The coordination of phytoglobin-nitric oxide cycle and alternative oxidase in plant adaptation to hypoxia

The non-energy conserving protein alternative oxidase (AOX) of the mitochondrial respiratory electron transport chain (ETC) is hypothesized to regulate nitric oxide (NO), reactive oxygen species (ROS), and ethylene levels in plants under stress. The purpose of this research is to provide direct evidence in favor of this hypothesis, as well as to investigate the implications of this regulation during plant adaptation to hypoxic stress. We studied NO metabolism and the involvement of key components of the phytoglobin (Pgb1) -NO cycle in imbibed transgenic barley seeds with altered Pgb1 levels during germination process as well as in transgenic tobacco seedlings with altered AOX levels exposed to nitrogen atmosphere. NO emission increased more in overexpressing lines of tobacco under hypoxia, although the quantity of nitrosylated proteins was higher in AOX knockdown plants. There was a significant increase in Pgb1 expression which upregulates the turnover of the Pgb1-NO cycle in imbibed barley seeds and tobacco leaves under hypoxic condition. The cycle's operation not only controls NO metabolism and redox homeostasis, but it improves the efficiency of energy metabolism. The current study demonstrates that AOX which contributes positively to the operation of the Pgb1-NO cycle, regulates NO generation under hypoxia and leads to a shift towards biosynthesis of amino acids. It demonstrates that hypoxia results in the upregulation of fermentation pathways in the plants expressing AOX. The plants lacking AOX exhibited the increased levels of ROS as compared to wild type and AOX overexpression plants during hypoxia, suggesting that AOX induces a fine-tuned balance in ROS levels by the regulation of ROS production and scavenging. I found that ethylene biosynthesis genes are induced during hypoxia and correlate with AOX and NO levels. I conclude that AOX is involved in NO turnover and plays a protective role by reducing ROS, regulating the ethylene levels, and sustaining energy requirements during hypoxia

Transcriptional and Metabolic Changes Associated with Phytoglobin Expression during Germination of Barley Seeds

To understand how the class 1 phytoglobin is involved in germination process via the modulation of the nitric oxide (NO) metabolism, we performed the analysis of physiological and molecular parameters in the embryos of transgenic barley (Hordeum vulgare L. cv Golden Promise) plants differing in expression levels of the phytoglobin (Pgb1) gene during the first 48 h of germination. Overexpression of Pgb1 resulted in a higher rate of germination, higher protein content and higher ATP/ADP ratios. This was accompanied by a lower rate of NO emission after radicle protrusion, as compared to the wild type and downregulating line, and a lower rate of S-nitrosylation of proteins in the first hours postimbibition. The rate of fermentation estimated by the expression and activity of alcohol dehydrogenase was significantly higher in the Pgb1 downregulating line, the same tendency was observed for nitrate reductase expression. The genes encoding succinate dehydrogenase and pyruvate dehydrogenase complex subunits were more actively expressed in embryos of the seeds overexpressing Pgb1. It is concluded that Pgb1 expression in embryo is essential for the maintenance of redox and energy balance before radicle protrusion, when seeds experience low internal oxygen concentration and exerts the effect on metabolism during the initial development of seedlings

The Role of Alternative Oxidase in the Interplay between Nitric Oxide, Reactive Oxygen Species, and Ethylene in Tobacco (<i>Nicotiana tabacum</i> L.) Plants Incubated under Normoxic and Hypoxic Conditions

The transgenic tobacco (Nicotiana tabacum L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating nitro-oxidative stress and metabolic changes after exposing plants to hypoxia for 6 h. Under normoxia, AOX expression resulted in the decrease of nitric oxide (NO) levels and of the rate of protein S-nitrosylation, while under hypoxia, AOX overexpressors exhibited higher NO and S-nitrosylation levels than knockdowns. AOX expression was essential in avoiding hypoxia-induced superoxide and H2O2 levels, and this was achieved via higher activities of catalase and glutathione reductase and the reduced expression of respiratory burst oxidase homolog (Rboh) in overexpressors as compared to knockdowns. The AOX overexpressing lines accumulated less pyruvate and exhibited the increased transcript and activity levels of pyruvate decarboxylase and alcohol dehydrogenase under hypoxia. This suggests that AOX contributes to the energy state of hypoxic tissues by stimulating the increase of pyruvate flow into fermentation pathways. Ethylene biosynthesis genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene-responsive factors (ERFs) were induced during hypoxia and correlated with AOX and NO levels. We conclude that AOX controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia

The Role of Alternative Oxidase in the Interplay between Nitric Oxide, Reactive Oxygen Species, and Ethylene in Tobacco (Nicotiana tabacum L.) Plants Incubated under Normoxic and Hypoxic Conditions

The transgenic tobacco (Nicotiana tabacum L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating nitro-oxidative stress and metabolic changes after exposing plants to hypoxia for 6 h. Under normoxia, AOX expression resulted in the decrease of nitric oxide (NO) levels and of the rate of protein S-nitrosylation, while under hypoxia, AOX overexpressors exhibited higher NO and S-nitrosylation levels than knockdowns. AOX expression was essential in avoiding hypoxia-induced superoxide and H2O2 levels, and this was achieved via higher activities of catalase and glutathione reductase and the reduced expression of respiratory burst oxidase homolog (Rboh) in overexpressors as compared to knockdowns. The AOX overexpressing lines accumulated less pyruvate and exhibited the increased transcript and activity levels of pyruvate decarboxylase and alcohol dehydrogenase under hypoxia. This suggests that AOX contributes to the energy state of hypoxic tissues by stimulating the increase of pyruvate flow into fermentation pathways. Ethylene biosynthesis genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene-responsive factors (ERFs) were induced during hypoxia and correlated with AOX and NO levels. We conclude that AOX controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia.</jats:p

Nitric Oxide Turnover Under Hypoxia Results in the Rapid Increased Expression of the Plastid-Localized Phosphorylated Pathway of Serine Biosynthesis

The plant mitochondrial electron transport chain influences carbon and nitrogen metabolism under near anoxic conditions through its involvement in the phytoglobin-nitric oxide cycle, where the respiratory chain reduces nitrite to nitric oxide (NO), followed by NO conversion to nitrate by class 1 phytoglobin. Wild type (WT) and transgenic tobacco (Nicotiana tabacum L.) with differing amounts of alternative oxidase (AOX) were used to manipulate NO generation under hypoxia, and to examine whether this in turn influenced the gene expression of two stress-related amino acid biosynthetic pathways, the plastid-localized phosphorylated pathway of serine biosynthesis (PPSB), and the γ-aminobutyric acid (GABA) shunt. Under hypoxia, leaf NO emission rate was highest in AOX overexpressors and lowest in AOX knockdowns, with WT showing an intermediate rate. In turn, the rate of NO emission correlated with the degree to which amino acids accumulated. This amino acid accumulation was associated with the increased expression of the enzymes of the stress-related amino acid biosynthetic pathways. However, induction of the PPSB occurred much earlier than the GABA shunt. This work shows that high rates of NO turnover associate with rapid gene induction of the PPSB, establishing a clear link between this pathway and the maintenance of carbon, nitrogen and energy metabolism under hypoxia.</jats:p