Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.

SummaryNitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes. A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation.Here we show that endogenous NO accumulation in Arabidopsis, resulting from loss-offunction mutations in NO Overexpression 1 (NOX1), led to disabled Resistance (R) gene-mediated protection, basal resistance and defence against nonadapted pathogens. In nox1 plants both salicylic acid (SA) synthesis and signalling were suppressed, reducing SA-dependent defence gene expression.Significantly, expression of a GSNOR1 transgene complemented the SNO-dependent phenotypes of paraquat resistant 2-1 (par2-1) plants but not the NO-related characters of the nox1-1 line. Furthermore, atgsnor1-3 nox1-1 double mutants supported greater bacterial titres than either of the corresponding single mutants.Our findings imply that GSNO and NO, two pivotal redox signalling molecules, exhibit additive functions and, by extension, may have distinct or overlapping molecular targets during both immunity and development.

Switches between global sleep and wakefulness states are believed to be dictated by top-down influences arising from subcortical nuclei. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterized by a substantially reduced propensity to transition between wake and sleep states with an especially pronounced deficit in initiating rapid eye movement (REM) sleep episodes. The causative mutation, an Ile102Asn substitution in the synaptic vesicular protein, VAMP2, was associated with morphological synaptic changes and specific behavioral deficits, while in vitro electrophysiological investigations with fluorescence imaging revealed a markedly diminished probability of vesicular release in mutants. Our data show that global shifts in the synaptic efficiency across brain-wide networks leads to an altered probability of vigilance state transitions, possibly as a result of an altered excitability balance within local circuits controlling sleep-wake architecture.

Pulmonary hypertension (PH) is a progressive fatal disease that currently has no specific therapeutic approaches. In this study, dsRNA-dependent protein kinase (PKR) was considered a candidate molecule in PH. We demonstrated that PKR is activated in the endothelium of experimental PH models. Deletion of PKR or treatment with the PKR activation inhibitor C16 inhibited the development of PH. To explore the mechanism of PKR in PH, we detected its downstream signaling and found that PKR knockout represses apoptosis-associated speck-like protein containing CARD (ASC) activation to inhibit high mobility group box 1 (HMGB1) and Interleukin-1 beta (IL-1β) release. To further explore whether ASC mediate the pro-PH role of PKR, we used ASC deletion mice and found that ASC deletion inhibits the development of PH and the release of HMGB1 and IL-1β. Furthermore, we cocultured pulmonary arterial endothelial cells (PAECs) and pulmonary arterial smooth muscle cells (PASMCs) and found that endothelial PKR promotes PASMCs proliferation through the release of HMGB1 and IL-1β. In conclusion, these data indicate that endothelial PKR promotes the excessive proliferation of PASMCs by inducing ASC activation to release HMGB1 and IL-1β, which lead to the development of PH. Our study will provide a novel insight that PKR is a potential target in the future treatment of PH.

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