Rapid Alkalinization Factor Peptides Research Articles

Fusarium graminearum rapid alkalinization factor peptide negatively regulates plant immunity and cell growth via the FERONIA receptor kinase.

The plant rapid alkalinization factor (RALF) peptides function as key regulators in cell growth and immune responses through the receptor kinase FERONIA (FER). In this study, we report that the transcription factor FgPacC binds directly to the promoter of FgRALF gene, which encodes a functional homologue of the plant RALF peptides from the wheat head blight fungus Fusarium graminearum (FgRALF). More importantly, FgPacC promotes fungal infection via host immune suppression by activating the expression of FgRALF. The FgRALF peptide also exhibited typical activities of plant RALF functions, such as inducing plant alkalinization and inhibiting cell growth, including wheat (Triticum aestivum), tomato (Solanum lycopersicum) and Arabidopsis thaliana. We further identified the wheat receptor kinase FERONIA (TaFER), which is capable of restoring the defects of the A. thaliana FER mutant. In addition, we found that FgRALF peptide binds to the extracellular malectin-like domain (ECD) of TaFER (TaFERECD) to suppress the PAMP-triggered immunity (PTI) and cell growth. Overexpression of TaFERECD in A. thaliana confers plant resistance to F. graminearum and protects from FgRALF-induced cell growth inhibition. Collectively, our results demonstrate that the fungal pathogen-secreted RALF mimic suppresses host immunity and inhibits cell growth via plant FER receptor. This establishes a novel pathway for the development of disease-resistant crops in the future without compromising their yield potential.

Growth-inhibiting effects of the unconventional plant APYRASE 7 of Arabidopsis thaliana influences the LRX/RALF/FER growth regulatory module.

Plant cell growth involves coordination of numerous processes and signaling cascades among the different cellular compartments to concomitantly enlarge the protoplast and the surrounding cell wall. The cell wall integrity-sensing process involves the extracellular LRX (LRR-Extensin) proteins that bind RALF (Rapid ALkalinization Factor) peptide hormones and, in vegetative tissues, interact with the transmembrane receptor kinase FERONIA (FER). This LRX/RALF/FER signaling module influences cell wall composition and regulates cell growth. The numerous proteins involved in or influenced by this module are beginning to be characterized. In a genetic screen, mutations in Apyrase 7 (APY7) were identified to suppress growth defects observed in lrx1 and fer mutants. APY7 encodes a Golgi-localized NTP-diphosphohydrolase, but opposed to other apyrases of Arabidopsis, APY7 revealed to be a negative regulator of cell growth. APY7 modulates the growth-inhibiting effect of RALF1, influences the cell wall architecture and -composition, and alters the pH of the extracellular matrix, all of which affect cell growth. Together, this study reveals a function of APY7 in cell wall formation and cell growth that is connected to growth processes influenced by the LRX/RALF/FER signaling module.

Genome-Wide Identification and Comparative Analysis of RALF Gene Family in Legume and Non-Legume Species.

Rapid alkalinization factor (RALF) are small secreted peptide hormones that can induce rapid alkalinization in a medium. They act as signaling molecules in plants, playing a critical role in plant development and growth, especially in plant immunity. Although the function of RALF peptides has been comprehensively analyzed, the evolutionary mechanism of RALFs in symbiosis has not been studied. In this study, 41, 24, 17 and 12 RALFs were identified in Arabidopsis, soybean, Lotus and Medicago, respectively. A comparative analysis including the molecular characteristics and conserved motifs suggested that the RALF pre-peptides in soybean represented a higher value of isoelectric point and more conservative motifs/residues composition than other species. All 94 RALFs were divided into two clades according to the phylogenetic analysis. Chromosome distribution and synteny analysis suggested that the expansion of the RALF gene family in Arabidopsis mainly depended on tandem duplication, while segment duplication played a dominant role in legume species. The expression levels of most RALFs in soybean were significantly affected by the treatment of rhizobia. Seven GmRALFs are potentially involved in the release of rhizobia in the cortex cells. Overall, our research provides novel insights into the understanding of the role of the RALF gene family in nodule symbiosis.

RALF peptide signaling controls the polytubey block in Arabidopsis.

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana, the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

New paradigms in cell adaptation: decades of discoveries on the CrRLK1L receptor kinase signalling network.

