Characterization of APX and APX-R gene family in Brassica juncea and B. rapa for tolerance against abiotic stresses.

KEY MESSAGE: APX and APX-R gene families were identified and characterized in two important oilseed species of Brassica. Gene expression under abiotic stress conditions, recombinant protein expression, and analysis further divulged their drought, heat, and salt-responsive behavior. Ascorbate peroxidases (APX) are heme-dependent enzymes that rid the cells of H2O2 and regulate diverse biological processes. In the present study, we performed APX gene family characterization in two Brassica sp. (B. juncea and B. rapa) as these are commercially important oilseed crops and affected severely by abiotic stresses. We identified 16 BjuAPX and 9 BraAPX genes and 2 APX-R genes each in B. juncea and B. rapa genomes, respectively. Phylogenetic analysis divided the APX genes into five distinct clades, which exhibited conservation in the gene structure, motif organization, and sub-cellular location within the clade. Structural analysis of APX and APX-R proteins revealed the amino acid substitutions in conserved domains of APX-R proteins. The expression profiling of BjuAPX and BraAPX genes showed that 3 BjuAPX, 7BraAPX, and 2 BraAPX-R genes were drought and heat responsive. Notably, BjuAAPX1a, BjuAPX1d, BjuAAPX6, BraAAPX1a, BraAAPX2, and BraAAPX3b showed high expression levels in RT-qPCR. Cis-regulatory elements in APX and APX-R gene promoters supported the differential behavior of these genes. Further, two stress-responsive genes BjuAPX1d and BraAAPX2 were cloned, characterized, and their roles were validated under heat, drought, salt, and cold stress in bacterial expression system. This study for the first time reports the presence of APX activity in dimeric and LMW form of purified BraAAPX2 protein. The study may help pave way for developing abiotic stress-tolerant Brassica crops.