FERONIA Research Articles

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.

Extracellular matrix sensing by FERONIA and Leucine‐Rich Repeat Extensins controls vacuolar expansion during cellular elongation in Arabidopsis thaliana

Cellular elongation requires the defined coordination of intra‐ and extracellular processes, but the underlying mechanisms are largely unknown. The vacuole is the biggest plant organelle, and its dimensions play a role in defining plant cell expansion rates. Here, we show that the increase in vacuolar occupancy enables cellular elongation with relatively little enlargement of the cytosol in Arabidopsis thaliana. We demonstrate that cell wall properties are sensed and impact on the intracellular expansion of the vacuole. Using vacuolar morphology as a quantitative read‐out for intracellular growth processes, we reveal that the underlying cell wall sensing mechanism requires interaction of extracellular leucine‐rich repeat extensins (LRXs) with the receptor‐like kinase FERONIA (FER). Our data suggest that LRXs link plasma membrane‐localised FER with the cell wall, allowing this module to jointly sense and convey extracellular signals to the cell. This mechanism coordinates the onset of cell wall acidification and loosening with the increase in vacuolar size.

FERONIA regulates auxin-mediated lateral root development and primary root gravitropism.

The Arabidopsis FERONIA (FER) receptor kinase is a key hub of cell signaling networks mediating various hormone, stress, and immune responses. Previous studies have shown that FER functions correlate with auxin responses, but the underlying molecular mechanism is unknown. Here, we demonstrate that the primary root of the fer-4 mutant displays increased lateral root branching and a delayed gravitropic response, which are associated with polar auxin transport (PAT). Our data suggest that aberrant PIN2 polarity is responsible for the delayed gravitropic response in fer-4. Furthermore, the diminished F-actin cytoskeleton in fer-4 implies that FER modulates F-actin-mediated PIN2 polar localization. Our findings provide new insights into the function of FER in PAT.

Leucine-rich repeat extensin proteins regulate plant salt tolerance in Arabidopsis.

The perception and relay of cell-wall signals are critical for plants to regulate growth and stress responses, but the underlying mechanisms are poorly understood. We found that the cell-wall leucine-rich repeat extensins (LRX) 3/4/5 are critical for plant salt tolerance in Arabidopsis The LRXs physically associate with the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23, which in turn interact with the plasma membrane-localized receptor-like protein kinase FERONIA (FER). The lrx345 triple mutant as well as fer mutant plants display retarded growth and salt hypersensitivity, which are mimicked by overexpression of RALF22/23 Salt stress promotes S1P protease-dependent release of mature RALF22 peptides. Treatment of roots with mature RALF22/23 peptides or salt stress causes the internalization of FER. Our results suggest that the LRXs, RALFs, and FER function as a module to transduce cell-wall signals to regulate plant growth and salt stress tolerance.

EBP1: A crucial growth regulator downstream of receptor kinases across kingdoms.

Controlling organ growth and development is crucial for all multicellular organisms and is controlled by plasma membrane localized receptor kinases (RKs) across kingdoms, including animals and plants. A central RK in plants is FERONIA (FER), which perceives endogenous rapid alkalinization factor (RALF) peptides to regulate a plethora of biological responses, including growth and development. However, it remained largely unknown how RALF sensing by FER at the plasma membrane is translated into a nuclear response. A key step forward is presented by Li and colleagues, who show that FER increases ERBB3 binding protein 1 (EBP1) mRNA translation and directly phosphorylates EBP1 to shift its subcellular localization from the cytoplasm to the nucleus where it controls growth and development through its regulation of transcription. Importantly, EBP1 is described as a transcriptional and translational regulator in mammals by acting downstream of epidermal growth factor receptor (EGFR) signaling, suggesting that animals and plants use similar conserved pathways to fine-tune growth and development. Furthermore, this work highlights the importance of protein translation as a direct output of RK signaling, a mechanism that is largely unknown in plants.

