Radhika Desikan

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.

A new role for an old enzyme: Nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana

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.

SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling.

Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone — an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation and climate change. The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes. Despite the vital role of guard cells in controlling plant water loss, ozone sensitivity and CO2 supply, the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli. However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO2, abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca2+ and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca2+ channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate suggest a vital role for SLAC1 in the function of S-type anion channels.

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.

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.

Hydrogen peroxide is a common signal for darkness- and ABA-induced stomatal closure in Pisum sativum

The requirement for hydrogen peroxide (H2O2) generation and action during stomatal closure induced by darkness and abscisic acid (ABA) was investigated in pea (Pisum sativum L.). Stomatal closure induced by darkness or ABA was inhibited by the H2O2-scavenging enzyme catalase or the antioxidant N-acetyl cysteine (NAC), or by diphenylene iodonium (DPI), an inhibitor of the H2O2-generating enzyme NADPH oxidase. Exogenous H2O2 induced stomatal closure in a dose- and time-dependent manner, and H2O2 was also required for ABA-inhibition of stomatal opening in the light. H2O2 accumulation in guard cells was increased by darkness or ABA, as assessed with the fluorescent dye dichlorodihydrofluorescein diacetate (H2-DCFDA) and confocal microscopy. Such increases were inhibited by catalase, NAC or DPI, consistent with the effects of these compounds on stomatal apertures. Employing polymerase chain reaction (PCR) with degenerate oligonucleotide primers, several NADPH oxidase homologues were identified from pea genomic DNA that had substantial identity to the Arabidopsis thaliana (L.) Heynh. rboh (respiratory burst oxidase homologue) genes. Furthermore, an antibody raised against the tomato rboh identified immunoreactive proteins in epidermal, mesophyll and guard cells.

Cell signalling following plant/pathogen interactions involves the generation of reactive oxygen and reactive nitrogen species

It is now clear that reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), and reactive nitrogen species, such as nitric oxide (NO), are produced by plant cells in response to a variety of stresses, including pathogen challenge. Such molecules may be involved in direct defence mechanisms, such as cross-linking of plant cell walls, or as antimicrobial agents. However, it is also apparent that cells generate such reactive species as signalling molecules, produced at controlled levels, and leading to defined responses. Signalling responses to ROS and NO include the activation of mitogen-activated protein kinases, and the up- and down-regulation of gene expression, often leading to localised programmed cell death, characteristic of the hypersensitive response. Therefore, ROS and NO are key molecules which may help to orchestrate events following pathogen challenge. Here we review the generation and role of both reactive oxygen and reactive nitrogen species in plant cells.

Does the redox status of cytochrome C act as a fail-safe mechanism in the regulation of programmed cell death?

It has now become recognized that one of the key events in the induction of apoptosis, or programmed cell death, in both plants and animals is the release of cytochrome c from mitochondria. It is also known that oxidative stress imposed on cells can have a profound effect on the onset or progression of apoptosis. Here, we discuss how the redox status of cytochrome c, and thus its structure, can be altered by the presence of reactive oxygen species (ROS) and reduced glutathione (GSH). We suggest that cytochrome c will only induce programmed cell death if present in the cytoplasm in the oxidized state, and that the presence of high levels of cytoplasmic GSH maintain cytochrome c in an inactive (reduced) state, thus behaving as a fail-safe mechanism if cytochrome c is released by mitochondria when programmed cell death is not the required outcome. If the redox status of the cell is disturbed however, perhaps in the presence of hydrogen peroxide, GSH concentrations will drop, the cellular E(h) will rise, and cytochrome c will tend towards the oxidized state, allowing programmed cell death to proceed. Therefore, we propose that the redox state of cytoplasmic cytochrome c may be a key regulator of programmed cell death.