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Small secreted peptides have been described as key contributors to complex signalling networks that control plant development and stress responses. The Brassicaceae-specific PROSCOOP family encodes precursors of Serine riCh endOgenOus Peptides (SCOOPs). In Arabidopsis SCOOP12 has been shown to promote the defence response against pathogens and to be involved in root development. Here, we explore its role as a moderator of Arabidopsis primary root development. We show that the PROSCOOP12 null mutation leads to longer primary roots through the development of longer differentiated cells while PROSCOOP12 overexpression induces dramatic plant growth impairments. In comparison, the exogenous application of synthetic SCOOP12 peptide shortens roots through meristem size and cell length reductions. Moreover, superoxide anion (O2·−) and hydrogen peroxide (H2O2) production in root tips vary according to SCOOP12 abundance. By using reactive oxygen species scavengers that suppress the proscoop12 phenotype, we showed that root growth regulation by SCOOP12 is associated with reactive oxygen species metabolism. Furthermore, our results suggest that peroxidases act as potential SCOOP12 downstream targets to regulate H2O2 production, which in turn triggers cell wall modifications in root. Finally, a massive transcriptional reprogramming, including the induction of genes from numerous other pathways, including ethylene, salicylic acid, and glucosinolates biosynthesis, was observed, emphasizing its dual role in defence and development. [ABSTRACT FROM AUTHOR]

Summary: Signaling peptides play crucial roles in plant growth, development and defense. We report here that the Arabidopsis thaliana secreted peptide, ROOT MERISTEM GROWTH FACTOR 7 (RGF7), functions as an endogenous elicitor to trigger plant immunity.Expression of the RGF7 precursor‐encoding gene (preRGF7) is highly induced in Arabidopsis leaves upon infection by the bacterial pathogen Pseudomonas syringae. The pathogen‐responsive preRGF7 expression is regulated by the transcription factor WRKY33 and its upstream mitogen‐activated protein kinases MPK3/MPK6 and calcium‐dependent protein kinases CPK5/CPK6. In the absence of pathogen attack, chemically induced expression of preRGF7 in transgenic Arabidopsis plants was sufficient to trigger immune responses. Pre‐induction of preRGF7 expression in transgenic Arabidopsis also led to enhanced immune responses and increased resistance to P. syringae infection.Biochemical and genetic analyses demonstrated that RGF7 is perceived by the leaf‐expressed RGF1 INSENSITIVE (RGI) family receptors RGI4 and RGI5. The SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) BAK1 and SERK4 are also involved in RGF7 perception via forming RGF7‐induced receptor‐complexes with RGI4 and RGI5.These results indicate that the pathogen‐induced RGF7 peptide, perceived by the RGI4/RGI5‐BAK1/SERK4 receptor complexes, acts as a new damage‐associated molecular pattern (DAMP) and plays a significant role in Arabidopsis immunity. [ABSTRACT FROM AUTHOR]

Plant stomata which consist of a pair of guard cells, are not only finely controlled to balance water loss as transpiration and CO2 absorption for photosynthesis, but also serve as the major sites to defend against pathogen attack, thus allowing plants to respond appropriately to abiotic and biotic stress conditions. The regulatory signaling network for stomatal movement is complex in nature, and plant peptides have been shown to be involved in signaling processes. Arabidopsis secreted peptide PIP1 was previously identified as an endogenous elicitor, which induced immune response through its receptor, RLK7. PIP1-RLK7 can activate stomatal immunity against the bacterial strain Pst DC3118. However, the molecular mechanism of PIP1 in stomatal regulation is still unclear and additional new factors need to be discovered. In this study, we further clarified that PIP1 could function as an important regulator in the induction of stomatal closure. The results showed that PIP1 could promote stomata to close in a certain range of concentrations and response time. In addition, we uncovered that PIP1-RLK7 signaling regulated stomatal response by activating S-type anion channel SLAC1. PIP1-induced stomatal closure was impaired in bak1, mpk3, and mpk6 mutants, indicating that BAK1 and MPK3/MPK6 were required for PIP1-regulated stomatal movement. Our research further deciphered that OST1 which acts as an essential ABA-signaling component, also played a role in PIP1-induced stomatal closure. In addition, ROS participated in PIP1-induced stomatal closure and PIP1 could activate Ca2+ permeable channels. In conclusion, we reveal the role of peptide PIP1 in triggering stomatal closure and the possible mechanism of PIP1 in the regulation of stomatal apertures. Our findings improve the understanding of the role of PIP1 in stomatal regulation and immune response.

