Abstract Rapid Alkalinization Factor (RALF) peptides regulate plant growth and cell wall signaling, but their roles in fruit development remain unclear. Here, we characterized tomato ( Solanum lycopersicum ) fruit-associated RALF peptides and their interactions with leucine-rich repeat extensins (LRXs). Expression analyses identified SlRALF5 , SlRALF7 , and SlRALF10 as the main fruit-expressed RALFs. SlRALF10 was associated with early fruit development, whereas SlRALF5 and SlRALF7 remained expressed during ripening. Sequence analyses showed that SlRALF5/7 retain conserved motifs of canonical RALFs, while SlRALF10 displays divergent structural features and altered charge distribution. Synthetic SlRALF5 and SlRALF7 inhibited root growth and induced extracellular alkalinization, whereas SlRALF10 lacked both activities. Co-immunoprecipitation assays showed that all three peptides interact with the fruit-expressed proteins SlLRX2 and SlLRX5. Structural modeling predicted distinct electrostatic properties for the SlLRX5/SlRALF10 complex compared with SlRALF5. These results reveal structural and functional specialization among tomato fruit RALF peptides and suggest that distinct SlRALFs may differentially respond to cell wall remodeling during fruit development and ripening.

Systemin, the first signalling peptide identified in plants, mediates induced resistance against insect herbivores and necrotrophic pathogens in tomato1–3. Initially, systemin was conceived as a hormone-like, long-distance messenger that triggers systemic defence responses far from the site of insect attack. It was later found to rather act as a phytocytokine, amplifying the local wound response for the production of downstream signals that activate defence gene expression in distant tissues4. Systemin perception and signalling rely on the systemin receptor SYR15. However, the specifics of SYR1-dependent signalling and how systemin signalling differs from other immune signalling pathways remain largely unknown. Here we report that systemin activates the poltergeist-like phosphatase PLL2 in a SYR1-dependent manner. PLL2, in turn, regulates early systemin responses at the plasma membrane, including the rapid inhibition of proton pumps through dephosphorylation of their regulatory C-termini. PLL2 was found to be essential for downstream defence gene induction, ultimately contributing to insect resistance. The phosphatase PLL2 was identified as a specific component in systemin-induced immune signalling. PLL2 regulates proton pump activity at the plasma membrane and is required for downstream defence gene induction and insect resistance.

Proteases degrade proteins that are damaged, misfolded, or no longer needed. This enables plants to repurpose their proteomes in response to environmental cues and adapt to adverse conditions. Proteases also play key roles in plant growth and development by the selective degradation of regulatory proteins. However, there is much more to proteases than just protein degradation. They can also act in a highly site-specific manner, ensuring protein maturation after subcellular targeting, regulating protein activity and function, and releasing or modulating peptide signals. Since their activity is irreversible and potentially deleterious, proteases are tightly controlled by endogenous inhibitors and other regulatory mechanisms. The reviews and research articles in this Special Issue put a timely spotlight on the fascinating diversity, function, and regulation of proteases in plants, discuss current challenges and technical developments, and provide a perspective on their use for crop protection.

Tight regulation of immune activation is crucial for plant health. How plants control the actions of their immunostimulatory phytocytokines is largely unknown. Here, we identify antiSYS as a natural inhibitor of the tomato cytokine systemin. AntiSYS is a systemin-like peptide encoded in a gene cluster with four additional paralogs, three of which comprise newly identified agonistic systemins. AntiSYS is a potent and specific antagonist of the systemin receptor. Tomato mutants lacking antiSYS show aberrant growth and reduced reproductive fitness. These symptoms of antiSYS deficiency are not observed in plants lacking functional systemin receptors, suggesting a role of antiSYS in counterbalancing agonistic systemins. Thus, reminiscent of antagonistic interleukins controlling immune homeostasis in animals, antiSYS serves a crucial role in the regulation of phytocytokine activity in tomato plants.