FERONIA mutation induces high levels of chloroplast-localized Arabidopsides which are involved in root growth.

The FERONIA (FER) signaling pathway is known to have diverse roles in Arabidopsis thaliana, such as growth, reproduction, and defense, but how this receptor kinase is involved in various biological processes is not well established. In this work, we applied multiple mass spectrometry techniques to identify metabolites involved in the FER signaling pathway and to understand their biological roles. A direct infusion Fourier transform ion cyclotron resonance (FT-ICR)-MS approach was used for initial screening of wild-type and feronia (fer) mutant plant extracts, and Arabidopsides were found to be significantly enriched in the mutant. As Arabidopsides are known to be induced by wounding, further experiments on wounded and non-wounded leaf samples were carried out to investigate these oxylipins as well as related phytohormones using a quadrupole-time-of-flight (Q-TOF) MS by direct injection and LC-MS/MS. In a root growth bioassay with Arabidopside A isolated from fer mutants, the wild-type showed significant root growth inhibition compared with the fer mutant. Our results therefore implicated Arabidopsides, and Arabidopside A specifically, in FER functions and/or signaling. Finally, matrix-assisted laser desorption/ionization MS imaging (MALDI-MSI) was used to visualize the localization of Arabidopsides, and we confirmed that Arabidopsides are highly abundant at wounding sites in both wild-type and fer mutant leaves. More significantly, five micron high-spatial resolution MALDI-MSI revealed that Arabidopsides are localized to the chloroplasts where many stress signaling molecules are made.

EBP1 nuclear accumulation negatively feeds back on FERONIA-mediated RALF1 signaling.

FERONIA (FER), a plasma membrane receptor-like kinase, is a central regulator of cell growth that integrates environmental and endogenous signals. A peptide ligand rapid alkalinization factor 1 (RALF1) binds to FER and triggers a series of downstream events, including inhibition of Arabidopsis H+-ATPase 2 activity at the cell surface and regulation of gene expression in the nucleus. We report here that, upon RALF1 binding, FER first promotes ErbB3-binding protein 1 (EBP1) mRNA translation and then interacts with and phosphorylates the EBP1 protein, leading to EBP1 accumulation in the nucleus. There, EBP1 associates with the promoters of previously identified RALF1-regulated genes, such as CML38, and regulates gene transcription in response to RALF1 signaling. EBP1 appears to inhibit the RALF1 peptide response, thus forming a transcription–translation feedback loop (TTFL) similar to that found in circadian rhythm control. The plant RALF1-FER-EBP1 axis is reminiscent of animal epidermal growth factor receptor (EGFR) signaling, in which EGF peptide induces EGFR to interact with and phosphorylate EBP1, promoting EBP1 nuclear accumulation to control cell growth. Thus, we suggest that in response to peptide signals, plant FER and animal EGFR use the conserved key regulator EBP1 to control cell growth in the nucleus.

FERONIA Receptor Kinase Contributes to Plant Immunity by Suppressing Jasmonic Acid Signaling in Arabidopsis thaliana

Bacterial pathogens use effectors and phytotoxins to facilitate infection of host plants. Coronatine (COR) is one of the phytotoxins produced in bacterial pathogens, such as Pseudomonas syringae pv. tomato DC3000 (pst DC3000). COR structurally and functionally mimics the active form of the plant hormone jasmonic acid (JA), JA-isoleucine (JA-Ile), and can hijack the host JA-signaling pathway to achieve host disease susceptibility [1]. COR utilizes the transcription factor MYC2, a master regulator of JA signaling, to activate NAC transcription factors, which functions to inhibit accumulation of salicylic acid (SA) and thus compromise host immunity [2]. Ithas been demonstrated that SA can antagonizeJAsignaling through NONEXPRESSOR of PATHOGENESIS-RELATED GENE1 (NPR1) [3] and downstream transcription factors TGAs [4] and WRKYs [5, 6]. However, the detailed mechanism by which host plants counteract COR-mediated susceptibility is largely unknown. Here, we show that the receptor kinase FERONIA (FER) functions to inhibit JA and COR signaling by phosphorylating and destabilizing MYC2, thereby positively regulating immunity. Conversely, the peptide ligand RALF23 acts through FER to stabilize MYC2 and elevate JA signaling, negatively contributing to plant immunity. Our results establish the RALF23-FER-MYC2 signaling module and provide a previously unknown mechanism by which host plants utilize FER signaling to counteract COR-mediated host disease susceptibility.

