Activation of ethylene biosynthetic gene <i>MaACS1</i> by <scp>RING</scp> ‐ <scp>H2</scp> ‐type <scp>E3</scp> ligase <scp>MaRHA2A1</scp> ‐mediated ubiquitin‐proteasomal degradation of <scp>MaMYB73</scp> during banana fruit ripening

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a central and rate-limiting enzyme in ethylene biosynthesis, but the regulation of ACS in response to endogenous signals and environmental stimuli remains largely unknown, especially in fruit crops such as bananas. In this study, we used various methods, including genome-wide identification, molecular dynamics, comparative genomics, DNase-seq, DAP-seq, RNA-seq, and molecular assays, to elucidate the mechanism by which the transcription factor MaMYB73 regulates the expression of the ethylene biosynthesis-related gene MaACS1. Fourteen MaACS genes were identified in banana, among which MaACS1 plays crucial roles in ethylene biosynthesis. Molecular dynamics showed that MaACS1 binds S-adenosyl-L-methionine (SAM) at residue R220. Comparative genomics revealed that MaACS1 has three orthologs in the tomato genome (SlACS2, SlACS4, and SlACS1B). This orthogroup underwent a statistically significant expansion event along the evolutionary lineage leading to tomato, correlating with its increased ethylene production compared with that of banana. DNase-seq and DAP-seq revealed that MaMYB73 directly binds to the MaACS1 promoter. The overexpression of MaMYB73 in both banana and tomato resulted in reduced expression of MaACS1 and its homologs, respectively, leading to delayed ethylene production. Conversely, silencing MaMYB73 through virus-induced gene silencing (VIGS) significantly increased MaACS1 expression and ethylene production in banana fruit. Furthermore, the RING-H2-type E3 ubiquitin ligase MaRHA2A1, an ethylene accelerator, targets MaMYB73 for ubiquitination and subsequent degradation via the 26S proteasome pathway. Transient overexpression of MaRHA2A1 in banana increased MaACS1 expression and ethylene levels possibly by reducing the accumulation of MaMYB73, whereas silencing of MaRHA2A1 had the opposite effect. These findings provide new insights into the regulatory mechanisms controlling ethylene biosynthesis during banana fruit ripening and establish a new strategy for characterizing the transcriptional regulators of ethylene biosynthesis using various bioinformatics technologies.