Phytocytokines are hormone-like plant peptides that modulate immune homeostasis and development. Phytosulfokine (PSK) mediates plant growth and attenuates activation of plant pattern-triggered immunity (PTI). We show that the small cysteine-containing effector VdSCP8 from Verticillium dahliae (Vd) is a virulence-promoting protein that suppresses PTI in Arabidopsis thaliana and Nicotiana benthamiana. Apoplastic SCP8 suppresses immune activation through leucine-rich repeat ectodomain pattern recognition receptors. SCP8 virulence and immunosuppressive activities require PHYTOSULFOKINE RECEPTOR 1 (PSKR1), which binds PSK and forms a complex with co-receptor BAK1 for PTI suppression. We find that PSK, like SCP8, suppresses PTI, SCP8 stimulates PSKR1-BAK1 complex formation, and that Vd requires PSK signaling for host infection. SCP8 interacts with an apoplastic subtilase, and co-expression of SCP8 and subtilase inhibitors reduces PTI suppression. Our findings suggest that a multi-host plant pathogen manipulates PTI by enhancing immunosuppressive PSK signaling, likely through plant subtilase activity.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Plants respond to wounding such as caused by insect herbivores by activating their defenses and thus increasing their resistance against attacking pests and opportunistic pathogens(Howe and Schaller,2008).The phenomenon was first described more than 50 years ago by Green and Ryan,who reported that wounding triggers tomato and potato plants to produce proteinase inhibitors that inhibit digestive proteases in the gut of leaf-eating in-sects to starve them of nutrients(Green and Ryan,1972).

Extracellular alkalinization mediated by the inhibition of plasma membrane-located proton pumping ATPases hallmarks the initiation of defense signaling in plant cells. Early defense responses also include depolarization of the plasma membrane, increase in cytosolic Ca2+ concentration, and an oxidative burst. Together these early signaling events lead to the activation of plant immunity. The transient alkalinization response is triggered by well-studied pathogen-derived and plant endogenous elicitors, including, for example, bacterial flagellin, fungal chitin, and tomato systemin in both model and agronomic species. Employing cell suspension cultures, extracellular alkalinization can be easily assessed by measuring the elicitor-induced pH changes of the cultivating medium. Here, we provide a protocol for an improved alkalinization assay in a system which is able to simultaneously monitor multiple samples, and fully automatically transfer customizable real-time pH records. In this system flagellin, chitin and systemin elicit robust time- and dose-dependent responses, proving a powerful tool for assessing plant early defense signaling.

Phtheirospermum japonicum is a hemiparasitic plant of the Orobanchaceae, the largest family of parasitic plants. It extracts water and nutrients from other plants through haustoria along its roots. Haustoriogenesis, the formation of haustoria, is initiated by host-derived haustorium-inducing factors (HIFs). The first step in haustoriogenesis is the development of parasitically inactive protohaustoria. Here, we report that an endogenous peptide hormone, CLAVATA3/Embryo Surrounding Region 1 ( Pj CLE1), is sufficient to induce protohaustorium formation. Pj CLE1 hyperactivated HIF-responses and caused prolific protohaustoria formation. PjCLE1 expression and activation by the subtilisin-type protease Pj SBT1.2.3 occur in fully developed, mature haustoria, suggesting that Pj CLE1 acts as an internal signal produced by mature haustoria to stimulate additional protohaustorium formation for effective extraction of resources from hosts. Pj CLE1 is similar in sequence to CLEs regulating nodulation in legumes and part of a regulatory system for haustoria formation in parasitic plants.

The rapid accumulation of extracellular "reactive oxygen species" (ROS), also known as the "oxidative burst", is an early plant immune response triggered by pathogen-derived microbe-associated molecular patterns and by endogenous plant signaling molecules. The oxidative burst is often used as a readout for the activation of defense signaling. Here, we present a detailed protocol for the continuous measurement of ROS production in leaf discs of tomato plants, using a chemiluminescence-based assay in a microtiter plate format. We also include recommendations for data analysis and for the quantitative assessment of differences in ROS burst dynamics, as caused by different types of elicitors, or in different tomato genotypes.

Systemin, the first signaling peptide identified in plants, mediates induced resistance against insect herbivores in tomato1. Initially, systemin was perceived as a hormone-like, long-distance messenger that triggers systemic defense responses far from the site of insect attack. It was later found to rather act as a phytocytokine, amplifying the local wound response for the production of downstream signals that activate defense gene expression in distant tissues2. Systemin perception and signaling rely on the systemin receptor SYR13. However, the specifics of SYR1-dependent signaling and how systemin signaling differs from other phytocytokine signaling pathways remain largely unknown. Here, we report that systemin activates the poltergeist-like phosphatase PLL2 in a SYR1-dependent manner. PLL2, in turn, regulates early systemin responses at the plasma membrane, including the rapid inhibition of proton pumps through the dephosphorylation of their regulatory C-termini. PLL2 was found to be essential for downstream defense gene induction, ultimately contributing to insect resistance.