Abstract Background Many small peptides regulate eukaryotic cell biology. In fungi, some of these peptides are produced after KEX2 protease activity on proteins displaying repetitions of identical or nearly identical motifs. Following this endoprotease activity, peptides are released in the extracellular space. This type of protein maturation is involved in the production of the α-type sexual pheromone in Ascomycota. In other cases, this processing allows the production of secreted peptides regulating fungal cell wall structure or acting as mycotoxins. In this work, we report for the first time a genome-wide search of KEX2-processed repeat proteins that we call KEPs. We screened the secreted proteins of 250 fungal species to compare their KEP repertoires with regard to their lifestyle, morphology or lineage. Results Our analysis points out that nearly all fungi display putative KEPs, suggesting an ancestral origin common to all opisthokonts. As expected, our pipeline identifies mycotoxins but also α-type sexual pheromones in Ascomycota that have not been explored so far, and unravels KEP-derived secreted peptides of unknown functions. Some species display an expansion of this class of proteins. Interestingly, we identified conserved KEPs in pathogenic fungi, suggesting a role in virulence. We also identified KEPs in Basidiomycota with striking similarities to Ascomycota α-type sexual pheromones, suggesting they may also play alternative roles in unknown signalling processes. Conclusions We identified putative, new, unexpected secreted peptides that fall into different functional categories: mycotoxins, hormones, sexual pheromones, or effectors that promote colonization during host-microbe interactions. This wide survey will open new avenues in the field of small-secreted peptides in fungi that are critical regulators of their intimate biology and modulators of their interaction with the environment.

Plant stomata which consist of a pair of guard cells, are not only finely controlled to balance water loss as transpiration and CO2 absorption for photosynthesis, but also serve as the major sites to defend against pathogen attack, thus allowing plants to respond appropriately to abiotic and biotic stress conditions. The regulatory signaling network for stomatal movement is complex in nature, and plant peptides have been shown to be involved in signaling processes. Arabidopsis secreted peptide PIP1 was previously identified as an endogenous elicitor, which induced immune response through its receptor, RLK7. PIP1-RLK7 can activate stomatal immunity against the bacterial strain Pst DC3118. However, the molecular mechanism of PIP1 in stomatal regulation is still unclear and additional new factors need to be discovered. In this study, we further clarified that PIP1 could function as an important regulator in the induction of stomatal closure. The results showed that PIP1 could promote stomata to close in a certain range of concentrations and response time. In addition, we uncovered that PIP1-RLK7 signaling regulated stomatal response by activating S-type anion channel SLAC1. PIP1-induced stomatal closure was impaired in bak1 , mpk3 , and mpk6 mutants, indicating that BAK1 and MPK3/MPK6 were required for PIP1-regulated stomatal movement. Our research further deciphered that OST1 which acts as an essential ABA-signaling component, also played a role in PIP1-induced stomatal closure. In addition, ROS participated in PIP1-induced stomatal closure and PIP1 could activate Ca2+ permeable channels. In conclusion, we reveal the role of peptide PIP1 in triggering stomatal closure and the possible mechanism of PIP1 in the regulation of stomatal apertures. Our findings improve the understanding of the role of PIP1 in stomatal regulation and immune response. [ABSTRACT FROM AUTHOR]

As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders.

