The interaction of CaM7 and CNGC14 regulates root hair growth in Arabidopsis

Oscillations in cytosolic free calcium determine the polarity of tip‐growing root hairs. The Ca²⁺ channel cyclic nucleotide gated channel 14 (CNGC14) contributes to the dynamic changes in Ca²⁺ concentration gradient at the root hair tip. However, the mechanisms that regulate CNGC14 are unknown. In this study, we detected a direct interaction between calmodulin 7 (CaM7) and CNGC14 through yeast two‐hybrid and bimolecular fluorescence complementation assays. We demonstrated that the third EF‐hand domain of CaM7 specifically interacts with the cytosolic C‐terminal domain of CNGC14. A two‐electrode voltage clamp assay showed that CaM7 completely inhibits CNGC14‐mediated Ca²⁺ influx, suggesting that CaM7 negatively regulates CNGC14‐mediated calcium signaling. Furthermore, CaM7 overexpressing lines phenocopy the short root hair phenotype of a cngc14 mutant and this phenotype is insensitive to changes in external Ca²⁺ concentrations. We, thus, identified CaM7‐CNGC14 as a novel interacting module that regulates polar growth in root hairs by controlling the tip‐focused Ca²⁺ signal.     

AGD1, a class 1 ARF-GAP, acts in common signaling pathways with phosphoinositide metabolism and the actin cytoskeleton in controlling Arabidopsis root hair polarity

The Arabidopsis thaliana AGD1 gene encodes a class 1 adenosine diphosphate ribosylation factor‐gtpase‐activating protein (ARF‐GAP). Previously, we found that agd1 mutants have root hairs that exhibit wavy growth and have two tips that originate from a single initiation point. To gain new insights into how AGD1 modulates root hair polarity we analyzed double mutants of agd1 and other loci involved in root hair development, and evaluated dynamics of various components of root hair tip growth in agd1 by live cell microscopy. Because AGD1 contains a phosphoinositide (PI) binding pleckstrin homology (PH) domain, we focused on genetic interactions between agd1 and root hair mutants altered in PI metabolism. Rhd4, which is knocked‐out in a gene encoding a phosphatidylinositol‐4‐phosphate (PI‐4P) phosphatase, was epistatic to agd1. In contrast, mutations to PIP5K3 and COW1, which encode a type B phosphatidylinositol‐4‐phosphate 5‐kinase 3 and a phosphatidylinositol transfer protein, respectively, enhanced the root hair defects of agd1. Enhanced root hair defects were also observed in double mutants to AGD1 and ACT2, a root hair‐expressed vegetative actin isoform. Consistent with our double‐mutant studies, targeting of tip growth components involved in PI signaling (PI‐4P), secretion (RABA4b) and actin regulation (ROP2), were altered in agd1 root hairs. Furthermore, tip cytosolic calcium ([Ca²⁺]_cyt) oscillations were disrupted in root hairs of agd1. Taken together, our results indicate that AGD1 links PI signaling to cytoskeletal‐, [Ca²⁺]_cyt?, ROP2‐, and RABA4b‐mediated root hair development.     

Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana

In this study, confocal ratio analysis was used to image the relationship between cytoplasmic free calcium concentration ([Ca²⁺]_c) and the development of root hairs of Arabidopsis thaliana. Although a localized change in [Ca²⁺]_c that preceded or predicted the site of root hair initiation could not be detected, once initiated the majority of emerging root hairs showed an elevated [Ca²⁺]_c (>1 μM) in their apical cytoplasm, compared with 100– 200 nM in the rest of the cell. These emerging root hairs then moved into a 3–5 h phase of sustained elongation during which they showed variable growth rates. Root hairs that were rapidly elongating exhibited a highly localized, elevated [Ca²⁺]_c at the tip. Non-growing root hairs did not exhibit the [Ca²⁺]_c gradient. The rhd-2 mutant, which is defective in sustained root hair growth, showed an altered [Ca²⁺]_c distribution compared with wild-type. These results implicate [Ca²⁺]_c in regulating the tip growth process. Treatment of elongating wild-type root hairs with the Ca²⁺ channel blocker verapamil (50 μM) caused dissipation of the elevated [Ca²⁺]_c at the tip and cessation of growth, suggesting a requirement for Ca²⁺ channel activity at the root hair tip to maintain growth. Manganese treatment also preferentially quenched Indo-1 fluorescence in the apical cytoplasm of the root hair. As manganese is thought to enter cells through Ca²⁺-permeable channels, this result also suggests increased Ca²⁺ channel activity at the tip of the growing hair. Taken together, these data suggest that although Ca²⁺ does not trigger the initiation of root hairs, Ca²⁺ influx at the tip of the root hair leads to an elevated [Ca²⁺]_c that may be required to sustain root hair elongation.     

