B. B. Vartapetian

Plant anaerobic stress as a novel trend in ecological physiology, biochemistry, and molecular biology: 2. Further development of the problem

This review is a logical development of a previous publication, which summarized the main results of the early period of the systematic and active studying of hypoxic and anoxic stresses in plants. These studies laid a foundation for a new scientific discipline in biology, the investigation relevant to plant anaerobic stress. This review considers a further development of this trend when the investigations embraced a wider set of topics and the discipline acquired an international recognition. The results obtained during last decades by physiologists, biochemists, and molecular biologists engaged in the problem of plant anaerobic stress confirmed the correctness of a concept of the two principal strategies of plant adaptation to hypoxia and anoxia conditions. They are “true” tolerance manifesting at the molecular level under conditions of oxygen deficiency or its absence and “apparent” tolerance, which is realized by avoidance of anaerobiosis due to the long-distance oxygen transport. Therefore, experimental material available now is considered and discussed in this review mainly in the light of these principal notions. Especial attention is paid to the role of stress proteins, which synthesis is induced under hypoxia and anoxia. The results of these experiments confirmed earlier conclusions about the key role of energy (glycolysis and alcoholic fermentation) and carbohydrate (mobilization and utilization of reserved carbohydrates) metabolism in plant adaptation to oxygen deficiency or its absence from the environment. The phenomenon of hypoxic acclimation and its role in plant adaptation to anoxia are also considered. Along with these topics, a further development of pH-stat theory is discussed. A special attention is paid to plant strategy realized by the formation of the net of air-filled spaces (aerenchyma) and long-distance oxygen transport from aerated plant parts to those located in anaerobic environment (apparent tolerance). Among other important aspects, we consider (1) post-anaerobic plant injury by free oxygen radicals; (2) the physiological role of alternative pathways of plant adaptation (nitrate reduction and lipid synthesis); (3) the phenomenon of the adaptation syndrome in plants and possible molecular mechanisms of its realization; and (4) some biotechnological advances in the field of genetic and cell engineering used for the creation of plants more tolerant to anaerobic stress.

Protective effect of exogenous nitrate on the mitochondrial ultrastructure ofOryza sativa coleoptiles under strict anoxia

SummaryThe effect of exogenous KNO3, the terminal acceptor of electrons in oxygen-free medium, on mitochondrial ultrastructure and on the growth rate of 4-day-old rice coleoptiles under strictly anoxic conditions was studied. Exogenous nitrate (10 mM) did not exert any significant effect on the growth rate of coleoptiles of intact seedlings compared to their growth in KNO3-free medium. Anaerobic incubation of detached coleoptiles in KNO3-free medium for 48 h resulted in the complete destruction of mitochondrial and other cell membranes. In the presence of KNO3, no mitochondrial-membrane destruction was observed even after 48 h anoxia although the mitochondrial ultrastructure was modifed. Cristae were arranged in parallel rows and elongated dumbbell-shaped mitochondria appeared in some cells. The data obtained indicate a protective role of exogenous nitrate as electron acceptors in oxygen-free medium. The results of the present investigation are discussed and compared with reports of either markedly damaging or favorable effects of exogenous nitrate on the growth, metabolism, and energetics of rice and other plants under hypoxic and anoxic conditions.

Plant Anaerobic Stress as a Novel Trend in Ecological Physiology, Biochemistry, and Molecular Biology: 1. Establishment of a New Scientific Discipline

This review attempted to follow the establishment of a novel branch of biology arisen at the interfaces between plant physiology, biochemistry, and molecular biology—plant anaerobic stress. Most attention was given to the early period of these investigations, the activity of the members of International Society for Plant Anaerobiosis in particular, and the contribution of Russian scientists, who played a significant role at that time in the establishment and international recognition of this new trend. In this connection, the following points are considered: (1) Crawfords metabolic theory, which could not withstand experimental verification but induced an active discussion, thus stimulating further investigations in this field; (2) a concept of two main strategies of plant adaptation to anaerobic stress (true and apparent adaptation), which was put forward based on the following experimental data: (a) a discovery of a paradoxical phenomenon of hyper-sensitivity, but not hyper-resistance to anoxia, of the flood-tolerant plant roots (“apparent” tolerance); (b) the elucidation of the physiological role of oxygen transported from aerated organs of flood-tolerant plants to the roots inhabiting anaerobic environment; (c) demonstration of the key role of both energy metabolism, and (d) substrate providing for glycolysis and ethanolic fermentation in plants manifesting “true” tolerance to oxygen deprivation; (3) the discovery of plant stress proteins; and finally (4) pH-stat theory put forward by Davies.

