Extracellular ATP plays an important role in systemic wound response activation

Mechanical wounding occurs in plants during biotic (e.g., herbivore or pathogen attack) or abiotic (e.g., wind damage or freezing) stresses and is associated with the activation of multiple signaling pathways. These initiate many wound responses at the wounded tissues, as well as trigger long-distance signaling pathways that activate wound responses in tissues that were not affected by the initial wounding event (termed systemic wound response). Among the different systemic signals activated by wounding are electric signals, calcium and reactive oxygen species (ROS) waves, and different plant hormones such as jasmonic acid. The release of glutamate from cells at the wounded tissues was recently proposed to trigger several different systemic signal transduction pathways via glutamate-like receptors (GLRs). However, the role of another important compound released from cells during wounding (i.e., extracellular ATP; eATP) in triggering systemic responses is not clear. Here we show that eATP that accumulates in wounded leaves and is sensed by the purinoreceptor kinase P2K is required for the activation of the ROS wave during wounding. Application of eATP to unwounded leaves triggered the ROS wave, and the activation of the ROS wave by wounding or eATP application was suppressed in mutants deficient in P2K (i.e., p2k1-3, p2k2, and p2k1-3p2k2). In addition, the expression of several systemic wound response transcripts was suppressed in mutants deficient in P2K during wounding. Our findings reveal that in addition to sensing glutamate via GLRs, eATP sensed by P2Ks is playing a key role in the triggering of systemic wound responses in plants.

Pvr and distinct downstream signaling factors are required for hemocyte spreading and epidermal wound closure at Drosophila larval wound sites

Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. Here, we discover a role for the Platelet-derived growth factor (PDGF)/Vascular endothelial growth factor (VEGF)-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, we performed a genetic screen in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure, another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. Our data reveals that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal wound closure or hemocyte spreading.

Spatial and temporal patterns of wound periderm development in Cryptomeria japonica bark

Limited investigations have been carried out on the physiological and growth responses of bark to wounding, even though wound periderms play crucial roles in tree defenses. To understand the mechanisms of wound periderm formation, we studied the growth responses and structural changes of wounded bark of three Cryptomeria japonica individuals. We observed the developmental time frame and morphology of wound periderms around mechanically induced wounds in summer. The wound responses included discoloration, lignification, and suberization in tissues present at the time of wounding, followed by wound periderm formation and secondary metabolite deposition. The trees had developed wound periderms approximately 4 weeks after wounding. The wound periderms were within 3 mm in the axial directions and within 1 mm in the lateral directions from the wound surfaces. The distinct patterns of wound periderm formation in the axial and lateral regions resulted from the arrangement and anatomical features of the cells adjacent to the wounds. The wound phellem cells were tangentially narrower and axially shorter in the side and upper/lower regions, respectively, of the wounds. Therefore, the cell division frequencies in the planes parallel to the wound surface may be greater than those in the other directions. Wound reactions in bark might initially be triggered by microenvironmental changes, such as the spread of desiccation, which depends directly on the morphology of phloem cell complexes.

Signatures of plant defense response specificity mediated by herbivore-associated molecular patterns in legumes

Chewing herbivores activate plant defense responses through a combination of mechanical wounding and elicitation by herbivore associated molecular patterns (HAMPs). HAMPs are wound response amplifiers; however, specific defense outputs may also exist that strictly require HAMP-mediated defense signaling. To investigate HAMP-mediated signaling and defense responses, we characterized cowpea transcriptome changes following elicitation by inceptin, a peptide HAMP common in Lepidoptera larvae oral secretions. Following inceptin treatment, we observed large-scale reprogramming of the transcriptome consistent with 3 different response categories: 1) amplification of mechanical wound responses, 2) temporal extension through accelerated or prolonged responses, and 3) examples of inceptin-specific elicitation and suppression. At both early and late timepoints, namely 1 and 6 hours, large sets of transcripts specifically accumulated following inceptin elicitation but not wounding alone. Further inceptin-regulated transcripts were classified as reversing changes induced by wounding alone. Within key signaling and defense related gene families, inceptin-elicited responses commonly targeted select subsets of wound-induced transcripts. Transcripts displaying the largest inceptin-elicited fold-changes included terpene synthases (TPS) and peroxidases (POX) that correspond with induced volatile production and increased peroxidase activity in cowpea. Characterization of inceptin-elicited cowpea defenses via heterologous expression in Nicotiana benthamiana demonstrated that specific cowpea TPS and POX were able to confer terpene emission and the reduced growth of beet armyworm (Spodoptera exigua) herbivores, respectively. Collectively, our present findings in cowpea support a model where HAMP-elicitation both amplifies concurrent wound responses and specifically contributes to the activation of selective outputs associated with direct and indirect anti-herbivore defenses.

