Suitability of Arabidopsis for studies on intracellular pH regulation: correlation between H⁺ pump activity, cytosolic pH and malate level in wild‐type and in a mutant partially insensitive to fusicoccin

Abstract

Data are presented on the suitability of Arabidopsis thaliana seedlings for studies on intracellular pH regulation. In this material, grown in the dark in liquid medium, the determination of weak acid distribution at equilibrium provides an adequate method for calculating cytosolic pH values, in spite of the failure of benzylamine as a vacuolar pH probe. The stimulation of the H+ pump by K+ or K+ and fusicoccin (FC) is associated with a marked alkalinization of both cytosol and cell sap, and with a strong increase in malate level, whereas its inhibition by erythrosin B (EB) leads to the opposite effects. A good quantitative correlation is evident between the changes in net H+ extrusion and those in intracellular pH and malate content, in particular, with FC+K+. Cell sap buffer capacity is strongly influenced by the different treatments, its changes being substantially accounted for by changes in malate level. A comparison between the values of intracellular pH and malate level in wt and in the 5‐2 mutant shows that in the mutant the cytosolic pH is always more acidic, and the intracellular alkalinization induced by FC+K+ and also by K+ alone is significatively lower. These results support the view that the partial insensitivity of 5‐2 to FC is due to a reduced functionality of the H+‐extruding system on which FC acts, and that the depression of the H+ pump activity in the mutant does not depend on a possible regulation by constitutively higher cytosolic pH values.

Fusicoccin‐induced catalase inhibitor is produced independently of H+‐ATPase activation and behaves as an organic acid

“…The production of transgenic catalase‐deficient tobacco lines allowed the study of the role of H 2 O 2 in pathogen defence (Chamnongpol et al 1996); in this context, of particular interest was the finding that transgenic Cat1‐deficient tobacco leaf disks were proved to be strongly impaired in their ability to remove extracellular H 2 O 2 , suggesting that peroxisomal catalase can also functionally protect cells against H 2 O 2 produced at a distant location (Willekens et al 1997). These data indeed supported the belief that for FC‐treated cells, in which the strong cytoplasmic pH (pH cyt ) increase following H + ‐ATPase activation (Beffagna and Romani 1991, 1994, Marrè et al 1987) allows only a low NADPH oxidase‐mediated ROS production (Beffagna et al 2005), the achievement of the threshold apoplastic H 2 O 2 level needed for triggering the defence responses might be provided by a fall in catalase activity.…”

“…A w/v ratio of 100 mg FW 3 ml −1 was also maintained for all the following treatments. Unless otherwise specified, treatments were routinely carried out in a basal medium made of 0.2 m M CaSO 4 , 2 m M K + (supplied as K 2 SO 4 ) and 2 m M MES/Li (pH 6), namely, a condition of partial stimulation of the PM H + ‐ATPase (Beffagna and Romani 1991, 1994, Marrè et al 1987). The data reported are the mean of different numbers of experiments ± se , as indicated in the figure legends.…”

“…Most commonly, the rise in apoplastic H 2 O 2 content occurring in plants exposed to both biotic and abiotic stresses reflects an increased production of reactive oxygen species (ROS), an event substantially mediated by an NADPH oxidase complex operating at plasma membrane (PM) level (Cazalè et al 1998, Desikan et al 1996, Lamb and Dixon 1997, Mathieu et al 1991, Pugin et al 1997). Conversely, no increase in ROS production but a decreased cell's capability for H 2 O 2 scavenging was recently proposed to be responsible for the apoplastic H 2 O 2 accumulation elicited in Arabidopsis thaliana cells by fusicoccin (FC), a fungal phytotoxin mainly known for its capacity to strongly activate the P‐type H + ‐ATPase (Beffagna and Romani 1994, Beffagna et al 1977, Marrè 1979, Rasi‐Caldogno et al 1986), namely, the major ion pump in the PM of all plant tissues (Palmgren 2001). This peculiar FC effect was apparently due to the ability of FC to promote the production of an as yet unknown factor behaving as a non‐competitive catalase inhibitor, which could be easily detected even in the incubation media of FC‐treated cells (Beffagna and Lutzu 2007).…”

“…Among the organic acids, for instance, ascorbate (AA), although actually considered the major apoplastic antioxidant playing a pivotal role in maintaining the ideal redox balance in plant tissues (Foyer and Noctor 2005), was also reported to reversibly inhibit catalase activity (Davison et al 1986); moreover, AA efflux from the cytosol was found to be progressively raised by increasing FC concentrations (Takahama 1996). Several effectors eliciting a stimulation of H + extrusion, among which FC, were proved to cause also a marked increase in cytosolic malate level (Beffagna and Romani 1994), reflecting the biochemical pH‐stat response to the pH cyt increase due to the activation of the PM H + ‐ATPase (Davies 1973, Raven and Smith 1974). An important role for citrate excretion into the apoplast in response to environmental stresses such as P i deficiency and exposure to high Al 3+ levels was also established.…”

Sugar Retrieval by Coats of Developing Seeds of Phaseolus vulgaris L. and Vicia faba L.

“…In another test of whether the proton motive force (pmf) was generated by a plasma membrane H + -ATPase (P-type H + -ATPase), excised seed coats were treated with erythrosin B (EB). At the concentrations employed, EB is an inhibitor of plasma membrane H + -ATPases in seed coats (Walker et al 1995) and in other plant systems (see Beffagna and Romani 1994, and references cited therein). Under these conditions, sugar influx was slowed by 10-34% (Table 3).…”