Auxins and Cytokinins as Antipodal Modulators of Elasticity within the Actin Network of Plant Cells

The cytoskeleton of plant and animal cells serves as a transmitter, transducer, and effector of cell signaling mechanisms. In plants, pathways for proliferation, differentiation, intracellular vesicular transport, cell-wall biosynthesis, symbiosis, secretion, and membrane recycling depend on the organization and dynamic properties of actin- and tubulin-based structures that are either associated with the plasma membrane or traverse the cytoplasm. Recently, a new in vivo cytoskeletal assay (cell optical displacement assay) was introduced to measure the tension within subdomains (cortical, transvacuolar, and perinuclear) of the actin network in living plant cells. Cell optical displacement assay measurements within soybean (Glycine max [L.]) root cells previously demonstrated that lipophilic signals, e.g. linoleic acid and arachidonic acid or changes in cytoplasmic pH gradients, could induce significant reductions in the tension within the actin network of transvacuolar strands. In contrast, enhancement of cytoplasmic free Ca2+ resulted in an increase in tension. In the present communication we have used these measurements to show that a similar antipodal pattern of activity exists for auxins and cytokinins (in their ability to modify the tension within the actin network of plant cells). It is suggested that these growth substances exert their effect on the cytoskeleton through the activation of signaling cascades, which result in the production of lipophilic and ionic second messengers, both of which have been demonstrated to directly effect the tension within the actin network of soybean root cells.     

Actin-filament-dependent remodeling of the vacuole in cultured mesophyll protoplasts

Summary. The ability of plant cells to dedifferentiate represents an important survival strategy invoked in a range of situations from repair mechanisms following wounding to apomixis. Dedifferentiation requires that somatic cells reprogram and enter the cell division cycle. This in turn necessitates the accurate partitioning of nuclear content and organelles, such as chloroplasts, to daughter cells, thereby ensuring continuity of cellular information systems. The distribution of cytoplasm and its organelle content in mature plant cells is governed by a large, central vacuole, with connections between distant cortical and perinuclear cytoplasmic domains mediated by transvacuolar strands. Here we examined the changes to vacuolar architecture in Arabidopsis thaliana protoplasts expressing a green-fluorescent protein fusion to a δ-tonoplast-intrinsic protein (δTIP). We found that vacuolar architecture became increasingly intricate during protoplast culture with the development of numerous transvacuolar strands. The development of an intricate vacuolar architecture was an actin filament- and not microtubule-dependent process, as is the case in interphase plant cells. Furthermore, we show that myosin is required for this increased complexity of vacuolar architecture and the formation of subcortical actin filament arrays. Despite the likelihood that increased vacuolar invagination would allow better redistribution of cytoplasmic organelles, we found that repositioning of chloroplasts from cortical to perinuclear cytoplasm was not dependent on transvacuolar strands. Our findings indicate that the vacuole is a dynamic entity that develops a complex architecture before dedifferentiating plant cells enter cell division. Supplementary material to this paper is available in electronic form at Correspondence and reprints: School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.     

Roles of actin filaments in cytoplasmic streaming and organization of transvacuolar strands in root hair cells ofHydrocharis

Summary Effects of cytochalasin B and mycalolide-B on cytoplasmic streaming, organizations of actin filaments and the transvacuolar strand were studied in root hair cells ofHydrocharis, which shows reverse fountain streaming. Both toxins inhibited cytoplasmic streaming and destroyed the organizations of actin filaments and transvacuolar strands. However, we found a great difference between these toxins with respect to reversibility. The effects of cytochalasin B were reversible but not those of mycalolide B. The present results suggest that actin filaments work as a track of cytoplasmic streaming and as a cytoskeleton to maintain the transvacuolar strand. The usefulness of root hair cells ofHydrocharis in studying the dynamic organization of actin filaments of plant is discussed.Abbreviations CB cytochalasin B - DMSO dimethylsulfoxide - ML-B mycalolide B     

Four-dimensional imaging of transvacuolar strand dynamics in tobacco BY-2 cells

The vacuole is a characteristic organelle of plant cells and fulfills several important functions related to metabolism and growth of the cell. To shed light on the details of vacuolar structural changes in plant cells, we explored the three-dimensional organization and dynamics of living Nicotiana tabacum L. cv. Bright Yellow 2 cell vacuoles by real-time confocal time-lapse imaging. For imaging, the cells were pulse-labeled with the amphipathic styryl dye FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), which is delivered to the plant vacuole by endocytic uptake and then incubated overnight. Imaging of the membrane-labeled vacuole revealed a complex vacuole morphology underlaid by constant remodeling. The vacuole is traversed by multiple transvacuolar strands which move along each other and fuse in multiple manners. New strands were created by fission of large membrane sheets. Endocytic vesicle trafficking was followed within the dynamic transvacuolar strands. The movement occurred in a stop-and-go fashion with an average vesicle velocity of 0.46 microm/s and a peak velocity of 0.82 microm/s. Transvacuolar-strand reduction and creation is a characteristic event observed during mitosis. Here we propose a mechanistic model for the alteration of the number of transvacuolar strands, on the basis of their fusion and fission.     

