Vorsicht, Verwundung! Reizweiterleitung mit mechanosensitiven Proteinen

Being sessile, plants are exposed to adverse stresses, including wounding by insects. Albeit lacking experimental evidence, one hypothesis predicted involvement of hydro-electric signals in wound signaling. Now, we could show that the mechanosensitive anion channel MSL10 is necessary for wound-induced long-distance signaling in plants. By linking mechano-sensing, ion fluxes, membrane depolarization and electrical signal propagation, MSL10 might integrate hydraulic and electric wound signals.

Zones of cellular damage around pulsed-laser wounds

After a tissue is wounded, cells surrounding the wound adopt distinct wound-healing behaviors to repair the tissue. Considerable effort has been spent on understanding the signaling pathways that regulate immune and tissue-resident cells as they respond to wounds, but these signals must ultimately originate from the physical damage inflicted by the wound. Tissue wounds comprise several types of cellular damage, and recent work indicates that different types of cellular damage initiate different types of signaling. Hence to understand wound signaling, it is important to identify and localize the types of wound-induced cellular damage. Laser ablation is widely used by researchers to create reproducible, aseptic wounds in a tissue that can be live-imaged. Because laser wounding involves a combination of photochemical, photothermal and photomechanical mechanisms, each with distinct spatial dependencies, cells around a pulsed-laser wound will experience a gradient of damage. Here we exploit this gradient to create a map of wound-induced cellular damage. Using genetically-encoded fluorescent proteins, we monitor damaged cellular and sub-cellular components of epithelial cells in living Drosophila pupae in the seconds to minutes following wounding. We hypothesized that the regions of damage would be predictably arrayed around wounds of varying sizes, and subsequent analysis found that all damage radii are linearly related over a 3-fold range of wound size. Thus, around laser wounds, the distinct regions of damage can be estimated after measuring any one. This report identifies several different types of cellular damage within a wounded epithelial tissue in a living animal. By quantitatively mapping the size and placement of these different types of damage, we set the foundation for tracing wound-induced signaling back to the damage that initiates it.

Zones of Cellular Damage Around Pulsed-Laser Wounds

After a tissue is wounded, cells surrounding the wound adopt distinct wound-healing behaviors to repair the tissue. Considerable effort has been spent on understanding the signaling pathways that regulate immune and tissue-resident cells as they respond to wounds, but these signals must ultimately originate from the physical damage inflicted by the wound. Tissue wounds comprise several types of cellular damage, and recent work indicates that different types of cellular damage initiate different types of signaling. Hence to understand wound signaling, it is important to identify and localize the types of wound-induced cellular damage. Laser ablation is widely used by researchers to create reproducible, aseptic wounds in a tissue that can be live-imaged. Because laser wounding involves a combination of photochemical, photothermal and photomechanical mechanisms, each with distinct spatial dependencies, cells around a pulsed-laser wound will experience a gradient of damage. Here we exploit this gradient to create a map of wound-induced cellular damage. Using genetically-encoded fluorescent proteins, we monitor damaged cellular and sub-cellular components of epithelial cells in living Drosophila pupae in the seconds to minutes following wounding. We hypothesized that the regions of damage would be predictably arrayed around wounds of varying sizes, and subsequent analysis found that all damage radii are linearly related o ver a 3-fold range of wound size . Thus, around laser wounds, the distinct regions of damage can be estimated after measuring any one. This report identifies several different types of cellular damage within a wounded epithelial tissue in a living animal. By quantitatively mapping the size and placement of these different types of damage, we set the foundation for tracing wound-induced signaling back to the damage that initiates it.

A triresidue motif in the GLUTAMATE RECEPTOR-LIKE 3.3 C-tail interacts with IMPAIRED SUCROSE INDUCTION 1 and controls long distance wound signaling

Arabidopsis Clade 3 GLUTAMATE RECEPTOR-LIKE (GLRs) genes are primary players in wound-induced electrical signaling and jasmonate-activated defense responses. As cation-permeable ion channels, previous studies have focused on resolving their gating properties and structures. However, little is known regarding to the regulatory mechanism of these channel proteins. Here, we report that the C-tail of GLR3.3 contains key elements that control its function in long distance wound signaling. GLR3.3 without its C-tail failed to rescue the glr3.3a mutant. To further investigate the underlying mechanism, we performed a yeast two-hybrid screen. IMPAIRED SUCROSE INDUCTION 1 (ISI1) was identified as an interactor with both the C-tail and the full-length GLR3.3 in planta. Reduced function isi1 mutants had enhanced electrical activity and jasmonate-regulated defense responses. Furthermore, we found that a triresidue motif RFL (R884, F885 and L886) in the GLR3.3 C-tail is essential for interacting with ISI1. RFL mutation abolished GLR3.3 function in electrical signaling and jasmonate-mediated defense gene activation. Our study shows the importance of the C-tail in GLR3.3 function, and reveals parallels with the ipnotropic glutamate receptor regulation in animal cells.

Two glutamate- and pH-regulated Ca2+ channels are required for systemic wound signaling in Arabidopsis

Plants defend against herbivores and nematodes by rapidly sending signals from the wounded sites to the whole plant. We investigated how plants generate and transduce these rapidly moving, long-distance signals referred to as systemic wound signals. We developed a system for measuring systemic responses to root wounding in Arabidopsis thaliana. We found that root wounding or the application of glutamate to wounded roots was sufficient to trigger root-to-shoot Ca2+ waves and slow wave potentials (SWPs). Both of these systemic signals were inhibited by either disruption of both GLR3.3 and GLR3.6, which encode glutamate receptor–like proteins (GLRs), or constitutive activation of the P-type H+-ATPase AHA1. We further showed that GLR3.3 and GLR3.6 displayed Ca2+-permeable channel activities gated by both glutamate and extracellular pH. Together, these results support the hypothesis that wounding inhibits P-type H+-ATPase activity, leading to apoplastic alkalization. This, together with glutamate released from damaged phloem, activates GLRs, resulting in depolarization of membranes in the form of SWPs and the generation of cytosolic Ca2+ increases to propagate systemic wound signaling.

The structural bases for agonist diversity in anArabidopsis thalianaglutamate receptor-like channel

Arabidopsis thalianaglutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+increase inArabidopsisseedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference forl-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology.