Extracellular pH sensing by plant cell-surface peptide-receptor complexes.

The extracellular pH is a vital regulator of various biological processes in plants. However, how plants perceive extracellular pH remains obscure. Here, we report that plant cell-surface peptide-receptor complexes can function as extracellular pH sensors. We found that pattern-triggered immunity (PTI) dramatically alkalinizes the acidic extracellular pH in root apical meristem (RAM) region, which is essential for root meristem growth factor 1 (RGF1)-mediated RAM growth. The extracellular alkalinization progressively inhibits the acidic-dependent interaction between RGF1 and its receptors (RGFRs) through the pH sensor sulfotyrosine. Conversely, extracellular alkalinization promotes the alkaline-dependent binding of plant elicitor peptides (Peps) to its receptors (PEPRs) through the pH sensor Glu/Asp, thereby promoting immunity. A domain swap between RGFR and PEPR switches the pH dependency of RAM growth. Thus, our results reveal a mechanism of extracellular pH sensing by plant peptide-receptor complexes and provide insights into the extracellular pH-mediated regulation of growth and immunity in the RAM.

Lateral Root Initiation in Cucumber (Cucumis sativus): What Does the Expression Pattern of Rapid Alkalinization Factor 34 (RALF34) Tell Us?

In Arabidopsis, the small signaling peptide (peptide hormone) RALF34 is involved in the gene regulatory network of lateral root initiation. In this study, we aimed to understand the nature of the signals induced by RALF34 in the non-model plant cucumber (Cucumis sativus), where lateral root primordia are induced in the apical meristem of the parental root. The RALF family members of cucumber were identified using phylogenetic analysis. The sequence of events involved in the initiation and development of lateral root primordia in cucumber was examined in detail. To elucidate the role of the small signaling peptide CsRALF34 and its receptor CsTHESEUS1 in the initial stages of lateral root formation in the parental root meristem in cucumber, we studied the expression patterns of both genes, as well as the localization and transport of the CsRALF34 peptide. CsRALF34 is expressed in all plant organs. CsRALF34 seems to differ from AtRALF34 in that its expression is not regulated by auxin. The expression of AtRALF34, as well as CsRALF34, is regulated in part by ethylene. CsTHESEUS1 is expressed constitutively in cucumber root tissues. Our data suggest that CsRALF34 acts in a non-cell-autonomous manner and is not involved in lateral root initiation in cucumber.

Isolation and functional diversity of Bowman-Birk type serine proteinase inhibitors from Hyacinthus orientalis.

Bowman-Birk inhibitors (BBIs) are plant-derived serine proteinase inhibitors. Endogenously, they function as defense molecules against pathogens and insects, but they also have been explored for applications in cancer treatment and inflammatory disorders. Here, we isolated 15 novel BBIs from the bulb of Hyacinthus orientalis (termed HOSPIs). These isoinhibitors consisted of two or three chains, respectively, that are linked by disulfides bonds based on proposed cleavage sites in the canonical BBI reactive site loop. They strongly inhibited trypsin (Ki = 0.22-167 nM) and α-chymotrypsin (Ki = 19-1200 nM). Notably, HOSPI-B4 contains a six-residue reactive loop, which appears to be the smallest such motif discovered in BBIs to date. HOSPI-A6 and -A7 contain an unusual reactive site, i.e. Leu-Met at the P1-P1' position and have strong inhibitory activity against trypsin, α-chymotrypsin, and elastase. Analysis of the cDNA encoding HOSPIs revealed that the precursors have HOSPI-like domains repeated at least twice with a defined linker sequence connecting individual domains. Lastly, mutational analysis of HOSPIs suggested that the linker sequence does not affect the inhibitory activity, and a Thr residue at the P2 site and a Pro at the P3' site are crucial for elastase inhibition. Using mammalian proteases as representative model system, we gain novel insight into the sequence diversity and proteolytic activity of plant BBI. These results may aid the rational design of BBI peptides with potent and distinct inhibitory activity against human, pathogen, or insect serine proteinases.

Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation.

Disulfide-rich plant peptides with molecular masses of 2-6 kDa represent an expanding class of peptidyl-type natural products with diverse functions. They are structurally compact, hyperstable, and underexplored as cell-penetrating agents that inhibit intracellular functions. Here, we report the discovery of an anionic, 34-residue peptide, the disulfide-rich roseltide rT7 from Hibiscus sabdariffa (of the Malvaceae family) that penetrates cells and inhibits their proteasomal activities. Combined proteomics and NMR spectroscopy revealed that roseltide rT7 is a cystine-knotted, six-cysteine hevein-like cysteine-rich peptide. A pair-wise comparison indicated that roseltide rT7 is >100-fold more stable against protease degradation than its S-alkylated analog. Confocal microscopy studies and cell-based assays disclosed that after roseltide rT7 penetrates cells, it causes accumulation of ubiquitinated proteins, inhibits human 20S proteasomes, reduces tumor necrosis factor-induced IκBα degradation, and decreases expression levels of intercellular adhesion molecule-1. Structure-activity studies revealed that roseltide rT7 uses a canonical substrate-binding mechanism for proteasomal inhibition enabled by an IIML motif embedded in its proline-rich and exceptionally long intercysteine loop 4. Taken together, our results provide mechanistic insights into a novel disulfide-rich, anionic, and cell-penetrating peptide, representing a potential lead for further development as a proteasomal inhibitor in anti-cancer or anti-inflammatory therapies.

Sulfated plant peptide hormones.

Sulfated peptides are plant hormones that are active at nanomolar concentrations. The sulfation at one or more tyrosine residues is catalysed by tyrosylprotein sulfotransferase (TPST), which is encoded by a single-copy gene. The sulfate group is provided by the co-substrate 3´-phosphoadenosine 5´-phosphosulfate (PAPS), which links synthesis of sulfated signaling peptides to sulfur metabolism. The precursor proteins share a conserved DY-motif that is implicated in specifying tyrosine sulfation. Several sulfated peptides undergo additional modification such as hydroxylation of proline and glycosylation of hydroxyproline. The modifications render the secreted signaling molecules active and stable. Several sulfated signaling peptides have been shown to be perceived by leucine-rich repeat receptor-like kinases (LRR-RLKs) but have signaling pathways that, for the most part, are yet to be elucidated. Sulfated peptide hormones regulate growth and a wide variety of developmental processes, and intricately modulate immunity to pathogens. While basic research on sulfated peptides has made steady progress, their potential in agricultural and pharmaceutical applications has yet to be explored.

Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design.

Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.

A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis.

High salinity has negative impacts on plant growth through altered water uptake and ion-specific toxicities. Plants have therefore evolved an intricate regulatory network in which plant hormones play significant roles in modulating physiological responses to salinity. However, current understanding of the plant peptides involved in this regulatory network remains limited. Here, we identified a salt-regulated peptide in Arabidopsis. The peptide was 11 aa and was derived from the C terminus of a cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily. This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal. In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity. We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production. Moreover, although PROAtCAPE1 was expressed mainly in the roots, AtCAPE1 was discovered to be upregulated systemically upon salt treatment. The salt-induced AtCAPE1 negatively regulated salt tolerance by suppressing several salt-tolerance genes functioning in the production of osmolytes, detoxification, stomatal closure control, and cell membrane protection. This discovery demonstrates that AtCAPE1, a homologue of tomato immune regulator CAPE1, plays an important role in the regulation of salt stress responses. Our discovery thus suggests that the peptide may function in a trade-off between pathogen defence and salt tolerance.

Genome-wide annotation and characterization of CLAVATA/ESR (CLE) peptide hormones of soybean (Glycine max) and common bean (Phaseolus vulgaris), and their orthologues of Arabidopsis thaliana.

CLE peptides are key regulators of cell proliferation and differentiation in plant shoots, roots, vasculature, and legume nodules. They are C-terminally encoded peptides that are post-translationally cleaved and modified from their corresponding pre-propeptides to produce a final ligand that is 12-13 amino acids in length. In this study, an array of bionformatic and comparative genomic approaches was used to identify and characterize the complete family of CLE peptide-encoding genes in two of the world's most important crop species, soybean and common bean. In total, there are 84 CLE peptide-encoding genes in soybean (considerably more than the 32 present in Arabidopsis), including three pseudogenes and two multi-CLE domain genes having six putative CLE domains each. In addition, 44 CLE peptide-encoding genes were identified in common bean. In silico characterization was used to establish all soybean homeologous pairs, and to identify corresponding gene orthologues present in common bean and Arabidopsis. The soybean CLE pre-propeptide family was further analysed and separated into seven distinct groups based on structure, with groupings strongly associated with the CLE domain sequence and function. These groups provide evolutionary insight into the CLE peptide families of soybean, common bean, and Arabidopsis, and represent a novel tool that can aid in the functional characterization of the peptides. Transcriptional evidence was also used to provide further insight into the location and function of all CLE peptide-encoding members currently available in gene atlases for the three species. Taken together, this in-depth analysis helped to identify and categorize the complete CLE peptide families of soybean and common bean, established gene orthologues within the two legume species, and Arabidopsis, and provided a platform to help compare, contrast, and identify the function of critical CLE peptide hormones in plant development.

Molecular Mechanisms of Regulation of Root Development by Plant Peptides.

Peptides perform many functions, participating in the regulation of cell differentiation, regulating plant growth and development, and also involved in the response to stress factors and in antimicrobial defense. Peptides are an important class biomolecules for intercellular communication and in the transmission of various signals. The intercellular communication system based on the ligand-receptor bond is one of the most important molecular bases for creating complex multicellular organisms. Peptide-mediated intercellular communication plays a critical role in the coordination and determination of cellular functions in plants. The intercellular communication system based on the receptor-ligand is one of the most important molecular foundations for creating complex multicellular organisms. Peptide-mediated intercellular communication plays a critical role in the coordination and determination of cellular functions in plants. The identification of peptide hormones, their interaction with receptors, and the molecular mechanisms of peptide functioning are important for understanding the mechanisms of both intercellular communications and for regulating plant development. In this review, we drew attention to some peptides involved in the regulation of root development, which implement this regulation by the mechanism of a negative feedback loop.

Brt9SIDA/IDALs as peptide signals mediate diverse biological pathways in plants.

As signal molecules, plant peptides play key roles in intercellular communication during growth and development, as well as stress responses. The 14-amino-acid (aa) INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide was originally identified to play an essential role in the floral organ abscission of Arabidopsis. It is synthesized from its precursor, a small protein containing 77-aa residues with an N-terminal signal peptide sequence. Recently, the IDA/IDA-like (IDLs) genes are isolated in several angiosperms and are highly conserved in land plants. In addition, IDA/IDLs are not only involved in organ abscission but also function in multiple biological processes, including biotic and abiotic stress responses. Here, we summarize the post-translational modification and proteolytic processing, the evolutionary conservation, and the potential regulatory function of IDA/IDLs, and also present future perspectives to investigate the IDA/IDLs signaling pathway. We anticipate that this detailed knowledge will help to improve the understanding of the molecular mechanism of plant peptide signaling.

Root-Knot-Nematode-Encoded CEPs Increase Nitrogen Assimilation.

C-terminally encoded peptides (CEPs) are plant developmental signals that regulate growth and adaptive responses to nitrogen stress conditions. These small signal peptides are common to all vascular plants, and intriguingly have been characterized in some plant parasitic nematodes. Here, we sought to discover the breadth of root-knot nematode (RKN)-encoded CEP-like peptides and define the potential roles of these signals in the plant-nematode interaction, focusing on peptide activity altering plant root phenotypes and nitrogen uptake and assimilation. A comprehensive bioinformatic screen identified 61 CEP-like sequences encoded within the genomes of six root-knot nematode (RKN; Meloidogyne spp.) species. Exogenous application of an RKN CEP-like peptide altered A. thaliana and M. truncatula root phenotypes including reduced lateral root number in M. truncatula and inhibited primary root length in A. thaliana. To define the role of RKN CEP-like peptides, we applied exogenous RKN CEP and demonstrated increases in plant nitrogen uptake through the upregulation of nitrate transporter gene expression in roots and increased 15N/14N in nematode-formed root galls. Further, we also identified enhanced nematode metabolic processes following CEP application. These results support a model of parasite-induced changes in host metabolism and inform endogenous pathways to regulate plant nitrogen assimilation.

PSY-PSYR peptide-receptor pairs control the trade-off between plant growth and stress response.

Leucine-rich repeat-receptor kinases (LRR-RKs) perceive various endogenous peptide hormones that control plant growth and development. However, the majority of corresponding ligands and their direct ligand-binding receptors have not been identified yet. A recent study demonstrated that three LRR-RK PLANT PEPTIDE CONTAINING SULFATED TYROSINE RECEPTORS (PSYRs) act as ligand-receptors of the PSY family peptides that mediate the trade-off between the optimal plant growth and stress tolerance responses. The genetic, biochemical, and transcriptome analyses suggested that PSYR1, PSYR2, and PSYR3 function as negative regulators of plant growth in the absence of PSY peptides and induce stress tolerance responses, whereas the PSY family peptides repress PSYR signaling, allowing plant growth. This trade-off mechanism between plant growth and stress responses mediated by the PSY-PSYR signaling module allows plants to survive under ever changing environmental stresses.

