Intracellular pH in rice and wheat root tips under hypoxic and anoxic conditions

Abstract

The influence of anoxia and hypoxia on dynamic of intracellurar pH and ATP content in rice and wheat root tips was investigated with ³¹P-NMR spectroscopy. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6-7.7 to 7.1), which was followed by slow partial recovery (0.3 units). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6-7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. Anoxic wheat root tips were deficient in ADH induction, whereas increased activity of alcoholic fermentation enzymes took place in anoxic rice root tips, as well as in both species after hypoxic treatment. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]cyt = 10–^pHcyt) for both hypoxic and anoxic conditions. In this addendum we want to focus the reader’s attention on the importance of adequate experimental design when hypoxia is under investigation and on some further perspectives of intracellular pH regulation in plants under anaerobic conditions.

Introduction

Intracellular pH regulation under oxygen deprivation stress is a complex interaction of biophysical (active H⁺ transport from the cytoplasm, accompanied with concurrent influxes of cations, or effluxes of anions to maintain the charge balance) and biochemical (H⁺ buffering and H⁺ producing/consuming reactions) pH_cyt-stator mechanisms.1,2 Previously we have shown that there was a 6-fold decrease in malic acid content in rice roots after 6 h of anoxia, whereas in wheat roots malate decreased gradually during 24 h of exposure in oxygen-free conditions (Fig. 1).3 Our recent NMR investigations4 brought to light the difference in pH_cyt dynamics between rice and wheat root tips at the first hours of anoxic treatment: the initial cytoplasmic acidification in rice root tips was followed by partial recovery, which was absent in wheat root tips. The response to hypoxia was similar and recovery took place for both cereal species. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt=10^−pHcyt) for both hypoxic and anoxic conditions.4 Anoxic wheat root tips displayed the lowest pH_cyt value and the least NTP content throughout the experiment. Further investigation showed that wheat root tips were deficient in ADH induction under anoxia, whereas in hypoxic wheat and in rice root tips in both anoxia and hypoxia the increase of ADH activity took place (Fig. 2, unpublished data).

The rapid decrease in endogenous malic acid content in anoxic rice roots reflects the operation of biochemical pH-stat, which seems not to be effective in wheat roots. Nevertheless, the response to hypoxia in both pH_cyt dynamics and ADH induction was similar in rice and wheat root tips.

Low-oxygen state is of great interest because of hypoxia induced anoxia tolerance.5 Previous investigations have concentrated predominantly on the differences between hypoxically pretreated and nonpretreated plant and tissues subjected to anoxia, whereas the investigations of hypoxic state itself are few in numbers. Hypoxia may be characterized as a condition, when oxygen concentration in the medium is lower than the critical O₂ pressure for respiration (COP). This is a wide range of oxygen concentrations from zero oxygen (anoxia) to COP, where oxygen concentration is a determinant of the physiological state of cell/tissue/plant. The main O₂-dependent physiological characteristic of plant cell under anaerobic condition is the energy state—ATP concentration and adenylate energy charge (AEC)—which determines the ability to maintain membrane transport and biosynthetic events. This is why in the experiments, where hypoxic conditions are applied, the degree of hypoxia should be assessed. Without such assessment it will be difficult to compare the data with any previous and future research results.

For this reason, we would like to focus the reader's attention in this addendum on the importance of adequate experimental design, especially when hypoxic state is under investigation. Also, we would like to discuss some further perspectives of investigations on intracellular pH regulation under anaerobic condition.

Hypoxic and Anoxic Experiments: The Importance of Experimental Design

The simplest approach to characterize hypoxia is to investigate the rate of O₂ consumption of cell, tissue, or plant as a function pO₂ followed by mathematical processing of experimental data to determine maximal rate of O₂ consumption (V_max) and COP. We have applied this approach in our investigation.4 As a degree of hypoxia we used the ratio of oxygen consumption rate at exact O₂ concentration to V_max. The ratio is a dimensionless quantity and allows the comparison of plants, tissues, and conditions with different COP and V_max.

Another characteristic of oxygen deprivation is the energy state - ATP concentration, ATP/ADP ratio or AEC. It has been shown long ago, that the value ATP/ADP ratio and AEC are controlled by pO₂.6 A decrease in oxygen concentration below COP lead to a decrease in the ATP content, ATP/ADP ratio and AEC value simultaneously with the rate of O₂ consumption, but in tissues having a high glycolytic capacity the correlation between ATP/ADP ratio and the respiratory rate breaks down as O₂ tension decreases, due to the contribution of fermentative processes. Replacement of ATP by inorganic pyrophosphate (PP_i) on some steps of glycolytic cleavage under anaerobic condition may also lead to increased ATP yield of glycolysis7 and affect the correlation. Hence, the measurement of O₂ consumption in dependence on pO₂ may not be enough to characterize experimental condition. In some cases, the measurements of ATP/ADP ratio and AEC should also to be done.

Anaerobic conditions may also be created by inhibiting the cytochrome c oxidase (COX) with azide or cyanide, rather than by removing O₂ from the medium. This treatment is known as “chemical anoxia”. Despite the visual consequence of inhibitor application is similar with the real anaerobic treatment, the background could be different, as has been shown by earlier.8 Sometimes inhibitor application leads to the same degree of ATP hydrolysis as anoxia, but without strong cytoplasmic acidification.9 This fact reflects more site-specific effect of respiratory inhibitors compared with oxygen deprivation. Another difference between real and “chemical” anoxia is that in the latter oxygen is still present at relatively high concentrations in the medium and in the cell. When COX is inhibited, the key components of mitochondrial electron transport chain will be reduced leading to increased production of reactive oxygen species (ROS). There is evidence that ROS are signaling molecules and can regulate gene expression.10 The ROS-signalling system may be expected to act in another way to the application of respiratory inhibitors compared with real anoxia and/or hypoxia.

pH_cyt Regulation Under Oxygen Deprivation: Further Perspectives

To reveal the origin of cytoplasmic acidification and the mechanism of pH_cyt regulation, an assessment of relative contribution from all H⁺-producing and consuming mechanisms should be made.11 Our data show that the biochemical pH-stat plays an important role at the initial stages of low-oxygen conditions. A metabolomics approach12 would allow the determination of the full set of anaerobic end-products and intermediates and to define their contribution to H⁺ balance in the cytoplasm. An application of metabolic control analysis (MCA)13 to the processing of metabolomic data obtained, may help in the determination of enzyme reactions critical for biochemical pH_cyt regulation under anaerobic condi tions. Metabolomic data combined with data of ion fluxes between the external medium, cytoplasm and vacuole, will create a full picture of pH_cyt regulation in the plant tissue under investigation.

Abbreviations

ADH	=	alcohol dehydrogenase
AEC	=	adenylate energy charge: [(ATP) + 0.5(ADP)] / [(ATP) + (ADP) + (AMP)]
ATP	=	adenosine triphosphate
ADP	=	adenosine diphosphate
COP	=	critical oxygen pressure for respiration
COX	=	cytochrome c oxidase
NMR	=	nuclear magnetic resonance spectroscopy
pHcyt	=	cytoplasmic pH
pO2	=	partial oxygen pressure
Vmax	=	maximal rate of oxygen consumption

Figures and Tables

Figure 1 The influence of anoxia on malate content in roots of intact rice and wheat seedlings.

Figure 2 The influence of anoxia and hypoxia on ADH activity in isolated rice and wheat root tips. Stars show significant differences between experimental and control values.

Acknowledgements

This investigation has been supported by grants from Academy of Finland (no: 171987, 178918 and 1207898).

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