Nitrogen source interacts with ROP signalling in root hair tip‐growth

Root hairs elongate in a highly polarized manner known as tip growth. Overexpression of constitutively active Rho of Plant (ROP)/RAC GTPases mutants induces swelling of root hairs. Here, we demonstrate that Atrop11^CA‐induced swelling of root hairs depends on the composition of the growth medium. Depletion of ammonium allowed normal root hair elongation in Atrop11^CA plants, induced the development of longer root hairs in wild‐type plants and suppressed the effect of Atrop11^CA expression on actin organization and reactive oxygen species distribution, whereas membrane localization of the protein was not affected. Ammonium at concentrations higher than 1 mM and the presence of nitrate were required for induction of swelling. Oscillations in wall and cytoplasmic pH are known to accompany tip growth in root hairs, and buffering of the growth medium decreased Atrop11^CA‐induced swelling. Fluorescence ratio imaging experiments revealed that in wild‐type root hairs, the addition of NH₄NO₃ to the growth medium induced an increase in the amplitude of extracellular and intracellular pH oscillations and an overall decrease in cytoplasmic pH at the cell apex. Based on these results, we suggest a model in which ROP GTPases and nitrogen‐dependent pH oscillations function in parallel pathways, creating a positive feedback loop during root hair growth.     

Comparative analysis of the reactive oxygen species‐producing enzymatic activity of Arabidopsis NADPH oxidases

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, ‐D, ‐F, ‐H and ‐J) are synergistically activated by Ca²⁺‐binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS‐producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, ‐H and ‐J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or ‐J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.     

Nod factors modulate the concentration of cytosolic free calcium differently in growing and non-growing root hairs of Medicago sativa L.

Using Ca²⁺-selective microelectrodes, the concentration of free calcium ([Ca²⁺]) in the cytosol has been measured in root hair cells of Medicago sativa L. in the presence of nodulation (Nod) factors. Growing root hairs of M. sativa displayed a steep apical [Ca²⁺] gradient, i.e. 604–967 nM in the tip compared with 95–235 nM in the basal region. When tested within the first 5 to 10 μm of the tip, addition of NodRm-IV(C16:2,S) decreased the cytosolic [Ca²⁺], whereas an increase was observed when tested behind the tip. Overall, this led to a partial dissipation of the [Ca²⁺] gradient. The Ca²⁺ response was specific: it was equally well observed in the presence of NodRm-IV(Ac,C16:2,S), reduced with NodRm-IV(C16:0,S), but not with chitotetraose, the nonactive glucosamine backbone. In contrast to growing root hairs, non-growing root hairs without a tip-to-base cytosolic [Ca²⁺] gradient responded to NodRm-IV(C16:2,S) with an increase in cytosolic [Ca²⁺] at the tip as well as at the root hair base. We suggest that the response to Nod factors depends on the stage of development of the root hairs, and that changes in cytosolic [Ca²⁺] may play different roles in Nod-factor signaling: changes of cytosolic [Ca²⁺] in the apical part of the root hair may be related to root hair deformation, while the increase in [Ca²⁺] behind the tip may be essential for the amplification of the Nod signal, for its propagation and transduction to trigger downstream events. Received: 5 January 1999 / Accepted: 14 April 1999     

