Use of a novel R2R3-MYB transcriptional activator of anthocyanin biosynthesis as visual selection marker for rubber tree (Hevea brasiliensis) transformation

Abstract

Hevea brasiliensis is presently the only commercial source of natural rubber. The use of anthocyanin production as a visual and non-destructive selection marker may be a viable strategy for increasing the transformation efficiency of this species. In this study, high performance liquid chromatography (HPLC) analysis confirmed that the cyanidin-type anthocyanin is a major determinant of red color in Hevea tissues. It was also found that the anthocyanin production in Hevea somatic embryos could be activated by methyl jasmonate (MeJA) treatment. By sequence alignment and phylogenetic analysis, a novel Hevea R2R3-MYB transcription factor (TF) was isolated and designated here as Hevea brasiliensis Anthocyanin 2 (HbAN2). The expressions of HbAN2 correlate well with the anthocyanin levels in Hevea tissues. The regulatory roles of HbAN2 in anthocyanin biosynthesis were functionally characterized in the native and heterologous systems. In tobacco suspension culture cells, the expression of HbAN2 alone or in combination with two HbbHLH genes activated the promoter of NtDFR, a key gene responsible for anthocyanin biosynthesis. In tobacco plants and Hevea embryos, overexpression of HbAN2 upregulated the late stage anthocyanin pathway genes, resulting in increased anthocyanin accumulation in the transformed parts of the explants, which were therefore easily distinguishable from the non transformed parts. Although transgenic Hevea shoots failed to regenerate into whole plants, these results suggest that HbAN2 is a regulator of anthocyanin biosynthesis and has the potential to be used as visual selection marker for Hevea transformation.