Receptor-like kinases (RLKs), which constitute the largest receptor family in plants, are essential for perceiving and relaying information about various environmental stimuli. Tremendous progress has been made in the past few decades towards elucidating the mechanisms of action of several RLKs, with emerging paradigms pointing to their roles in cell adaptations. Among these paradigms, Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) proteins and their rapid alkalinization factor (RALF) peptide ligands have attracted much interest. In particular, FERONIA (FER) is a CrRLK1L protein that participates in a wide array of physiological processes associated with RALF signalling, including cell growth and monitoring cell wall integrity, RNA and energy metabolism, and phytohormone and stress responses. Here, we analyse FER in the context of CrRLK1L members and their ligands in multiple species. The FER working model raises many questions about the role of CrRLK1L signalling networks during cell adaptation. For example, how do CrRLK1Ls recognize various RALF peptides from different organisms to initiate specific phosphorylation signal cascades? How do RALF-FER complexes achieve their specific, sometimes opposite, functions in different cell types? Here, we summarize recent major findings and highlight future perspectives in the field of CrRLK1L signalling networks.

Family-wide evaluation of RAPID ALKALINIZATION FACTOR peptides.

Plant peptide hormones are important players that control various aspects of the lives of plants. RAPID ALKALINIZATION FACTOR (RALF) peptides have recently emerged as important players in multiple physiological processes. Numerous studies have increased our understanding of the evolutionary processes that shaped the RALF family of peptides. Nevertheless, to date, there is no comprehensive, family-wide functional study on RALF peptides. Here, we analyzed the phylogeny of the proposed multigenic RALF peptide family in the model plant Arabidopsis (Arabidopsis thaliana), ecotype Col-0, and tested a variety of physiological responses triggered by RALFs. Our phylogenetic analysis reveals that two of the previously proposed RALF peptides are not genuine RALF peptides, which leads us to propose a revision to the consensus AtRALF peptide family annotation. We show that the majority of AtRALF peptides, when applied exogenously as synthetic peptides, induce seedling or root growth inhibition and modulate reactive oxygen species (ROS) production in Arabidopsis. Moreover, our findings suggest that alkalinization and growth inhibition are, generally, coupled characteristics of RALF peptides. Additionally, we show that for the majority of the peptides, these responses are genetically dependent on FERONIA, suggesting a pivotal role for this receptor kinase in the perception of multiple RALF peptides.

Evidence for multiple receptors mediating RALF-triggered Ca2+ signaling and proton pump inhibition.

Acidification of the apoplastic space facilitates cell wall loosening and is therefore a key step in cell expansion. PSY1 is a growth-promoting secreted tyrosine-sulfated glycopeptide whose receptor directly phosphorylates and activates the plasma membrane H+ -ATPase, which results in acidification and initiates cellular expansion. Although the mechanism is not clear, the Rapid Alkalinization Factor (RALF) family of small, secreted peptides inhibits the plasma membrane H+ -ATPase, leading to alkalinization of the apoplastic space and reduced growth. Here we show that treating Arabidopsis thaliana roots with PSY1 induced the transcription of genes encoding the RALF peptides RALF33 and RALFL36. A rapid burst of intracellular Ca2+ preceded apoplastic alkalinization in roots triggered by RALFs, with peptide-specific signatures. Ca2+ channel blockers abolished RALF-induced alkalinization, indicating that the Ca2+ signal is an obligatory part of the response and that it precedes alkalinization. As expected, fer mutants deficient in the RALF receptor FERONIA did not respond to RALF33. However, we detected both Ca2+ and H+ signatures in fer mutants upon treatment with RALFL36. Our results suggest that different RALF peptides induce extracellular alkalinization by distinct mechanisms that may involve different receptors.

Overlapping functions and protein-protein interactions of LRR-extensins in Arabidopsis.

Plant cell growth requires the coordinated expansion of the protoplast and the cell wall, which is controlled by an elaborate system of cell wall integrity (CWI) sensors linking the different cellular compartments. LRR-eXtensins (LRXs) are cell wall-attached extracellular regulators of cell wall formation and high-affinity binding sites for RALF (Rapid ALkalinization Factor) peptide hormones that trigger diverse physiological processes related to cell growth. LRXs function in CWI sensing and in the case of LRX4 of Arabidopsis thaliana, this activity was shown to involve interaction with the transmembrane Catharanthus roseus Receptor-Like Kinase1-Like (CrRLK1L) protein FERONIA (FER). Here, we demonstrate that binding of RALF1 and FER is common to most tested LRXs of vegetative tissue, including LRX1, the main LRX protein of root hairs. Consequently, an lrx1-lrx5 quintuple mutant line develops shoot and root phenotypes reminiscent of the fer-4 knock-out mutant. The previously observed membrane-association of LRXs, however, is FER-independent, suggesting that LRXs bind not only FER but also other membrane-localized proteins to establish a physical link between intra- and extracellular compartments. Despite evolutionary diversification of various LRX proteins, overexpression of several chimeric LRX constructs causes cross-complementation of lrx mutants, indicative of comparable functions among members of this protein family. Suppressors of the pollen-growth defects induced by mutations in the CrRLK1Ls ANXUR1/2 also alleviate lrx1 lrx2-induced mutant root hair phenotypes. This suggests functional similarity of LRX-CrRLK1L signaling processes in very different cell types and indicates that LRX proteins are components of conserved processes regulating cell growth.

