Isolation and characterization of a novel anthocyanin-promoting MYBA gene family in Citrus

MYB transcription factors play an important role in the activation and regulation of anthocyanin biosynthesis. Many such transcription factor genes from various plant species were well characterized at the molecular level. However, none were reported in Citrus . Here, we demonstrated the isolation, molecular characterization, and functional evaluation of a large number of Citrus genes belonging to a previously unidentified MYBA gene family. All these genes were recovered from up to nine genotypes in the genus Citrus by PCR amplification using primers corresponding to VvMybA1 of grapevine ( Vitis vinifera ). Among the 56 genes analyzed, 35 genes contained full-length coding sequences and showed a 98.3% to 100% amino acid sequence homology with VvMybA1 (referred to as MybA1 genes), whereas 21 genes contained a 209-bp deletion in the exon-3 region similar to a related mutant gene VvMybA3 of grapevine. Citrus MybA1genes are composed of three exons ranging in sizes from 115, 130, to 508bp and two introns of 87 and 119bp, respectively, and encode a polypeptide of 250 residues. Unique single-residue substitution mutations allowed the categorization of these genes into three groups. Transient transgene expression analysis using grapevine somatic embryos revealed that genes from group a failed to induce anthocyanin production. Many members from groups b and c were fully functional and capable of activating anthocyanin biosynthesis in non-anthocyanic explants. Reversion mutagenesis experiments indicate that an R93K substitution unique to group a genes is the single loss-of-function mutation. This study confirmed, for the first time, the existence of highly homologous regulatory genes involved in the anthocyanin pathway among taxonomically distant species.