Effects of Deuterium Oxide on Growth, Proton Extrusion, Potassium Influx, and in Vitro Plasma Membrane Activities in Maize Root Segments

Abstract

Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by 2H20 in the incubation medium. Short-term exposure (30 min) to 2H20 slightly reduced 02 uptake and significantly increased ATP levels. 2H20 inhibited H' extrusion in the presence of both low (0.05 mM) and high (5 mM) external concentrations of K' (about 30 and 53%, respectively at 50% [v/v] 2H20).Experiments on plasma membrane vesicles showed that H+-pumping and ATPase activities were greatly inhibited by 2H20 (about 35% at 50% [v/v] 2H20); NADH-ferricyanide reductase and 1,3-B-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by 2H20. Therefore, the effect of short incubation time and low concentration of 2H20 is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H -ATPase. K+ uptake was inhibited by 2H20 only when K+ was present at a low (0.05 mM) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (Em) was slightly hyperpolarized by 2H20 at low K+, but was not affected at the higher K+ concentrations. These results suggest a relationship between H+ extrusion and K' uptake at low K+ external concentration.plast, probably through a direct or chaotropic effect of 2H20 (7). The effect of 2H20 on membrane functions has not yet been specifically investigated in plant materials. An effect of 2H20 on plant membrane functions might be hypothesized on the basis of the consideration that H+ translocating activities, which play a central role in the function of plant membranes (27), could also utilize 2H' even if to a lesser extent.The experiments described in this paper were aimed at studying the effects of 2H20 on H+ extrusion and related transport activities in subapical maize (Zea mays) root segments. Plasma membrane vesicles of high purity from maize roots were used to compare the in vivo effect of 2H20 on H+ transport with the in vitro effect of 2H20 on H+ pumping and ATPase activities and to clarify whether 2H20 inhibits specifically the H+-translocating activities or membrane activities as a whole. Finally, the possibility of using 2H20 as a tool to investigate the involvement of H+ in membrane transport has been analyzed. MATERIALS AND METHODS Plant MaterialIncubation in 2H20 of animal and plant tissues and organs has been used extensively for proton density-labeling experiments to study synthesis, degradation, and turnover of proteins (8), to investigate the role of water in the structure and function of biosystems (29), and to suppress solvent signal from the water in proton NMR (11). However, a toxic effect of 2H20 has been observed on growth of Escherichia coli (4), growth and development of plants (2,6,28), and seed germination (12). It has been suggested that these effects, obtained at high 2H20 concentrations and after long exposur...

Cadmium-Induced Sulfate Uptake in Maize Roots

“…An actual effect of Cd 2ϩ on the electric component of ⌬ H ϩ can be excluded because the values of E m in root cells were only slightly more negative in Cd-treated than in control plants (Table II). Similar to sulfate, K ϩ and Pi transports are dependent on H ϩ electrochemical gradient across the plasma membrane (UllrichEberius et al, 1984;Thibaud et al, 1988;Sacchi and Cocucci, 1992;Daram et al, 1998), but unlike sulfate, their uptake was inhibited or unaffected, respectively, by Cd treatment (Fig. 4, B and C).…”

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein

“…Movement of label from H 2 18 O into amino acids is rather slow (Zhou et al, 2012). Label moves rapidly from 2 H 2 O into amino acids and proteins (Mitra et al, 1976;Yang et al, 2010), but discrimination against 2 H 2 O can lead to changes in metabolite pools and fluxes, to changes in gene expression indicative of stress, and to inhibition of growth (Thomson et al, 1963;Sacchi and Cocucci, 1992;Kushner et al, 1999;Yang et al, 2010). Yang et al (2010) showed that inhibitory effects on growth can be decreased, although not totally prevented, by decreasing enrichment to 30% and that the stressrelated changes in transcript levels were less marked when a protocol was used in which seedlings were pulsed with 2 H 2 O and the labeling kinetics were analyzed in a chase with water.…”

The plant plasma membrane proton pump ATPase: a highly regulated P-type ATPase with multiple physiological roles

“…The facts that the size of this ion is close to that of other ions transported by P-type ATPases and that the enzyme can be inhibited by D 2 O support this hypothesis [19]. Furthermore, homology modeling suggests the presence of a hydronium binding site contributed by the fourth, fifth, and sixth transmembrane segments of the Arabidopsis H + -ATPase AHA2 [20], and Ileu282 was shown to be involved in this binding pocket [21].…”