As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro , we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders. [ABSTRACT FROM AUTHOR]

Small secreted peptides are important players in plant development and stress response. Using a targeted in silico approach, we identified a family of 14 Arabidopsis genes encoding precursors of serine-rich endogenous peptides (PROSCOOP). Transcriptomic analyses revealed that one member of this family, PROSCOOP12, is involved in processes linked to biotic and oxidative stress as well as root growth. Plants defective in this gene were less susceptible to Erwinia amylovora infection and showed an enhanced root growth phenotype. In PROSCOOP12 we identified a conserved motif potentially coding for a small secreted peptide. Exogenous application of synthetic SCOOP12 peptide induces various defense responses in Arabidopsis. Our findings show that SCOOP12 has numerous properties of phytocytokines, activates the phospholipid signaling pathway, regulates reactive oxygen species response, and is perceived in a BAK1 co-receptor-dependent manner. [ABSTRACT FROM AUTHOR]

Background: Many small peptides regulate eukaryotic cell biology. In fungi, some of these peptides are produced after KEX2 protease activity on proteins displaying repetitions of identical or nearly identical motifs. Following this endoprotease activity, peptides are released in the extracellular space. This type of protein maturation is involved in the production of the α-type sexual pheromone in Ascomycota. In other cases, this processing allows the production of secreted peptides regulating fungal cell wall structure or acting as mycotoxins. In this work, we report for the first time a genome-wide search of KEX2-processed repeat proteins that we call KEPs. We screened the secreted proteins of 250 fungal species to compare their KEP repertoires with regard to their lifestyle, morphology or lineage. Results: Our analysis points out that nearly all fungi display putative KEPs, suggesting an ancestral origin common to all opisthokonts. As expected, our pipeline identifies mycotoxins but also α-type sexual pheromones in Ascomycota that have not been explored so far, and unravels KEP-derived secreted peptides of unknown functions. Some species display an expansion of this class of proteins. Interestingly, we identified conserved KEPs in pathogenic fungi, suggesting a role in virulence. We also identified KEPs in Basidiomycota with striking similarities to Ascomycota α-type sexual pheromones, suggesting they may also play alternative roles in unknown signalling processes. Conclusions: We identified putative, new, unexpected secreted peptides that fall into different functional categories: mycotoxins, hormones, sexual pheromones, or effectors that promote colonization during host-microbe interactions. This wide survey will open new avenues in the field of small-secreted peptides in fungi that are critical regulators of their intimate biology and modulators of their interaction with the environment. [ABSTRACT FROM AUTHOR]

The endogenous peptides from peanut hairy root culture were induced upon elicitor treatment with chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT+MeJA+CD. The peptides secreted into the liquid culture medium play an important role in plant signaling and stress responses. By performing gene ontology (GO) analysis, a number of plant proteins involved in biotic and abiotic defense responses were identified, such as endochitinase, defensin, antifungal protein, cationic peroxidase and Bowman-Birk type protease inhibitor A-II. The bioactivity of 14 peptides synthesized from secretome analysis was determined. Peptide BBP1–4, derived from the diverse region of Bowman-Birk type protease inhibitor, displayed high antioxidant activity and mimicked the property of chitinase and β-1,3-glucanase enzymes. The antimicrobial activity against S. aureus , S. typhimurium , and E. coli was evidenced with different peptide concentrations. Additionally, peptide BBP1–4 has the potential to be a useful candidate for an immune response property, as it was found to increase the expression of some pathogenesis-related (PR) proteins and stilbene biosynthesis genes in peanut hairy root tissues. The findings indicate that secreted peptides may play a role in plant responses to both abiotic and biotic stresses. These peptides, which possess bioactive properties, could be considered as potential candidates for use in the pharmaceutical, agricultural, and food industries. [Display omitted] • Elicitors induce the production of endogenous peptides in peanut hairy roots. • BBP peptides possess both antioxidant and antimicrobial properties. • BBP peptides trigger the induction of PR protein and stilbene biosynthesis genes. • BBP peptides are involved in plant immune response. [ABSTRACT FROM AUTHOR]

