During oxygen limitation in higher plants, energy metabolism switches from respiration to fermentation. As part of this anaerobic response the expression of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) is strongly induced. In addition there is ample evidence for post‐translational regulation. In order to understand this multi‐level regulation of the anaerobic response, we provided tobacco with the constitutive capacity of ethanolic fermentation by expressing a PDC gene derived from the obligate anaerobe Zymomonas mobilis. The protein accumulated to high levels and was active in an in vitro assay. During the first 2‐4 h of anoxia, acetaldehyde accumulated to 10‐ to 35‐fold and ethanol to 8‐ to 20‐fold higher levels than in wild‐type. Under normoxic conditions no accumulation of acetaldehyde and ethanol could be measured. Instead, the two products may be immediately re‐metabolized in tobacco leaf tissue. We show that aerobic fermentation takes place when the respiratory system is inhibited. Although these conditions enhance ethanolic fermentation under normoxia, they fail to increase ADH transcript levels. These results indicate that anaerobic transcription is triggered not by the metabolic consequences of oxygen limitation, but directly through an oxygen‐sensing system.
SummaryPlant survival during flooding relies on ethanolic fermentation for energy production. The available literature indicates that the first enzyme of the ethanolic fermentation pathway, pyruvate decarboxylase (PDC), is expressed at very low levels and is likely to be rate-limiting during oxygen deprivation. The authors expressed high levels of bacterial PDC in tobacco to study the modulation of PDC activity in vivo, and assess its impact on the physiology of ethanolic fermentation and survival under oxygen stress. In contrast to leaves, wild-type normoxic roots contained considerable PDC activity, and overexpression of the bacterial PDC caused only a moderate increase in acetaldehyde and ethanol production under anoxia compared to wild-type roots. No significant lactate production could be measured at any time, making it unlikely that lactate-induced acidification (LDH/PDC pH-stat) triggers the onset of ethanol synthesis. Instead, the authors favour a model in which the flux through the pathway is regulated by substrate availability. The increased ethanolic flux in the transgenics compared to the wild-type did not enhance anoxia tolerance. On the contrary, rapid utilisation of carbohydrate reserves enhanced premature cell death in the transgenics while replenishment of carbohydrates improved survival under anoxia.
SynopsisAll plants are able to survive anoxic periods, but the degree of tolerance shows large variation. The main injuries related to anoxia are eventually due to changes in energy metabolism. Low energy charge values indicate a cessation of many ATP consuming processes. Sugar starvation, lactic acid fermentation and proton release from leaky vacuoles are responsible for cell death. Long-term anoxia tolerance is dependent on storage products in the vicinity of sinks, on an adequate control of glycolysis, synthesis of essential proteins, and stability of membranes and organelles. However, no fundamental differences between the metabolic pathways of tolerant and non-tolerant tissues are known. It is rather a question of minor changes and the regulation of anaerobic metabolism.Re-exposure of anoxic tissues to air may even be more detrimental than anoxia itself. These injuries are mainly due to enhanced radical generation. Lipid peroxidation processes lead to membrane damage, disintegration, and leakage of solutes. Under natural conditions plants are equipped with radicaldetoxifying systems (SOD, peroxidases and antioxidants). Natural detoxifying systems can be reduced in non-adapted plants under anoxia and they become more sensitive to post-anoxic damage. In addition, the rapid conversion of ethanol to extremely toxic acetaldehyde seems to be a cause of tissue injury and death.
The importance of lipid peroxidation and its contributing pathways (via reactive oxygen species and lipoxygenase) during post-anoxia was evaluated with respect to the biphasic behavior of membrane lipids under anoxia (A. Rawyler, D. Pavelic, C. Gianinazzi, J. Oberson, R. Brändle [1999] Plant Physiol 120: 293-300), using potato (Solanum tuberosum cv Bintje) cell cultures. When anoxic cells in the pre-lytic phase were re-oxygenated for 2 h, superoxide anion was not detectable, the hydrogen peroxide (H 2 O 2 ) level remained small and similar to that of controls, and cell viability was preserved. Lipids were intact and no lipid hydroperoxides were detected. However, small amounts of lipid hydroperoxides accumulated upon feeding anoxic cells with H 2 O 2 and incubation for an additional 2 h under anoxia. When cells that entered the lytic phase of anoxia were re-oxygenated for 2 h, the H 2 O 2 and superoxide anion levels were essentially unchanged. However, cell respiration decreased, reflecting the extensive lipid hydrolysis that had already started under anoxia and continued during post-anoxia. Simultaneous with the massive release of free polyunsaturated fatty acids, small amounts of lipid hydroperoxides were formed, reaching 1% to 2% of total fatty acids. Catalase and superoxide dismutase activities were not greatly affected, whereas the amount and activity of lipoxygenase tended to increase during anoxia. Lipid peroxidation in potato cells is therefore low during post-anoxia. It is mainly due to lipoxygenase, whereas the contribution of reactive oxygen species is negligible. But above all, it is a late event that occurs only when irreversible damage is already caused by the anoxia-triggered lipid hydrolysis.
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