Transcriptional activation of antioxidant enzyme genes by PtMYB14 underlies mycorrhiza-enhanced manganese tolerance in trifoliate orange
Manganese (Mn) toxicity severely limits plant growth in acidic soils. Although arbuscular mycorrhizal fungi (AMF) can enhance plant tolerance to heavy metal stress, the underlying transcriptional regulatory mechanisms remain unknown. This study aimed to identify key transcription factors (TFs) involved in AMF (Rhizoglomus intraradices)-mediated Mn tolerance in trifoliate orange (Poncirus trifoliata) and to characterize their regulatory functions. Phenotypic analysis showed that R. intraradices inoculation significantly alleviated Mn-induced growth inhibition, increasing biomass accumulation compared with Mn treatment alone, while maintaining mycorrhizal colonization rates of 70%. Transcriptome analysis identified 498 TFs commonly enriched in response to both AMF inoculation and Mn stress, with PtMYB14 (Pt3g030770) showing the highest expression levels under AMF inoculation treatments. Subcellular localization confirmed that PtMYB14 is localized to the nucleus. Molecular interaction assays (Y1H, LUC, and EMSA) demonstrated that PtMYB14 directly binds to the MYB-binding sequences in the promoters of key antioxidant genes including PtSOD1, PtCAT2, PtGPXL6, and PtFSD2, thereby activating their expression. Functional validation using PtMYB14 knockout and overexpression transgenic lines confirmed that PtMYB14 positively regulated antioxidant enzyme activities, promoted the accumulation of osmotic regulatory substances (proline, soluble sugar, and soluble protein), mitigated oxidative damage, and protected photosynthetic capacity under Mn stress. Collectively, this study identifies PtMYB14 as a central transcriptional regulator orchestrating AMF-mediated Mn tolerance by directly activating antioxidant defense systems and preserving photosynthetic function.