabidopsis,all identified BAG genes were named Sl BAG1～10.By constructing phylogenetic tree of BAG in tomato,Arabidopsis,rice,tobacco and analyzing amino acid sequences,we found Sl BAGs mainly contained BAG domains,UBL domains and Ca M binding motifs.Further analysis of protein information and structures showed that most Sl BAGs were composed of 300～400 amino acids,and there were differences in types and number of domains contained in proteins.Transcriptional regulation analysis showed that there were cold stress response elements in the antisense strand of BAG8 promoter and heat shock response elements in BAG9.These results indicate the members of Sl BAGs have rich structures and biological functions,which may be widely involved in response to abiotic stress through various pathways,especially BAG8 and BAG9 may play key roles in resisting temperature stress in tomato.2.The main genes BAG8 and BAG9 in tomato responding to cold stress were screened,and their positive regulation effects on cold resistance were confirmed.After cold treatment,it was found six genes were up-regulated,among which BAG8and BAG9 were significantly.Silencing plants of the six up-regulated genes were constructed by VIGS and subjected to cold treatment,we found p TRV-BAG8 and p TRV-BAG9 showed significantly weakened resistance and the expression of CBF1was significantly inhibited.Further,net photosynthetic rate(Pn),stomatal conductance(G_S)and transpiration rate(Gr)of p TRV-BAG8 and p TRV-BAG9 were significantly inhibited.The increases of genes expressions and activities of antioxidant enzymes were significantly inhibited.The number of autophagosomes decreased significantly and the expression of ATG8a was inhibited.The accumulation of ubiquitinated proteins was increased.These results suggest that BAG8 and BAG9 play key roles in resisting cold stress in all up-regulated genes.They may be involved in positive regulation of cold resistance pathway by CBF and may also play roles in maintaining the normal operation of photosynthesis and the homeostasis of antioxidant defense mechanism,affecting the formation of autophagosomes and slowing down the accumulation of protein ubiquitination,thus positively regulating the cold resistance of tomato.3.The expression levels of BAG family in response to heat stress and positive regulation effects of BAG8 and BAG9 on heat resistance in tomato were confirmed.After heat treatment,BAG6,BAG8 and BAG9 were significantly up-regulated,indicating that BAG6,BAG8 and BAG9 might play roles in response to heat stress.In order to improve the research on effects of BAG8 and BAG9,p TRV-BAG8 and p TRV-BAG9 were constructed by VIGS.After heat treatment,p TRV-BAG8 and p TRV-BAG9showed obvious heat sensitivity and Pn,Gs,Gr were significantly inhibited.The number of autophagosomes was significantly reduced and the level of protein ubiquitination was significantly increased.These results indicate BAG8 and BAG9positively regulate the heat resistance of tomato,and they may play roles in maintaining normal photosynthesis and antioxidant defense mechanisms,affecting autophagosomes formation and slowing down protein ubiquitination accumulation induced by heat stress.4.The localization and interaction proteins of BAG8 and BAG9 were defined,the biological functions of BAG8 and BAG9 were preliminarily identified.Subcellular localization showed that both BAG8 and BAG9 were localized in nucleus and cytoplasm.Yeast library screening showed the interaction proteins that might be involved in the response to temperature stress were calmodulins(Ca M1～6),Fe S proteins(PGR1,Fd C1,Fd2)and small heat shock proteins(HSP17.7A,HSP17.7B,HSP17.6B,HSP17.6C).Y2H and Bi FC showed BAG8 interacted with PGR1 and Ca M2～4 in cytoplasm,BAG9 interacted with PGR1,Fd C1,Fd2 and Ca M2～4 in nucleus and cytoplasm.Further verification showed both BAG8 and BAG9 interacted with small heat shock protein HSP20 family,which contained HSP17.7A,HSP17.7B,HSP17.6B,HSP17.6C.These results suggest that BAG8 and BAG9 may positively regulate resistance to temperature stress through binding with calmodulins,Fe S proteins or small heat shock proteins in tomato,but the specific mechanism remains to be further explored.