FERONIA phosphorylates E3 ubiquitin ligase ATL6 to modulate the stability of 14-3-3 proteins in response to the carbon/nitrogen ratio.

The ratio between carbon (C) and nitrogen (N) utilization must be precisely coordinated to enable plant growth. Although numerous physiological studies have examined carbon/nitrogen (C/N) ratios, the mechanisms of sensing the C/N balance and C/N signaling remain elusive. Here, we report that a mutation of FERONIA (FER), a receptor kinase that plays versatile roles in plant cell growth and stress responses, caused hypersensitivity to a high C/N ratio in Arabidopsis. In contrast, FER-overexpressing plants displayed more resistant phenotypes. FER can interact with and phosphorylate ATL6, an E3 ubiquitin ligase that has been shown to regulate plant C/N responses. FER-mediated ATL6 phosphorylation enhanced the interaction between ATL6 and its previously identified target 14-3-3 proteins, thus decreasing 14-3-3 protein levels, leading to an increased insensitivity to high C/N ratios. Further analyses showed that the rapid alkalinization factor peptide (RALF1), which is a ligand of FER, also influenced the stability of 14-3-3 proteins via a FER-ATL6-mediated pathway. These findings reveal a novel regulatory mechanism that links the RALF1/FER-ATL6 pathway to whole-plant C/N responses and growth.

Mechanisms of RALF peptide perception by a heterotypic receptor complex.

Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment1. Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides2 have previously been proposed as ligands for several members of the CrRLK1L family1. However, the mechanistic basis of this perception is unknown. Here we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23-LLG1-FER and RALF23-LLG2-FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Our work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LREandLLG family.

Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple.

The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol purple, treated with the peptide and photographed after 30 min. Herein the protocol is optimized for evaluation of exogenous treatment, described in detail and expected results are presented.

FERONIA Receptor Kinase at the Crossroads of Hormone Signaling and Stress Responses.

Plant receptor-like kinases (RLKs) are involved in nearly all aspects of plant life including growth, development and stress response. Recent studies show that FERONIA (FER), a CrRLK1L subfamily member, is a versatile regulator of cell expansion and serves as a signaling node mediating cross-talk among multiple phytohormones. As a receptor for the RALF (Rapid Alkalinization Factor) peptide ligand, FER triggers a downstream signaling cascade that leads to a rapid cytoplasmic calcium increase and inhibition of cell elongation in plants. Moreover, FER recruits and activates small G proteins through the guanine nucleotide exchange factor-Rho-like GTPase (GEF-ROP) network to regulate both auxin and ABA responses that cross-talk with the RALF signaling pathway. One of the downstream processes is NADPH oxidase-dependent ROS (reactive oxygen species) production that modulates cell expansion and responses to both abiotic and biotic stress responses. Intriguingly, some pathogenic fungi produce RALF-like peptides to activate the host FER-mediated pathway and thus increase their virulence and cause plant disease. Studies so far indicate that FER may serve as a central node of the cell signaling network that integrates a number of regulatory pathways targeting cell expansion, energy metabolism and stress responses. This review focuses on recent findings and their implications in the context of FER action as a modulator that is crucial for hormone signaling and stress responses.

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides.

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen-activated protein kinase activation). Gene expression analysis confirmed that a RALF-encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant-pathogen interactions.

FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis

Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)-A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals.

Phosphoproteomics-based peptide ligand-receptor kinase pairing. Commentary on: "A peptide hormone and its receptor protein kinase regulate plant cell expansion".

Phosphoproteomics-based peptide ligand-receptor kinase pairing. Commentary on: "A peptide hormone and its receptor protein kinase regulate plant cell expansion".

Understanding the RALF family: a tale of many species.

