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.     

Multiple cyclic nucleotide‐gated channels coordinate calcium oscillations and polar growth of root hairs

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca²⁺‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca²⁺ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca²⁺ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca²⁺ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca²⁺‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.     

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase

 Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination. Received: 28 September 1999 / Accepted: 27 December 1999     

Annexin 1 regulates the H2O2‐induced calcium signature in Arabidopsis thaliana roots

Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a second messenger, with activation of plasma membrane Ca²⁺‐permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca²⁺‐permeable Stelar K⁺ Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide. We show here that the ROS‐regulated Ca²⁺ transport protein Annexin 1 in Arabidopsis thaliana (AtANN1) is involved in regulating the root epidermal [Ca²⁺]_cyt response to stress levels of extracellular hydrogen peroxide. Peroxide‐stimulated [Ca²⁺]_cyt elevation (determined using aequorin luminometry) was aberrant in roots and root epidermal protoplasts of the Atann1 knockout mutant. Similarly, peroxide‐stimulated net Ca²⁺ influx and K⁺ efflux were aberrant in Atann1 root mature epidermis, determined using extracellular vibrating ion‐selective microelectrodes. Peroxide induction of GSTU1 (Glutathione‐S‐Transferase1 Tau 1), which is known to be [Ca²⁺]_cyt‐dependent was impaired in mutant roots, consistent with a lesion in signalling. Expression of AtANN1 in roots was suppressed by peroxide, consistent with the need to restrict further Ca²⁺ influx. Differential regulation of annexin expression was evident, with AtANN2 down‐regulation but up‐regulation of AtANN3 and AtANN4. Overall the results point to involvement of AtANN1 in shaping the root peroxide‐induced [Ca²⁺]_cyt signature and downstream signalling.     

Arabidopsis PCaP2 modulates the phosphatidylinositol 4,5‐bisphosphate signal on the plasma membrane and attenuates root hair elongation

Phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P₂] serves as a subcellular signal on the plasma membrane, mediating various cell‐polarized phenomena including polar cell growth. Here, we investigated the involvement of Arabidopsis thaliana PCaP2, a plant‐unique plasma membrane protein with phosphoinositide‐binding activity, in PtdIns(4,5)P₂ signaling for root hair tip growth. The long‐root‐hair phenotype of the pcap2 knockdown mutant was found to stem from its higher average root hair elongation rate compared with the wild type and to counteract the low average rate caused by a defect in the PtdIns(4,5)P₂‐producing enzyme gene PIP5K3. On the plasma membrane of elongating root hairs, the PCaP2 promoter‐driven PCaP2–green fluorescent protein (GFP), which complemented the pcap2 mutant phenotype, overlapped with the PtdIns(4,5)P₂ marker 2xCHERRY‐2xPH^PLC in the subapical region, but not at the apex, suggesting that PCaP2 attenuates root hair elongation via PtdIns(4,5)P₂ signaling on the subapical plasma membrane. Consistent with this, a GFP fusion with the PCaP2 phosphoinositide‐binding domain PCaP2^N23, root hair‐specific overexpression of which caused a low average root hair elongation rate, localized more intense to the subapical plasma membrane than to the apical plasma membrane similar to PCaP2–GFP. Inducibly overexpressed PCaP2–GFP, but not its derivative lacking the PCaP2^N23 domain, replaced 2xCHERRY‐2xPH^PLC on the plasma membrane in root meristematic epidermal cells, and suppressed FM4‐64 internalization in elongating root hairs. Moreover, inducibly overexpressed PCaP2 arrested an endocytic process of PIN2–GFP recycling. Based on these results, we conclude that PCaP2 functions as a negative modulator of PtdIns(4,5)P₂ signaling on the subapical plasma membrane probably through competitive binding to PtdIns(4,5)P₂ and attenuates root hair elongation.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

α1‐adrenergic stimulation mediates Ca2+‐dependent inositol phosphate formation through the α1B‐like adrenoceptor subtype in adult rat cardiac myocytes

We studied the effects of increased Ca²⁺ influx on α₁‐adrenoceptor‐stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific α₁‐adrenoceptor subtype. [³H]InsP responses to adrenaline were dependent on extracellular Ca²⁺ concentration, from 0.1 μM to 2 mM, and were completely blocked by Ca²⁺ removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca²⁺ concentrations higher than 1 μM had no effect on adrenaline‐stimulated [³H]InsP formation. Taken together these results suggest that [³H]InsP formation induced by α₁‐adrenergic stimulation is in part mediated by increased Ca²⁺ influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [³H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the α_1B‐adrenoceptor alkylating agent, CEC, [³H]InsP formation remained unaffected by increased Ca²⁺ concentrations, a pattern similar to that observed when intracellular Ca²⁺ was chelated with BAPTA. In contrast, addition of the α_1A‐subtype antagonist, 5′‐methyl urapidil, did not affect the Ca²⁺ dependence of [³H]InsP formation. Neither nifedipine, a voltage‐dependent Ca²⁺ channel blocker nor the inorganic Ca²⁺ channel blockers, Ni²⁺ and Co²⁺, had any effect on adrenaline stimulated [³H]InsP, at concentrations that inhibit Ca²⁺ channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein‐mediated response, α₁‐adrenergic‐stimulated [³H]InsP formation is activated by increased Ca²⁺ influx mediated by the α_1B‐subtype. J. Cell. Biochem. 84: 201–210, 2002. © 2001 Wiley‐Liss, Inc.     

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.     

The Arabidopsis calmodulin‐like protein,CML39, functions during early seedling establishment

During Ca²⁺ signal transduction, Ca²⁺‐binding proteins known as Ca²⁺ sensors function to decode stimulus‐specific Ca²⁺ signals into downstream responses. Plants possess extended families of unique Ca²⁺ sensors termed calmodulin‐like proteins (CMLs) whose cellular roles are not well understood. CML39 encodes a predicted Ca²⁺ sensor whose expression is strongly increased in response to diverse external stimuli. In the present study, we explored the biochemical properties of recombinant CML39, and used a reverse genetics approach to investigate its physiological role. Our data indicate that Ca²⁺ binding by CML39 induces a conformational change in the protein that results in an increase in exposed‐surface hydrophobicity, a property that is consistent with its predicted function as a Ca²⁺ sensor. Loss‐of‐function cml39 mutants resemble wild‐type plants under normal growth conditions but exhibit persistent arrest at the seedling stage if grown in the absence of sucrose or other metabolizable carbon sources. Under short‐day conditions, cml39 mutants display increased sucrose‐induced hypocotyl elongation. When grown in the dark, cml39 mutants show impaired hypocotyl elongation in the absence of sucrose. Promoter–reporter data indicate that CML39 expression is prominent in the apical hook in dark‐grown seedlings. Collectively, our data suggest that CML39 functions in Arabidopsis as a Ca²⁺ sensor that plays an important role in the transduction of light signals that promote seedling establishment.     

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.