Apoplastic pH modulates gene expression, proteome, and ABA content in <i>Vicia faba</i> guard cells, accompanied by reduced stomatal aperture under salt stress

Although the regulatory role of apoplastic pH (pHapo) changes in response to environmental conditions is well documented in root signalling, the contribution of pHapo to guard cell (GC) regulation and NaCl stress response remains poorly understood. To determine whether alterations in leaf pHapo modulate physiological responses in GC 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 pHapo monitoring was coupled to transcriptomic, proteomic and phytohormone profiling of GC-enriched samples. Leaf apoplastic alkalinisation triggered distinct transcriptomic and proteomic responses in GC. Among over 57,000 transcripts, 1,562 were associated with pHapo, including 577 previously uncharacterised reading frames ('NOVEL regions'). 18 genes and 2 proteins responded consistently in both treatments (e.g. HSC70, SOAR1, OMT1, EDGP), highlighting their responsiveness to shifts in leaf pHapo. These molecular changes accompanied NaCl-induced stomata closure and rising GC-intrinsic ABA. Apoplastic alkalinisation in response to NaCl is closely linked to increased ABA levels, along with transcriptomic and proteomic shifts in GC. This temporal pattern suggests that the rise in pHapo functions as an initiating signal that triggers downstream molecular changes, which unfolds before becoming physiologically visible as stomata closure.