A kinase-dead version of FERONIA receptor-like kinase has dose-dependent impacts on rosette morphology and RALF1-mediated stomatal movements.

The receptor-like kinase FERONIA (FER) pleiotropically affects plant reproduction, development, and stress tolerance. We recently showed that the FER ligand RALF1 promotes stomatal closure and inhibits stomatal opening in a G-protein-dependent manner. FER responses have been designated as kinase-dependent or kinase-independent, based largely on fer complementation assays employing a kinase-dead FERK565R. Our quantification of FERK565R-GFP transcript and FERK565R-GFP protein in fer complementation lines reveal that, even within individual complementation lines, different levels of FERK565R expression prevail. FERK565R-GFP expression comparable to that of FER in Col-0 plants fail to elicit complementation of either fer rosette phenotypes or RALF1-elicited stomatal movements, whereas overexpression levels of FERK565R-GFP result in complementation. These results suggest possible alternative interpretations of previous conclusions regarding kinase-independent FER signaling.

Probing a Plant Plasma Membrane Receptor Kinase's Three-Dimensional Structure Using Mass Spectrometry-Based Protein Footprinting.

FERONIA (FER), one of the 17 malectin-like receptor-like kinases encoded in the Arabidopsis genome, acts as a receptor for a 5 kDa growth-inhibiting secreted protein hormone, rapid alkalinization factor 1 (RALF1). Upon binding the peptide ligand, FER is involved in a variety of signaling pathways eliciting ovule fertilization and vegetative root cell expansion. Here, we report the use of mass spectrometry-based, carbodiimide-mediated protein carboxyl group (aspartic and glutamic acid) footprinting to map solvent accessible amino acids of the ectodomain of FER (ectoFER), including those involved in RALF1 binding and/or allosteric changes. Aspartate and glutamate residues labeled in this procedure were located in various regions, including the N-terminus, malectin-like domains, and juxtamembrane region, and these correlated well with a three-dimensional structural model of ectoFER predicted from the crystal structure of a related receptor. Covalent cross-linking experiments also revealed the N-terminus of ectoFER linked to the highly conserved C-terminus of RALF1. RALF1 binding assays performed with truncation mutants of ectoFER further implicated the receptor N-terminal and juxtamembrane regions in the binding of RALF1. In conclusion, our results of mass spectrometry-based footprinting methods provide a framework for understanding ligand-induced changes in solvent accessibility and their positions within the three-dimensional structure of a plant receptor kinase.

BZR1 Transcription Factor Regulates Heat Stress Tolerance Through FERONIA Receptor-Like Kinase-Mediated Reactive Oxygen Species Signaling in Tomato.

BRASSINAZOLE RESISTANT 1 (BZR1), the critical regulator of brassinosteroid (BR) response, participates in various BR-mediated developmental processes. However, the roles of BZR1 in stress tolerance are less clear. Here, we found that BZR1-like protein in tomato controls BR response and is involved in thermotolerance by regulating the FERONIA (FER) homologs. The CRISPR-bzr1 mutant showed reduced growth and was not responsive to 24-epibrassinolide (EBR) with regard to the promotion of plant growth. Mutation in BZR1 impaired the induction of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1), production of H2O2 in the apoplast and heat tolerance. Exogenous H2O2 recovered the heat tolerance of the tomato bzr1 mutant. Overexpression of BZR1 enhanced the production of apoplastic H2O2 and heat stress responses. However, silencing of RBOH1 abolished the BZR1-mediated heat tolerance. Further analysis showed that BZR1 bound to the promoters of FERONIA2 (FER2) and FER3 and induced their expression. Silencing of FER2/3 suppressed BZR1-dependent BR signaling for the induction of RBOH1 transcripts, accumulation of apoplastic H2O2 and heat tolerance. These results indicate that BZR1 regulates heat stress responses in tomato through RBOH1-dependent reactive oxygen species (ROS) signaling, which is at least partially mediated by FER2 and FER3.