ACTIN BINDING PROTEIN 29 from Lilium pollen plays an important role in dynamic actin remodeling

Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca(2+)- and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.     

A Translocation Hypothesis based on the Structure of Plant Cytoplasm

Two types of linear structures have been seen in plant cytoplasm,largely by phase-contrast microscopy. Microscopic fine threads,o.Iµ to Iµ in diameter, were revealed in hair cells,where they formed endoplasmic systems along streaming pathwaysin the parietal cytoplasm and in transvacuolar strands. Duringcirculation streaming, small plastids and mitochondria-likeparticles were observed moving along the fine threads. Similarfine threads, together with small plastids and mitochondria-likeparticles occurred in phloem exudate and transcellular microscopicstrands. The transcellular strands, Iµ to 7µ indiameter, were seen in sieve-tube elements, phloem parenchyma,border parenchyma, and cortical cells. The movement of small plastids across end walls in border-parenchymacells, the appearance of the same structures within strandsin phloem cells, and the undiminished occurrence of small plastidsin successive drops of phloem exudate are collectively takenas evidence for their participation in translocation. Particlemovement is thought to occur through transcellular strands inassociation with fine threads, and to be motivated by a transcellularform of protoplasmic streaming.     

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.     

Use of phenylboronic acids to investigate boron function in plants. Possible role of boron in transvacuolar cytoplasmic strands and cell-to-wall adhesion

The only defined physiological role of boron in plants is as a cross-linking molecule involving reversible covalent bonds with cis-diols on either side of borate. Boronic acids, which form the same reversible bonds with cis-diols but cannot cross-link two molecules, were used to selectively disrupt boron function in plants. In cultured tobacco (Nicotiana tabacum cv BY-2) cells, addition of boronic acids caused the disruption of cytoplasmic strands and cell-to-cell wall detachment. The effect of the boronic acids could be relieved by the addition of boron-complexing sugars and was proportional to the boronic acid-binding strength of the sugar. Experiments with germinating petunia (Petunia hybrida) pollen and boronate-affinity chromatography showed that boronic acids and boron compete for the same binding sites. The boronic acids appear to specifically disrupt or prevent borate-dependent cross-links important for the structural integrity of the cell, including the organization of transvacuolar cytoplasmic strands. Boron likely plays a structural role in the plant cytoskeleton. We conclude that boronic acids can be used to rapidly and reversibly induce boron deficiency-like responses and therefore are useful tools for investigating boron function in plants.     

The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis

In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells. Received: 16 September 1999 / Accepted: 3 December 1999     

Serotonin modifies cytoskeleton and brush-border membrane architecture in human intestinal epithelial cells

Serotonin or 5-hydroxytryptamine (5-HT) influences numerous functions in the gastrointestinal tract. We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited Na(+)/H(+) exchangers (NHE) and Cl(-)/OH(-) exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 muM, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular Ca(2+) and PKCalpha inhibition (Go6976). In vitro assays revealed that PKCalpha interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause Ca(2+) and PKCalpha-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT.     

Capturing in vivo dynamics of the actin cytoskeleton stimulated by auxin or light

We present here a transient expression system that allows the response of actin microfilaments to physiological stimuli (changes in auxin content, light) to be observed in single cells in vivo. Etiolated, intact rice seedlings are attached to glass slides, transfected biolistically with talin fused to yellow-fluorescent protein to visualize actin microfilaments, and either treated with auxin or irradiated. The talin marker labels distinct populations of actin that are differentially expressed depending on the physiological state of the coleoptile (active elongation versus ceased elongation). Whereas longitudinal transvacuolar bundles prevail in cells that have ceased to elongate, fine cortical strands are characteristic for elongating cells. The visualized actin structures remain dynamic and responsive to signals. Exogenous auxin triggers a loosening of the bundles and an extension of the cortical strands, whereas irradiation reorientates cortical strands into longitudinal arrays. These responses correspond in quality and timing to the signal responses inferred previously from fixed specimens and biochemical studies. In big advantage over those methods it is now possible to observe them directly at the single cell level. Thus, the rice coleoptile system can be used as a convenient model to study actin dynamics in vivo, in response to physiologically relevant stimuli.     

