Comparative Transcriptome Analysis Revealed Two Alternative Splicing bHLHs Account for Flower Color Alteration in Chrysanthemum.

'Jimba' is a white chrysanthemum cultivar, which occasionally and spontaneously produces red flower petals under natural cultivation due to cyanidin-based anthocyanin accumulation. To investigate the underlying mechanism of this process, a comparative transcriptome was analyzed between white and turning red 'Jimba'. The structural and regulatory genes of anthocyanin pathway were significantly up-regulated in turning red 'Jimba'. Among them, two alternative splicings, CmbHLH2 and CmbHLH2.1, showed the most significantly up-regulated in turning red tissue. Transiently over-expressed 35S::CmMYB6-CmbHLH2 strongly induced anthocyanin accumulation in 'Jimba' flower petals, while moderate amount of anthocyanin was detected when over-expressed 35S::CmMYB6-CmbHLH2.1. Both CmbHLH2 and CmbHLH2.1 could interact with CmMYB6 to activate CmDFR promoter according to Yeast two-hybrid and dual-luciferase assay. Moreover, CmMYB6-CmbHLH2 but not CmMYB6-CmbHLH2.1 could activate the CmbHLH2 promoter to provide positive feedback loop regulation. Taken together, it suggested that both CmbHLH2 and CmbHLH2.1 involved in regulation flower color alteration in turning red 'Jimba', and CmbHLH2 played a predominant role in this process.