PhytoSelectDBT: A database for the molecular models of anti-diabetic targets docked with bioactive peptides from selected ethno-medicinal plants.

It is of interest to assess the effectiveness of bioactive peptides derived from 41 ethno-medicinal plants, classify them according to their anti-diabetic protein targets (DPP-IV, alpha-amylase, alpha-glucosidase, GRK2, GSK3B, GLP-1R, and AdipoR1), and create a web tool named PhytoSelectDBT by using the top seven peptides per target. If one of the target-based medicinal plant suggestions made by PhytoSelectDBT is unsuccessful, alternative target-based possibilities are presented by PhytoSelectDBT for treating the condition and any other related complications. The results provide a useful resource for the management of type 2 diabetes and emphasize the significance of utilising ethnomedical knowledge for the identification of potent anti-diabetic plants and their peptides. We used molecular docking to investigate interactions between anti-diabetic targets (DPP-IV, alpha-amylase, alpha-glucosidase, GRK2, GSK3B, GLP-1R, and AdipoR1) and projected bioactive peptides from 41 ethnomedicinal plants. All bioactive peptides were cross-checked against several databases to determine their allergenicity, toxicity, and cross-reactivity. The presence of B and T cell epitopes was also examined in all simulated digested bioactive peptides for reference. This data is archived at the PhytoselectDBT database.

Discovery of a cysteine-rich peptide with glycation modification from Achyranthes bidentata Blume.

Cysteine-rich peptides (CRPs) are stable molecules that contain multiple disulphide bonds. Various CRPs are found in plants and animals, representing potential compounds for drug development with diverse activities. Modification of CRPs, such as glycation, has attracted increased attention due to its special structural and functional properties. Hence, this study explored a CRP isolated from the Chinese herb Achyranthes bidentata Blume, which contains a glycation modification. Herein, a reverse phase high-performance liquid chromatography system with mobile phases was used to extract and purify the peptide. The eluted peptide was detected using high resolution mass spectrometry and structurally identified using high resolution mass spectrometry and nuclear magnetic resonance. The effect of the peptide on the viability of N-methyl-D-aspartic acid (NMDA)-induced HT22 cells was determined using a cell assay. Here, a new cysteine-rich glycation peptide, termed glycation-bidentatide (Gly-BTP), with three pairs of disulphide bonds and a glycation modification at the N-terminus linked to cysteine, was discovered. Cell bioactivity assay results suggested that Gly-BTP might be a potential therapeutic and provide a neuroprotective effect in NMDA-induced HT22 murine hippocampal neuronal cells. The discovery of Gly-BTP will promote the understanding of the role of CRPs in neuroprotection.

The INFLORESCENCE DEFICIENT IN ABSCISSION-LIKE6 Peptide Functions as a Positive Modulator of Leaf Senescence in Arabidopsis thaliana.

Leaf senescence is a highly coordinated process and has a significant impact on agriculture. Plant peptides are known to act as important cell-to-cell communication signals that are involved in multiple biological processes such as development and stress responses. However, very limited number of peptides has been reported to be associated with leaf senescence. Here, we report the characterization of the INFLORESCENCE DEFICIENT IN ABSCISSION-LIKE6 (IDL6) peptide as a regulator of leaf senescence. The expression of IDL6 was up-regulated in senescing leaves. Exogenous application of synthetic IDL6 peptides accelerated the process of leaf senescence. The idl6 mutant plants showed delayed natural leaf senescence as well as senescence included by darkness, indicating a regulatory role of IDL6 peptides in leaf senescence. The role of IDL6 as a positive regulator of leaf senescence was further supported by the results of overexpression analysis and complementation test. Transcriptome analysis revealed differential expression of phytohormone-responsive genes in idl6 mutant plants. Further analysis indicated that altered expression of IDL6 led to changes in leaf senescence phenotypes induced by ABA and ethylene treatments. The results from this study suggest that the IDL6 peptide positively regulates leaf senescence in Arabidopsis thaliana.

Molecular mechanisms of plant peptide binding to receptors.

