Characterization of erythroferrone oligomerization and its impact on BMP antagonism.

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

In vitro metabolism of synthetic Elabela/Toddler (ELA-32) peptide in human plasma and kidney homogenates analyzed with mass spectrometry and validation of endogenous peptide quantification in tissues by ELISA.

BACKGROUND: Elabela/Toddler (ELA) is a novel endogenous ligand of the apelin receptor, whose signalling has emerged as a therapeutic target, for example, in cardiovascular disease and cancer. Shorter forms of ELA-32 have been predicted, including ELA-21 and ELA-11, but metabolism and stability of ELA-32 in humans is poorly understood. We, therefore, developed an LC-MS/MS assay to identify ELA-32 metabolites in human plasma and tissues. METHOD: Human kidney homogenates or plasma were incubated at 37 °C with ELA-32 and aliquots withdrawn over 2-4 h into guanidine hydrochloride. Proteins were precipitated and supernatant solid-phase extracted. Peptides were extracted from coronary artery, brain and kidney by immunoprecipitation or solid-phase extraction following acidification. All samples were reduced and alkylated before analysis on an Orbitrap mass spectrometer in high and nano flow mode. RESULTS: The half-life of ELA-32 in plasma and kidney were 47.2 ±â€¯5.7 min and 44.2 ±â€¯3 s, respectively. Using PEAKS Studio and manual data analysis, the most important fragments of ELA-32 with potential biological activity identified were ELA-11, ELA-16, ELA-19 and ELA-20. The corresponding fragments resulting from the loss of C-terminal amino acids were also identified. Endogenous levels of these peptides could not be measured, as ELA peptides are prone to oxidation and poor chromatographic peaks. CONCLUSIONS: The relatively long ELA plasma half-life observed and identification of a potentially more stable fragment, ELA-16, may suggest that ELA could be a better tool compound and novel template for the development of new drugs acting at the apelin receptor.

A Pilot Screen of a Novel Peptide Hormone Library Identified Candidate GPR83 Ligands.

The identification of novel peptide hormones by functional screening is challenging because posttranslational processing is frequently required to generate biologically active hormones from inactive precursors. We developed an approach for functional screening of novel potential hormones by expressing them in endocrine host cells competent for posttranslational processing. Candidate preprohormones were selected by bioinformatics analysis, and stable endocrine host cell lines were engineered to express the preprohormones. The production of mature hormones was demonstrated by including the preprohormones insulin and glucagon, which require the regulated secretory pathway for production of the active forms. As proof of concept, we screened a set of G-protein-coupled receptors (GPCRs) and identified protein FAM237A as a specific activator of GPR83, a GPCR implicated in central nervous system and regulatory T-cell function. We identified the active form of FAM237A as a C-terminally cleaved, amidated 9 kDa secreted protein. The related protein FAM237B, which is 64% homologous to FAM237A, demonstrated similar posttranslational modification and activation of GPR83, albeit with reduced potency. These results demonstrate that our approach is capable of identifying and characterizing novel hormones that require processing for activity.

Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

Bacteria-Derived Recombinant Human ANGPTL8/Betatrophin Significantly Increases the Level of Triglyceride.

ANGPTL8/Betatrophin has been implicated in the regulation of both glucose and triglyceride metabolism. However, its role in regulating glucose metabolism by promoting ß cell proliferation remains controversial, and its physiological functions and molecular targets are largely unknown. Hence, it is of great importance to make recombinant protein and test its effects on ß cell mass directly. In this study, the mature form gene of human ANGPTL8/betatrophin was obtained through chemical synthesis on to the vector pUCE, and the fusion protein was expressed in the Transetta (DE3)/pEASY-E2-betatrophin strain. The inclusion bodies were solubilized in urea and purified by Ni-NTA affinity chromatography. The yield of purified ANGPTL8/betatrophin was approximately 20 mg per liter of culture medium. In vitro studies revealed that the recombinant ANGPTL8/betatrophin had no proliferation effect on MIN6 cells but promoted TG levels in HepG2 cells. This method to generate bioactive ANGPTL8/betatrophin is a simple, practical and user-friendly protocol.

On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity.