The Ca2+‐binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer

Plasma membrane‐associated Ca²⁺‐binding protein–2 (PCaP2) of Arabidopsis thaliana is a novel‐type protein that binds to the Ca²⁺/calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca²⁺. Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N–terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca²⁺/calmodulin complex, and that the residual domain of PCaP2 binds to free Ca²⁺. In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild‐type. To examine the function of each domain in root hair cells, we over‐expressed PCaP2 and its variants using the root hair cell‐specific EXPANSIN A7 promoter. Transgenic lines over‐expressing PCaP2, PCaP2^G2A (second glycine substituted by alanine) and ?23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over‐expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP‐tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca²⁺ and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.     

Role of calcium and other ions in directing root hair tip growth in Limnobium stoloniferum

The magnitude and spatial localization of Ca²⁺, K⁺ and H⁺ fluxes in growing and non-growing Limnobium stoloniferum root hairs was determined using non-invasive, ion-selective vibrating microelectrodes. Both the spatial pattern and magnitude of the ionic flux was dependent on the particular ion in question. Both H⁺ and Ca²⁺ influx was localized almost exclusively to the tips of growing root hairs, suggesting that these fluxes may be involved in directing growth. Influx of K⁺ showed no distinct localization and uptake appeared uniform along the length of the root hair. Competitive inhibition of Ca²⁺ influx using a range of Mg⁺ concentrations indicated that the magnitude of the Ca²⁺ flux entering the root hair tip did not determine growth rate; however, the presence of Ca²⁺ on the external face of the membrane was implicit for root hair integrity. Aluminum proved to be a potent inhibitor of root hair growth. At an exogenous Al concentration of 20 M a complete blockage of Ca²⁺ influx into root hair tips was observed, suggesting that Al blockage of Ca²⁺ influx could be involved in Al toxicity. However, at a lower Al concentration (2 M), Ca²⁺ fluxes were unaffected while inhibition of growth was still observed along with a distinct swelling of the root hair tip. The swelling at the root hair tips was identical in appearance to that seen in the presence of microtubule inhibitors, suggesting that Al could influence a number of different sites at the plasma-membrane surface and within the cell. The possible role(s) of Ca²⁺ and H⁺ fluxes in directing tip growth are discussed.     

Root hair growth in Arabidopsis thaliana is directed by calcium and an endogenous polarity

Tip growth of plant cells has been suggested to be regulated by a tip-focused gradient in cytosolic calcium concentration ([Ca²⁺]_c). However, whether this gradient orients apical growth or follows the driving force for this process remains unknown. Using localized photoactivation of the caged calcium ionophore Br-A23187 we have been able to artificially generate an asymmetrical calcium influx across the root hair tip. This led to a change in the direction of tip growth towards the high point of the new [Ca²⁺]_c gradient. Such reorientation of growth was transient and there was a return to the original direction within 15 min. Root hairs forced to change the direction of their growth by placing a mechanical obstacle in their path stopped, reoriented growth to the side, and grew past the mechanical blockage. However, as soon as the growing tip had cleared the obstacle, growth returned to the original direction. Confocal ratio imaging revealed that a tip-focused [Ca²⁺]_c gradient was always centered at the site of active growth. When the root hair changed direction the gradient also reoriented, and when growth returned to the original direction, so did the [Ca²⁺]_c gradient. This normal direction of apical growth of Arabidopsis thaliana (L.) Heynh. root hairs was found to be at a fixed angle from the root of 85 ± 6.7 degrees. In contrast, Tradescantia virginiana (L.) pollen tubes that were induced to reorient by touch or localized activation of the caged ionophore, did not return to the original growth direction, but continued to elongate in their new orientation. These results suggest that the tip-focused [Ca²⁺]_c gradient is an important factor in localizing growth of the elongating root hair and pollen tube to the apex. However, it is not the primary determinant of the direction of elongation in root hairs, suggesting that other information from the root is acting to continuously reset the growth direction away from the root surface. Received: 22 April 1997 / Accepted: 14 May 1997     

