PtAHA-dependent H⁺-ATPase activation underlies mycorrhiza-induced drought tolerance in trifoliate orange

Plasma membrane (PM) H⁺-ATPase plays a crucial role in plant stress adaptation, while its involvement in mycorrhiza-mediated drought tolerance remains incompletely understood. This study investigated how inoculation with Rhizophagus intraradices affects drought (50–55 % of field maximum capacity for seven weeks) responses in potted trifoliate orange ( Poncirus trifoliata ) through comprehensive analysis of growth, photosynthetic performance, nutrient dynamics, and PM H⁺-ATPase activity and gene expression. Under drought conditions, mycorrhizal inoculation significantly enhanced plant growth parameters and root volume, despite drought-induced reductions in root colonization rate and soil mycelium length. Under drought conditions, mycorrhizal plants showed mitigated declines in water use efficiency and photosynthetic efficiency, particularly through enhanced net photosynthetic rate and photochemical quantum efficiency (both maximum and steady-state), accompanied by elevated leaf phosphorus and iron as well as root phosphorus and calcium levels. Mechanistically, mycorrhizal plants exhibited significantly higher root H⁺ efflux, increased NH 4 + levels, and elevated H⁺-ATPase activity in both leaves and roots, alongside a notable decrease in soil pH, regardless of moisture regime. Nine PtAHA genes encoding PM H⁺-ATPases were identified, displaying distinct tissue-specific expression patterns, with PtAHA1 / 4 / 8 / 9 predominantly expressed in roots. Both mycorrhizal colonization and drought triggered tissue-specific expression of these genes, especially PtAHA8 , which was upregulated in leaves and roots under drought. Correlation analysis revealed that most leaf PtAHAs were closely associated with mycorrhizal colonization and H⁺-ATPase activity, contributing to improved growth and nutrient acquisition. Nevertheless, in roots, seven PtAHAs were linked to H⁺-ATPase activity, supporting NH 4 + assimilation and soil acidification. Specifically, PtAHA8 was identified as a central player associated with root H⁺-ATPase activity under combined drought and mycorrhizal conditions. It concluded that mycorrhizas enhance drought tolerance in trifoliate orange by PtAHA -driven proton extrusion, highlighting the potential of R . intraradices inoculation to bolster drought resilience in citrus. • AMF improved growth, photosynthesis, and plant nutrients under drought stress. • AMF elevated root H⁺ efflux, NH₄⁺, and H⁺-ATPase activity. • AMF upregulate specific PtAHA homologs in leaves/roots under different treatment. • PtAHA genes with tissue-specific expression exhibited distinct expression patterns. • PtAHA8 linked to H⁺-ATPase activity, and drought and AMF affected its expression.