Feeling the danger: local wound signaling in plants

Local wound signaling in plants informs the surrounding tissues about an injury and initiates the regeneration process. In a recent paper published in Cell, Yang and colleagues show the involvement of a single Pep family member from tomato in wound signaling and how exogenous application of this regeneration factor enhances transformation efficiency in crops.

Wounding represents a major challenge to plant tissues as plants cannot actively evade damage and lack the mobile cells that close and heal wounds in animals. Nonetheless, plants have evolved remarkably robust, yet simple mechanisms to deal with wounds quickly and efficiently. The outstanding plant wound healing abilities have been utilized for centuries for agricultural improvement, e.g., in the development of grafting or pruning. Wound healing in plants starts with the rapid sensing of the injury and is then accomplished mainly by the targeted proliferation of cells in the undamaged surrounding tissue and directional growth of the restoring tissue towards the wound.^1,2,3 Additionally, wounded tissues trigger defense responses in adjacent cells and in tissues or organs further away, constituting local and systemic defenses, respectively, that protect the damaged plants from entry of pathogens and further damage.⁴ While the regeneration and defense processes are well described now, the nature of the initial wound signal and its detection in the surrounding cells, here referred to as wound signaling, remain enigmatic. Various compounds have been postulated to be involved in the initial wound signaling, triggering downstream signal transduction in perceiving cells. These components include so-called damage-associated molecular patterns (DAMPs) like cell wall fragments, lipids or the ATP released from ruptured cells; short-range signals including reactive oxygen species (ROS) and Ca²⁺; and phytohormones, mainly jasmonate or ethylene.^5,6 As localized responses to wounding occur only in a single or a couple of cells adjacent to the wound site, classical genetic or omics approaches in whole plants fall short in observing involvement of any of these components. Nonetheless, a study employing microscopy and pharmacological screens managed a major breakthrough regarding the mechanism of wound perception in the roots of Arabidopsis thaliana.⁷ Following membrane rupture, Ca²⁺-dependent metacaspases execute the maturation of plant elicitor peptide 1 (Pep1) that is perceived by neighboring cells via Pep receptors (PEPRs) to trigger local defense responses.

A recent study published in Cell by Yang et al. used tomato to investigate local and systemic wound signaling.⁸ While systemic wound signaling is mediated by the small peptide systemin, little is known about the components required for local wound signaling. The authors identified a novel wound-induced peptide, an ortholog of Arabidopsis Pep1 called REGENERATION FACTOR1 (REF1). They demonstrated its involvement in both systemic and local wound signaling responses, necessary for callus and shoot regeneration from tomato explants. They showed that REF1, which is the only Pep family member in tomato, is perceived by the PEPR1/2-ORTHOLOG RECEPTOR KINASE1 (PORK1). REF1 boosts regeneration capacity dependent on the receptor PORK1, while both ref1 and pork1 loss-of-function mutants displayed decreased regeneration capacity. The authors also found that a key regeneration component SlWIND1, a homolog of WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) from Arabidopsis, is induced in tomato by wounding and this is partly mediated by REF1 signaling. Conversely, REF1 expression is also activated after wounding and this is dependent on SlWIND1, which binds to the REF1 promoter. Hence, the authors proposed that REF1 and SlWIND1 act in a positive feedback loop for propagating wound responses within the local tissue context and mediating regeneration (Fig. 1).

Fig. 1: Schematic representation of local and systemic wound signaling for regeneration.

a Wounding triggers the release of peptides systemin (yellow) and REF1 (red) in tomato. Systemin triggers systemic wound responses in distant tissues including defense and immune responses. REF1 acts locally in the vicinity of the wound and enhances systemic wound signaling promoting regeneration and wound healing. Rectangular inset represents cells drawn in b. b Cell rupture induces maturation of Pep family member REF1, which is perceived by the receptor PORK1. PORK1 perception of REF1 partly mediates activation of SlWIND1, which drives the expression of REF1 itself. This contributes to the propagation of local wound signaling in cells surrounding the wound site. c Exogenous treatment with REF1 dramatically enhances transformation efficiency in recalcitrant accessions of tomato, soybean, wheat and maize. Removed leaves indicate explant preparation required for crop transformation. Circle with orange arrow indicates a plasmid. Color change indicates successful transformation.

A limiting factor for transformation of crops like tomato is the regeneration capacity of explants. The authors used REF1 to enhance regeneration capacity and tested transformation efficiency in various crop species. They found significant increases of 4–12-fold in transformation efficiency within recalcitrant accessions of tomato, soybean, wheat and maize. This represents a promising, novel approach to enhance transformation efficiency as traditional methods rely on endogenous overexpression of genes involved in regeneration or somatic embryogenesis.⁹ However, besides the need of tedious generation of transgenic lines, such overexpression lines often show pleiotropic defects and abnormal development, limiting their applicability. Enhancing transformation efficiency by mere application of an exogenous regeneration factor is not only much easier but also allows the subsequent cultivation without developmental defects. This approach also significantly simplifies genetic studies and the generation of fluorescently tagged marker lines for in vivo microscopy in crops. Notably, it may also allow the construction of novel genetically modified crop lines with improved yield or resistance. Moreover, if the application of only one regeneration factor already significantly elevates transformation efficiency, then the discovery of so far unknown additional molecular players may have a great potential for enhancing transformation efficiency and may enable the transgenesis of species that could not have been studied so far.

The findings of this study have a number of further important implications. Firstly, the study suggests that REF1 acts in accordance with SlWIND1 to establish a feedback loop that propagates the wound signaling and activates defense and regeneration responses. However, the authors’ results show that SlWIND1 expression is only partly dependent on REF1 and REF1 is activated only transcriptionally by SlWIND1. Additionally, the maturation of REF1 from the propeptide has not been studied in their setup. Hence, the full clarification of the suggested feedback loop requires further investigation. It would be interesting to observe the Pep–WIND1 interplay in Arabidopsis, where the Pep peptide maturation and signaling has been characterized by microscopic analyses.⁷

Secondly, the authors showed that REF1 is a single member of the Pep family in tomato. The mutants show reduced, but not abolished wound responses suggesting that there exist parallel, so far unknown mechanisms in local wound signaling acting in conjunction with Peps. These components may include other wound-triggered peptides, Ca²⁺, ROS, ATP or other secondary signals. Recent studies suggested that cell expansion and cell displacement act as purely mechanical signals critical for healing and regeneration of single-cell wounds.^2,3 The tomato system with its single Pep member represents an intriguing model system to test the involvement and importance of these additional components and the mechanistic signaling, which can be investigated with genetic and microscopic techniques.