Transactivation of the epidermal growth factor receptor mediates muscarinic stimulation of focal adhesion kinase in intestinal epithelial cells

We have previously shown that the Gq protein coupled receptor (GqPCR) agonist, carbachol (CCh), transactivates and recruits epidermal growth factor receptor (EGFr)-dependent signaling mechanisms in intestinal epithelial cells. Increasing evidence suggests that GqPCR agonists can also recruit focal adhesion-dependent signaling pathways in some cell types. Therefore, the aim of the present study was to investigate if CCh stimulates activation of the focal adhesion-associated protein, focal adhesion kinase (FAK), in intestinal epithelia and, if so, to examine the signaling mechanisms involved. Experiments were carried out on monolayers of T84 cells grown on permeable supports. CCh rapidly induced tyrosine phosphorylation of FAK in T84 cells. This effect was accompanied by phosphorylation of another focal adhesion-associated protein, paxillin, and association of FAK with paxillin. CCh-stimulated FAK phosphorylation was inhibited by a chelator of intracellular Ca2+, BAPTA/AM (20 microM), and was mimicked by thapsigargin (2 microM), which mobilizes intracellular Ca2+ in a receptor-independent fashion. CCh also induced association of FAK with the EGFr and FAK phosphorylation was attenuated by an EGFr inhibitor, tyrphostin AG1478, and an inhibitor of Src family kinases, PP2. The actin cytoskeleton disruptor, cytochalasin D (20 microM), abolished FAK phosphorylation in response to CCh but did not alter CCh-induced EGFr or ERK MAPK activation. In summary, these data demonstrate that agonists of GqPCRs have the ability to induce FAK activation in intestinal epithelial cells. GqPCR-induced FAK activation is mediated by via a pathway involving transactivation of the EGFr and alterations in the actin cytoskeleton.     

Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle

Intact cardiac cells from the adult rat or rabbit ventricle were isolated by enzymatic digestion with a progressive increase of the [free Ca2+] in the solution. These cells were electrically stimulated in the presence of 2.50 mM free Ca2+, and a twitch of maximum amplitude was elicited by the positive inotropic interventions that were found to be optimum. Then the cells were chemically skinned, and the maximum tension induced by a saturating [free Ca2+] was used as a reference to express the tension developed during the twitch of the intact cells. The myoplasmic [free Ca2+] reached during the twitch was inferred from the tension-pCa curve. In mechanically skinned cells of the same animal species, the myoplasmic [free Ca2+] reached during Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR) was inferred by two methods using (a) the tension-pCa curve and (b) a direct calibration of the transients of aequorin bioluminescence. The induction of a maximum Ca2+ release from the SR required a larger Ca2+ preload of the SR and a higher [free Ca2+] trigger in the rabbit than in the rat skinned cells. However, the results obtained with the two methods of inference of the myoplasmic [free Ca2+] suggest that in both animal species a maximum myoplasmic [free Ca2+] of pCa approximately 5.40 was reached during both the optimum Ca2+-induced release of Ca2+ from the SR of the skinned cells and the optimum twitch of the intact cells. This was much lower than the [free Ca2+] necessary for the full activation of the myofilaments (pCa approximately 4.90).     

Host cell invasion mediated by Trypanosoma cruzi surface molecule gp82 is associated with F-actin disassembly and is inhibited by enteroinvasive Escherichia coli

The target cell F-actin disassembly, induced by a Ca2+-signaling Trypanosoma cruzi factor of unknown molecular identity, has been reported to promote parasite invasion. We investigated whether the metacyclic trypomastigote stage-specific surface molecule gp82, a Ca2+-signal-inducing molecule implicated in host cell invasion, displayed the ability to induce actin cytoskeleton disruption, using a recombinant protein (J18) containing the full-length gp82 sequence fused to GST. J18, but not GST, induced F-actin disassembly in HeLa cells, significantly reducing the number as well as the length of stress fibers. The number of cells with typical stress fibers scored approximately 70% in untreated and GST-treated cells, as opposed to approximately 30% in J18-treated samples, which also showed decreased F-actin content. J18, but not GST, inhibited approximately 6-fold the HeLa cell entry of enteroinvasive Escherichia coli (EIEC), which depends on actin cytoskeleton. Not only were fewer cells infected with bacteria in the presence of J18, there were also fewer bacteria per cell. The inhibitory activity of J18 was Ca2+ dependent. In co-infection experiments, preincubation of HeLa cells with EIEC drastically reduced gp82-dependent internalization of T. cruzi metacyclic forms. All these data, plus the finding that gp82-mediated penetration of metacyclic forms was associated with disrupted HeLa cell cytoskeletal architecture, indicate that gp82 promotes parasite invasion by disassembling the cortical actin cytoskeleton.     

