Stress is basic: ABA alkalinizes both the xylem sap and the cytosol of Arabidopsis vascular bundle sheath cells by inhibiting their P-type H⁺-ATPase and stimulating their V-type H⁺-ATPase

Abstract

BACKGROUND AND HYPOTHESIS•Under water deprivation, in many perennial species, the stress hormone, ABA, appears in the xylem sap in the shoot (including leaf) veins and the xylem sap pH (pHEXT) increases. This study aimed to test the hypothesis that ABA is the signal for an altered proton balance of the leaf-vein-enwrapping bundle sheath cells (BSCs).METHODS•Plant Material. We used a few Arabidopsis thaliana (L.) Heynh. genotypes: wildtype (WT) of two accessions, Landsberg erecta (Ler) and Columbia (Col), and a few mutants and transformants in these backgrounds.•H+-Pumps activities. We monitored ABA effects on the H+-pump activities in the BSCs cytosol-delimiting membranes (plasma membrane and tonoplast) by monitoring the cytosol and the xylem pH, and the membrane potential (EM), by imaging the fluorescence of pH- and membrane potential (EM)-reporting probes: (a) the BSCs’ pHEXT – with the ratiometric fluorescent dye FITC-dextran petiole-fed into detached leaves in unbuffered xylem perfusion solution (XPS), (b) the BSCs’ pHCYT – with the ratiometric dye SNARF1 loaded into BSCs isolated protoplasts, and (c) the BSCs’ EM – with the ratiometric dye di- 8-ANEPPS.RESULTS•ABA increased the pHEXT; this response was abolished in an abi1-1 mutant with impaired signaling via a PP2C (ABI1) and in an aha2-4 mutant with knocked-down AHA2;•ABA depolarized the WT BSCs;•ABA increased pHCYT irrespective of AHA2 activity (i.e., whether or not AHA was inhibited by vanadate, or in the aha2-4 mutant);•The ABA-induced cytosol alkalinization was abolished in the absence of VHA activity (i.e., when VHA was inhibited by bafilomycin A1, or in the vha-a2 vha-a3 double mutant with inactive VHA);•All these results resemble the ABA effect on GCs;•In contrast to GCs, AHA2 and not AHA1 is the ABA major target in BSCs;•Blue light (BL) enabled the response of the BSCs’ VHA to ABA;•The ABA- and BL-signaling pathways acting on both BSCs’ pumps, AHA2 and VHA, are likely to be BSCs autonomous, based on (a) the presence in the BSCs of many genes of the ABA- and BL-signaling pathways and (b) ABA responses (depolarization and pHCYT elevation) demonstrated under BL in isolated protoplasts.SIGNIFICANCE STATEMENTWe reveal here an alkalinizing effect of the plant drought-stress hormone ABA on the pH on both sides of the plasmalemma of the vein-enwrapping bundle sheath cells (BSCs), due to ABA inhibition of the BSCs’ AHA2, the plasmalemma H+- ATPase and stimulation of VHA, their vacuolar H+-ATPase. Since pH affects the BSCs’ selective regulation of solute and water fluxes into the leaf, these H+- pumps may be attractive targets for manipulations aiming to improve plant drought response.

Out of the blue: Phototropins of the leaf vascular bundle sheath mediate the regulation of leaf hydraulic conductance by blue light

“…In particular, Prado et al ( 2019) demonstrated a contribution of the abundantalso in the leaf veinsaquaporin AtPIP2;1 to the diurnal and circadian rhythms of the hydraulic conductance of the Arabidopsis rosette, K ros , which peaked before midday. Thus, in addition to the likely Pf-promoting effect of the slightly acidic pH (6) perfusion solution (a link established by Grunwald et al, 2021), that Prado et al, (2019 used for their Kros measurements, the changes that they observed in Kros also reflected the effects of light and circadian rhythms (Prado et al, 2019). Thus, light-induced changes in aquaporin activity underlying the effects of light on Kros are consistent with light-induced increase of Pf that we observed.…”

“…Plant genotypes. We used WT (wild type) Arabidopsis thaliana plants ecotype Colombia, Col-0 (aka Col), the T-DNA insertion AHA2 mutants aha2-4 (SALK_ 082786) (Col) and aha2-4 complemented with SCR: AHA2 (line 56) (Col), and SCR:GFP (Col) as described previously (Grunwald et al, 2021). In addition, we used WT Arabidopsis (Col) with the glabrous mutation (WT Col-gl), phot1-5 (nph1-5) (Col-gl), phot 2-1 (npl1-1 or cav1-1) (Col-gl) and the double mutant phot1-5phot2-1 (nph1-5 npl1-1) (Col-gl) (Kagawa et al, 2001), which were kindly provided by the Ken-Ichiro Shimazaki lab (Tokyo, Japan).…”

“…Following the light treatments, the leaves were immediately placed in a LI-COR 6400XT for measurements of stomatal conductance (gS) and transpiration (E) similar to Grunwald et al (2021), with the following changes: All of the experiments were conducted in the dark at 22°C and the illumination in the LI-COR cuvette was adjusted to match the preceding light treatments. Then, the water potential (Ψleaf) was determined in a pressure chamber (ARIMAD-3000; MRC Israel) and Kleaf was calculated (as in Grunwald et al, 2021) as:…”

“…The leaves were imaged using an inverted microscope (Olympus-IX8, as detailed by Grunwald et al, 2021). Image capture and image analysis of the intra-xylem pH were as already described (ibid.…”

“…In the past decade, it has been established that bundle sheath cells (BSCs), which form a parenchymal layer that tightly enwraps the entire leaf vasculature, can act as a dynamic and selective xylem-mesophyll barrier to water and ions, and participate in Kleaf regulation (Shapira et al, 2009;Shatil-Cohen and Moshelion, 2012). Moreover, we recently discovered that the BSCs' H + -ATPase proton pump, AHA2, participates in regulating Kleaf via changes in the xylem pH and that the positive correlation observed between AHA2driven xylem acidification and Kleaf is due to an increase in the osmotic water permeability of BSCs' membranes (Grunwald et al, 2021). AHA2 is particularly abundant in BSCs (present at a >3-fold higher level than that seen in neighboring mesophyll cells; Wigoda et al, 2017) which explains how such acidification is possible.…”