The use of eukaryotic expression systems facilitates the heterologous expression of complex eukaryotic proteins in their post-translationally modified and biologically active state, as a prerequisite for subsequent biochemical characterization and functional analysis. Here we describe the complete workflow for the expression of Arabidopsis thaliana prolyl-4-hydroxylases (P4Hs) in the methylotrophic yeast Pichia pastoris (renamed as Komagataella phaffii), for the extraction of the recombinant enzymes, purification by affinity chromatography, and characterization of P4H activity and specificity toward oligopeptide substrates by mass spectrometry. We expressed eight of the 13 Arabidopsis P4Hs and show that they are all active against proline-rich extensin-derived peptides. However, three of them differed in substrate specificity and were also able to hydroxylate the CLEL9 signaling peptide, featuring a single proline within its mature peptide sequence.

Post-translationally modified peptides are now recognized as important regulators of plant stress responses. We recently identified the sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in dark-grown and in light-stressed Arabidopsis seedlings. The function of CLEL6 depends on proteolytic processing by subtilisin-like serine proteinase SBT6.1, and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST), and CLEL6 signaling relies on the ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptor family. In this chapter, we describe in detail how to quantify peptide-regulated and stress-induced anthocyanin biosynthesis. We include protocols for peptide treatment of Arabidopsis seedlings and growth under different stress conditions, for the extraction and quantification of anthocyanins, and for the expression analysis of anthocyanin biosynthetic genes.

Posttranslationally modified peptides are now recognized as important regulators of plant stress responses. Here, we identified the small sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in etiolated and in light-stressed Arabidopsis (Arabidopsis thaliana) seedlings. CLEL6 function depends on proteolytic processing of the CLEL6 precursor by subtilisin-like serine proteinase 6.1 (SBT6.1) and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST). Loss-of-function mutants of either sbt6.1 or tpst showed significantly higher anthocyanin accumulation than the wild type upon light stress. The anthocyanin overaccumulation phenotype of sbt6.1 and tpst was suppressed by application of mature CLEL6. Overexpression and external application of CLEL6 inhibited the expression of anthocyanin biosynthesis genes in etiolated and light-stressed seedlings, confirming the role of CLEL6 as an inhibitor of anthocyanin biosynthesis. Small posttranslationally modified peptides are perceived by leucine-rich repeat receptor-like kinases. Using a quintuple mutant of ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors, we showed the essential function of the RGI receptor family in CLEL6 signaling. Our data indicate that overexpression or application of CLEL6 inhibits anthocyanin biosynthesis through RGI receptors. We propose that CLEL6 inhibits anthocyanin biosynthesis in etiolated seedlings, and that anthocyanin biosynthesis is derepressed when CLEL6 expression is downregulated upon light exposure. Hyperaccumulation of anthocyanins in light-stressed tpst and sbt6.1 mutant seedlings suggests that CLEL6, or related sulfopeptides, continues to act as negative regulators to limit pigment accumulation in the light.

A critical step in the functional characterization of proteases is the identification of physiologically relevant substrates, which often starts with a collection of candidate proteins. To test these candidates and identify specific processing sites, in vitro cleavage assays are typically used, followed by polyacrylamide gel electrophoresis (SDS-PAGE) to separate and visualize the cleavage products. For the identification of cleavage sites, the sequences at the N- or C-terminal ends of the cleavage products need to be identified, which is the most challenging step in this procedure. Here, we describe a method for the reliable identification of the N-termini of polypeptides after separation by SDS-PAGE. The procedure relies on in-gel labeling of the N-terminal-free amino group by reductive dimethylation, followed by tryptic digestion and analysis of resulting peptides by mass spectrometry. N-terminal peptides are readily identified by the 28 Da mass dimethyl tag linked to their first amino acid.