Plants have evolved diverse peptide hormones and cognate receptors to orchestrate plant growth and development. Secreted peptide ligands are mainly sensed by membrane receptor kinases that mediate cell-cell communication. The secreted peptides are categorized into two groups: small linear post-translationally modified peptides and cysteine-rich peptides. The small linear peptides are recognized by the corresponding receptors and co-receptors in a conserved manner. By contrast, the cysteine-rich peptides are perceived by various types of receptor proteins using diverse binding modes. Recent studies have revealed the molecular and mechanistic origins of peptide recognition and receptor activation. This review summarizes plant-peptide binding modes and receptor-activation mechanisms that have been structurally characterized in recent studies.

Identification of Ipomoea batatas anti-cancer peptide (IbACP)-responsive genes in sweet potato leaves.

IbACP, Ipomoea batatas anti-cancer peptide, a sixteen-amino-acid peptide isolated from sweet potato leaves, is capable of mediating a rapid alkalinization of growth media in plant suspension cells. However, the biological roles of IbACP as a defense peptide have not been studied. The objective of this study was to investigate the effect of IbACP on the accumulation of reactive oxygen species (ROS) and the expression of the defense-related genes. IbACP treatment of sweet potato leaves resulted in marked accumulation of both superoxide ion (O2-) and hydrogen peroxide (H2O2). The activity of peroxidase (POD) was significantly enhanced by IbACP treatment, suggesting that high levels of POD antioxidant activity may be used to scavenge the excess H2O2 in sweet potato plants. The IbACP-related genes were identified by suppression subtractive hybridization (SSH), and were then classified and assigned to the following categories: defense, development, metabolism, signaling, gene expression, and abiotic stress. H2O2 acts as a second messenger for gene activation in some of the IbACP-triggered gene expressions. These results demonstrated that IbACP is part of an integrated strategy for genetic regulation in sweet potato. Our work highlights the function of IbACP and its potential use for enhancing stress tolerance in sweet potato, in an effort to improve our understanding of defense-response mechanisms.

Progress toward sourcing plants for new bioconjugation tools: a screening evaluation of a model peptide ligase using a synthetic precursor.

In the present study, leaves from 39 phylogenetically distant plant species were sampled and screened for asparaginyl endopeptidase ligase activity using mass spectrometry to test the generality of peptide ligases in plants. A modified version of the sunflower trypsin inhibitor-1 precursor was used as the substrate for reactions with leaf crude extracts and protein fractions. Masses consistent with products of asparaginyl endopeptidase activities that cleave and ligate the substrate into cyclic peptide following the reactions were detected in 8 plants: Nerium oleander and Thevetia peruviana of the family Apocynaceae; Bauhinia variegata, Dermatophyllum secundiflorum, Pithecellobium flexicaule, and Prosopis chilensis of the family Fabaceae; Morus alba of the family Moraceae; and Citrus aurantium of the family Rutaceae. This screening result represents a 20% hit rate for finding asparaginyl endopeptidase ligase activity from the arbitrary plants sampled. Analysis following a 2-h reaction of the substrate with the crude extract of D. secundiflorum leaves showed that the yield of cyclic peptide remained stable around 0.5 ± 0.1% of the substrate over the course of the reaction.

Plant peptides in plant defense responses.

Plant peptides secreted as signal molecular to trigger cell-to-cell signaling are indispensable for plant growth and defense processes. Preciously, it is regraded some plant peptides function in plant growth and development, whereas others regulate defense response in plant-microbe interactions. However, this prejudice is got rid due to more and more evidence showed growth-related plant peptides also exhibit bifunctional roles in plant defense response against different microbial pathogens. Here we provide a mini-review of reported types of plant peptides, including their basic information, reported receptor ligands, and especially direct or indirect roles in plant immune responses.

The Structural Basis of Ligand Perception and Signal Activation by Receptor Kinases.

Plants have evolved a family of unique membrane receptor kinases to orchestrate the growth and development of their cells, tissues, and organs. Receptor kinases also form the first line of defense of the plant immune system and allow plants to engage in symbiotic interactions. Here, we discuss recent advances in understanding, at the molecular level, how receptor kinases with lysin-motif or leucine-rich-repeat ectodomains have evolved to sense a broad spectrum of ligands. We summarize and compare the established receptor activation mechanisms for plant receptor kinases and dissect how ligand binding at the cell surface leads to activation of cytoplasmic signaling cascades. Our review highlights that one family of plant membrane receptors has diversified structurally to fulfill very different signaling tasks.