Analyses of functional conservation and divergence reveal requirement of b HLH010/089/091 for pollen development at elevated temperature in Arabidopsis

The Arabidopsis b HLH010/089/091(basic helix-loop-helix)genes are functionally redundant and are required for both anther development and normal expression of DYT1-activated anther-related genes.These three genes are conserved in Brassicaceae,suggesting that each of them is under selection pressure;however,little is known about the possible functional differences among these b HLH genes and between the b HLH and DYT1 genes.Here,we compared novel anther transcriptomic data sets from b HLH010/089/091 single and double mutants,with an anther transcriptomic data set from the wild type(WT)and a previously obtained anther transcriptomic data set from the bhlh010 bhlh089 bhlh091 triple mutant.The results revealed molecular phenotypes that support the functional redundancy and divergence of b HLH010,b HLH089,and b HLH091,as well as the functional overlap and difference between them and DYT1.DNA-binding analyses revealed that DYT1 and b HLH089 specifically recognize the TCATGTGC box to activate the expression of target genes,including ATA20,EXL4,and MEE48.In addition,among genes whose expression was affected in the bhlh010 bhlh089 double and bhlh010 bhlh089 bhlh091 triple mutants,genes that are involved in the stress response and cell signaling were enriched,which included256 genes whose expression was preferentially induced by heat during early flower development.Moreover,the bhlh double mutants exhibited defective pollen development when the plants were grown under elevated temperature,suggesting that b HLH genes are important for anther gene expression under such conditions.These results are consistent with the observation that the heat-induced expression of several genes is less in the bhlh mutants than that in the WT.Therefore,our results provide important insights into the molecular mechanism underlying the activation of direct targets by DYT1-b HLH089 heterodimers and demonstrate the protective roles of b HLH010/089/091 in maintaining fertility upon heat stress.