Angiopoietin-like (ANGPTL) 8 is a secreted inhibitor of LPL, a key enzyme in plasma triglyceride metabolism. It was previously reported that ANGPTL8 requires another member of the ANGPTL family, ANGPTL3, to act on LPL. ANGPTL3, much like ANGPTL4, is a physiologically relevant regulator of LPL activity, which causes irreversible inactivation of the enzyme. Here, we show that ANGPTL8 can form complexes with either ANGPTL3 or ANGPTL4 when the proteins are refolded together from their denatured states. In contrast to the augmented inhibitory effect of the ANGPTL3/ANGPTL8 complex on LPL activity, the ANGPTL4/ANGPTL8 complex is less active compared with ANGPTL4 alone. In our experiments, all three members of the ANGPTL family use the same mechanism to inactivate LPL, which involves dissociation of active dimeric LPL to monomers. This inactivation can be counteracted by the presence of glycosylphosphatidylinositol-anchored HDL binding protein 1, the endothelial LPL transport protein previously known to protect LPL from spontaneous and ANGPTL4-catalyzed inactivation. Our data demonstrate that ANGPTL8 may function as an important metabolic switch, by forming complexes with ANGPTL3, or with ANGPTL4, in order to direct the flow of energy from triglycerides in blood according to the needs of the body.

Structure-function analyses reveal a triple ß-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple ß-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

Phoenixin-14: detection and novel physiological implications in cardiac modulation and cardioprotection.

Phoenixin-14 (PNX) is a newly identified peptide co-expressed in the hypothalamus with the anorexic and cardioactive Nesfatin-1. Like Nesfatin-1, PNX is able to cross the blood-brain barrier and this suggests a role in peripheral modulation. Preliminary mass spectrography data indicate that, in addition to the hypothalamus, PNX is present in the mammalian heart. This study aimed to quantify PNX expression in the rat heart, and to evaluate whether the peptide influences the myocardial function under basal condition and in the presence of ischemia/reperfusion (I/R). By ELISA the presence of PNX was detected in both hypothalamus and heart. In plasma of normal, but not of obese rats, the peptide concentrations increased after meal. Exposure of the isolated and Langendorff perfused rat heart to exogenous PNX induces a reduction of contractility and relaxation, without effects on coronary pressure and heart rate. As revealed by immunoblotting, these effects were accompanied by an increase of Erk1/2, Akt and eNOS phosphorylation. PNX (EC50 dose), administered after ischemia, induced post-conditioning-like cardioprotection. This was revealed by a smaller infarct size and a better systolic recovery with respect to those detected on hearts exposed to I/R alone. The peptide also activates the cardioprotective RISK and SAFE cascades and inhibits apoptosis. These effects were also observed in the heart of obese rats. Our data provide a first evidence on the peripheral activity of PNX and on its direct cardiomodulatory and cardioprotective role under both normal conditions and in the presence of metabolic disorders.

Immunoaffinity techniques coupled to mass spectrometry for the analysis of human peptide hormones: advances and applications.

INTRODUCTION: The accurate and comprehensive determination of peptide hormones from biological fluids has represented a considerable challenge to analytical chemists for decades. Besides long-established bioanalytical ligand binding assays (or ELISA, RIA, etc.), more and more mass spectrometry-based methods have been developed recently for purposes commonly referred to as targeted proteomics. Eventually the combination of both, analyte extraction by immunoaffinity and subsequent detection by mass spectrometry, has shown to synergistically enhance the test methods' performance characteristics. Areas covered: The review provides an overview about the actual state of existing methods and applications concerning the analysis of endogenous peptide hormones. Here, special focus is on recent developments considering the extraction procedures with immobilized antibodies, the subsequent separation of target analytes, and their detection by mass spectrometry. Expert commentary: Key aspects of procedures aiming at the detection and/or quantification of peptidic analytes in biological matrices have experienced considerable improvements in the last decade, particularly in terms of the assays' sensitivity, the option of multiplexing target compounds, automatization, and high throughput operation. Despite these advances and progress as expected to be seen in the near future, immunoaffinity purification coupled to mass spectrometry is not yet a standard procedure in routine analysis compared to ELISA/RIA.

Challenges in Modern Anti-Doping Analytical Science.

The challenges facing modern anti-doping analytical science are increasingly complex given the expansion of target drug substances, as the pharmaceutical industry introduces more novel therapeutic compounds and the internet offers designer drugs to improve performance. The technical challenges are manifold, including, for example, the need for advanced instrumentation for greater speed of analyses and increased sensitivity, specific techniques capable of distinguishing between endogenous and exogenous metabolites, or biological assays for the detection of peptide hormones or their markers, all of which require an important investment from the laboratories and recruitment of highly specialized scientific personnel. The consequences of introducing sophisticated and complex analytical procedures may result in the future in a change in the strategy applied by the Word Anti-Doping Agency in relation to the introduction and performance of new techniques by the network of accredited anti-doping laboratories.

Orally active hypoglycemic protein from Costus igneus N. E. Br.: an in vitro and in vivo study.

