Hydrogen peroxide (H2O2) was detected cytochemically in plant tissues during anoxia and re-oxygenation by transmission electron microscopy using its reaction with cerium chloride to produce electron dense precipitates of cerium perhydroxides. Anoxia-tolerant yellow flag iris (Iris pseudacorus) and rice (Oryza sativa), and anoxia-intolerant wheat (Triticum aestivum) and garden iris (Iris germanica) were used in the experiments. In all plants tested, anoxia and re-oxygenation increased H2O2 in plasma membranes and the apoplast. In the anoxia-tolerant species the response was delayed in time, and in highly tolerant I. pseudacorus plasma membrane associated H2O2 was detected only after 45 d of oxygen deprivation. Quantification of cerium precipitates showed a statistically significant increase in the amount of H2O2 caused by anoxia in wheat root meristematic tissue, but not in the anoxia-tolerant I. pseudacorus rhizome parenchyma. Formation of H2O2 under anoxia is considered mainly an enzymatic process (confirmed by an enzyme inhibition analysis) and is due to the trace amount of dissolved oxygen (below 10(-5) M) present in the experimental system. The data suggest oxidative stress is an integral part of oxygen deprivation stress, and emphasize the importance of the apoplast and plasma membrane in the development of the anoxic stress response.

The redox potential of the cell, as well as the antioxidant status of the tissue, are considered to be important regulatory constituents in an adaptive response in plants. Here the involvement of active antioxidants ascorbic acid (AA), reduced glutathione (GSH) and α‐ and β‐tocopherols in reactive oxygen species scavenging, and the effect of anoxic stress on their reduction state were studied in 4 anoxia‐tolerant and ‐intolerant plant species: Iris germanica L., Iris pseudacorus L., wheat (Triticum aestivum L. cv. Leningradka) and rice (Oryza sativa L. cv. VNIIR). The initial antioxidant content (both AA and GSH) was higher in the rhizomes of the more anoxia‐tolerant Iris spp., as compared with that of the roots of the cereals. The predominant form of ascorbate was dehydroascorbic acid (DHA) in the cereals and AA in the Iris spp. Imposition of anoxia with subsequent reoxygenation resulted in an overall depletion of the reduced forms of antioxidants. No concurrent increase in oxidised forms (DHA and conjugated glutathione) was observed in anoxic samples. α‐tocopherol content in Iris spp. was in the range 1–2 μg g−1 fresh weight, while β‐tocopherol content was higher in the anoxia‐intolerant I. germanica (7.2 μg g−1 fresh weight) as compared with the tolerant I. pseudacorus (1.5 μg g−1 fresh weight). In I. pseudacorus, a significant decrease in α‐ and β‐tocopherol levels was observed only after long‐term (45 days) anoxia. The results suggested exclusion of AA and GSH from the redox cycling under prolonged anoxia, and a concomitant decrease in the redox state, as well as an anoxia‐induced depletion of α‐ and β‐tocopherols.

The anoxia-dependent elevation of cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca(2+)](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca(2+)](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca(2+) concentration (to 0.1 mM) resulted in considerable decrease of the [Ca(2+)](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La(3+) and EGTA) and intracellular membrane Ca(2+)-channel antagonists (Li(+), ruthenium red and cyclosporine A) reduced the anoxic Ca(2+)-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca(2+)](cyt). In wheat leaf protoplasts, the amplitude of the Ca(2+)-shift little depended on the external level of Ca(2+). Wheat root protoplasts were characterized by a small shift of [Ca(2+)](cyt) under anoxia. Plasmalemma Ca(2+)-channel blockers had little effect on the elevation of cytosolic Ca(2+) in wheat protoplasts. Intact rice seedlings absorbed Ca(2+) from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca(2+). Verapamil abolished the Ca(2+) influx in rice roots and Ca(2+) efflux from wheat roots. Anoxia-induced [Ca(2+)](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca(2+) stores are important for anoxic [Ca(2+)](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca(2+)](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.

Hydrogen peroxide (H2O2) was detected cytochemically in plant tissues during anoxia and re-oxygenation by transmission electron microscopy using its reaction with cerium chloride to produce electron dense precipitates of cerium perhydroxides. Anoxia-tolerant yellow flag iris (Iris pseudacorus) and rice (Oryza sativa), and anoxia-intolerant wheat (Triticum aestivum) and garden iris (Iris germanica) were used in the experiments. In all plants tested, anoxia and re-oxygenation increased H2O2 in plasma membranes and the apoplast. In the anoxia-tolerant species the response was delayed in time, and in highly tolerant I. pseudacorus plasma membrane associated H2O2 was detected only after 45 d of oxygen deprivation. Quantification of cerium precipitates showed a statistically significant increase in the amount of H2O2 caused by anoxia in wheat root meristematic tissue, but not in the anoxia-tolerant I. pseudacorus rhizome parenchyma. Formation of H2O2 under anoxia is considered mainly an enzymatic process (confirmed by an enzyme inhibition analysis) and is due to the trace amount of dissolved oxygen (below 10(-5) M) present in the experimental system. The data suggest oxidative stress is an integral part of oxygen deprivation stress, and emphasize the importance of the apoplast and plasma membrane in the development of the anoxic stress response.