A fast, nondestructive method for the detection of disease-related lesions and wounded leaves

Trypan blue staining is a classic way of visualizing leaf disease and wound responses in plants, but it involves working with toxic chemicals and is time-consuming (2–3 days). Here, the investigators established near-infrared scanning with standard lab equipment as a fast and nondestructive method for the analysis of leaf injuries compared with trypan blue staining. Pathogen-inoculated and wounded leaves from potato, tomato, spinach, strawberry, and arabidopsis plants were used for proof of concept. The results showed that this newly developed protocol with near-infrared scanning gave the same results as trypan blue staining. Furthermore, a macro in FIJI was made to quantify the leaf damage. The new protocol was time-efficient, nondestructive, chemical-free and may be used for high-throughput studies.

Pvr and downstream signaling factors are required for spreading of Drosophila hemocytes at larval wound sites

Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. Here, we discover a role for the Platelet-derived growth factor (PDGF)/ Vascular endothelial growth factor (VEGF)-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, we performed a genetic screen in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure, another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. Our data reveals that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal wound closure or hemocyte spreading.

Identification of functionally distinct macrophage subpopulations in Drosophila

Vertebrate macrophages are a highly heterogeneous cell population, but whileDrosophilablood is dominated by a macrophage-like lineage (plasmatocytes), until very recently these cells were considered to represent a homogeneous population. Here, we present our identification of enhancer elements labelling plasmatocyte subpopulations, which vary in abundance across development. These subpopulations exhibit functional differences compared to the overall population, including more potent injury responses and differential localisation and dynamics in pupae and adults. Our enhancer analysis identified candidate genes regulating plasmatocyte behaviour: pan-plasmatocyte expression of one such gene (Calnexin14D) improves wound responses, causing the overall population to resemble more closely the subpopulation marked by theCalnexin14D-associated enhancer. Finally, we show that exposure to increased levels of apoptotic cell death modulates subpopulation cell numbers. Taken together this demonstrates macrophage heterogeneity inDrosophila, identifies mechanisms involved in subpopulation specification and function and facilitates the use ofDrosophilato study macrophage heterogeneity in vivo.

Differential Regulation of the Ribosomal Association of mRNA Transcripts in an Arabidopsis Mutant Defective in Jasmonate-Dependent Wound Response

Jasmonoyl-L-isoleucine (JA-Ile) is a powerful oxylipin responsible for the genome-wide transcriptional reprogramming in plants that results in major physiological shifts from growth to defense. The double T-DNA insertion Arabidopsis mutant, cyp94b1cyp94b3 (b1b3), defective in cytochrome p450s, CYP94B1 and CYP94B3, which are responsible for oxidizing JA-Ile, accumulates several fold higher levels of JA-Ile yet displays dampened JA-Ile–dependent wound responses—the opposite of what is expected. Transcriptomic and proteomic analyses showed that while the transcriptional response to wounding was largely unchanged in b1b3 compared to wild type (WT), many proteins were found to be significantly reduced in the mutant, which was verified by immunoblot analyses of marker proteins. To understand this protein phenotype and their hypothesized contribution to the b1b3 phenotypes, wounded rosette leaf samples from both WT and b1b3 were subject to a translating ribosome affinity purification RNA sequencing analysis. More than 1,600 genes whose transcripts do not change in abundance by wounding changed their association with the ribosomes after wounding in WT leaves. Consistent with previous observations, the total pool of mRNA transcripts was similar between WT and b1b3; however, the ribosome-associated pool of transcripts was changed significantly. Most notably, fewer transcripts were associated with the ribosome pool in b1b3 than in WT, potentially explaining the reduction of many proteins in the mutant. Among those genes with fewer ribosome-associated transcripts in b1b3 were genes relating to stress response, specialized metabolism, protein metabolism, ribosomal subunits, and transcription factors, consistent with the biochemical phenotypes of the mutant. These results show previously unrecognized regulations at the translational level that are affected by misregulation of JA homeostasis during the wound response in plants.

Foreign body responses in mouse central nervous system mimic natural wound responses and alter biomaterial functions

Biomaterials hold promise for therapeutic applications in the central nervous system (CNS). Little is known about molecular factors that determine CNS foreign body responses (FBRs) in vivo, or about how such responses influence biomaterial function. Here, we probed these factors in mice using a platform of injectable hydrogels readily modified to present interfaces with different physiochemical properties to host cells. We found that biomaterial FBRs mimic specialized multicellular CNS wound responses not present in peripheral tissues, which serve to isolate damaged neural tissue and restore barrier functions. We show that the nature and intensity of CNS FBRs are determined by definable properties that significantly influence hydrogel functions, including resorption and molecular delivery when injected into healthy brain or stroke injuries. Cationic interfaces elicit stromal cell infiltration, peripherally derived inflammation, neural damage and amyloid production. Nonionic and anionic formulations show minimal levels of these responses, which contributes to superior bioactive molecular delivery. Our results identify specific molecular mechanisms that drive FBRs in the CNS and have important implications for developing effective biomaterials for CNS applications.