Functional Validation of miR319a and Its Target Gene ArMYB33 in Acer rubrum

Acer rubrum has become an important ornamental tree species due to its spectacular autumn leaf color. However, its leaf color change has problems such as a short color transition period and unstable effects, and the related molecular mechanisms are not clear. To understand this mechanism, this study used Acer rubrum 'Autumn Fantasy' as the experimental material. By combining transcriptome and small RNA sequencing data, and performing differential expression analysis and target gene prediction, the key regulatory factor miR319a and its target gene ArMYB33 were identified.The study found that under different light and temperature conditions, miR319a negatively regulates the increase of anthocyanins, while ArMYB33 positively regulates anthocyanin accumulation. Through the combined analysis of transcriptome and small RNA, a total of 554 differentially expressed miRNAs and 433 target genes were found to form 2460 targeting interactions. GUS histochemical staining confirmed that miR319a and ArMYB33 interact in tobacco leaves after co-transformation. RLM 5'RACE determined the cleavage site when miR319a binds to its target gene ArMYB33. Subcellular localization showed that ArMYB33 is located in the nucleus and has transcriptional activation activity. In transgenic tobacco, overexpression of ArMYB33 can promote anthocyanin accumulation and upregulate genes in the phenylpropanoid pathway (PAL, UFGT); in contrast, overexpression of miR319a inhibits the expression of ArMYB33, leading to a decrease in anthocyanin content.This study revealed that under low temperature and high light conditions, the downregulation of miR319a relieves the inhibition of ArMYB33. The latter then drives anthocyanin synthesis by activating phenylpropanoid metabolism, thus regulating leaf color redness. By integrating multi-omics data and multi-level functional verification experiments, this study constructed a cascade regulation model of "environmental signal perception - miRNA post-transcriptional regulation - transcription factor network activation - metabolic pathway response". This model enriches the theoretical system of molecular regulation of plant leaf color change and provides an important theoretical basis for studying the molecular mechanism of leaf color redness.