Exogenous Nitrate as a Terminal Acceptor of Electrons in Rice (Oryza sativa) Coleoptiles and Wheat (Triticum aestivum) Roots under Strict Anoxia

To elucidate the physiological role of exogenous nitrate under anaerobic conditions, we studied the effect of 10 mM KNO3 on the mitochondrial ultrastructure in rice (Oryza sativa L.) coleoptiles and in wheat (Triticum aestivum L.) roots, detached from four-day-old seedlings, under strict anoxia. In wheat roots, following 6-h-long anoxia in the absence of exogenous nitrate, the mitochondrial membranes were partially degraded and, after 9 h under anoxia, the mitochondrial membranes and the membranes of other organelles were completely destroyed. In rice coleoptiles, the partial membrane degradation was observed only after 24 h and their complete breakdown after 48 h of anaerobiosis. In the presence of exogenous nitrate, no membrane destruction was noticed even after 9 and 48 h of anaerobiosis in wheat roots and rice coleoptiles, respectively. These results indicate that exogenous nitrate exerts protective action as a terminal electron acceptor, alternative to the molecular oxygen. Our findings are compared with the results of other researchers concerning the adverse or favorable nitrate action on plant growth, metabolism, and energy status under anaerobic stress.

Induction of alcoholic and lactic fermentation in the early stages of anaerobic incubation of higher plants

Abstract The dynamics of ethanol and lactate accumulation in detached roots of pea ( Pisum sativum ) and of rice ( Oryza sativa ), embryo of pea, discs of maple tree leaves ( Acer platanoides ), and in apple fruit tissues ( Malus domestica ) were investigated on transferring them from aerobic to anoxic conditions. In each of the tissues studied, the induction of lactic and alcoholic fermentations was a characteristic of the tissue under investigation: in pea roots both ethanol and lactate were formed within several min after the creation of anaerobic conditions, ethanol accumulation being much greater than that of lactate; no accumulation of lactate was observed in pea embryo, although ethanol synthesis took place rapidly from the very beginning of anaerobic incubation; in contrast to roots and embryo of peas; in rice roots, anaerobiosis immediately induced lactic fermentation, whereas alcoholic fermentation was much less pronounced. Tissues of apple fruit reacted to anaerobic conditions in a similar way to rice roots, although the rate of lactate and of ethanol accumulation was much lower. Maple leaves produced only ethanol under anaerobic conditions. The results obtained and findings of other investigators show that there is no single universal mechanism of induction of lactic and alcoholic fermentation in the organs of higher plants when they are transferred from aerobic to anaerobic conditions.

On the Physiological Role of Anaerobically Synthesized Lipids in Oryza sativa Seedlings

The objective of this work was to elucidate a possible adaptive role of lipid biosynthesis and unsaturated fatty acids (FAs), esterified to lipids, as terminal acceptors of electrons, alternative to molecular oxygen, in the shoots of rice seedlings (Oryza sativa L.) under conditions of strict anoxia. Biosynthesis of lipids and their accumulation, as well as the reduction of double bonds in unsaturated FAs, were studied by electron microscopic observation of the accumulation of lipid bodies in the cytoplasm and by the biochemical analysis of FAs in shoot lipids before and after anaerobic incubation of the shoots. The experiments were carried out with intact coleoptiles after 5 and 8 days of anaerobic germination of seeds (primary anoxia) and with detached shoots, preliminarily grown in air and then subjected to anoxia in the presence of 2% glucose for 48 h (secondary anoxia). In these experiments, lipid bodies did not accumulate in the cytoplasm under anoxic conditions. Lipid bodies appeared only during 48-h anaerobic incubation of detached coleoptiles in the absence of exogenous glucose, when mitochondria degraded. There was no change either in the double bond index of FAs, or in the qualitative and quantitative composition of FAs during shoot anaerobic incubation. We conclude that neither lipids synthesized under anaerobic conditions nor esterified unsaturated FAs are involved in plant adaptation to anaerobiosis as terminal acceptors of electrons, alternative to molecular oxygen. Lipid biosynthesis under anoxic conditions, which was demonstrated for anoxia-tolerant seedlings of Oryza sativa and Echinochloa phyllopogon in experiments with radioactive precursors, 14C-acetate and 3H-glycerol, is only the manifestation of a turnover of saturated FAs and various classes of lipids, which stabilizes cell membranes under adverse conditions of strict anoxia.

