The phytoglobin-nitric oxide cycle: a transgenic approach to studying hypoxic stress in plants

Cochrane, Devin W. (2020) The phytoglobin-nitric oxide cycle: a transgenic approach to studying hypoxic stress in plants. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Hypoxic stress is one of the most common abiotic stresses plants face. Without sufficient oxygen to drive mitochondrial respiration, plants undergo an energy crisis. To combat this, a form of nitrite driven respiration called the phytoglobin-nitric oxide cycle is induced to maintain minimal ATP production. I studied the effects of overexpression and knockdown of the class-1 phytoglobin (Pgb) gene in barley (Hordeum vulgare L.) and the alternative oxidase (AOX) gene in tobacco (Nicotiana tabacum) in low-oxygen conditions to gain a better understanding of their roles in the hypoxic stress response. NO production was significantly lower in AOX knockdown plants compared to wild type and AOX overexpressing plants. The knockdown of AOX resulted in the increased level of protein S-nitrosylation under normoxia and the decreased S-nitrosylation under hypoxia. Under both conditions, there were complex differences in the pool size of amino and organic acids, depending upon AOX amount. Specifically under hypoxia, AOX amount strongly influenced the mitochondrial, but not cytosolic, activity of aconitase, which is known to be inactivated by NO and superoxide. Under normoxia and during reoxygenation after hypoxia, the knockdown of AOX increased the amount of superoxide, lipid peroxidation (malondialdehyde content) and total antioxidant reducing power. Hence, AOX has pervasive and oxygen concentration-dependent effects on NO production and protein S-nitrosylation, respiratory carbon and nitrogen flow, as well as on the metabolism of reactive oxygen species. The overexpression of phytoglobin lowered the amount of NO released, while knockdown significantly stimulated NO emission. The overexpression of phytoglobin corresponded to higher ATP/ADP ratios, pyrophosphate levels and aconitase activity under anoxia, while knockdown of phytoglobin resulted in the increased level of protein nitrosylation, elevation of alcohol dehydrogenase and nitrosoglutathione reductase activities. The overexpressing plants showed various signs of stunted growth under normoxia, but were the only type to germinate and survive under hypoxia. These results show that overexpression of phytoglobin protects plant cells via NO scavenging and improves their low-oxygen stress survival. However, it may not be useful for cereal crop improvement since it comes with a significant interference with normoxic NO signalling pathways.

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