Assembly of the enveloped bacteriophage phi 6 in environments which perturb the host cell membranes.

The effects of known membrane-perturbing agents (pH, Na+, Ca2+, and a small lipid-soluble molecule) on the enveloped bacteriophage phi 6 host cell system were investigated at the levels of cellular growth, virus assembly and stability, and the physical and chemical properties of host cell membranes. Spin-label probes of cellular membranes indicate that growth in high levels of Na+ or the small spherical hydrophobic molecule adamantanone results in membranes having increased "fluidity," while growth in high levels of Ca2+ results in slightly greater rigidity of the membranes. In addition, the phospholipid composition of the cellular membranes is dependent on the NaCl concentration in the growth medium. None of these membrane alterations, however, prevent the production of infectious phi 6 virus particles.     

Actin polymerization processes in plant cells

Growing evidence shows that the actin cytoskeleton is a key effector of signal transduction, which controls and maintains the shape of plant cells, as well as playing roles in plant morphogenesis. Recently, several signaling pathways, including those triggered by hormones, Ca(2+), and cAMP, have been reported to be connected to the reorganization of the actin cytoskeleton. The molecular mechanisms involved in such signaling cascades are, however, largely unknown. The Arabidopsis genome sequence is a valuable tool for identifying some of the highly conserved molecules that are involved in such signaling cascades. Recent work has begun to unravel these complex pathways using a panoply of techniques, including genetic analysis, live-cell imaging of intracellular actin dynamics, in vivo localization of factors that are involved in the control of actin dynamics, and the biochemical characterization of how these factors function.     

Disruption of the cortical actin cytoskeleton does not affect store operated Ca2+ channels in human T-cells

Lymphocyte signaling and activation leads to the influx of extracellular Ca(2+) via the activation of Ca(2+) release activated Ca(2+) (CRAC) channels in the plasma membrane. Activation of CRAC channels occurs following emptying of the endoplasmic reticulum intracellular Ca(2+) stores. One model to explain the coupling of store-emptying to CRAC activation is the secretion-like conformational coupling model. This model proposes that store depletion increases junctions between the endoplasmic reticulum and the plasma membrane in a manner that could be regulated by the cortical actin cytoskeleton. Here, we show that stabilization or depolymerization of the actin cytoskeleton failed to affect CRAC activation. We therefore conclude that rearrangement of the actin cytoskeleton is dispensable for store-operated Ca(2+) entry in T-cells.     

Analysis of nuclear degradation during lens cell differentiation

Lens cells demonstrate a terminal differentiation process with loss of their organelles including nuclei. Chromatin disappearance is characterised by the same changes as most apoptotic cells, i.e. condensation of chromatin and cleavage into high molecular weight fragments and oligonucleosomes. The endo-deoxyribonucleases (bicationic (Ca2+, Mg2+), mono-cationic (Ca2+ or Mg2+) and acidic non-cationic dependent nucleases) are present in lens fibre cells. Our results suggest that the acidic non-cationic nuclease (DNase II) plays a major role in chromatin cleavage. This nuclease, known to be lysosomal, is found in lens fibre nuclei and only an antibody directed against DNase II inhibits the acidic DNA cleavage of lens fibre nuclei. In addition, there must be another DNase implicated in the process which is not DNase I but appears to be a Ca2+, Mg2+ dependent molecule. Regulation of these DNase activities may be accomplished by the effect of post-translational modifications, acidic pH, mitochondrial release molecules, growth factors or oncogenes. Finally, fibre cells lose organelles without cytoplasmic elimination. The survival of these differentiated cells might be due to the action of survival factors such as FGF 1.     

L-Cysteine Desulfhydrase 1 modulates the generation of the signaling molecule sulfide in plant cytosol

Consistent with data in animal systems, experimental evidence highlights sulfide as a signaling molecule of equal importance to NO and H₂O₂ in plant systems. In mammals, two cytosolic enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), have been shown to be responsible for the endogenous production of sulfide. L-cysteine desulfhydrase 1 (DES1) has been recently established as the only enzyme that is involved in the generation of hydrogen sulfide in plant cytosol. Although plants have an available source of sulfide within chloroplasts, the basic stromal pH prevents sulfide release into the cytosol. Therefore, DES1 is essential for the production of sulfide for signaling purposes.     

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.