MdBZR6 activates MdBAM17-mediated guard cell starch degradation during ALA-induced stomatal opening in apple leaves

5-Aminolevulinic acid (ALA), an emerging plant growth regulator, functions to modulate stomatal aperture, which is crucial for regulating plant growth and development. Although we have previously demonstrated that the β-amylase gene, MdBAM17, mediates ALA-induced guard cell starch degradation and stomatal aperture enlargement, the detailed underlying regulatory mechanisms remain elusive. Here, MdBZR6, a transcription factor (TF) localized in the nucleus, was identified as an upstream regulator of MdBAM17 through bioinformatic predictions, a yeast one-hybrid (Y1H) cDNA library screen, and subsequent Y1H verification. RT-qPCR analysis and GUS (β-glucuronidase) staining revealed that ALA upregulated MdBZR6 expression, which positively correlated with increased MdBAM17 transcription, enhanced BAM activity, reduced guard cell starch content, and enlarged stomatal aperture. Further investigation confirmed that MdBZR6 specifically bound to the BRRE element of the MdBAM17 promoter and transcriptionally activated its expression. Overexpression (OE) of MdBZR6 elevated MdBAM17 expression and BAM activity, resulting in accelerated guard cell starch breakdown and stomatal aperture enlargement, which was further enhanced by exogenous ALA. Conversely, RNA interference (RNAi)-mediated silencing of MdBZR6 resulted in the opposite phenotype. Additionally, neither OE nor RNAi of MdBZR6 influenced ALA-regulated flavonols accumulation or H₂O₂ elimination in guard cells, suggesting that MdBZR6 operates independently of the ALA-regulated reactive oxygen species (ROS) signaling pathway during ALA-induced stomatal opening. Collectively, our findings reveal that MdBZR6 is a transcriptional activator of ALA-regulated guard cell starch degradation and stomatal movement, providing new mechanistic insights into ALA-induced stomatal opening.