Cytoplasmic acidification as an early phosphorylation-dependent response of tobacco cells to elicitors

Abstract

Abstract. Elicitor-induced cytoplasmic pH changes of tobacco (Nicotiana tabacum L. cv. Xanthi) cells grown in suspension cultures were explored under a variety of conditions by using a flexible technique based on the distribution of [14C] benzoic acid between the intracellular and extracellular compartments. Comparison of data obtained by this technique and by 31p-nuclear magnetic resonance spectrometry qualifies the benzoic acid distribution method as a convenient and reliable way to probe cytoplasmic pH variations. Various elicitors shown to induce several defense-related responses in tobacco cells, namely oligogalacturonides of degree of polymerization 7-20, pectolyase from Aspergillusjaponicus, Phytophthora megasperma crude elicitors and purified cryptogein, triggered cytoplasmic acidifications differing in intensity and kinetics according to the signal molecule. In contrast, no changes in cytoplasmic protons and external pH were observed in cells treated with short galacturonide oligomers, or with soybean-specific hepta 13-glucoside from P. megasperma, which are devoid of elicitor activity in tobacco cells. The oligogalacturonide-induced cytoplasmic acidification was inhibited by two structurally unrelated protein kinase inhibitors, staurosporine and 6-dimethylaminopurine, which both reduced the external alkalinization response to the elicitor. The protein phosphatase inhibitor calyculin A alone behaved as an elicitormimicking molecule in triggering cytoplasmic acidification, again associated with extracellular alkalinization. These results indicate that the increase in the cytoplasmic concentration of protons may be considered as a common early intracellular response of tobacco cells to elicitors, associated with the extracellular alkalinization response and controlled by protein phosphorylation.Abbreviations: BA(H) = benzoic acid (protonated form); 6-DMAP = 6-dimethylaminopurine; DP = degree of polymerization; Mes = 2-(N-morpholino)ethanesulfonic acid; OG = oligogalacturonide; pHc = cytoplasmic pH; 31p-NMR = nuclear magnetic resonance spectroscopy of 31p atoms Correspondence to: Y. Mathieu; FAX: 33 (1) 69 82 37 68

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells1

“…The effect of a change in pH c on auxin transport has been less documented. Potassium propionate has been shown to provoke a dose-dependent acidification of the cytosol in suspensioncultured tobacco cells, as shown by 31 P-NMR spectroscopy experiments and determination of distribution of [ 14 C]BA (Mathieu et al, 1996a). Cytosolic acidification was maximal after 5 to 10 min of treatment and increased more than 1 pH unit at the highest concentrations used in our work.…”

“…2). This treatment allows ⌬pH changes without modifying intracellular pH (approximately 7.5), at least in the short term (Mathieu et al, 1996a). At all of the pH e values, [ 14 C]2,4-D accumulated less in the presence than in the absence of 50 m 2,4-D (Fig.…”

“…3A). Yet, the treatment pro- voked a strong decrease in pH i , as observed by Mathieu et al (1996a) (Fig. 3B) by 50% at 7.5 mm (approximately 0.8 mm PAH) and in totality at 20 mm (approximately 2.2 mm PAH), as well as the carrier-mediated efflux of [ 3 H]NAA, whether this latter component was uncorrected (Fig.…”

“…The inhibitors were expected to permeate the PM according to their chemical structure and their ability to elicit rapid defense responses in plant cells (MacKintosh et al, 1994;Mathieu et al, 1996a;Smith and Walker, 1996). (Table III), whereas calyculin A (0.2 m, 30 min) and cantharidin (50 m, 60 min) inhibited the activity of the influx carrier by 16 and 55% (not shown) and that of the efflux carrier by 57 and 77% (Table III).…”

Induction of defense responses in tobacco by the protein Nep1 from Fusarium oxysporum

“…Mathieu et al [46] have characterized an increase in cytoplasmic H + as a common early response of tobacco cells to elicitors and also showed that the uptake of H + by tobacco cells was mediated by protein phosphorylation. Proton-ATPases and G proteins may also be involved in the elicitor-induced import of H + [47], and the H + import observed with the 24-kDa protein is likely linked to the K + efflux (Fig.…”