Characterization of the PtPHT family in Poncirus trifoliata and identification of a regulatory module, PtRap2.12–PtPHT1;2, under drought stress

Abstract Phosphate transporters (PHTs) are H 2 PO 4 ⁻ /H + transporters that facilitate inorganic phosphate uptake from the soil and its distribution within plants. In citrus, seasonal summer drought constrains yield and limits phosphate uptake. However, the roles of PHT genes in drought response remain poorly understood. In this study, we identified 20 PtPHTs in the Poncirus trifoliata genome and classified them into five subgroups (I to V) based on phylogenetic relationships. Promoter analysis revealed abundant cis- acting elements associated with abiotic stress and phytohormone responses, while synteny analysis indicated that gene duplication contributed to PtPHT family expansion. Expression profile showed that several genes, including PtPHT1;1 , PtPHT1;2 , PtPHT1;3 , PtPHT3;3 , PtPHT4;3 , PtPHT5;1 , and PtPHT5;2 were significantly upregulated by drought, with PtPHT1;2 exhibiting the strongest response (about 16.4 folds). Silencing PtPHT1;2 markedly reduced phosphorus (P) accumulation in transgenic citrus calli, confirming its functional role in inorganic phosphate uptake. Yeast one-hybrid (Y1H) screening identified Related to AP2.12 (PtRap2.12), an APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor, as an upstream regulator of PtPHT1;2 . This interaction was validated by Y1H and dual-luciferase assays, demonstrating that PtRap2.12 directly binds to and activates the PtPHT1;2 promoter. Functional analysis further showed that overexpression of PtRap2.12 enhances drought tolerance of transgenic plants, as indicated by improved growth, reduced membrane damage, elevated antioxidant enzyme activities, and increased P accumulation. Moreover, transgenic plants displayed upregulated expression of NtPHT1;1 in shoots and NtPHT1;4 in roots of tobacco ( Nicotiana tabacum ) under drought conditions. In summary, this study provides a comprehensive analysis of PtPHT genes in P. trifoliata and highlights the role of the PtRap2.12– PtPHT1;2 module in regulating both phosphate uptake and drought tolerance, offering a theoretical basis for breeding citrus rootstocks with enhanced resistance to drought and phosphate deficiency.