Receptor Kinase THESEUS1 Is a Rapid Alkalinization Factor 34 Receptor in Arabidopsis

The growth of plants, like that of other walled organisms, depends on the ability of the cell wall to yield without losing its integrity. In this context, plant cells can sense the perturbation of their walls and trigger adaptive modifications in cell wall polymer interactions. Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) THESEUS1 (THE1) was previously shown in Arabidopsis to trigger growth inhibition and defense responses upon perturbation of the cell wall, but so far, neither the ligand nor the role of the receptor in normal development was known. Here, we report that THE1 is a receptor for the peptide rapid alkalinization factor (RALF) 34 and that this signaling module has a role in the fine-tuning of lateral root initiation. We also show that RALF34-THE1 signaling depends, at least for some responses, on FERONIA (FER), another RALF receptor involved in a variety of processes, including immune signaling, mechanosensing, and reproduction [1]. Together, the results show that RALF34 and THE1 are part of a signaling network that integrates information on the integrity of the cell wall with the coordination of normal morphogenesis.

Inter-relationships between the heterotrimeric Gβ subunit AGB1, the receptor-like kinase FERONIA, and RALF1 in salinity response.

Plant heterotrimeric G proteins modulate numerous developmental stress responses. Recently, receptor-like kinases (RLKs) have been implicated as functioning with G proteins and may serve as plant G-protein-coupled-receptors. The RLK FERONIA (FER), in the Catharantus roseus RLK1-like subfamily, is activated by a family of polypeptides called rapid alkalinization factors (RALFs). We previously showed that the Arabidopsis G protein β subunit, AGB1, physically interacts with FER, and that RALF1 regulation of stomatal movement through FER requires AGB1. Here, we investigated genetic interactions of AGB1 and FER in plant salinity response by comparing salt responses in the single and double mutants of agb1 and fer. We show that AGB1 and FER act additively or synergistically depending on the conditions of the NaCl treatments. We further show that the synergism likely occurs through salt-induced ROS production. In addition, we show that RALF1 enhances salt toxicity through increasing Na+ accumulation and decreasing K+ accumulation rather than by inducing ROS production, and that the RALF1 effect on salt response occurs in an AGB1-independent manner. Our results indicate that RLK epistatic relationships are not fixed, as AGB1 and FER display different genetic relationships to RALF1 in stomatal versus salinity responses.

The Protein Phosphatases ATUNIS1 and ATUNIS2 Regulate Cell Wall Integrity in Tip-Growing Cells.

Fast tip-growing plant cells such as pollen tubes (PTs) and root hairs (RHs) require a robust coordination between their internal growth machinery and modifications of their extracellular rigid, yet extensible, cell wall (CW). Part of this essential coordination is governed by members of the Catharanthus roseus receptor-like kinase1-like (CrRLK1L) subfamily of RLKs with FERONIA (FER) and its closest homologs, ANXUR1 (ANX1) and ANX2, controlling CW integrity during RH and PT growth, respectively. Recently, Leucine-Rich Repeat Extensin 8 (LRX8) to LRX11 were also shown to be important for CW integrity in PTs. We previously reported an anx1 anx2 suppressor screen in Arabidopsis thaliana that revealed MARIS (MRI) as a positive regulator of both FER- and ANX1/2-dependent CW integrity pathways. Here, we characterize a suppressor that exhibits a weak rescue of the anx1 anx2 PT bursting phenotype and a short RH phenotype. The corresponding suppressor mutation causes a D94N substitution in a Type One Protein Phosphatase we named ATUNIS1 (AUN1). We show that AUN1 and its closest homolog, AUN2, are nucleocytoplasmic negative regulators of tip growth. Moreover, we demonstrate that AUN1D94N and AUN1H127A harboring mutations in key amino acids of the conserved catalytic site of phosphoprotein phosphatases function as dominant amorphic variants that repress PT growth. Finally, genetic interaction studies using the hypermorph MRIR240C and amorph AUN1D94N dominant variants indicate that LRX8-11 and ANX1/2 function in distinct but converging pathways to fine-tune CW integrity during tip growth.

