| Summary: | Nitric oxide (NO) is emerging as an important signalling molecule in plants. A major source of NO in planta is through the activity of nitrate reductase (NR),an enzyme required for nitrate reduction that can also reduce nitrite to NO. The model plant Arabidopsis thalianahas been extensively used to study mechanisms of NO production and signalling. Arabidopsis contains two isoforms of NR, NITRATE REDUCTASE 1 (NIAl) and NITRATE REDUCTASE 2 (NIA2). Increasing evidence suggests diverse roles for these proteins in NO production modulating plant development, biotic and abiotic stress tolerance. Low-oxygen or hypoxic stress, induced by periods of waterlogging or 'submergence, is a significant factor impacting plant and crop growth and survival globally. Terrestrial plants have evolved diverse mechanisms to cope with these stresses including alterations in gene expression, metabolism, morphology and physiology. In this thesis, Arabidopsis was used to as a model plant to investigate the putative regulation of these processes via NR and NO signalling. This investigation has focused primarily on analysing the initiation of stomatal closure, the formation of aerenchyma, and the induction of hypoxic gene expression in wild- type Arabidopsis plants and NIA gene knockout mutants. Waterlogging stress induced hypoxia in the root environment within 4 days, at which time stomatal aperture was significantly reduced in wild-type plants. In NIAI gene mutants, stomatal closure was completely abolished whilst closure occurred normally in the NIA2 mutant. Ethylene signal transduction and reactive oxygen species (ROS) production, mediated by ERECTA, ETRl, RBOHF, EIN2 and EIN3 were also shown to be critical for this response. The data suggest a role for NIAl in waterlogging stress induced guard cell NO generation. Under waterlogging stress conditions, stomata of wild-type plants were insensitive to exogenous NO, which functionally restored stomatal closure in NIAI and other mutants compromised in the stomatal response. A model is proposed whereby waterlogging stress induces ethylene and ROS signalling which acts upstream of NO production to regulate stomatal aperture during rhizospheric hypoxia. Aerenchyma is made up of gas spaces (lacunae) formed by apoptotic cell death of localised cells and is proposed to facilitate gas-exchange in submerged plant tissues. In Arabidopsis hypocotyls, prolonged waterlogging induced cell death leading to aerenchymous lacunae, as measured by anatomical analyses of hypocotyl sections. NR was not suggested to be required for cell death leading to aerenchyma, since NIA gene mutants exhibited equivalent lacunae formation to wild-type plants. A hydroponic cultivation system was employed to study hypoxic root gene expression. The upregulation of hypoxic tolerance genes, ALCOHOL DEHYDROGENASE 1 (ADHI) and HAEMOGLOBIN I(AHBI) was attenuated in a NIAI mutant and to a lesser extent in a NIA2 mutant over a 24 hour period of hypoxic stress, although whether this was attributed to NO production could not be confirmed. AffymetrixGeneChiptranscriptome profiling identified hypoxia-induced alterations in gene expression at 24 hours hypoxia in wild-type and NIA 1 mutant roots. Functional analysis of differentially expressed transcripts was performed utilising gene ontology annotations for the Arabidopsis genome. The analysis identified known hypoxia-associated processes significantly enriched amongst up or downregulated genes, and identified novel pathways directly or indirectly regulated by NIAl function. These cumulative data support an important role of NR and specifically NIAl in the regulation of hypoxic stress tolerance mechanisms at the physiological and molecular level.
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