Apoplastic pH modulates gene expression, the proteome, and ABA content in Vicia faba guard cells and is accompanied by reduced stomatal aperture under salt stress

Although the regulatory role of changes in apoplastic pH (pHapo) in response to environmental conditions is well documented in root signalling, its contribution to guard cell (GC) regulation and the stress response to NaCl remains poorly understood. To determine whether alterations in leaf pHapo modulate physiological responses in GCs under NaCl stress, hydroponically grown Vicia faba plants were subjected to root-applied NaCl stress or alkaline-buffered infiltration of the leaf apoplast. In vivo monitoring of pHapo was coupled with transcriptomic, proteomic, and phytohormone profiling of samples enriched for GCs. Leaf apoplastic alkalinisation triggered distinct transcriptomic and proteomic responses in GCs. Among over 57 000 transcripts, 1562 were associated with pHapo, including 577 previously uncharacterised reading frames. Eighteen genes and two proteins responded consistently in both treatments, highlighting their responsiveness to shifts in leaf pHapo. These molecular changes accompanied NaCl-induced stomatal closure and rising GC-intrinsic ABA. Apoplastic alkalinisation in response to NaCl was closely linked to increased ABA levels, along with transcriptomic and proteomic shifts in the GCs. The temporal pattern suggests that the rise in pHapo functions as an initiating signal that triggers downstream molecular changes that occur before the stress becomes physiologically visible as stomatal closure.