RESEARCH PAPER Effect of NO 2 3 transport and reduction on intracellular pH: an in vivo NMR study in maize roots Luca Espen, Fabio F. Nocito and Maurizio Cocucci* Dipartimento di Produzione Vegetale, University of Milan, Via Celoria 2, I-20133 Milano, Italy Received 8 January 2004; Accepted 18 June 2004 Abstract The effect of NO 2 3 uptake on cellular pH was studied in maize roots by an in vivo 31 P-NMR technique. In order to separate the effects on cytoplasmic pH due to NO 2 3 uptake from those due to NO 2 3 reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. 15 N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither NO 2 3 uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during NO 2 3 uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that NO 2 3 uptake is an acidifying process and suggest a possible involvement of NO 2 3 reduction in pH homeostasis. In the presence of NO 2 3 , a transient de- polarization of transmembrane electric potential differ- ence (E m ) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolar- ization accompanied by a greater ability to recover E m was found. This was related to a higher activity of the plasma membrane (PM) H 1 -ATPase. When NO 2 3 was administered as potassium salt, its uptake increased and a greater depolarization of E m took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K 1 in the control of plasma membrane activities and cell pH homeo- stasis. A possible involvement of cytoplasmic pH in the control of PM H 1 -ATPase expression during nitrate exposure is suggested. Key words: Cytoplasmic pH, maize, nitrate reductase, nitrate uptake, NMR spectroscopy, tungstate. Introduction Absorption of nitrate (NO ÿ 3 ) by roots occurs by means of at least three types of NO ÿ 3 transport systems operating at the plasma membrane, which consist of an inducible high- affinity (IHATS), a constitutive high-affinity (CHATS), and a low-affinity (LATS) transport system (Glass and Siddiqi, 1995; Crawford and Glass, 1998; Forde and Clarkson, 1999; Forde, 2000; Glass et al., 2001). Current evidence strongly suggests that all these systems use the H + electrochemical gradient to drive the uptake of NO ÿ 3 into the cells by means of a cotransport mechanism. According to this model, nitrate uptake increases as external pH de- creases, with transient depolarization of the plasma mem- brane (McClure et al., 1990; Ullrich and Novacky, 1990). In this context, one should therefore expect that such a transport mechanism would lead to acidification of the cytoplasm, at least in the earlier period after the addition of nitrate into the medium. Nevertheless, experimental evidence is controversial. It has been observed that, in roots of maize seedlings growing in nutrient solutions at different pH and supplemented with nitrate, only small changes in cytoplamic pH occurred (Gerenda ´s et al., 1990). These results are attributed by the authors to the presence of a tight regulatory mechanism of intracellular pH. Moreover, in Limnobium stoloniferum root hairs, nitrate uptake leads to an increase in cytoplasmic pH (Ulrich and Novacky, 1990). The authors speculate that this could be ascribed to nitrate assimilation which is a proton-consuming process (Raven, 1985, 1986; Ullrich and Novacky, 1990). The pH changes occurring in root cells during nitrate exposure could be directly related to the activities of both NO ÿ 3 transport and reduction and N assimilation, as well as to the effectiveness of the intracellular pH regulatory mechanisms (Kurkdjian and Guern, 1989; Li and Oaks, 1993; Sivansankar et al., 1996; Crawford and Glass, 1998). It is interesting to observe that in the chl1 mutant of * To whom correspondence should be addressed. Fax: +39 (0)2 50316521. E-mail: maurizio.cocucci@unimi.it Journal of Experimental Botany ª Society for Experimental Biology 2004; all rights reserved Journal of Experimental Botany, Page 1 of 9 DOI: 10.1093/jxb/erh231 Advance Access published August 13, 2004 by guest on June 2, 2013 http://jxb.oxfordjournals.org/ Downloaded from