Anthocyanin biosynthesis and accumulation is closely associated with tissue/organ coloring in plants. To gain insight into the physiological and molecular mechanisms of leaf coloring in Acer palmatum, a deciduous tree during autumnal senescence, we first investigated concentration dynamics of pigments (i.e., chlorophyll, carotenoid and anthocyanin) in leaves with differential coloring. It was found that compared to green leaves (GN), anthocyanins were accumulated actively in semi-red (SR) and total-red (TR) leaves, accompanied with chlorophyll and carotenoid degradation. Then transcriptional profiling on GN and SR leaves identified thousands of transcripts with differential expression in SR compared to GN leaves. An annotation search showed that the entire flavonoid/anthocyanin biosynthesis pathway from the production of naringenin chalcone to modification of flavonoid backbone was extensively activated at the transcriptional level in SR leaves. Phylogenetic analysis of putative MYB proteins identified ApMYB1 as a putative regulator promoting anthocyanin biosynthesis. Expression of ApMYB1 in leaves was induced by exogenous hormones including abscisic acid. Stable overexpression of ApMYB1 in tobacco resulted in leaves with higher accumulation of anthocyanins. Collectively, our results identified ApMYB1 as a positive regulator associated with leaf coloring in Acer palmatum during autumnal senescence, which may be regarded a potential target for breeding color-leafed plants.

Anthocyanin biosynthesis determines the leaf color of Acer palmatum as a widely-planted landscape tree. Previously, ApMYB1 has been characterized as a positive regulator of anthocyanin biosynthesis. To further elucidate the mechanism of leaf coloration, the present study identified a basic helix-loop-helix (bHLH) transcription factor (ApbHLH1) through the phylogenetic analysis of 156 putative bHLH proteins in Acer palmatum and eight reference bHLHs which were known to be involved in the anthocyanin biosynthesis of selected plants. Protein structure comparison showed that ApbHLH1 has a conserved bHLH domain, and its N-terminal contains an MYB-interacting region. The expression of ApbHLH1 in leaves was found to not be correlated with anthocyanin contents either in green, semi-red leaves or during leaf autumnal senescence when anthocyanin content increased. ApbHLH1 expression in detached leaves was induced by exogenous senescence-promoting chemicals, including H₂O₂, SA, MeJA, ACC and ABA, with certain durations. In particular, either high light or low temperature induced ApbHLH1 expression significantly, and combination of high light and low temperatures seemed more effective in inducing ApbHLH1 expression. Luciferase complementation imaging assays confirmed the physical interaction between ApbHLH1 and ApMYB1, which could be abolished by either the truncating MYB-interacting region of ApbHLH1 or the deleting bHLH interacting domain of ApMYB1. The transient expression of ApbHLH1 could not induce anthocyanin production, while the co-expression of ApbHLH1 and ApMYB1 resulted in a higher accumulation of anthocyanins compared to the expression of ApMYB1 alone in tobacco leaves. Collectively, our results revealed that ApbHLH1 participated in leaf coloration through binding with ApMYB1 and enhancing the ApMYB1 function of promoting anthocyanin biosynthesis during leaf autumnal reddening in Acer palmatum. ApbHLH1 could have the potential for breeding color-leafed plants through co-transformation with ApMYB1.