John T. Hancock

Hydrogen peroxide signalling

Recent biochemical and genetic studies confirm that hydrogen peroxide is a signalling molecule in plants that mediates responses to abiotic and biotic stresses. Signalling roles for hydrogen peroxide during abscisic-acid-mediated stomatal closure, auxin-regulated root gravitropism and tolerance of oxygen deprivation are now evident. The synthesis and action of hydrogen peroxide appear to be linked to those of nitric oxide. Downstream signalling events that are modulated by hydrogen peroxide include calcium mobilisation, protein phosphorylation and gene expression. Calcium and Rop signalling contribute to the maintenance of hydrogen peroxide homeostasis.

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

Nitric oxide (NO) and hydrogen peroxide (H2 O2 ) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H2 O2 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 H2 O2 in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H2 O2 production. Stomatal closure induced by H2 O2 is inhibited by the removal of NO with NO scavenger, and both ABA and H2 O2 stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H2 O2 synthesis nor does NO treatment induce H2 O2 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 H2 O2 and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H2 O2 synthesis. In the NR double mutant nia1, nia2 both ABA and H2 O2 fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H2 O2 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 H2 O2 production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H2 O2 and NO-induced stomatal closure.

Nitric oxide, stomatal closure, and abiotic stress

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.

Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH oxidase-like enzyme

Suspension‐cultured cells of Arabidopsis thaliana generated active oxygen species (AOS) (measured by luminol‐dependent chemiluminescence) following challenge with the bacterial protein elicitor harpin or the protein kinase activator phorbol 12‐myristate 13‐acetate. These responses were blocked by inhibitors of superoxide dismutase (SOD), NADPH oxidase and protein kinase. Harpin treatment also resulted in an increase in cell death, a response reduced by inhibitors of AOS generation or AOS scavengers. Extracellular SOD activity was found to be present in cell culture medium. Immunoblotting of Arabidopsis extracts revealed the presence of proteins immunologically related to the human neutrophil NADPH oxidase comples, and cell‐free reconstitution assays showed that human neutrophil cytosol combined with Arabidopsis membranes could initiate superoxide generation. These data suggest that the enzyme catalysing the generation of superoxide in elicited Arabidopsis cells is similar to the mammalian NADPH oxidase and that a signalling cascade leading to AOS generation involves protein phosphorylation.

NADPH oxidase: a universal oxygen sensor?

NADPH oxidase is classically regarded as a key enzyme of neutrophils, where it is involved in the pathogenic production of reactive oxygen species. However, NADPH oxidase-like enzymes have recently been identified in non-neutrophil cells, supporting a separate role for NADPH-oxidase derived oxygen species in oxygen sensitive processes. This article reviews the current literature surrounding the potential role of NADPH oxidase in the oxygen sensing processes which underlie hypoxic pulmonary vasoconstriction, systemic vascular smooth muscle proliferation, carotid and airways chemoreceptor activation, erythropoietin gene expression, and oxytropic responses of plant cells.

A role for ETR1 in hydrogen peroxide signaling in stomatal guard cells

Signaling through the redox active molecule hydrogen peroxide (H2O2) is important for several processes in plants, such as stomatal closure, root growth, gravitropism, and responses to pathogen challenge ([Neill et al., 2002][1]; [Laloi et al., 2004][2]). Although oxidative modification of reactive

Hydrogen peroxide–induced gene expression in Arabidopsis thaliana

Hydrogen peroxide (H(2)O(2)) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H(2)O(2) generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H(2)O(2). These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H(2)O(2)-induced defense responses are discussed.

A novel hydrogen sulfide donor causes stomatal opening and reduces nitric oxide accumulation

Effects of hydrogen sulfide (H(2)S) on plant physiology have been previously studied, but such studies have relied on the use of NaSH as a method for supplying H(2)S to tissues. Now new compounds which give a less severe H(2)S shock and a more prolonged exposure to H(2)S have been developed. Here the effects of one such compound, GYY4137, has been investigated to determine its effects on stomatal closure in Arabidopsis thaliana. It was found that both NaSH and GYY4137 caused stomatal opening in the light and prevented stomatal closure in the dark. Nitric oxide (NO) has been well established as a mediator of stomatal movements and here it was found that both NaSH and GYY4137 reduced the accumulation of NO in guard cells, perhaps suggesting a mode of action for H(2)S in this system. GYY4137, and future related compounds, will be important tools to unravel the effects of plant exposure to H(2)S and to determine how H(2)S may fit into plant cell signalling pathways.

Hydrogen sulfide: environmental factor or signalling molecule?

Hydrogen sulfide (H₂S) has traditionally been thought of as a phytotoxin, having deleterious effects on the plant growth and survival. It is now recognized that plants have enzymes which generate H₂S, cysteine desulfhydrase, and remove it, O-acetylserine lyase. Therefore, it has been suggested that H₂S is considered as a signalling molecule, alongside small reactive compounds such as hydrogen peroxide (H₂O₂) and nitric oxide (NO). Exposure of plants to low of H₂S, for example from H₂S donors, is revealing that many physiological effects are seen. H₂S seems to have effects on stomatal apertures. Intracellular effects include increases in glutathione levels, alterations of enzyme activities and influences on NO and H₂O₂ metabolism. Work in animals has shown that H₂S may have direct effects on thiol modifications of cysteine groups, work that will no doubt inform future studies in plants. It appears therefore, that instead of thinking of H₂S as a phytotoxin, it needs to be considered as a signalling molecule that interacts with reactive oxygen species and NO metabolism, as well as having direct effects on the activity of proteins. The future may see H₂S being used to modulate plant physiology in the field or to protect crops from postharvest spoilage.

Role of Xanthine Oxidoreductase as an Antimicrobial Agent

Xanthine oxidoreductase (XOR) is widely distributed in mammalian tissues and has long been known to be a major constituent of the milk fat globule membrane (MFGM), which surrounds fat globules in cows milk (36, 50). This source has been exploited for isolation and characterization of the bovine enzyme for many decades (44). XOR is a complex enzyme comprising two identical 147,000-Mr subunits, each of which contains one molybdenum, one flavin adenine dinucleotide, and two nonidentical iron-sulfur redox centers (8, 32, 33). While the enzymology of XOR is well documented, its physiological role is unclear. The enzyme occurs in most mammalian tissues, and although it has a broad specificity for reducing substrates, its conventionally accepted role is in purine catabolism, catalyzing the oxidation of hypoxanthine to xanthine and the oxidation of xanthine to uric acid. Despite its wide tissue distribution, the enzyme is believed to be largely concentrated in endothelial and epithelial cells. Such a specific cellular location implies a physiological role apart from that of a simple housekeeping enzyme, and other functions have been sought. Indeed, the role of XOR in milk has long been a puzzle.