Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2005.02615.x

ABA‐induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Contents

Summary  11

1Introduction  12
2Why does NO make a good signal?  12
3NO biosynthesis  13
4NO biology  17
5NO signal transduction  23
6Conclusion  30

Acknowledgements  31


References  31



Summary
Recently nitric oxide (NO) has emerged as a key signalling molecule in plants. Here we review the potential sources of endogenous NO, outline the biological processes likely to be mediated by NO, and discuss the downstream signalling processes by which NO exerts its cellular effects. It will be important to develop methods to quantify intracellular NO synthesis and release. Clasification of the biosynthetic origins of NO is also required. NO can be synthesised from nitrite via nitrate reductase (NR) and although biochemical and immunological data indicate the presence of enzyme(s) similar to mammalian nitric oxide synthase (NOS), no NOS genes have been identified. NO can induce various processes in plants, including the expression of defence-related genes and programmed cell death (PCD), stomatal closure, seed germination and root development. Intracellular signalling responses to NO involve generation of cGMP, cADPR and elevation of cytosolic calcium, but in many cases, the precise biochemical and cellular nature of these responses has not been detailed. Research priorities here must be the reliable quantification of downstream signalling molecules in NO-responsive cells, and cloning and manipulation of the enzymes responsible for synthesis and degradation of these molecules.

https://nph.onlinelibrary.wiley.com/doi/epdf/10.1046/j.1469-8137.2003.00804.x

Nitric oxide signalling in plants

(I) Inorganic components . . . . . . . (a) Nitrate . . . . . . . . . (b) Cation-anionuptake . . . . . . (2) Organic compounds . . . . . . . (a) Amino acids and other organic nitrogenous compounds (b) Organic acids . . . . . . . . (3) Effects on flowering . . . . . . . (4) Ammonium plus nitrate as a nitrogen source . . . ( 5 ) Metabolic changes characteristic of ammonium toxicity . VII . Summary . . . . . . . . . . VI . Physiological effects of nitrogen sources . . . .

Ammonium and nitrate nutrition of plants

Increased synthesis and redistribution of the phytohormone abscisic acid (ABA) in response to water deficit stress initiates an intricate network of signalling pathways in guard cells leading to stomatal closure. Despite the large number of ABA signalling intermediates that are known in guard cells, new discoveries are still being made. Recently, the reactive oxygen species hydrogen peroxide (H2O2) and the reactive nitrogen species nitric oxide (NO) have been identified as key molecules regulating ABA-induced stomatal closure in various species. As with many other physiological responses in which H2O2 and NO are involved, stomatal closure in response to ABA also appears to require the tandem synthesis and action of both these signalling molecules. Recent pharmacological and genetic data have identified NADPH oxidase as a source of H2O2, whilst nitrate reductase has been identified as a source of NO in Arabidopsis guard cells. Some signalling components positioned downstream of H2O2 and NO are calcium, protein kinases and cyclic GMP. However, the exact interaction between the various signalling components in response to H2O2 and NO in guard cells remains to be established.

ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells

Publisher Summary This chapter describes the preparation and extraction of nitrate reductase from higher plants. For preparation of nitrate reductase, chlorophyllous lamina tissue from illuminated plants well supplied with nitrate is normally used as a source material because of its high activity. However, monchlorophyllous organs, such as corn scutella or roots, have been used. Soybean leaves, corn scutella, and cultured rice seedlings are the best known sources of NAD(P)H nitrate reductase. For extraction of the enzyme, the material is homogenized for 30 to 90 seconds in a medium of 1 mM EDTA, 1 to 25 mM cysteine, and 25 mM potassium phosphate, adjusted to a final pH of 8.8 with KOH. Six milliliters of grinding medium are added for each gram of fresh weight of tissue. The homogenate is pressed through four layers of cheesecloth or a single layer of Miracloth and the filtrate centrifuged for 15 minutes at 30,000 g. The supernatant fluid is then decanted through glass wool and used for assays. The homogenates and extracts are kept cold (2° to 3°) throughout. The optimum concentration of cysteine must be verified for each type of tissue and homogenization technique. Glutathione and dithiothreitol are usually slightly more effective (10%) than cysteine.

E.J. Hewitt

Papers

The role of tungsten in the inhibition of nitrate reductase activity in spinach (spinacea oleracea L.) leaves

Oxidation of hydroxylamine by plant enzyme systems.

Molecular exclusion chromatography of nitrite and hydroxylamine reductases from plants with reference to electron donor systems