Global investigation into the CqCYP76AD and CqDODA families in Chenopodium quinoa: Identification, evolutionary history, and their functional roles in betalain biosynthesis.

Abstract

Betalains are water-soluble pigments mainly distributed in the core Caryophyllales plants. Betalains provide plant with striking colors to attract pollinators and are beneficial to human health due to the strong antioxidant activity. To date, many studies regarding to betalain biosynthesis have been exerted in sugar beet (Beta vulgaris) and four-O-clock (Mirabilis jalapa), however, the key regulators in betalain pigmentation of quinoa (Chenopodium quinoa) remain to be elucidated. CYP76AD and DODA genes encode core enzymes converting L-DOPA to cyclo-DOPA and betalamic acid, respectively, in betalain biosynthesis. In this study, 44 CqCYP76AD (5 α-clade, 6 β-clade and 33 γ-clade homologs) and 18 CqDODA (10 α-clade, 2 β-clade and 6 γ-clade homologs) members were identified in quinoa genome. Expression analysis and cis-element analysis indicated that light and ABA are involved in the regulation of CqCYP76AD and CqDODA. We found application of exogenous ABA and darkness repressed the betalain production in quinoa seedlings. Tandem duplication is the major driving force for CqCYP76AD and CqDODA family expansion. Evolutionary history analysis on the duplication events of quinoa and its close relatives, sugar beet, C. pallidicaule, C. suecicum and C. formosanum, identified the quinoa-specific tandem duplications CqCYP76AD-α2/-α3, CqDODA-α1/-α6 in Chr04, and CqCYP76AD-α1/-α4/-α5, CqDODA-α3/-α4/-α5 in Chr03, which are absent in sugar beet. The close co-location of the CqCYP76AD-α-CqDODA-α gene clusters suggests they are putative enhanced regulatory units for betalain biosynthesis in quinoa, similar to the operon BvCYP76AD1-BvDODA1 in sugar beet. The functions of α-, β- and γ-clade CqCYP76ADs and CqDODAs were investigated by transient expression system in tobacco leaves and hairy root transformation in quinoa. The results indicated that CqCYP76AD-α1, CqCYP76AD-β3, CqDODA-α1, CqDODA-α3 and CqDODA-α5 are the important positive regulators for betalain accumulation in quinoa. Correlation between pigment contents and expression levels at different developmental stages indicates their roles in pigmentation of leaf, stem and spike tissues of in betalain-enriched quinoa. Overall, this study performed genome-wide identification and functional characterization of the important functional enzymes of CqCYP76ADs and CqDODAs for betalain biosynthesis in quinoa, which will deep our understanding of the mechanisms of betalain pigmentation in quinoa.