Cell-to-cell signaling is essential for many processes in plant growth and development, including coordination of cellular responses to developmental and environmental cues. Cumulative studies have demonstrated that peptide signaling plays a greater-than-anticipated role in such intercellular communication. Some peptides act as signals during plant growth and development, whereas others are involved in defense responses or symbiosis. Peptides secreted as signals often undergo posttranslational modification and proteolytic processing to generate smaller peptides composed of approximately 10 amino acid residues. Such posttranslationally modified small-peptide signals constitute one of the largest groups of secreted peptide signals in plants. The location of the modification group incorporated into the peptides by specific modification enzymes and the peptide chain length defined by the processing enzymes are critical for biological function and receptor interaction. This review covers 20 years of research into posttranslationally modified small-peptide signals in plants. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study was to evaluate the capacity of Lactobacillus reuteri CRL1098 soluble factors (Lr-S) to modulate TNF-α production in peripheral blood mononuclear cells (PBMC) and to study lipid rafts participation in this response. PBMC treated with Lr-S showed a reduced production of TNF-α. In addition, Lr-S treatment activated ERK and p38 MAPK pathways in PBMC. Lipid rafts participation in the reduced production of TNF-α by PBMC induced by Lr-S was verified by lipid rafts disruption with methyl-β-cyclodextrin and the reduction of the Src-tyrosine kinase Lck localization in rafts. Moreover, PBMC pre-treatment with Lck inhibitors blocked the effect of Lr-S on TNF-α production suggesting that activation and mobilization of Lck from lipid rafts would be involved in the modulatory effect of L. reuteri CRL1098. A secreted peptide of 5785Da would be responsible of the modulatory effect of CRL1098 strain. This study demonstrated for the first time the lipid rafts participation in a response induced by a beneficial bacterium. Also, these results open new possibilities for investigating the molecular mechanisms involved in the interaction of probiotic bacterial extracellular compounds with immune cells. [Copyright &y& Elsevier]

Leucine-rich repeat receptor kinases (LRR-RKs) are the largest sub-family of transmembrane receptor kinases in plants. In several LRR-RKs, a loop-out region called an 'island domain', which intercepts the extracellular tandem LRRs at a position near the transmembrane domain, constitutes the ligand-binding pocket, but the absence of the island domain in numerous LRR-RKs raises questions about which domain recognizes the ligand in non-island domain LRR-RKs. Here, we used photoaffinity labeling followed by chemical and enzymatic digestion to show that BAM1, a CLV1/BAM-family LRR-RK whose extracellular domain comprises 22 consecutive LRRs, directly interacts with the small peptide ligand CLE9 at the LRR6-LRR8 region that is relatively distal from the transmembrane domain. Multiple sequence alignment and homology modeling revealed that the inner concave side of LRR6-LRR8 of CLV1/BAM-family LRR-RKs deviates slightly from the LRR consensus. In support of our findings, the clv1-4 mutant carries a missense mutation at the inner concave side of LRR6 of CLV1, and introduction of the corresponding mutation in BAM1 resulted in complete loss of ligand binding activity. Our results indicate that the ligand recognition mechanisms of plant LRR-RKs are more complex and diverse than anticipated. [ABSTRACT FROM AUTHOR]

Plants use a variety of small peptides for cell to cell communication during growth and development. Leguminous plants are characterized by their ability to develop nitrogen-fixing nodules via an interaction with symbiotic bacteria. During nodule organogenesis, several so-called nodulin genes are induced, including large families that encode small peptides. Using a three-hybrid approach in yeast cells, we identified two new small nodulins, MtSNARP1 and MtSNARP2 (for small nodulin acidic RNA-binding protein), which interact with the RNA of MtENOD40, an early induced nodulin gene showing conserved RNA secondary structures. The SNARPs are acidic peptides showing single-stranded RNA-binding activity in vitro and are encoded by a small gene family in Medicago truncatula. These peptides exhibit two new conserved motifs and a putative signal peptide that redirects a GFP fusion to the endoplasmic reticulum both in protoplasts and during symbiosis, suggesting they are secreted. MtSNARP2 is expressed in the differentiating region of the nodule together with several early nodulin genes. MtSNARP2 RNA interference (RNAi) transgenic roots showed aberrant early senescent nodules where differentiated bacteroids degenerate rapidly. Hence, a functional symbiotic interaction may be regulated by secreted RNA-binding peptides. [ABSTRACT FROM AUTHOR]

