Cellular pH homeostasis shapes root system architecture by modulating auxin-mediated developmental responses

Abstract Cell expansion relies on turgor pressure and acidification-dependent loosening of the rigid cell wall. Distinct cell surface-based and intracellular auxin signaling pathways synergistically activate plasma membrane H+-ATPases, acidifying the apoplast, a prerequisite for cell elongation. Unlike in shoots, auxin inhibits cell elongation in roots. This auxin paradox highlights a largely unknown antagonistic pathway, driving root apoplast alkalinization. Auxin fluxes, regulated by the TINY ROOT HAIR 1 (TRH1)/POTASSIUM (K+) UPTAKE 4 (KUP4) transporter, modulate root gravitropism and root hair morphogenesis through the acropetal and basipetal auxin transport pathways, respectively. Here, we show that under acidic conditions, wild-type Arabidopsis (Arabidopsis thaliana) seedlings develop shorter root hairs and exhibit an agravitropic response, a defect that is even more pronounced in trh1/kup4 roots. Acidic conditions also distort auxin responses in wild-type roots, with these effects further exacerbated in trh1/kup4 roots. Remarkably, exogenous auxin application restores the trh1-like developmental defects in wild-type roots, suggesting that acidity chemiosmotically impairs active auxin transport. Advanced compartmental pH imaging combined with pharmacological applications revealed cytoplasmic and vacuolar acidification in trh1/kup4 root cells, which activates AHA2, the predominant plasma membrane H+-ATPase in roots. Proton efflux leads to apoplast acidification and rhizotoxicity, thereby inhibiting primary root elongation of trh1/kup4 seedlings. Our results demonstrate that as a proton-coupled potassium transporter, TRH1/KUP4 maintains a balance between cytosolic and apoplastic proton gradients, facilitating cytoplasm neutralization and apoplast alkalization in roots. Through this regulatory mechanism, we postulate that TRH1/KUP4 enables pH-driven intracellular auxin transport and modulates cell surface pH, driving root cell elongation and shaping root system architecture.