firmed the formation of dienes. However, determination of Peroxidation was studied in anoxically treated plant tissues TBARS in Iris spp. showed no lipid peroxidation in the anoxia and quantified as conjugated dienes/trienes in the total lipid tolerant I. pseudacorus. In the rhizomes of the anoxia intoler-fraction and as the production of thiobarbituric acid reactive ant I. germanica, elevated levels of TBARS correlated posi-substances (TBARS). Oxidative stress caused by re-exposure tively with conjugated diene/triene formation. The results of plants to oxygen led to an increase of conjugated diene/ triene formation in rhizomes of Iris germanica and roots of suggest that anoxic stress may induce qualitative changes in membrane lipids, as indicated by lipid peroxidation after wheat (Triticum aesti7um L.) and oats (A7ena sati7a L.), and after a long anoxic exposure (45 days) in the rhizomes of the restoration of aerobic conditions. The rate of lipid peroxidation correlated negatively with anoxic stress tolerance. very anoxia tolerant Iris pseudacorus. Second derivative (SD) spectrophotometry of the UV spectrum of lipid extracts con-

Hypoxic pretreatment is known to induce anoxia tolerance in plant species sensitive to oxygen deprivation. However, we still do not have detailed information on changes in cytoplasmic and vacuolar pH (pH cyt and pH vac ) in plants under low-oxygen availability (hypoxia) and under anoxia. To investigate this, we have studied the influence of hypoxia and anoxia on pH cyt and pH vac , glucose-6-phosphate (Glc-6-P) and nucleotide triphosphate (NTP) contents in rice (Oryza sativa L.) root tips in comparison with those of wheat (Triticum aestivum L.) with in vivo 31 P-nuclear magnetic resonance. 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 after 6 h). 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. In both plants, NTP content followed the dynamics of pH cyt . There was a strong correlation between NTP content and cytoplasmic H 1 activity ([H 1 ] cyt ¼ 10 2pH cyt ) for both hypoxic and anoxic conditions. Glc-6-P content increased in rice under anoxia and hypoxia. In wheat, Glc-6-P was not detectable under anoxia but increased under hypoxia. In this study, rice root tips were shown to behave as anoxia tolerant tissues. Our results suggest that the initial cytoplasmic acidification and subsequent pH cyt are differently regulated in anoxia tolerant and intolerant plants and depend on the external oxygen concentration.

The first studies on plant anaerobiosis started at the Department of Plant Physiology at St. Petersburg University in the beginning of the 20 th century, but interest in this subject became most intensive during the investigations of the ecological plant physiology group under the supervision of Prof. T. V. Chirkova. Their first step was focused on the mechanisms of transport of gases from the aerated aboveground part of the plant to the flooded root system. Further interest shifted towards clarifying the biochemistry of respiratory metabolism, pathways of reoxidation of the reduced cofactors, and protein and lipid metabolism of plants under anoxic conditions. The group's studies have always distinguished the comparative approach, in which the changes taking place in plants differing in resistance to oxygen deficiency were analyzed. In many ways, this research was pioneering and was recognized throughout the world. For the first time the possibility of hydrogen peroxide formation in plants under total anoxia was demonstrated. The role of cell membranes in adaptation processes was revealed. Pioneering investigations distinguished the features of photosynthesis in an oxygen-free environment and the work of an antioxidant system under conditions of anoxia and post-anoxic oxidative effects. Now, the plant ecophysiology group of the Department of Plant Physiology and Biochemistry of St. Petersburg State University concentrates on the mechanisms of anaerobic signal transduction and reveals how plant hormones regulate adaptation to anoxic and post-anoxic stresses.

Both ion fluxes and changes of cytosolic pH take an active part in the signal transduction of different environmental stimuli. Here we studied the anoxia-induced alteration of cytosolic K+ concentration, [K+]cyt, and cytosolic pH, pHcyt, in rice and wheat, plants with different tolerances to hypoxia. The [K+]cyt and pHcyt were measured by fluorescence microscopy in single leaf mesophyll protoplasts loaded with the fluorescent potassium-binding dye PBFI-AM and the pH-sensitive probe BCECF-AM, respectively. Anoxic treatment caused an efflux of K+ from protoplasts of both plants after a lag-period of 300–450 s. The [K+]cyt decrease was blocked by tetraethylammonium (1 mM, 30 min pre-treatment) suggesting the involvement of plasma membrane voltage-gated K+ channels. The protoplasts of rice (a hypoxia-tolerant plant) reacted upon anoxia with a higher amplitude of the [K+]cyt drop. There was a simultaneous anoxia-dependent cytosolic acidification of protoplasts of both plants. The decrease of pHcyt was slower in wheat (a hypoxia-sensitive plant) while in rice protoplasts it was rapid and partially reversible. Ion fluxes between the roots of intact seedlings and nutrient solutions were monitored by ion-selective electrodes and revealed significant anoxia-induced acidification and potassium leakage that were inhibited by tetraethylammonium. The K+ efflux from rice was more distinct and reversible upon reoxygenation when compared with wheat seedlings.