Unprecedented evolution and transcriptional reprogramming of CYP81E subfamily in Carthamus tinctorius during flavonoid biosynthesis

Abstract Cytochrome P450s are widely known as an important class of enzymes involved in multi-dimensional metabolic reactions which facilitate both primary and secondary metabolism in plants. Recent advances in genome sequencing of new plant species have greatly influenced our knowledge of the evolution of gene families. Herein, we present the extensive genome-wide identification study and early experimental groundwork of CtCYP81E subfamily extracted from safflower genome. The evolutionary divergence and several other molecular aspects of CtCYP81E enzymes were described with the help of phylogenetic reconstruction and robust in silico analysis. A total of 15 CtCYP81E candidate enzymes were identified and clustered together with A-type CYP71 clan of the model plant. The detail overview of their gene structures organization, conserved signatures motif, cis regulatory elements, Go functional categorization and protein-protein interaction network, respectively suggested novel insights for physiological and biosynthetic implications. Following multiple recombinant DNA approaches combined with the development of GPF fusion, heterologous expression, and transcriptional regulation network of CtCYP81E8 under normal and fluctuating environments, further functional validation was performed. The transient expression system using onion epidermal cells revealed the candidate protein's subcellular position to cell membrane. Similarly, the biochemical assay of recombinant CtCYP81E8 protein, effectively produced during heterologous expression, verified 2,4-dimethylphenol activity over different time periods. Moreover, the results of RNA-transcriptomic data and qRT-PCR analysis of 15 CtCYP81Es at different flowering stages indicated a differential expression levels defining their potential roles during safflower metabolite biosynthesis.Consequently, the transcriptional regulation of CtCYP81E exploited with various stress conditions indicated considerable susceptibility against these environmental drifts. Furthermore, the correlation analysis of CtCYP81E8 transcription and metabolite accumulation pattern in wild and mutant safflower lines also suggested positive outcomes during flower development. Although presumably, these results may be helpful in determining the fundamental idea of transcriptional regulation channels that strategically turn on the secondary metabolic pool of plant system in response cute environmental falls.