Peptidomics is a challenging field in which to create a link between genomic information and biological function through biochemical analysis of expressed peptides, including precise identification of post-translational modifications and proteolytic processing. We found that secreted peptides in Arabidopsis plants diffuse into the medium of whole-plant submerged cultures, and can be effectively identified by o-chlorophenol extraction followed by LC-MS analysis. Using this system, we first confirmed that a 12-amino-acid mature CLE44 peptide accumulated at a considerable level in the culture medium of transgenic plants overexpressing CLE44. Next, using an in silico approach, we identified a novel gene family encoding small secreted peptides that exhibit significant sequence similarity within the C-terminal short conserved domain. We determined that the mature peptide encoded by At1g47485, a member of this gene family, is a 15-amino-acid peptide containing two hydroxyproline residues derived from the conserved domain. This peptide, which we have named CEP1, is mainly expressed in the lateral root primordia and, when overexpressed or externally applied, significantly arrests root growth. CEP1 is a candidate for a novel peptide plant hormone. [ABSTRACT FROM AUTHOR]

We performed high-throughput in situ hybridization screening of sections of mouse epidermis using an equalized skin cDNA library as probes and identified a novel gene giving rise to two splicing variants, both of which are expressed in the spinous layer. This gene was mapped between two genes encoding keratinocyte-related peptides, suprabasin and keratinocyte differentiation-associated protein (Kdap), on human chromosome 19q13.1. These gene products appeared to carry functional signal sequences. We then designated these two splicing variants as dermokine-α and -β. Northern blotting and quantitative RT-PCR revealed that dermokine-α/-β, suprabasin, and Kdap were highly expressed in stratified epithelia. In mouse embryonic development, dermokine-α/-β began to be expressed during the period of stratification. Also, in differentiating primary cultured human keratinocytes, transcription of dermokine-α/-β, suprabasin, and Kdap was induced. These findings indicated that dermokine-α/-β, suprabasin, and Kdap are secreted from the spinous layer of the stratified epithelia and that these genes form a novel gene complex on the chromosome. [Copyright &y& Elsevier]

The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to-cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post-translational modification. This review summarizes how we discovered these peptide hormone-receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation.

Background: Antimicrobial peptides could be used in several fields of application, and large quantities of antimicrobial peptides would be required. However, their production is very expensive; this is why a suitable production method, alternative to traditional chemical synthesis is necessary. Production of recombinant antimicrobial peptides in prokaryotic systems has demonstrated the viability of this approach. Nevertheless, expression of antimicrobial peptides in Escherichia coli an others microorganisms is potentially limited due to their toxicity to host cells and susceptibility to proteolytic degradation. As an alternative, we describe a successful antimicrobial peptide production system in Streptomyces lividans which showed to be effective for the secretion of large quantities of cationic antimicrobial peptides., Objective: Therefore, as a solution to the difficulties for heterologous expression of CAP we demonstrate efficient production by S. lividans., Method: In this study, a strategy for CAP overexpression is presented based on the construction of an expression cassette for Streptomyces lividans TK24. For the construction of this cassette, the peptide of interest was fused to the vsi promoter and signal sequence (vsi-ss) of the subtilisin inhibitor from Streptomyces venezuelae CBS762.70, which is a signal peptide with a proven high secretion efficiency. The cloning vector used was pIJ486, which includes a transcription terminator sequence and a thiostrepton resistance marker. This system contains elements that allow the increase of the efficiency of the peptide's expression., Results: The production system allows the efficient secretion of the peptide to the growth medium, thereby simplifying its recovery and avoiding its toxic effect on the producing organism. The production obtained demonstrated the system's efficiency by achieving a peptide concentration of 11.61 mg/ml. This represents at least a 10-fold increase compared to previously established strategies., Conclusion: The expression system constructed may facilitate the production of large amounts of peptides with antimicrobial activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)