Context-specific dependence on FERONIA kinase activity.

Read the Original article at doi: 10.1002/1873‐3468.13157

The common bean COK-4 and the Arabidopsis FER kinase domain share similar functions in plant growth and defence.

Receptor-like kinases are membrane proteins that can be shared by diverse signalling pathways. Among them, the Arabidopsis thaliana FERONIA (FER) plays a role in the balance between distinct signals to control growth and defence. We have found that COK-4, a putative kinase encoded in the common bean anthracnose resistance locus Co-4, which is transcriptionally regulated during the immune response, is highly similar to the kinase domain of FER. To assess whether COK-4 is a functional orthologue of FER, we expressed COK-4 in the wild-type Col-0 and the fer-5 mutant of Arabidopsis and evaluated FER-associated traits. We observed that fer-5 plants show an enhanced apoplastic and stomatal defence against Pseudomonas syringae. In addition, the fer-5 mutant shows reduced biomass, smaller guard cell size, greater number of stomata per leaf area, fewer leaves, faster transition to reproductive stage and lower seed weight per plant than the wild-type Col-0. Except for the stomatal complex length and number of stomata, COK-4 expression in fer-5 lines partially or completely rescued both defence and developmental defects of fer-5 to the wild-type level. Notably, COK-4 may have an additive effect to FER, as the expression of COK-4 in Col-0 resulted in enhanced defence and growth phenotypes in comparison with wild-type Col-0 plants. Altogether, these findings indicate that the common bean COK-4 shares at least some of the multiple functions of the Arabidopsis FER kinase domain, acting in both the induction of plant growth and regulation of plant defence.

The FERONIA Receptor Kinase Maintains Cell-Wall Integrity during Salt Stress through Ca2+ Signaling

SUMMARYCells maintain integrity despite changes in their mechanical properties elicited during growth and environmental stress. How cells sense their physical state and compensate for cell-wall damage is poorly understood, particularly in plants. Here we report that FERONIA (FER), a plasma-membrane-localized receptor kinase from Arabidopsis, is necessary for the recovery of root growth after exposure to high salinity, a widespread soil stress. The extracellular domain of FER displays tandem regions of homology with malectin, an animal protein known to bind diglucose in vitro and important for protein quality control in the endoplasmic reticulum. The presence of malectin-like domains in FER and related receptor kinases has led to widespread speculation that they interact with cell-wall polysaccharides and can potentially serve a wall-sensing function. Results reported here show that salinity causes softening of the cell wall and that FER is necessary to sense these defects. When this function is disrupted in the fer mutant, root cells explode dramatically during growth recovery. Similar defects are observed in the mur1 mutant, which disrupts pectin cross-linking. Furthermore, fer cell-wall integrity defects can be rescued by treatment with calcium and borate, which also facilitate pectin cross-linking. Sensing of these salinity-induced wall defects might therefore be a direct consequence of physical interaction between the extracellular domain of FER and pectin. FER-dependent signaling elicits cell-specific calcium transients that maintain cell-wall integrity during salt stress. These results reveal a novel extracellular toxicity of salinity, and identify FER as a sensor of damage to the pectin-associated wall.

The G Protein β-Subunit, AGB1, Interacts with FERONIA in RALF1-Regulated Stomatal Movement.