Small secreted peptides are gaining importance as signalling molecules in plants. Among the 1000 open reading frames (ORFs) in the Arabidopsis (Arabidopsis thaliana) genome potentially encoding small secreted peptides, the members of the RAPID ALKALINIZATION FACTOR (RALF) family of peptides have been linked to several physiological and developmental processes. Here, we provide a comprehensive overview of current knowledge on the RALF family. Discovered in tobacco (Nicotiana tabacum), the role of RALF peptides has been investigated in numerous plant species. Together, these observations suggest that RALF peptides impact on acidification and cell expansion during growth and development. Although few components of the signalling pathway have been revealed, the recent identification of FERONIA (FER) as a RALF receptor and plasma membrane H(+)-ATPase 2 as a downstream target provide a major step forward.

SacRALF1, a peptide signal from the grass sugarcane (Saccharum spp.), is potentially involved in the regulation of tissue expansion

Rapid alkalinization factor (RALF) is part of a growing family of small peptides with hormone characteristics in plants. Initially isolated from leaves of tobacco plants, RALF peptides can be found throughout the plant kingdom and they are expressed ubiquitously in plants. We took advantage of the small gene family size of RALF genes in sugarcane and the ordered cellular growth of the grass sugarcane leaves to gain information about the function of RALF peptides in plants. Here we report the isolation of two RALF peptides from leaves of sugarcane plants using the alkalinization assay. SacRALF1 was the most abundant and, when added to culture media, inhibited growth of microcalli derived from cell suspension cultures at concentrations as low as 0.1 microM. Microcalli exposed to exogenous SacRALF1 for 5 days showed a reduced number of elongated cells. Only four copies of SacRALF genes were found in sugarcane plants. All four SacRALF genes are highly expressed in young and expanding leaves and show a low or undetectable level of expression in expanded leaves. In half-emerged leaf blades, SacRALF transcripts were found at high levels at the basal portion of the leaf and at low levels at the apical portion. Gene expression analyzes localize SacRALF genes in elongation zones of roots and leaves. Mature leaves, which are devoid of expanding cells, do not show considerable expression of SacRALF genes. Our findings are consistent with SacRALF genes playing a role in plant development potentially regulating tissue expansion.

Characterization of five RALF-like genes from Solanum chacoense provides support for a developmental role in plants.

Five RALF (rapid alkalinization factor)-like genes, named ScRALF1 to 5, were isolated from fertilized ovule and ovary cDNA libraries of Solanum chacoense. They showed high sequence similarities with the RALF protein sequence from Nicotiana tabacum, and exhibited the characteristic architecture of RALF polypeptides. All ScRALFs were moderately to highly expressed at some stage of fruit maturation. ScRALF1 and ScRALF3 were predominantly expressed in ovaries and larger fruits, while ScRALF2, ScRALF4, and ScRALF5 were also expressed in other tissues, indicating that while some RALFs may be involved in fruit maturation, others could be involved in other developmental processes. Wounding or treatment of plants with growth regulators involved in plant defense responses had no significant impact on the mRNA level of any of these genes. These results suggest and support previous data showing that RALF peptides are more likely to act as a small peptide involved in plant development than in defense responses.

Rapid alkalinization factors in poplar cell cultures. Peptide isolation, cDNA cloning, and differential expression in leaves and methyl jasmonate-treated cells.

A family of peptides inducing rapid pH alkalinization in hybrid poplar (Populus trichocarpa x Populus deltoides) cell culture medium was isolated from hybrid poplar leaves. Five related approximately 5-kD peptides were purified by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption ionization-mass spectrometry. The N-terminal sequence of one of the isolated peptides was very similar to a previously characterized peptide from tobacco (Nicotiana tabacum), rapid alkalinization factor (RALF), which causes a rapid increase in culture medium pH when added to tobacco cell cultures (G. Pearce, D.S. Moura, J. Stratmann, C.A. Ryan [2001] Proc Natl Acad Sci USA 98: 12843-12847). Two unique poplar RALF cDNAs (PtdRALF1 and PtdRALF2) were isolated from a poplar cDNA library and used to study RALF expression in poplar saplings and cultured poplar cells. Both genes were found to be expressed constitutively in poplar saplings and cultured cells. However, PtdRALF2 was expressed in leaves at very low levels, and its expression in suspension culture cells was transiently suppressed by methyl jasmonate (MeJa). Although the function of these novel peptides remains enigmatic, our experiments suggest their role may be developmental rather than stress related. Overall, our study confirms the presence of active RALF peptides in other plants, and provides new data on the complexity of the RALF gene family in poplar.