Damage on plants activates Ca2+-dependent metacaspases for release of immunomodulatory peptides

Rapid response to tissue damage Damaged plants are susceptible to microbial attack. In response to physical damage, plants proactively generate signal peptides to activate their immune systems. Hander et al. examined wound responses in the model plant Arabidopsis thaliana. They identified a metacaspase that releases an immunomodulatory signal peptide from its precursor form within 30 seconds of the damage. The metacaspase itself was activated by a burst of calcium released by tissue damage. Science, this issue p. eaar7486 The plant immune system is activated when a protease translates physical damage into a defense response. INTRODUCTION Cellular damage caused by wounding triggers signals to alert the surrounding tissue. As a universal process in all multicellular organisms, these signals activate the immune system to prevent infection and promote tissue regeneration, eventually leading to wound healing, but they have to be secured because aberrant immune stimulation can negatively affect health and growth. Plants, as sessile organisms, are regularly subject to chewing or sucking insects and physical damage inflicted by metazoans or exposure to the environment. In plants, short protein fragments or peptides can have immunomodulatory functions, such as those derived from the plant elicitor peptide (Pep) gene family. Peps are part of precursor proteins and have been proposed to act as wound signals that bind and activate the extracellular Pep receptors (PEPRs) to initiate an immune-like response. How Peps are produced and released upon wounding and how far this response extends from the harm site remain unclear. RATIONALE Proteases can generate peptides from precursor proteins, but the genome of the model plant, Arabidopsis thaliana, encodes approximately 600 different proteases. To understand the molecular mechanisms that control the immune-activating signals, it is essential to identify the immune response-contributing proteases. Furthermore, wound formation has to be observed with great speed and precision to delineate the extent of the overall process. Pep1 was studied here as a representative member of the gene family. RESULTS Pep1 generation was detected within 30 s after the injury, peaking at 5 min and lasting up to 1 hour in two wound model experiments on Arabidopsis seedlings: pinching with forceps or mechanical disruption of tissue integrity through grinding. Screening with various protease inhibitors revealed the involvement of metacaspases in Pep1 formation. Metacaspases are cysteine proteases, conserved in plants, fungi, protists, and bacteria that were historically named after caspases because of a certain degree of structural homology. However, they differ in activity and substrate specificity. Metacaspases require low-millimolar amounts of calcium (Ca2+) for in vitro activity and cleave their substrate proteins after the amino acids arginine and lysine. Physical damage activated the abundant METACASPASE4 (MC4) in both wound experiments. MC4 released Pep1 from its protein precursor PRECURSOR OF PEP1 (PROPEP1) by cleaving it behind a conserved arginine. A mutant lacking MC4 was unable to produce Pep1 in leaf tissue, whereas certain redundancy occurred with other metacaspase (or metacaspase-like) activities in root tissue. Inside the plant cell, PROPEP1 is attached to the cytosolic side of the vacuolar membrane. When undisturbed, cytosolic Ca2+ concentrations ([Ca2+]cyt) are too low to activate metacaspases that need unusually high [Ca2+] to function. When Arabidopsis root epidermis cells were damaged by means of multiphoton laser ablation, Pep1 release from the vacuolar membrane occurred only in the cytosol of directly hit cells, as observed with confocal microscopy. Loss of plasma membrane integrity in these cells led to a high and prolonged influx of extracellular [Ca2+] into the cytosol, sufficient to activate metacaspases, leading to the cleavage and release of Pep1 from PROPEP1. Application of exogenous PROPEP1 protein fragments, longer than the native Pep1, reduced root growth—a known negative effect of Pep1 overload—irrespective of MC4 cleavage. Accordingly, PROPEP1 cleavage to overcome retention of Pep1 at the vacuolar membrane seems more important than obtaining the mature Pep1 size. CONCLUSION Plants exploit highly conserved mechanisms—such as Ca2+-partitioning across intact membranes and maturation of immunomodulatory peptides by proteases—to rapidly trigger defense responses against tissue damage. Pep1 is released by activation of MC4 upon prolonged high levels of [Ca2+]cyt that occur only in directly damaged cells, and this response is safeguarded by subcellular retention of PROPEP1 at the vacuolar membrane in the absence of damage. Metacaspases, together with Peps and PEPRs, now emerge as potential targets for breeding and improving crop immunity. Metacaspases activate defense responses upon wounding. Undisturbed root epidermal cells contain inactive zymogen MC4 (zMC4) in the cytosol and PROPEP1 attached to the vacuolar membrane. Damage induces a prolonged influx of Ca2+ in the cytosol, triggering MC4 to cleave PROPEP1 and to release Pep1. Pep1 can then diffuse to neighboring cells and bind to the receptor, PEPR, to activate a defense response (bottom, light green cells). Pep1 release occurs only in cells that lose plasma membrane integrity (bottom, red cells). Physical damage to cells leads to the release of immunomodulatory peptides to elicit a wound defense response in the surrounding tissue. In Arabidopsis thaliana, the plant elicitor peptide 1 (Pep1) is processed from its protein precursor, PRECURSOR OF PEP1 (PROPEP1). We demonstrate that upon damage, both at the tissue and single-cell levels, the cysteine protease METACASPASE4 (MC4) is instantly and spatiotemporally activated by binding high levels of Ca2+ and is necessary and sufficient for Pep1 maturation. Cytosol-localized PROPEP1 and MC4 react only after loss of plasma membrane integrity and prolonged extracellular Ca2+ entry. Our results reveal that a robust mechanism consisting of conserved molecular components links the intracellular and Ca2+-dependent activation of a specific cysteine protease with the maturation of damage-induced wound defense signals.