Plants have been used for the treatment of diabetes since time immemorial. In the present study, insulin-like protein (ILP) is purified from Costus igneus belonging to family Costaceae from Western ghats of India. The ILP showed cross reactivity with murine anti-insulin antibodies hence was purified by affinity chromatography using anti-insulin antibodies. The characterization of ILP showed that it is structurally different from insulin but functionally similar. The ILP showed a hypoglycemic activity in an in vitro assay with insulin responsive cell line RIN 5f. Interestingly ILP showed significant decrease in blood glucose level when administered orally in oral glucose tolerance test. This was compared to insulin a positive control given intraperitoneally in streptozotocine induced diabetic mice. There was no toxic effect seen on animals after administrating the ILP. Therefore we conclude that the ILP purified in the present study from C. igneus is a novel protein having hypoglycemic activity.

A novel reproductive peptide, phoenixin.

Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones luteinising hormone and follicle-stimulating hormone is regulated primarily by hypothalamically-derived gonadotrophin-releasing hormone (GnRH), although other releasing factors (RF) have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotrophin secretion by modulating the expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using small interfering RNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretary vesicles to pituitary membranes.

Electrowetting on dielectric actuation of droplets with capillary electrophoretic zones for MALDI mass spectrometric analysis.

An automated fraction collection interface was developed for coupling CE with MALDI-MS. This fraction collection approach is based on the electrowetting on dielectric (EWOD) phenomenon performed on a digital microfluidic (DMF) board; it does not rely on a MALDI spotter. In this study, a four-peptide mixture was used as a sample test, and the separations were conducted in a portable CE instrument with a 150 µm o.d. × 50 µm i.d. capillary and a contactless conductivity detector. The CE instrument was interfaced with a robust DMF board. The CE fractions were directly deposited onto the DMF board at predetermined locations prior to MALDI analysis. The series of experiments determined the lowest concentration that produces a measurable MALDI signal. The concentrations were 0.25, 0.5, 0.05, and 0.05 nmol for bradykinin, angiotensin, ACTH (18-39), and insulin, respectively. The contactless conductivity detector limit of detection for the same analytes was 2.5 µmol.

Recombinant production, isotope labeling and purification of ENOD40B: a plant peptide hormone.

The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.

[Preparation and identification of monoclonal antibody against human GCRG213].

AIM: To prepare and characterize the monoclonal antibody against human GCRG213. METHODS: The HIS-GCRG213 fusion protein was expressed in E.coli. Mice were immunized with the purified HIS-GCRG213 protein. Hybridoma cell lines secreting monoclonal antibodies against GCRG213 were screened by regular cell fusion and subcloning approach. The titer and specificity of the antibody was characterized by ELISA and Western blot, respectively. The expression of GCRG213 was determined using immunohistochemistry technique on paraffin-embedded tissue sections from normal gastric mucosal tissues and advanced gastric cancer. RESULTS: The HIS-GCRG213 fusion protein with relative molecular mass of 20 800 was over expressed in E.coli. Two hybridoma cell lines which secreted monoclonal antibody specifically against human GCRG213 fusion protein were successfully obtained. The ascite titers of this monoclonal antibody reached 1:10(6);. Western blot analysis showed that the monoclonal antibody could bind to the recombinant HIS-GCRG213 protein specifically.The immunohistochemistry showed that GCRG213 were expressed higher in gastric cancer tissues than in normal ones. CONCLUSION: The monoclonal antibody against human GCRG213 with high titer and specificity has been successfully prepared, which could be utilized as a useful reagent for further studying the biological function of the GCRG213.

Immunoaffinity purification of peptide hormones prior to liquid chromatography-mass spectrometry in doping controls.

For most peptide hormones prohibited in elite sports the concentrations in plasma or urine are very low (pg/mL). Accordingly, hyphenated purification and enrichment steps prior to mass spectrometric detection are required to obtain sufficient doping control assays. Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to high resolution/high accuracy mass spectrometry was found to have the potential of providing the necessary sensitivity and unambiguous specificity to produce reliable results. With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra, Lantus DesB30-32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing hormone (GH-RH(1-29)) and CJC-1295 (GH-RH analogue), LongR(3)-IGF-1 and IFG-1) were simultaneously purified from plasma/serum or urine. With limits of detection for each target compound ranging in the low pg/mL level (urine), the method enables the determination of urinary peptides at physiologically relevant concentrations. For each class of peptides an appropriate antibody and a respective internal standard was implemented ensuring robust analysis conditions. Due to the fast and simple sample preparation procedure (∼25 samples per day) and the fact that all materials are commercial available, the implementation of the methodology to laboratories from other analytical fields (forensics, pharmacokinetic sciences, etc.) is enabled.

Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.

A generic LC-MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1-34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC-MS setup, large volumes of filtrates (e.g., 2×750 µL) were injected and the peptides were trapped on a 1mm i.d.×10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments.

