Overexpression of pineapple gibberellin 2-oxidase gene AcGA2ox6 in Arabidopsis induces delayed flowering, dwarfism, and enhanced leaf serration.

Despite the widespread use of ethephon to induce synchronous flowering in pineapple, the molecular mechanisms underlying its action are still not fully defined. Our previous RNA-seq analysis identified a gibberellin 2-oxidase (GA2ox) gene, AcGA2ox6, as being associated with flowering, but its functional role remained unconfirmed. In this study, we conducted a functional characterization of AcGA2ox6 through bioinformatics analysis, subcellular localization, and overexpression in Arabidopsis. Transgenic Arabidopsis lines exhibited striking phenotypes, including dwarfism, darker green and highly serrated leaves, significantly delayed flowering under both long-day and short-day conditions, with a more severe delay under the latter, and disrupted floral development leading to reduced fertility. Quantitative reverse transcription PCR (qRT-PCR) revealed that these phenotypic changes were likely driven by altered expression of genes related to flowering, including strong upregulation of FLC and downregulation of SOC1 and FT, as well as genes involved in leaf development and chlorophyll metabolism. Crucially, exogenous gibberellin A3 (GA3) application completely rescued the flowering delay under both photoperiod conditions and most other defects, establishing AcGA2ox6 as a GA-deactivation enzyme that negatively regulates GA-mediated growth and reproductive development by restricting bioactive GA availability. RNA-seq analysis further identified global transcriptomic changes underlying these phenotypes. Importantly, we found that this role is likely conserved in pineapple, where ethephon-induced flowering correlated with a downregulation of AcGA2ox6 in the shoot apex. Collectively, our results demonstrate that AcGA2ox6 negatively regulates GA-mediated growth and development, particularly flowering, through GA catabolism, and provide significant insights into the molecular mechanisms of ethylene-induced flowering in pineapple.