Arabidopsis PROTEIN S‐ACYL TRANSFERASE4 mediates root hair growth

Polar growth of root hairs is critical for plant survival and requires fine‐tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S‐acyl transferases (PATs), mediating the S‐acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site‐directed mutagenesis and genetic analysis with related root‐hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S‐acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild‐type but not an enzyme‐inactive PAT4. Membrane‐associated ROP2 was significantly reduced in pat4, similar to S‐acylation‐deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4‐mediated S‐acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.     

Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana

Root hairs are specialized cells that are important for nutrient uptake. It is well established that nutrients such as phosphate have a great influence on root hair development in many plant species. Here we investigated the role of nitrate on root hair development at a physiological and molecular level. We showed that nitrate increases root hair density in Arabidopsis thaliana. We found that two different root hair defective mutants have significantly less nitrate than wild‐type plants, suggesting that in A. thaliana root hairs have an important role in the capacity to acquire nitrate. Nitrate reductase‐null mutants exhibited nitrate‐dependent root hair phenotypes comparable with wild‐type plants, indicating that nitrate is the signal that leads to increased formation of root hairs. We examined the role of two key regulators of root hair cell fate, CPC and WER, in response to nitrate treatments. Phenotypic analyses of these mutants showed that CPC is essential for nitrate‐induced responses of root hair development. Moreover, we showed that NRT1.1 and TGA1/TGA4 are required for pathways that induce root hair development by suppression of longitudinal elongation of trichoblast cells in response to nitrate treatments. Our results prompted a model where nitrate signaling via TGA1/TGA4 directly regulates the CPC root hair cell fate specification gene to increase formation of root hairs in A. thaliana.     

Effect of aluminum on cytoplasmic Ca2+ homeostasis in root hairs of Arabidopsis thaliana (L.)

Aluminum inhibition of root growth is a major world agricultural problem where the cause of toxicity has been linked to changes in cellular calcium homeostasis. Therefore, the effect of aluminum ions (Al) on changes in cytoplasmic free calcium concentration ([Ca²⁺]_c) was followed in root hairs of wild-type, Al-sensitive and Al-resistant mutants of Arabidopsis thaliana (L.) Heynh. Generally, Al exposure resulted in prolonged elevations in tip-localized [Ca²⁺]_c in both wild-type and Al-sensitive root hairs. However, these Al-induced increases in [Ca²⁺]_c were not tightly correlated with growth inhibition, occurring up to 15 min after Al had induced growth to stop. Also, in 32% of root hairs examined growth stopped without a detectable change in [Ca²⁺]_c. In contrast, Al-resistant mutants showed little growth inhibition in response to AlCl₃ exposure and in no case was a change in [Ca²⁺]_c observed. Of the other externally applied stresses tested (oxidative and mechanical stress), both were found to inhibit root hair growth, but only oxidative stress (H₂O₂, 10 μM) caused a prolonged rise in [Ca²⁺]_c similar to that induced by Al. Again this increase occurred after growth had been inhibited. The lack of a tight correlation between Al exposure, growth inhibition and altered [Ca²⁺]_c dynamics suggests that although exposure of root hairs to toxic levels of Al causes an alteration in cellular Ca²⁺ homeostasis, this may not be a required event for Al toxicity. The elevation in [Ca²⁺]_c induced by Al also strongly suggests that the phytotoxic action of Al in root hairs is not through blockage of Ca²⁺-permeable channels required for Ca²⁺ influx into the cytoplasm. Received: 24 October 1997 / Accepted: 6 March 1998     

Time Course of Cell Biological Events Evoked in Legume Root Hairs by Rhizobium Nod Factors: State of the Art