The zebra finch neuropeptidome: prediction, detection and expression.

BACKGROUND: Among songbirds, the zebra finch (Taeniopygia guttata) is an excellent model system for investigating the neural mechanisms underlying complex behaviours such as vocal communication, learning and social interactions. Neuropeptides and peptide hormones are cell-to-cell signalling molecules known to mediate similar behaviours in other animals. However, in the zebra finch, this information is limited. With the newly-released zebra finch genome as a foundation, we combined bioinformatics, mass-spectrometry (MS)-enabled peptidomics and molecular techniques to identify the complete suite of neuropeptide prohormones and final peptide products and their distributions. RESULTS: Complementary bioinformatic resources were integrated to survey the zebra finch genome, identifying 70 putative prohormones. Ninety peptides derived from 24 predicted prohormones were characterized using several MS platforms; tandem MS confirmed a majority of the sequences. Most of the peptides described here were not known in the zebra finch or other avian species, although homologous prohormones exist in the chicken genome. Among the zebra finch peptides discovered were several unique vasoactive intestinal and adenylate cyclase activating polypeptide 1 peptides created by cleavage at sites previously unreported in mammalian prohormones. MS-based profiling of brain areas required for singing detected 13 peptides within one brain nucleus, HVC; in situ hybridization detected 13 of the 15 prohormone genes examined within at least one major song control nucleus. Expression mapping also identified prohormone messenger RNAs in areas associated with spatial learning and social behaviours. Based on the whole-genome analysis, 40 prohormone probes were found on a commonly used zebra finch brain microarray. Analysis of these newly annotated transcripts revealed that six prohormone probes showed altered expression after birds heard song playbacks in a paradigm of song recognition learning; we partially verify this result experimentally. CONCLUSIONS: The zebra finch peptidome and prohormone complement is now characterized. Based on previous microarray results on zebra finch vocal learning and synaptic plasticity, a number of these prohormones show significant changes during learning. Interestingly, most mammalian prohormones have counterparts in the zebra finch, demonstrating that this songbird uses similar biochemical pathways for neurotransmission and hormonal regulation. These findings enhance investigation into neuropeptide-mediated mechanisms of brain function, learning and behaviour in this model.

Separation and determination of peptide hormones by capillary electrophoresis with laser-induced fluorescence coupled with transient pseudo-isotachophoresis preconcentration.

A new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration was established in this study. The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT(8-13)), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. When the addition of 70% (v/v) methanol and 1% (m/v) sodium chloride (NaCl) to the sample matrix, and with borate buffer (110 mM, pH 9.5) and 20% (v/v) methanol as running buffer, a preconcentration based on the pseudo-tITP afforded 100-fold improvement in peak heights compared with the traditional hydrodynamic injection (2.3% capillary volume). The detection limits (signal/noise=3) based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT(8-13), NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT(8-13) in human cerebrospinal fluid sample.

A cytoplasmic Ca2+ functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings: identification of AtRALF1 peptide.

Transient increases in the cytoplasmic Ca(2+) concentration are key events that initiate many cellular signaling pathways in response to developmental and environmental cues in plants; however, only a few extracellular mediators regulating cytoplasmic Ca(2+) singling are known to date. To identify endogenous cell signaling peptides regulating cytoplasmic Ca(2+) signaling, Arabidopsis seedlings expressing aequorin were used for an in vivo luminescence assay for Ca(2+) changes. These seedlings were challenged with fractions derived from plant extracts. Multiple heat-stable, protease-sensitive peaks of calcium elevating activity were observed after fractionation of these extracts by high-performance liquid chromatography. Tandem mass spectrometry identified the predominant active molecule isolated by a series of such chromatographic separations as a 49-amino acid polypeptide, AtRALF1 (the rapid alkalinization factor protein family). Within 40 s of treatment with nanomolar concentrations of the natural or synthetic version of the peptides, the cytoplasmic Ca(2+) level increased and reached its maximum. Prior treatment with a Ca(2+) chelator or inhibitor of IP 3-dependent signaling partially suppressed the AtRALF1-induced Ca(2+) concentration increase, indicating the likely involvement of Ca(2+) influx across the plasma membrane as well as release of Ca(2+) from intracellular reserves. Ca(2+) imaging using seedlings expressing the FRET-based Ca(2+) sensor yellow cameleon (YC) 3.6 showed that AtRALF1 could induce an elevation in Ca(2+) concentration in the surface cells of the root consistent with the very rapid effects of addition of AtRALF1 on Ca(2+) levels as reported by aequorin. Our data support a model in which the RALF peptide mediates Ca(2+)-dependent signaling events through a cell surface receptor, where it may play a role in eliciting events linked to stress responses or the modulation of growth.