The nuclear lamina is a complex network of nuclear lamins and lamin-associated nuclear membrane proteins, which scaffold the nucleus to maintain structural integrity. In Arabidopsis thaliana , nuclear matrix constituent proteins (NMCPs) are essential components of the nuclear lamina and are required to maintain the structural integrity of the nucleus and specific perinuclear chromatin anchoring. At the nuclear periphery, suppressed chromatin overlapping with repetitive sequences and inactive protein-coding genes are enriched. At a chromosomal level, plant chromatin organization in interphase nuclei is flexible and responds to various developmental cues and environmental stimuli. On the basis of these observations in Arabidopsis , and given the role of NMCP genes ( CRWN1 and CRWN4 ) in organizing chromatin positioning at the nuclear periphery, one can expect considerable changes in chromatin–nuclear lamina interactions when the global chromatin organization patterns are being altered in plants. Here we report the highly flexible nature of the plant nuclear lamina, which disassembles substantially under various stress conditions. Focusing on heat stress, we reveal that chromatin domains, initially tethered to the nuclear envelope, remain largely associated with CRWN1 and become scattered in the inner nuclear space. By investigating the three-dimensional chromatin contact network, we further reveal that CRWN1 proteins play a structural role in shaping the changes in genome folding under heat stress. Also, CRWN1 acts as a negative transcriptional coregulator to modulate the shift of the plant transcriptome profile in response to heat stress.

Proteases control plant growth and development by limited proteolysis of regulatory proteins at highly specific sites. This includes the processing of peptide hormone precursors to release the bioactive peptides as signaling molecules. The proteases involved in this process have long remained elusive. Confirmation of a candidate protease as a peptide precursor-processing enzyme requires the demonstration of protease-mediated precursor cleavage in vitro. In vitro cleavage assays rely on the availability of suitable substrates and the candidate protease with high purity. Here, we provide a protocol for the expression, purification, and characterization of tomato (Solanum lycopersicum) phytaspases as candidate proteases for the processing of the phytosulfokine precursor. We also show how synthetic oligopeptide substrates can be used to demonstrate site-specific precursor cleavage. Graphical abstract.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

Plant signalling peptides are typically released from larger precursors by proteolytic cleavage to regulate plant growth, development and stress responses. Recent studies reported the characterization of a divergent family of Brassicaceae-specific peptides, SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs), and their perception by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2). Here, we reveal that the SCOOP family is highly expanded, containing at least 50 members in the Columbia-0 reference Arabidopsis thaliana genome. Notably, perception of these peptides is strictly MIK2-dependent. How bioactive SCOOP peptides are produced, and to what extent their perception is responsible for the multiple physiological roles associated with MIK2 are currently unclear. Using N-terminomics, we validate the N-terminal cleavage site of representative PROSCOOPs. The cleavage sites are determined by conserved motifs upstream of the minimal SCOOP bioactive epitope. We identified subtilases necessary and sufficient to process PROSCOOP peptides at conserved cleavage motifs. Mutation of these subtilases, or their recognition motifs, suppressed PROSCOOP cleavage and associated overexpression phenotypes. Furthermore, we show that higher-order mutants of these subtilases show phenotypes reminiscent of mik2 null mutant plants, consistent with impaired PROSCOOP biogenesis, and demonstrating biological relevance of SCOOP perception by MIK2. Together, this work provides insights into the molecular mechanisms underlying the functions of the recently identified SCOOP peptides and their receptor MIK2. The SCOOP signalling peptide family expands to 50 members, whose activities are strictly dependent upon the receptor kinase MIK2. Two subtilase classes process PROSCOOPs, generating bioactive SCOOP peptides. A subtilase mutant phenocopies the mik2 receptor mutant.

Many peptide hormones and growth factors in plants, particularly the small posttranslationally modified signaling peptides, are synthesized as larger precursor proteins. Proteolytic processing is thus required for peptide maturation, and additional posttranslational modifications may contribute to bioactivity. To what extent these posttranslational modifications impact on processing is largely unknown. Likewise, it is poorly understood how the cleavage sites within peptide precursors are selected by specific processing proteases, and whether or not posttranslational modifications contribute to cleavage site recognition. Here, we describe a mass spectrometry-based approach to address these questions. We developed a method using heavy isotope labeling to directly compare cleavage efficiency of different precursor-derived synthetic peptides by mass spectrometry. Thereby, we can analyze the effect of posttranslational modifications on processing and the specific sequence requirements of the processing proteases. As an example, we describe how this method has been used to assess the relevance of tyrosine sulfation for the processing of the Arabidopsis CIF4 precursor by the subtilase SBT5.4.