Summary: Ethylene is a plant hormone that regulates many aspects of growth and development. Despite the well‐known association between ethylene and stress signalling, its effects on stomatal movements are largely unexplored. Here, genetic and physiological data are provided that position ethylene into the Arabidopsis guard cell signalling network, and demonstrate a functional link between ethylene and hydrogen peroxide (H₂O₂). In wild‐type leaves, ethylene induces stomatal closure that is dependent on H₂O₂ production in guard cells, generated by the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase AtrbohF. Ethylene‐induced closure is inhibited by the ethylene antagonists 1‐MCP and silver. The ethylene receptor mutants etr1–1 and etr1–3 are insensitive to ethylene in terms of stomatal closure and H₂O₂ production. Stomata of the ethylene signalling ein2–1 and arr2 mutants do not close in response to either ethylene or H₂O₂ but do generate H₂O₂ following ethylene challenge. Thus, the data indicate that ethylene and H₂O₂ signalling in guard cells are mediated by ETR1 via EIN2 and ARR2‐dependent pathway(s), and identify AtrbohF as a key mediator of stomatal responses to ethylene.

Summary: Nitric oxide (NO) and hydrogen peroxide (H₂O₂) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H₂O₂ 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 H₂O₂ in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA‐mediated NO generation is in fact dependent on ABA‐induced H₂O₂ production. Stomatal closure induced by H₂O₂ is inhibited by the removal of NO with NO scavenger, and both ABA and H₂O₂ stimulate guard cell NO synthesis. Conversely, NO‐induced stomatal closure does not require H₂O₂ synthesis nor does NO treatment induce H₂O₂ 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 H₂O₂ and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA‐mediated H₂O₂ synthesis. In the NR double mutant nia1, nia2 both ABA and H₂O₂ fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H₂O₂ 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 H₂O₂ production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter‐relationship between ABA, endogenous H₂O₂ and NO‐induced stomatal closure.

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. Contents Summary 11 1 Introduction 12 2 Why does NO make a good signal? 12 3 NO biosynthesis 13 4 NO biology 17 5 NO signal transduction 23 6 Conclusion 30 Acknowledgements 31 References 31

Various data indicate that nitric oxide (NO) is an endogenous signal in plants that mediates responses to several stimuli. Experimental evidence in support of such signalling roles for NO has been obtained via the application of NO, usually in the form of NO donors, via the measurement of endogenous NO, and through the manipulation of endogenous NO content by chemical and genetic means. Stomatal closure, initiated by abscisic acid (ABA), is effected through a complex symphony of intracellular signalling in which NO appears to be one component. Exogenous NO induces stomatal closure, ABA triggers NO generation, removal of NO by scavengers inhibits stomatal closure in response to ABA, and ABA-induced stomatal closure is reduced in mutants that are impaired in NO generation. The data indicate that ABA-induced guard cell NO generation requires both nitric oxide synthase-like activity and, in Arabidopsis, the NIA1 isoform of nitrate reductase (NR). NO stimulates mitogen-activated protein kinase (MAPK) activity and cGMP production. Both these NO-stimulated events are required for ABA-induced stomatal closure. ABA also stimulates the generation of H₂O₂ in guard cells, and pharmacological and genetic data demonstrate that NO accumulation in these cells is dependent on such production. Recent data have extended this model to maize mesophyll cells where the induction of antioxidant defences by water stress and ABA required the generation of H₂O₂ and NO and the activation of a MAPK. Published data suggest that drought and salinity induce NO generation which activates cellular processes that afford some protection against the oxidative stress associated with these conditions. Exogenous NO can also protect cells against oxidative stress. Thus, the data suggest an emerging model of stress responses in which ABA has several ameliorative functions. These include the rapid induction of stomatal closure to reduce transpirational water loss and the activation of antioxidant defences to combat oxidative stress. These are two processes that both involve NO as a key signalling intermediate.

The plant hormone abscisic acid (ABA), synthesized in response to water-deficit stress, induces stomatal closure via activation of complex signaling cascades. Recent work has established that nitric oxide (NO) is a key signaling molecule mediating ABA-induced stomatal closure. However, the biosynthetic origin of NO in guard cells has not yet been resolved. Here, we provide pharmacological, physiological, and genetic evidence that NO synthesis in Arabidopsis guard cells is mediated by the enzyme nitrate reductase (NR). Guard cells of wild-type Arabidopsis generate NO in response to treatment with ABA and nitrite, a substrate for NR. Moreover, NR-mediated NO synthesis is required for ABA-induced stomatal closure. However, in the NR double mutant, nia1 , nia2 that has diminished NR activity, guard cells do not synthesize NO nor do the stomata close in response to ABA or nitrite, although stomatal opening is still inhibited by ABA. Furthermore, by using the ABA-insensitive (ABI) abi1–1 and abi2–1 mutants, we show that the ABI1 and ABI2 protein phosphatases are downstream of NO in the ABA signal-transduction cascade. These data demonstrate a previously uncharacterized signaling role for NR, that of mediating ABA-induced NO synthesis in Arabidopsis guard cells.

Summary: Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant–pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO‐induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase‐like activity in Arabidopsis cells, while the caspase‐1 inhibitor, Ac‐YVAD‐CMK, blocked NO‐induced cell death. A well‐established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO‐induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell‐permeable cGMP analogue, 8Br‐cGMP, although 8Br‐cGMP alone did not induce cell death or potentiate NO‐induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO‐induced cell death in Arabidopsis. In‐gel protein kinase assays showed that NO activates a potential mitogen‐activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H₂O₂‐induced cell death, did not inhibit the effects of NO.