Regulation of intracellular pH during anoxia in rice coleoptiles in acidic and near neutral conditions

Abstract

Rice coleoptiles, renowned for anoxia tolerance, were hypoxically pretreated, excised, ‘healed’, and then exposed to a combination of anoxia and pH 3.5. The putative acid load was confirmed by net effluxes of K+ to the medium, with concurrent net decreases of H+ in the medium, presumably mainly due to H+ influx. Yet the coleoptiles survived the combination of anoxia and pH 3.5 for at least 90 h, and even for at least 40 h when the energy crisis, inherent to anoxia, had been aggravated by supplying the coleoptiles with 2.5 mM rather than 50 mM glucose. Even in the case of coleoptiles with 2.5 mM glucose, an accumulation ratio of 6 for Cl– was attained at 4 h after the start of re-aeration, implying plasma membrane integrity was either maintained during anoxia, or rapidly restored after a return to aerated conditions. Cytoplasmic pH and vacuolar pH were measured using in vivo 31P nuclear magnetic resonance spectroscopy with 50 mM glucose in the basal perfusion medium. After 60 h in anoxia, external pH was suddenly decreased from 6.5 to 3.5, but cytoplasmic pH only decreased from 7.35 to 7.2 during the first 2 h and then remained steady for the next 16 h. During the first 3 h at pH 3.5, vacuolar pH decreased from 5.7 to 5.25 and then stabilized. After 18 h at pH 3.5, the initial values of cytoplasmic pH and vacuolar pH were rapidly restored, both upon a return to pH 6.5 while maintaining anoxia and after subsequent return to aerated solution. Summing up, rice coleoptiles exposed to a combination of anoxia and pH 3.5 retained pH regulation and cellular compartmentation, demonstrating tolerance to anoxia even during the acid load imposed by exposure to pH 3.5.

pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NO3−

“…However, 0, 0.2 or 2 mM β-alanine was used in one experiment, as a check, as it was neecessary to relate the present findings to earlier experiments using 31 P-nuclear magnetic resonance (NMR) spectroscopy, which used β-alanine to buffer the solutions at pH 3.5 (Kulichikhin et al , 2009). During preparation of the pH 3.5 solution, the pH adjustment was with H 2 SO 4 .…”

“…Excised coleoptile tips provide a simpler system than intact seedlings and the data also assisted in elucidating the mechanisms involved in the regulation of cytoplasmic and vacuolar pH in coleoptile tips measured in our previous [ 31 P]NMR spectroscopy experiments (Kulichikhin et al , 2009). …”

“…Assessment of participation of contributions of organic acids to buffering and to the biochemical pHstatContributions of organic acids to cellular buffering and the biochemical pH stat were based on the measured pH of the vacuole (Kulichikhin et al , 2009) and the p K a of each organic acid, using the Henderson–Hasselbach equation (both based on Conn and Stumpf, 1972). …”

Flooding tolerance: suites of plant traits in variable environments

“…Such damage can, however, be avoided (or substantially delayed) in anoxia-tolerant plants (e.g. rice coleoptiles, Kulichikhin et al 2009 and S 2-can accumulate to toxic levels in root tissues (Jackson and Drew 1984). In addition, volatile lower organic acids (e.g.…”

Protonation and pK changes in protein–ligand binding

“…At the cell level, different cell compartments (organelles) have different characteristic pH as outlined in Refs. 24,69 A short sample list of characteristic sub-cellular pH values is shown in Table 4.…”