Anthocyanin biosynthesis in blueberry is regulated by light and abscisic acid signaling via a VcABF2 ‐ miR156 ‐ VcSPL9 feedback loop

The crosstalk between light and abscisic acid (ABA) signaling orchestrates fruit coloration in horticultural crops, including blueberry (Vaccinium spp.). Although the miR156-SPL module is known to post-transcriptionally regulate anthocyanin biosynthesis, its role in integrating light and ABA signaling remains elusive. In this study, we treated blueberry fruits with fluridone (Flu), an ABA biosynthesis inhibitor, which compromised the induction of anthocyanin biosynthesis by high-light (HL) intensity in the fruits. We determined that the ABA-responsive transcription factor (TF) VcABF2 participates in HL-mediated anthocyanin accumulation by transcriptionally activating VcANS expression. We determined that the low-light (LL)-induced SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) TF VcSPL9 directly binds to the promoters of VcABF2, VcDFR, and VcUFGT and transcriptionally represses their expression. However, ABA treatment antagonized the negative regulatory function of VcSPL9 in anthocyanin biosynthesis. Small-RNA transcriptome analysis revealed that miR156 is targeted and activated by VcABF2. This microRNA specifically cleaves VcSPL9 transcripts to relieve VcSPL9-mediated inhibition of VcABF2, ultimately forming a feedback loop that regulates anthocyanin biosynthesis. Our findings elucidate a mechanism by which the VcABF2-miR156-VcSPL9 loop coordinates light and ABA signaling to precisely modulate anthocyanin biosynthesis in blueberry.