Function and stimulation of the transient increase of the leaf apoplastic pH during exposure to chlorine-stress

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The apoplast is an extracellular compartment that lies outside the plasma membrane (PM). External stress factors can influence the pH of the fluid film of the leaf apoplast. For instance, the apoplastic pH (pHapo) of field bean (Vicia faba L.) leaves increases minutes after exposing the roots to a stressful dose of NaCl. This increase is transient and lasts over a period of 1-3 hours. It is ascribable to the chloride (Cl−) but not to the sodium or the osmotic component of the NaCl-stress. Although indications increase that this rise of the pHapo could be part of a beneficial response to salt stress, it has received little attention in the previous research. On the one hand, it could be a secondary effect without functional implication, arising i.e. from Cl− movements across the PM. On the other hand, results derived from studies on Vicia faba show that the Cl−-induced transient increase of the leaf pHapo is instrumental for enriching guard cells (GCs) with abscisic acid (ABA). ABA is a phytohormone that controls stomatal aperture. Regarding this enrichment, it is unclear whether the transient increase of the leaf pHapo represents a response to Cl−-stress that alone is sufficient to induce an accumulation of ABA in the GCs. To clarify this, it is planned in the envisaged project to artificially increase the leaf pHapo to increase in a transient way. This is done via the infiltration of a pH buffer. By this means, the effect of the transient pHapo increase on the ABA concentration in the GCs can be investigated in a background in which plants are not Cl−-stressed. This investigation will provide information as to whether the sole presence of the transient Cl−-induced pHapo rise was sufficient to cause the accumulation of ABA in the GCs. If this is not the case, it is likely that the GC ABA enrichment is rather the outcome of an interaction between the Cl− stress-induced pHapo change and other stress responses that arise from Cl−-stress. Ratiometric in planta H+-imaging is used for the non-invasive quantification of the pHapo. Guard cells will be purified from leaves that have shown the transient increase of the pHapo. These GC samples are subjected to different analysis: First, ABA concentration is analyzed via mass spectrometry (MS). Moreover, MS-based shotgun proteomics is applied to investigate pHapo-based effects on abundances of proteins that catalyse the GC-autonomous ABA synthesis or mediate ABA influx into GCs. Furthermore, the project seeks to expand knowledge concerning anionic stimulants other than Cl− that can induce the leaf apoplast to increase pH, when applied at a stressful dose. This could be sulfate, which is a dominant anion in many saline soils. Lastly, the response of the root apoplastic pH to a stressful dose of Cl− is investigated, as our understanding about the regulation of the root pHapo during exposure to Cl−-stress is rudimentary.