Regulation of Respiration and Fermentation to Control the Plant Internal Oxygen Concentration

Plant internal oxygen concentrations can drop well below ambient even when the plant grows under optimal conditions. Using pea (Pisum sativum) roots, we show how amenable respiration adapts to hypoxia to save oxygen when the oxygen availability decreases. The data cannot simply be explained by oxygen being limiting as substrate but indicate the existence of a regulatory mechanism, because the oxygen concentration at which the adaptive response is initiated is independent of the actual respiratory rate. Two phases can be discerned during the adaptive reaction: an initial linear decline of respiration is followed by a nonlinear inhibition in which the respiratory rate decreased progressively faster upon decreasing oxygen availability. In contrast to the cytochrome c pathway, the inhibition of the alternative oxidase pathway shows only the linear component of the adaptive response. Feeding pyruvate to the roots led to an increase of the oxygen consumption rate, which ultimately led to anoxia. The importance of balancing the in vivo pyruvate availability in the tissue was further investigated. Using various alcohol dehydrogenase knockout lines of Arabidopsis (Arabidopsis thaliana), it was shown that even under aerobic conditions, alcohol fermentation plays an important role in the control of the level of pyruvate in the tissue. Interestingly, alcohol fermentation appeared to be primarily induced by a drop in the energy status of the tissue rather than by a low oxygen concentration, indicating that sensing the energy status is an important component of optimizing plant metabolism to changes in the oxygen availability.Plants are obligate aerobic organisms, with oxygen being an essential substrate for mitochondrial energy production. However, the poor distribution efficiency for oxygen through root, tuber, seed, or stem tissue of various species results in steep drops of the internal oxygen concentration, ranging from values near above zero to just below 40% of air saturation (e.g. Armstrong et al., 1994; Geigenberger et al., 2000; Rolletschek et al., 2002; van Dongen et al., 2003, 2004; Vigeolas et al., 2003).These low levels of internal oxygen strongly affect plant metabolism. Several studies showed that energy-consuming metabolic pathways are adjusted to the actual oxygen availability (for review, see Geigenberger, 2003; Bailey-Serres and Voesenek, 2008). By saving energy, the plant decreases the demand for respiratory oxygen consumption that could help to postpone or even prevent the tissue from becoming anoxic. Indeed, it was observed that the metabolic flux through glycolysis slows down, respiratory oxygen consumption decreases, and adenylate levels drop in response to low internal oxygen (Geigenberger et al., 2000; Bologa et al., 2003). This inhibition of respiration is not easily explained by substrate limitation of cytochrome c oxidase (COX). First, the reduction of respiration becomes apparent already at oxygen concentrations around 20% of air saturation, whereas the K_m value for oxygen of COX lies around 0.05% of air saturation (which equals 0.14 μm under standard conditions; Drew, 1997). Second, the inhibition of respiration could be clearly distinguished from the induction of fermentation, which did not occur until oxygen fell to levels close to zero (Geigenberger, 2003).Based on these studies, it is reasonable to assume that a sensitive tuning mechanism must exist that allows the plant to regulate oxygen consumption while simultaneously preventing anoxia. However, hardly anything is known about the mechanism by which a plant induces adaptive responses to low oxygen (Bailey-Serres and Chang, 2005). Vice versa, comparably little is known about how metabolic activity affects the plant internal oxygen concentration. Previous studies focused on the effect that feeding of different sugars had on the respiration rate of tissue slices (Loef et al., 2001) or used transgenic approaches to stimulate metabolism by introducing a more energy-consuming route of Suc degradation via invertase in growing tubers (Bologa et al., 2003). In the latter case, respiration rates were increased and internal oxygen concentrations fell to very low levels that were close to zero. This shows that plant internal oxygen concentrations respond very sensitively to changes in metabolic activities. However, the underlying mechanism remains unclear.Glycolysis is part of the central backbone of primary carbohydrate metabolism and respiration. Pyruvate serves as a key metabolite linking glycolysis in the cytosol with mitochondrial respiration. Under aerobic conditions, pyruvate is transported into mitochondria and is oxidized through the tricarboxylic acid (TCA) cycle into organic acids and NADH. Moreover, pyruvate has regulatory potential, as it was shown that alternative oxidase (AOX) becomes more active in the presence of α-keto acids such as pyruvate (Millar et al., 1993, 1996; Vanlerberghe et al., 1995; Vanlerberghe and McIntosh, 1997), thus affecting the efficiency of ATP production per unit of oxygen being respired. Under oxygen-limiting conditions, pyruvate can either be converted into ethanol by pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) or to lactate by lactate dehydrogenase. Pyruvate also serves as a precursor for the synthesis of Ala via the reversible reaction catalyzed by Ala amino transferase, which was shown to play a crucial role in the rapid conversion of Ala to pyruvate during recovery from low-oxygen stress (Miyashita et al., 2007). Furthermore, pyruvate is the substrate of acetolactate synthase, which is the first enzyme committed to the biosynthesis of the branched-chain amino acids Val, Leu, and Ile. Inhibition of acetolactate synthase by the herbicide imazethapyr induces aerobic fermentation in plants (Gaston et al., 2002). Despite its central role in energy metabolism under both oxygen-rich and oxygen-depleted conditions, no investigations were made, to our knowledge, until now on the impact pyruvate has on the plant internal oxygen concentration.The aim of this study was to investigate the regulation of respiration and its relation to the plant internal oxygen concentration. To investigate this, we changed the oxygen concentration of the nutrient solution of hydroponically grown pea (Pisum sativum) plants and tested the influence of several sugars and organic acids. We measured root internal oxygen concentrations as well as the energy status of the tissue and related this to the rate of oxygen consumption by both the cytochrome c pathway and the AOX. The results are investigated in relation to the function and regulation of fermentative metabolism.