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The role of nitric oxide in survival of low oxygen stress in plants

Final Report Summary - TRNOILOS (The role of nitric oxide in survival of low oxygen stress in plants)

Molecular oxygen is essential for the efficient production of ATP through oxidative phosphorylation in aerobic organisms, with oxygen serving as the terminal electron acceptor for the mitochondrial electron transport chain. Many plants encounter oxygen deprivation during flooding, and the deleterious effect that this has on energy production has a major impact on crop productivity. Plants respond to oxygen deprivation in many ways, including changes in anatomy and morphology, as well as alterations in gene expression and metabolism, but despite extensive research, the mechanisms that underpin the sensing and response to oxygen deprivation in plants have not been fully elucidated. One of the characteristic features of the response to oxygen deprivation in roots is the substantial increase in the production of nitric oxide (NO). This small, lipophilic free radical is synthesized in virtually all organisms and is used as a signalling molecule that directly interacts with cell thiols or the catalytic metal centres of proteins. In plants, there are several oxidative and reductive pathways for the production of NO, and it is the nitrate reductase and plant mitochondrial pathways that are the most likely sources for NO during hypoxia. In addition class 1 haemoglobins play an important role in scavenging NO levels during hypoxia. The project aimed to extend our understanding of the contribution of NO to the regulation of plant metabolism through three linked objectives:

1. What is the metabolic significance of NO production under aerobic and hypoxic conditions?

2. How do the hypoxically-induced nonsymbiotic class 1 haemoglobins, which have NO dioxygenase activity, contribute to the hypoxic response?

3. Are there other roles for mitochondria beyond mitochondrial NO production under anoxia?

• Metabolic significance of NO under aerobic conditions

The role of NO under aerobic conditions was investigated in barley roots by comparing wildtype (WT) plants with plants over-expressing non-symbiotic haemoglobin 1 (Hb+). Measurements of NO level, respiration, carbohydrate oxidation and oxygen level showed that under aerobic conditions, overexpression of non-symbiotic haemoglobin 1 decreased the NO level, increased respiration and carbohydrate oxidation, and reduced the internal oxygen level. It was also observed that the production of reactive oxygen species (ROS) increased in the Hb+ roots. This work led to the conclusion that NO is important for the homeostasis of oxygen and ROS under aerobic conditions, and thus established a regulatory role for NO beyond that which has been identified under hypoxia.

Changes in NO level can occur in response to abiotic stress under aerobic conditions and the metabolic significance of the increase in NO production that occurs in response to phosphate deprivation was investigated in Arabidopsis seedlings using a nitrate reductase double mutant (nia1,2). Phosphate starvation increased the production of NO, the contribution of the alternative oxidase (AOX) to respiration, and the AOX level in WT seedlings; whereas the same treatment failed to stimulate NO production and AOX expression in the mutant, and the plants had shorter roots than WT. The NO donor S-nitrosoglutathione rescued the growth phenotype of the nia mutants under phosphate starvation to some extent, and it also increased the contribution of AOX to respiration, thus demonstrating that NO is required for the induction of the AOX pathway when seedlings are grown under phosphate limiting conditions.

• Impact of NO and non-symbiotic hemoglobins under hypoxia

Gas phase chemiluminescence measurements of NO emissions from barley WT and Hb+ roots under 0.4% oxygen showed that over-expression of the non-symbiotic haemoglobin led to decreased NO production. Experiments were performed in which roots were supplied with positionally labelled [14C] glucose (at C1, C2, C3,4 and C6) for 6 h and the release of 14CO2 was monitored under hypoxia using alkaline KOH traps. Analysis of the ratios of 14CO2 release from the labelled substrates suggested that there was an increase in the flux through the oxidative pentose phosphate pathway (oxPPP) in Hb+ relative to WT. To obtain further evidence for this metabolic effect, roots were incubated with [1, 2-13C]glucose and the time-course for the redistribution of the label was analysed by gas chromatography mass spectrometry. The analysis of the labelling data is still underway, but it should provide definitive evidence for the apparent link between decreased NO levels and increased flux through the oxPPP under hypoxia.

• Effect of hypoxic NO on protection of plants during reoxygenation

Plants that have been transiently flooded have to pass through a potentially damaging reoxygenation phase in which levels of ROS can be elevated. The impact of hypoxically produced NO on this process was studied in Arabidopsis seedlings using the nitrate reductase double mutant to reduce the level of NO. Plants were grown on vertical agar medium, and after a 6 h hypoxic treatment (0.4% O2) the plants were reoxygenated for 2 h. NO levels were measured during normoxia, hypoxia and reoxygenation and it was found that imposing hypoxia led to increased production of NO in WT and reduction of NO in the nia mutant. The survival rate of the WT was greater than the nia mutant in the reoxygenation phase, and so extensive measurements were conducted on ROS levels (DCF fluorescence), hydrogen peroxide levels (DAB staining), and lipid peroxidation, all of which increased in the nia mutant. Peroxynitrite and superoxide levels were also higher in the nia mutant, and a mutant for manganese superoxide dismutase (SOD) was used to demonstrate the importance of SOD in determining the observed levels of peroxynitrite. Moreover the possibility that NO could reduce ROS production during the reoxygenation phase by inducing antioxidant defense mechanisms was investigated by measuring the activities of glutathione reductase, ascorbate peroxidase and catalase activities and it was found that these activities did indeed increase to a greater extent in the WT than the nia mutant during hypoxia and reoxygenation. This work has led to the important conclusion that NO generated under transient hypoxic conditions protects plants during the subsequent reoxygenation phase via the induction of antioxidant mechanisms.

• Other roles for mitochondria beyond NO production under hypoxia

The impact of mitochondrial NO production on the structure and functionality of mitochondria under hypoxia was investigated by isolating mitochondria and imposing hypoxia (0.4% O2) in the presence or absence of nitrite. The mitochondria were stained with Mitotracker orange and observed under a confocal microscope. Reduced lipid peroxidation and ROS production in the presence of nitrite correlated with increased nitrite reduction to NO and increased membrane potential and ATP. Increased levels of several mitochondrial complexes and super complex levels were also observed under hypoxia in the presence of NO2-. These observations imply that nitrite reduction to NO protects mitochondrial structure and functionality under hypoxia.

• Conclusion

Overall the successful completion of the project has provided new insight into the regulatory role of NO in aerobic and hypoxic plant metabolism. The protective role of NO is increasingly evident and this suggests that it might be possible to develop a synthetic biology approach to crop protection from flooding injury based on the controlled release of nitrite or NO into the affected plant tissues.