Effect of the ipt gene expression on wheat tolerance to root flooding

The effect of enhanced cytokinin synthesis due to expression of the ipt gene from Agrobacterium tumefaciens on plant tolerance to root flooding was studied. Transgenic wheat (Triticum aestivum L.) plants carrying the ipt gene were more tolerant to flooding than wild-type plants. The effect of transformation was manifested in the higher yield and less growth inhibition during flooding. The measurements of activities of antioxidant enzymes, superoxide dismutase and catalase, as well as MDA content during flooding revealed differences between wild-type and transgenic plants that correlated with their tolerance. These results point to the protective role of cytokinins during wheat root flooding.

The Response of Sugarcane (Saccharum officinarum) Cultured Cells to Anoxia and the Selection of a Tolerant Cell Line

The effect of anoxia on the sugarcane (Saccharum officinarum L.) cultured cells was studied in order to elaborate a technique for in vitro selection of cell lines, which would be tolerant to anaerobic stress. Inhibitory and lethal doses of anaerobic incubation were established from the state of the mitochondrial ultrastructure during the anaerobic incubation of cells either with or without exogenous glucose, as well as from the pattern of the post-anaerobic callus growth. An intact state of the mitochondrial ultrastructure and the viability of some cells in the presence of 3% glucose were shown to be maintained for at least 14 days of anaerobic incubation, while the index of post-anaerobic growth decreased by almost 50% even after 72-hour-long anaerobiosis. In the absence of exogenous glucose, a marked destruction of mitochondria and a twofold decrease in the callus growth index were observed as early as after six-hour-long anaerobic stress. A 48-hour-long incubation under these conditions resulted in the maintenance of the intact ultrastructure only in 7–10% of cells, while a 96-hour-long anaerobiosis brought about the complete degradation of the subcellular structure and cell death. A 48-hour-long anaerobiosis without exogenous glucose was chosen for selecting the anoxia-tolerant cell lines. After three cycles of selection, the anoxia tolerance of the selected cell line exceeded the respective index of the initial callus several-fold. In selected line, about 50% of cells retained viability and could resume growth even after 96-hour-long anaerobic incubation. The experimental results obtained were used to determine the possible causes of the heterogeneity of callus cells as regards their anoxia resistance.

Demonstration of plant adaptation syndrome in plants and possible molecular mechanisms of its realization under conditions of anaerobic stress

Electron-microscopic examination of mitochondrial membrane ultrastructure in detached leaves of four-day-old wheat (Triticum aestivum L.) seedlings incubated under conditions of strict anoxia in the presence of exogenous glucose and cycloheximide or in the absence of these compounds revealed a paradoxical phenomenon: in the absence of exogenous glucose and cycloheximide, even a short-term (15–30 min) anaerobiosis resulted in a pathological destruction of mitochondria (swelling and the loss of cristae); however, a longer uninterrupted anaerobiosis (3–4 h) did not induce further mitochondria degradation but, in contrast, resulted in the recovery of their initial ultrastructure. Irreversible mitochondria degradation was observed only during subsequent still longer leaf anaerobic treatment (24–48 h). When, under conditions of strict anoxia, leaves were fed with glucose to stimulate glycolysis and ethanolic fermentation, we did not observe any signs of early destruction of mitochondrial ultrastructure and their swelling. Blockage of anaerobic protein synthesis with cycloheximide resulted in early destruction and subsequent irreversible degradation of mitochondria without any indications of their structural recovery. Based on the results of the experiments, we concluded that cell energy metabolism controlled byboth the presence of utilizable carbohydrates and also by the induction of anaerobic protein synthesis played a key role during early mitochondria destruction under extreme conditions of anaerobic stress, their subsequent recovery, and irreversible degradation during continuous long-term strict anoxia.

Physiological role of nitrate under anaerobic stress in Saccharum officinarum callus cells tolerant and sensitive to anoxia

The physiological role of nitrate as a protective factor against anaerobic stress was studied in experiments with tolerant to anoxia sugarcane (Saccharum officibarum L.) callus lines obtained by in vitro selection in the absence of exogenous carbohydrates. Original cell lines, which were not subjected to selection and therefore more sensitive to oxygen shortage, served as a control. In these lines, anaerobic stress was created in the presence or absence of nitrate in nutrient medium. The presence of nitrate in nutrient medium increased markedly tolerance to anaerobic stress of both lines differing in their sensitivity to anaerobiosis. However, the degree of tolerance differed substantially in compared lines. In the presence of exogenous nitrate, in tolerant cells there were no signs of mitochondrial membrane destruction or degradation even after 72 h of anoxia, whereas in control cells 48-h anaerobic incubation led to the complete degradation of mitochondrial membranes and membranes of other organelles. It is concluded that significant increase in the tolerance of S. officinarum cells in the process of in vitro selection most likely occurred due to induction and stimulation of not only the processes of glycolysis and fermentation, but also nitrate and maybe nitrite utilization.