Transcription factor NnMYB5 controls petal color by regulating GLUTATHIONE S-TRANSFERASE2 in Nelumbo nucifera.

Abstract

Lotus (Nelumbo spp.) is an important aquatic ornamental genus in the family Nelumbonaceae comprising only two species: N. lutea with yellow flowers and N. nucifera with red or white flowers. The petal color variations between these two species have previously been associated with the potential activities of FLAVONOL SYNTHASE (FLS) and MYB5. However, the underlying genetic mechanisms of flower color divergence within the N. nucifera species remains unclear. Here, quantitative trait locus mapping led to the identification of MYB5, a candidate gene controlling petal color in N. nucifera. Genotyping of 213 natural lotus accessions revealed an 80-kb presence/absence variant (PAV) of the NnMYB5 gene that is associated with petal color variation. Transcriptome analysis, dual-luciferase and yeast one-hybrid assays showed that NnMYB5 could directly activate the anthocyanin transporter gene GLUTATHIONE S-TRANSFERASE2 (NnGST2). Heterologous expression of NnGST2 in Arabidopsis (Arabidopsis thaliana) and its overexpression in lotus petals induced anthocyanin accumulation. Deletion of the 80-kb PAV within NnMYB5 inactivated NnGST2 expression and blocked anthocyanin accumulation in white N. nucifera petals. In contrast, the anthocyanin deficiency of N. lutea occurred due to pseudogenized NlMYB5 alleles. Our results establish a regulatory link between NnMYB5 and NnGST2 in petal anthocyanin accumulation and demonstrate the independent mechanisms controlling flower coloration in Nelumbo.