In response to flooding/waterlogging, plants develop various anatomical changes including the formation of lysigenous aerenchyma for the delivery of oxygen to roots. Under hypoxia, plants produce high levels of nitric oxide (NO) but the role of this molecule in plant-adaptive response to hypoxia is not known. Here, we investigated whether ethylene-induced aerenchyma requires hypoxia-induced NO. Under hypoxic conditions, wheat roots produced NO apparently via nitrate reductase and scavenging of NO led to a marked reduction in aerenchyma formation. Interestingly, we found that hypoxically induced NO is important for induction of the ethylene biosynthetic genes encoding ACC synthase and ACC oxidase. Hypoxia-induced NO accelerated production of reactive oxygen species, lipid peroxidation, and protein tyrosine nitration. Other events related to cell death such as increased conductivity, increased cellulase activity, DNA fragmentation, and cytoplasmic streaming occurred under hypoxia, and opposing effects were observed by scavenging NO. The NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt) and ethylene biosynthetic inhibitor CoCl both led to reduced induction of genes involved in signal transduction such as phospholipase C, G protein alpha subunit, calcium-dependent protein kinase family genes CDPK, CDPK2, CDPK 4, Ca-CAMK, inositol 1,4,5-trisphosphate 5-phosphatase 1, and protein kinase suggesting that hypoxically induced NO is essential for the development of aerenchyma.

Plant tissues, particularly roots, can be subjected to periods of hypoxia due to environmental circumstances. Plants have developed various adaptations in response to hypoxic stress and these have been extensively described. Less wellappreciated is the body of evidence demonstrating that scavenging of nitric oxide (NO) and the reduction of nitrate/nitrite regulate important mechanisms that contribute to tolerance to hypoxia. Whilst ethylene controls hyponasty and aerenchyma formation, NO production apparently regulates hypoxic ethylene biosynthesis. In the hypoxic mitochondrion, cytochrome c oxidase, which is a major source of NO, is also inhibited by NO, thereby reducing the respiratory rate and enhancing local oxygen concentrations. Nitrite can maintain ATP generation under hypoxia by coupling its reduction to the translocation of protons from the inner side of mitochondria and generating an electrochemical gradient. This reaction can be further coupled to a reaction whereby non-symbiotic haemoglobin oxidizes NO to nitrate. In addition to these functions, nitrite has been reported to influence mitochondrial structure and supercomplex formation, as well as playing a role in oxygen sensing via the N-end rule pathway. These studies establish that nitrite and NO perform multiple functions during plant hypoxia and suggest that further research into the underlying mechanisms is warranted.

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Nitric oxide (NO) is now established as an important signalling molecule in plants where it influences growth, development, and responses to stress. Despite extensive research, the most appropriate methods to measure and localize these signalling radicals are debated and still need investigation. Many confounding factors such as the presence of other reactive intermediates, scavenging enzymes, and compartmentation influence how accurately each can be measured. Further, these signalling radicals have short half-lives ranging from seconds to minutes based on the cellular redox condition. Hence, it is necessary to use sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we summarize the current knowledge on NO measurement in plant samples, via various methods. We also discuss advantages, limitations, and wider applications of each method.

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