Molecular and biochemical rhythms in dihydroflavonol 4-reductase- mediated regulation of leucoanthocyanidin biosynthesis in Carthamus tinctorius L

Flavonoids are a distinctive class of phenolic compounds known to be involved in plant growth, development and floral pigmentation. During the natural phenylalanine pathway, dihydroflavonol-4-reductase is the first committed enzyme that catalyzes the stereo-specific reduction of dihydroflavonols into leucoanthocyanidins.Howsoever, less attention has been given to studies explaining the evolution and function of dihydroflavonol-4-reductases in Carthamus tinctorius L. This study explains the first comprehensive genome-wide identification and functional characterization of putative dihydroflavonol 4-reductase in Carthamus tinctorius L. Altogether, 20 CtCYP45082C enzyme encoding genes have been identified in the Carthamus tinctorius genome. Phylogeney analysis revealed the clustering of CtCYP45082C sequences into five major clades illustrating the significant effects of evolutionary divergence across the plant kingdom. Further in silico analyses indicated that all enzyme-encoding CtCYP45082Cs contain fundamental cis-regulatory units and protein domains/motifs that are specifically conserved throughout eukaryotic CYP450 s. In addition, the transient expression of CtCYP45082C1 fused with green fluorescent protein in onion epidermal cells and tobacco leaves confirmed a clearly distinct subcellular localization to plasma membrane. Biochemical characterization of CtCYP45082C1 using the heterologous protein expression assay indicated that CtCYP45082C1 effectively catalyzes the reduction of cis-3,4-leucopelargonidin and dihydromyricetin in leucoanthocyanidin biosynthesis. Moreover, the transcript expression of 20 Carthamus tinctorius derived CYP45082C genes has also been analyzed by real-time quantitative PCR. Each mRNA transcript was detected, in general, in all the investigated tissues, but with different patterns except for CtCYP45082C17-20, indicating the potential role of CtCYP45082C gene family in the secondary metabolite biosynthesis of Carthamus tinctorius. Additionally, the expression of CtCYP45082C1 and two downstream flavonoid pathway regulatory genes following induction of methyl jasmonate, cold, H₂0₂ and heat irradiation suggested that mRNA expressions of CtCYP45082C1, CtCHI and CtFLS are susceptible to various environmental changes. This study provides meaningful insights for further functional characterization studies of the CtCYP45082C encoding enzymes which may also be involved in biosynthesis of flavonoids.