Transcriptional and post-transcriptional insights into MYB113-associated anthocyanin regulation in Eucalyptus

Anthocyanin accumulation is an important adaptive response that helps Eucalyptus cope with environmental stresses. However, the transcriptional and post-transcriptional mechanisms regulating anthocyanin biosynthesis in Eucalyptus remain poorly understood. Here, PacBio full-length transcriptome sequencing, RNA sequencing, and functional analyses were combined to identify transcription factors and structural genes involved in anthocyanin biosynthesis in green leaves (GLs) and anthocyanin-rich red leaves (RLs). PacBio sequencing generated 25,953 and 32,821 high-quality transcripts from GLs and RLs, respectively. Among these, 10,766 transcripts in GLs and 11,637 transcripts in RLs represented novel isoforms of annotated genes, with alternative splicing (AS) being the primary source of transcript diversity. Notably, most structural genes involved in anthocyanin biosynthesis generated multiple AS isoforms, suggesting that AS may contribute to the regulation of this pathway. Twelve MYB genes were predicted to be associated with flavonoid biosynthesis, including three MYB113 homologs ( MYB113a , MYB113b , and MYB113c ). These genes displayed distinct expression responses to cytokinin, nitrogen availability, and high-light treatment. Although MYB113c exhibited relatively low sequence similarity to previously characterized MYB113 genes, functional analyses in Eucalyptus and tobacco demonstrated that it positively regulates anthocyanin accumulation. Our results reveal extensive alternative splicing in the Eucalyptus transcriptome and indicate that AS maybe an important regulatory layer in anthocyanin biosynthesis. In addition, the findings identify MYB113 genes, particularly MYB113c , as potential key regulators linking environmental signals to anthocyanin accumulation in Eucalyptus .