In many common legumes, when host-specific nodule bacteria meettheir legume root they attach to it and enter through root hairs.The bacteria can intrude these cells because they instigatein the hairs the formation of an inward growing tube, the infectionthread, which consists of wall material. Prior to infectionthread formation, the bacteria exploit the cell machinery forwall deposition by inducing the hairs to form a curl, in whichthe dividing bacteria become entrapped. In most species, Nodfactor alone (a lipochito-oligosaccharide excreted by bacteria)induces root hair deformation, though without curling, thusmost aspects of the initial effects of Nod factor can be elucidatedby studying root hair deformation. In this review we discussthe cellular events that host-specific Nod factors induce intheir host legume root hairs. The first event, detectable onlya few seconds after Nod factor application, is a Ca²⁺influxat the root hair tip, followed by a transient depolarizationof the plasma membrane potential, causing an increase in cytosolic[Ca²⁺] at the root hair tip. Also within minutes, Nod factorschange the cell organization by acting on the actin cytoskeleton,enhancing tip cell wall deposition so that root hairs becomelonger than normal for their species. Since the remodellingof the actin cytoskeleton precedes the second calcium event,Ca²⁺spiking, which is observed in the perinuclear area, we proposethat the initial cytoskeleton events taking place at the hairtip are related to Ca²⁺influx in the hair tip and that Ca²⁺spikingserves later events involving gene expression. Copyright 2001Annals of Botany Company Review, Nod factor, tip growth, root hair, Rhizobium, legume, cytoskeleton, calcium, symbiosis     

Morphological synergism in root hair length,density, initiation and geometry for phosphorus acquisition in Arabidopsis thaliana: A modeling approach

Low phosphorus availability regulates root hair growth in Arabidopsis by (1) increasing root hair length, (2) increasing root hair density, (3) decreasing the distance between the root tip and the point at which root hairs begin to emerge, and (4) increasing the number of epidermal cell files that bear hairs (trichoblasts). The coordinated regulation of these traits by phosphorus availability prompted us to speculate that they are synergistic, that is, that they have greater adaptive value in combination than they do in isolation. In this study, we explored this concept using a geometric model to evaluate the effect of varying root hair length (short, medium, and long), density (0, 24, 48, 72, 96, and 120 root hairs per mm of root length), tip to first root hair distance (0.5, 1, 2, and 4 mm), and number of trichoblast files (8 vs. 12) on phosphorus acquisition efficiency (PAE) in Arabidopsis. SimRoot, a dynamic three-dimensional geometric model of root growth and architecture, was used to simulate the growth of Arabidopsis roots with contrasting root hair parameters at three values of phosphorus diffusion coefficient (D _e=1×10^–7, 1×10^–8, and 1×10^–9 cm² s^–1) over time (20, 40, and 60 h). Depzone, a program that dynamically models nutrient diffusion to roots, was employed to estimate PAE and competition among root hairs. As D _e decreased from 1×10^–7 to 1×10^–9 cm² s^–1, roots with longer root hairs and higher root hair densities had greater PAE than those with shorter and less dense root hairs. At D _e=1×10^–9 cm² s^–1, the PAE of root hairs at any given density was in the order of long hairs > medium length hairs > short hairs, and the maximum PAE occurred at density = 96 hairs mm^–1 for both long and medium length hairs. This was due to greater competition among root hairs when they were short and dense. Competition over time decreased differences in PAE due to density, but the effect of length was maintained, as there was less competition among long hairs than short hairs. At high D _e(1×10^–7 cm² s^–1), competition among root hairs was greatest among long hairs and lowest among short hairs, and competition increased with increasing root hair densities. This led to a decrease in PAE as root hair length and density increased. PAE was also affected by the tip to first root hair distance. At low D _e values, decreasing tip to first root hair distance increased PAE of long hairs more than that of short hairs, whereas at high D _e values, decreasing tip to first root hair distance increased PAE of root hairs at low density but decreased PAE of long hairs at very high density. Our models confirmed the benefits of increasing root hair density by increasing the number of trichoblast files rather than decreasing the trichoblast length. The combined effects of all four root hair traits on phosphorus acquisition was 371% greater than their additive effects, demonstrating substantial morphological synergy. In conclusion, our data support the hypothesis that the responses of root hairs to low phosphorus availability are synergistic, which may account for their coordinated regulation.     

