Abstract

Plants are obligate aerobic organisms and, therefore, need oxygen for survival. However, unlike animals, plants do not possess an active oxygen transport system to supply their organs with oxygen. Hence, oxygen can fall to low levels inside plant tissues if the diffusion of oxygen cannot keep pace with the rate of oxygen consumption. As a consequence, plants have developed special mechanisms to save oxygen. Studies show that plants actively regulate their respiration and metabolism in relation to the internal oxygen concentration. Nevertheless, the sequences of metabolic events leading to this adaptation are not yet known. Therefore, an experiment was developed to treat potato tuber slices with 4% oxygen (v/v) and analyze the metabolic response in a time dependent manner. The low-oxygen treatment led to a rapid inhibition of respiration and a general metabolic depression at different sites, while fermentation was activated at a later point in time. Regulatory sites have been identified in glycolysis and in the tricarboxylic acid (TCA) cycle. The experiments also revealed cytosolic pyruvate kinase (PKc) as important control site in glycolysis. PKc is crucial under low-oxygen conditions as it provides pyruvate for respiration and for fermentation. To further investigate the role of PKc under low-oxygen conditions transgenic potato tubers with decreased expression of PKc mediated by RNA interference were treated with 4% oxygen, and a comprehensive metabolic profile was performed. Indeed, the results indicate that PKc regulates the availability of pyruvate for fermentation, thereby influencing the metabolic performance under low-oxygen. Whereas potato tuber discs are an easy system to manipulate the surrounding oxygen concentration, there are only limited tools available for genetic manipulation. Therefore, Arabidopsis thaliana was used as model system for reverse genetic studies. Transgenic Arabidopsis plants carrying a T-DNA insertion in the catalytic subunit of mitochondrial NAD-dependent isocitrate dehydrogenase (IDH) were used to investigate the importance of the mitochondrial alpha-ketoglutarate provision for the reorganization of the TCA cycle under low-oxygen. The idhv mutant showed an improved low-oxygen tolerance accompanied by specific alterations of hypoxic metabolism compared to wild type, thus, suggesting that mitochondrial alpha-ketoglutarate production through IDH is dispensable under low-oxygen conditions in Arabidopsis. Moreover, the experiments showed that an increased activity of extramitochondrial pathways for 2-oxoglutarate production is beneficial for plant survival under low-oxygen. In addition to the modifications in primary metabolism for an improved survival under low-oxygen, changes in the redox state are also common characteristics of hypoxia. NADPH-dependent thioredoxin reductases (NTRs) modulate the activity of redox-regulated enzymes depending on the cellular redox-state. To explore the role of the NTR system under low-oxygen, a knockout of the plastidial NADPH-dependent thioredoxin reductase (NTRC) and a double knockout of the extraplastidial NADPH-dependent thioredoxin reductase A (NTRA) and NADPH-dependent thioredoxin reductase B (NTRB) in Arabidopsis thaliana were treated with hypoxia, and the relevant redox related parameters were measured. The results show opposed effects of the low-oxygen treatment for the ntrc and the ntrantrb mutant. Whereas the ntrantrb mutant revealed an increased resistance to hypoxia, the ntrc mutant displayed the opposite behavior. Apparently, the plastidial and extraplastidial NTR systems play different roles in the adaptation to low-oxygen, although the underlying reasons for this phenomenon are not yet fully understood. A further area of plant metabolism being affected by low-oxygen is the cellular energy status. With falling oxygen concentrations inside the cell the production of ATP through respiration decreases and the energy status declines. This in turn affects the biosynthesis pathways and, ultimately, the plant growth which needs to be adjusted to the energy deficit. A possible regulator that connects energy homeostasis with plant growth is the sucrose non-fermenting-1-related protein kinase (SnRK1). Transgenic Arabidopsis plants with beta-estradiol inducible transcriptional silencing of the regulatory SNF4 subunit of SnRK1 were used to study the function of SnRK1 under low-oxygen. The transgenic plants displayed a lower anoxic survival rate, a decrease in hypoxia marker genes expression and alterations in primary metabolism compared to wild type. Altogether, these results suggest an important role of SnRK1 in the low-oxygen response in Arabidopsis thaliana.