Reductive stress triggers ANAC017-mediated retrograde signaling to safeguard the endoplasmic reticulum by boosting mitochondrial respiratory capacity.

Redox processes are at the heart of universal life processes, such as metabolism, signaling, or folding of secreted proteins. Redox landscapes differ between cell compartments and are strictly controlled to tolerate changing conditions and to avoid cell dysfunction. While a sophisticated antioxidant network counteracts oxidative stress, our understanding of reductive stress responses remains fragmentary. Here, we observed root growth impairment in Arabidopsis thaliana mutants of mitochondrial alternative oxidase 1a (aox1a) in response to the model thiol reductant dithiothreitol (DTT). Mutants of mitochondrial uncoupling protein 1 (ucp1) displayed a similar phenotype indicating that impaired respiratory flexibility led to hypersensitivity. Endoplasmic reticulum (ER) stress was enhanced in the mitochondrial mutants and limiting ER oxidoreductin capacity in the aox1a background led to synergistic root growth impairment by DTT, indicating that mitochondrial respiration alleviates reductive ER stress. The observations that DTT triggered nicotinamide adenine dinucleotide (NAD) reduction in vivo and that the presence of thiols led to electron transport chain activity in isolated mitochondria offer a biochemical framework of mitochondrion-mediated alleviation of thiol-mediated reductive stress. Ablation of transcription factor Arabidopsis NAC domain-containing protein17 (ANAC017) impaired the induction of AOX1a expression by DTT and led to DTT hypersensitivity, revealing that reductive stress tolerance is achieved by adjusting mitochondrial respiratory capacity via retrograde signaling. Our data reveal an unexpected role for mitochondrial respiratory flexibility and retrograde signaling in reductive stress tolerance involving inter-organelle redox crosstalk.

Mitochondria-derived reactive oxygen species are the likely primary trigger of mitochondrial retrograde signaling in Arabidopsis.

Besides their central function in respiration, plant mitochondria play a crucial role in maintaining cellular homeostasis during stress by providing "retrograde" feedback to the nucleus. Despite the growing understanding of this signaling network, the nature of the signals that initiate mitochondrial retrograde regulation (MRR) in plants remains unknown. Here, we investigated the dynamics and causative relationship of a wide range of mitochondria-related parameters for MRR, using a combination of Arabidopsis fluorescent protein biosensor lines, in vitro assays, and genetic and pharmacological approaches. We show that previously linked physiological parameters, including changes in cytosolic ATP, NADH/NAD+ ratio, cytosolic reactive oxygen species (ROS), pH, free Ca2+, and mitochondrial membrane potential, may often be correlated with-but are not the primary drivers of-MRR induction in plants. However, we demonstrate that the induced production of mitochondrial ROS is the likely primary trigger for MRR induction in Arabidopsis. Furthermore, we demonstrate that mitochondrial ROS-mediated signaling uses the ER-localized ANAC017-pathway to induce MRR response. Finally, our data suggest that mitochondrially generated ROS can induce MRR without substantially leaking into other cellular compartments such as the cytosol or ER lumen, as previously proposed. Overall, our results offer compelling evidence that mitochondrial ROS elevation is the likely trigger of MRR.

Selective optogenetic control of Gq signaling using human Neuropsin.

Gq proteins are universally important for signal transduction in mammalian cells. The underlying kinetics and transformation from extracellular stimuli into intracellular signaling, however could not be investigated in detail so far. Here we present the human Neuropsin (hOPN5) for specific and repetitive manipulation of Gq signaling in vitro and in vivo with high spatio-temporal resolution. Properties and G protein specificity of hOPN5 are characterized by UV light induced IP3 generation, Ca2+ transients and inhibition of GIRK channel activity in HEK cells. In adult hearts from a transgenic animal model, light increases the spontaneous beating rate. In addition, we demonstrate light induced contractions in the small intestine, which are not detectable after pharmacological Gq protein block. All-optical high-throughput screening for TRPC6 inhibitors is more specific and sensitive than conventional pharmacological screening. Thus, we demonstrate specific Gq signaling of hOPN5 and unveil its potential for optogenetic applications.