Calcium influx at the tip of growing root-hair cells of Arabidopsis thaliana

The role of extracellular Ca²⁺ in root-hair tip growth has been investigated in Arabidopsis thaliana (L.) Heynh. Root-hair length was found to be dependent on the concentration of Ca²⁺ in the growth medium, with maximum length achieved at [Ca²⁺] of 0.3–3.0 mM. Using a non-intrusive calcium-specific vibrating microelectrode, an extracellular Ca²⁺ gradient was detected at the tips of individual growing root-hair cells. The direction of the gradient indicated a net influx of Ca²⁺ into root-hair cells. No gradient was detected near the sides of the root hairs or at the tips of non-growing root hairs. When root hairs were exposed to the Ca²⁺-channel blocker nifedipine, tip growth stopped and the extracellular Ca²⁺ gradient was abolished. These results indicate that Ca²⁺ influx through plasma-membrane Ca²⁺ channels is required for normal root-hair tip growth.Abbreviation APW artificial pond water We thank L.F. Jaffe, W. Kuhtreiber and A. Miller of the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA for their technical assistance and helpful discussions. We also thank Liam Dolan, Martin Steer, and Susan Ford for helpful discussions. This research was supported by National Science Foundation grant PCM-9004568.     

Cell biology and genetics of root hair formation inArabidopsis thaliana

Summary In this review we integrate the information available on the cell biology of root hair formation with recent findings from the analysis of root hair mutants ofArabidopsis thaliana. The mature Arabidopsis root epidermis consists of root-hair-producing cells and non-root-hair-producing cells. Root hair growth begins with a swelling of the outer epidermal wall. It has been postulated that this is due to a pH-mediated localised cell wall loosening. From the bulge a single root hair emerges which grows by tip growth. The root hair tip consists of a vesicle-rich zone and an organelle-rich subapical zone. The vesicles supply new plasma membrane and cell wall material for elongation. The cytoskeleton and its associated regulatory proteins such as profilin and spectrin are proposed to be involved in the targeting of vesicles. Ca²⁺ influxes and gradients are present in hair tips, but their function is still unclear. Mutants have been isolated with lesions in various parts of the root hair developmental pathway from bulge identity and initiation, to control of tip diameter and extent and polarity of elongation.Abbreviations [Ca²⁺]_c cytosolic calcium concentration - MT microtubule - PM plasma membrane - VRZ vesicle-rich zone - WT wild type Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Fungal hypaphorine reduces growth and induces cytosolic calcium increase in root hairs of Eucalyptus globulus

Summary. Root hairs are tubular cells resulting from a tip-localized growth in which calcium ions play a key role. Hypaphorine, an indole alkaloid secreted by the fungus Pisolithus microcarpus during the formation of ectomycorrhizae with the host plant Eucalyptus globulus, inhibits root hair tip growth. Hypaphorine-induced inhibition is linked to a transient depolarization of the plasma membrane and a reorganization of the actin and microtubule cytoskeletons. Here we investigated the activity of hypaphorine on calcium distribution in E. globulus root hairs with the ratiometric fluorochrome calcium indicator Indo-1. In 85% of actively growing root hairs, a significant but modest calcium gradient between the apex and the base was observed due to an elevated cytoplasmic calcium concentration at the apical tip. Following exposure to 1 mM hypaphorine, the apical and basal cytoplasmic Ca²⁺ concentration increased in 70 and 77% of the hairs, respectively, 10 min after treatment. This led to a reduced calcium gradient in 81% of the cells. The hypothetical links between calcium concentration elevation, regulation of actin cytoskeleton dynamics, and root hair growth inhibition in response to hypaphorine treatment are discussed. Correspondence and reprints: UMR 1136 Interactions Arbres–Microorganismes, Faculté des Sciences, Université Nancy I, BP 239, 54506 Vandoeuvre Cedex, France.