Evolutionary expansion and expression dynamics of cytokinin-catabolizing CKX gene family in the modern amphidiploid mustard ( Brassica sp.)

Plant cytokinins (CKs) promote development and physiological processes, drought tolerance, root architecture, and ultimately crop productivity. Biologically active CKs ( iP , tZ , and cZ ) are precisely maintained in the vegetative and floral tissues through their irreversible degradation by developmentally regulated CK-catabolizing cytokinin oxidase/dehydrogenase (CKX) enzyme. A meta-analysis of CKX proteins was performed through an exhaustive exploration of multiple genome databases of cyanobacteria, bryophyte, monocot and eudicot plants to reveal the intricate evolutionary profiles of CKX enzymes specific to the family Brassicaceae. At least 175 unique paralogous/orthologous CKX sequences were successfully retrieved and phylogenetically clustered into distinct groups. Observations of structural divergences among paralogous sequences compared to their orthologs indicated that the progenitor CKX sequence had been subjected to massive structural modifications, possibly as a result of the evolutionary split between monocots and eudicots. An analysis of dN/dS comparisons of orthologous genes revealed that segmental CKX gene duplications have evolved primarily under purifying selection. Further, 24 CKX genes with conserved signature domain were identified in the amphidiploid Brassica juncea genome (AABB; 2 n = 36). Genetic evolution of paralogous and orthologous genes was largely responsible for the expansion of CKX homoeologs in the amphidiploid Brassica genomes. Also, comparative analyses of 1.5 kb-long upstream regulatory regions of BjCKX genes identified various development- and stress-responsive elements. Spatial and temporal expression profiles of CKX genes were primarily attributed to their structural diversity observed in the 5′-regulatory regions along with species evolution. This data suggested that CKX duplicate genes had partitioned their spatial expression (= function) during evolution. These findings illustrated the evolutionary importance of CKX genes during plant development, and also suggested their deployment for future crop improvement programs.