Heterotrimeric guanine nucleotide-binding (G) proteins are composed of Gα, Gβ, and Gγ subunits and function as molecular switches in signal transduction. In Arabidopsis (Arabidopsis thaliana), there are one canonical Gα (GPA1), three extra-large Gα (XLG1, XLG2, and XLG3), one Gβ (AGB1), and three Gγ (AGG1, AGG2, and AGG3) subunits. To elucidate AGB1 molecular signaling, we performed immunoprecipitation using plasma membrane-enriched proteins followed by mass spectrometry to identify the protein interactors of AGB1. After eliminating proteins present in the control immunoprecipitation, commonly identified contaminants, and organellar proteins, a total of 103 candidate AGB1-associated proteins were confidently identified. We identified all of the G protein subunits except XLG1, receptor-like kinases, Ca2+ signaling-related proteins, and 14-3-3-like proteins, all of which may couple with or modulate G protein signaling. We confirmed physical interaction between AGB1 and the receptor-like kinase FERONIA (FER) using bimolecular fluorescence complementation. The Rapid Alkalinization Factor (RALF) family of polypeptides have been shown to be ligands of FER. In this study, we demonstrate that RALF1 regulates stomatal apertures and does so in a G protein-dependent manner, inhibiting stomatal opening and promoting stomatal closure in Columbia but not in agb1 mutants. We further show that AGGs and XLGs, but not GPA1, participate in RALF1-mediated stomatal signaling. Our results suggest that FER acts as a G protein-coupled receptor for plant heterotrimeric G proteins.

Role of CrRLK1L Cell Wall Sensors HERCULES1 and 2, THESEUS1, and FERONIA in Growth Adaptation Triggered by Heavy Metals and Trace Elements.

Cell walls are not only a protective barrier surrounding protoplasts but serve as signaling platform between the extracellular environment and the intracellular physiology. Ions of heavy metals and trace elements, summarized to metal ions, bind to cell wall components, trigger their modification and provoke growth responses. To examine if metal ions trigger cell wall sensing receptor like kinases (RLKs) of the Catharanthus roseus RLK1-like (CrRLK1L) family we employed a molecular genetic approach. Quantitative transcription analyses show that HERCULES1 (HERK1), THESEUS1 (THE1), and FERONIA (FER) were differently regulated by cadmium (Cd), nickel (Ni), and lead (Pb). Growth responses were quantified for roots and etiolated hypocotyls of related mutants and overexpressors on Cd, copper (Cu), Ni, Pb, and zinc (Zn) and revealed a complex pattern of gene specific, overlapping and antagonistic responses. Root growth was often inversely affected to hypocotyl elongation. For example, both HERK genes seem to negatively regulate hypocotyl elongation upon Cd, Ni, Zn, and Pb while they support root growth on Cd, Cu, and Ni. The different THE1 alleles exhibited a similar effect between roots and hypocotyls on Ni, where the loss-of-function mutant was more tolerant while the gain of function mutants were hypersensitive indicating that THE1 is mediating Ni specific inhibition of hypocotyl elongation in the dark. In contrast hypocotyl elongation of the knock-out mutant, fer-4, was hypersensitive to Ni but exhibited a higher tolerance to Cd, Cu, Pb, and Zn. These data indicate an antagonistic action between THE1 and FER in relation to hypocotyl elongation upon excess of Ni. FERs function as receptor for rapid alkalinization factors (RALFs) was tested with the indicator bromocresol purple. While fer-4 roots strongly acidified control and metal ion containing media, the etiolated hypocotyls alkalized the media which is consistent with the already shorter hypocotyl of fer-4. No other CrRLK1L mutant exhibited this phenotype except of the THE1:GFP overexpressor on Ni suggesting that THE1 might be involved in Ni induced and hypocotyl specific RALF signaling and growth regulating pathway. Overall, our findings establish a molecular link between metal ion stress, growth and the cell wall integrity sensors of the CrRLK1L family.