Contents Summary901I.Introduction901II.Biochemistry and structure of plant SBTs902III.Phylogeny of plant SBTs and family organization903IV.Physiological roles of plant SBTs905V.Conclusions and outlook911Acknowledgements912References912 Summary Subtilases (SBTs) are serine peptidases that are found in all three domains of life. As compared with homologs in other Eucarya, plant SBTs are more closely related to archaeal and bacterial SBTs, with which they share many biochemical and structural features. However, in the course of evolution, functional diversification led to the acquisition of novel, plant‐specific functions, resulting in the present‐day complexity of the plant SBT family. SBTs are much more numerous in plants than in any other organism, and include enzymes involved in general proteolysis as well as highly specific processing proteases. Most SBTs are targeted to the cell wall, where they contribute to the control of growth and development by regulating the properties of the cell wall and the activity of extracellular signaling molecules. Plant SBTs affect all stages of the life cycle as they contribute to embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell death, organ abscission, senescence, and plant responses to their biotic and abiotic environments. In this article we provide a comprehensive picture of SBT structure and function in plants.

SummaryHydrolases such as subtilases, vacuolar processing enzymes (VPEs) and the proteasome play important roles during plant programmed cell death (PCD). We investigated hydrolase activities during PCD using activity-based protein profiling (ABPP), which displays the active proteome using probes that react covalently with the active site of proteins.We employed tomato (Solanum lycopersicum) seedlings undergoing synchronized hypersensitive cell death by co-expressing the avirulence protein Avr4 from Cladosporium fulvum and the tomato resistance protein Cf-4. Cell death is blocked in seedlings grown at high temperature and humidity, and is synchronously induced by decreasing temperature and humidity.ABPP revealed that VPEs and the proteasome are not differentially active, but that activities of papain-like cysteine proteases and serine hydrolases, including Hsr203 and P69B, increase before hypersensitive tissue collapse, whereas the activity of a carboxypeptidase-like enzyme is reduced. Similar dynamics were observed for these enzymes in the apoplast of tomato challenged with C. fulvum. Unexpectedly, these challenged plants also displayed novel isoforms of secreted putative VPEs.In the absence of tissue collapse at high humidity, the hydrolase activity profile is already altered completely, demonstrating that changes in hydrolase activities precede hypersensitive tissue collapse.

Summary• In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca 2+ , nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown.• In vivo phospholipid signaling upon extracellular ATP treatment was studied in 32 P i -labeled suspension-cultured tomato (Solanum lycopersicum) cells.• Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mM induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase.• Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C ⁄ diacylglycerol kinase activation.

Caspases are key regulators of apoptosis in animals. This correlation has driven plant researchers for decades to look for caspases regulating programmed cell death (PCD) in plants. These studies revealed caspase-like activities, caspase-related proteases, and cysteine (Cys) proteases regulating PCD in plants, but identified no caspases and no conserved, apoptosis-like death pathway. Here, we critically review the evidence for Cys proteases implicated in PCD in plants. We discuss the role of papain-like Cys proteases, vacuolar processing enzymes, and metacaspases in PCD during the development of tracheary elements, seed coat, suspensor, and tapetum, and during the hypersensitive response. There are several convincing cases where these Cys proteases are required for PCD, but this requirement is often not conserved across different plant species. There are also cases where Cys proteases contribute to the speed, but not the timing of PCD, while other Cys proteases are nonessential for PCD, but have other roles, e.g., in the clearance of cell remains after PCD. These data illustrate the need for caution when generalizing the role of Cys proteases in regulating PCD in plants, and call for studies that further investigate plant Cys proteases and other PCD regulators.

SummaryPlant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death.NRC1 (NB-LRR Required for HR-Associated Cell Death-1) is a tomato (Solanum lycopersicum) NB-LRR protein that participates in the signalling cascade leading to resistance to the pathogens Cladosporium fulvum and Verticillium dahliae.To identify mutations in NRC1 that cause increased signalling activity, we generated a random library of NRC1 variants mutated in their nucleotide-binding domain and screened them for the ability to induce an elicitor-independent HR in Nicotiana tabacum. Screening of 1920 clones retrieved 11 gain-of-function mutants, with 10 of them caused by a single amino acid substitution.All substitutions are located in or very close to highly conserved motifs within the nucleotide-binding domain, suggesting modulation of the signalling activity of NRC1. Three-dimensional modelling of the nucleotide-binding domain of NRC1 revealed that the targeted residues are centred around the bound nucleotide. Our mutational approach has generated a wide set of novel gain-of-function mutations in NRC1 and provides insight into how the activity of this NB-LRR is regulated.