Root Hair Tip Research Articles

NaRALF, a peptide signal essential for the regulation of root hair tip apoplastic pH in Nicotiana attenuata, is required for root hair development and plant growth in native soils

Rapid alkalinization factor (RALF) is a 49-amino-acid peptide that rapidly alkalinizes cultivated tobacco cell cultures. In the native tobacco Nicotiana attenuata, NaRALF occurs as a single-copy gene and is highly expressed in roots and petioles. Silencing the NaRALF transcript by transforming N. attenuata with an inverted-repeat construct generated plants (irRALF) with normal wild-type (WT) above-ground parts, but with roots that grew longer and produced trichoblasts that developed into abnormal root hairs. Most trichoblasts produced a localized 'bulge' without commencing root hair tip growth; fewer trichoblasts grew, but were only 10% as long as those of WT plants. The root hair phenotype was associated with slowed apoplastic pH oscillations, increased pH at the tips of trichoblasts and decreased accumulation of reactive oxygen species in the root hair initiation zone. The root hair growth phenotype was partially restored when irRALF lines were grown in a low-pH-buffered medium, and reproduced in WT plants grown in a high-pH-buffered medium. When irRALF plants were grown in pH 5.6, 6.7 and 8.1 soils together with WT plants in glasshouse experiments, they were out-competed by WT plants in basic, but not acidic, soils. When WT and irRALF lines were planted into the basic soils of the native habitat of N. attenuata in the Great Basin Desert, irRALF plants had smaller leaves, shorter stalks, and produced fewer flowers and seed capsules than did WT plants. We conclude that NaRALF is required for regulating root hair extracellular pH, the transition from root hair initiation to tip growth and plant growth in basic soils.

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

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 Ca(2+) 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.

ENOD40 Gene Expression and Cytokinin Responses in the Nonnodulating, Nonmycorrhizal (Nod−Myc−) Mutant, Masym3, of Melilotus alba Desr.

Several nonnodulating, nonmycorrhizal (Nod−Myc−) mutants of Melilotus alba Desr. (white sweetclover) have been described. However, the details of their responses to Sinorhizobium meliloti have not been fully elucidated. We investigated rhizobial entry and colonization using Confocal Scanning Laser Microscopy on the Masym1–5 mutants and isolated an early nodulin (ENOD40) gene from wild-type M. alba. We focused on Masym3, the least responsive of the mutants to S. meliloti and VA-fungi, to determine its response to cytokinin. Cytokinin appears to be a downstream signal in the nodule developmental pathway based not only on our previous observations whereby Nod−Myc− alfalfa roots treated with cytokinin accumulated several ENOD gene transcripts, but also on recent reports showing the importance of cytokinin receptors for nodulation. Here we show that applying 10−6 M 6-benzylaminopurine to uninoculated Masym3 roots elicited ENOD40 transcript accumulation. In addition, Masym3 root hairs inoculated with either wild-type S. meliloti or Nod− S. meliloti expressing the trans-zeatin synthase gene of Agrobacterium tumefaciens exhibited tip swelling, suggesting that cytokinin mediated this response. However, Masym3 root hair tips swelled following inoculation with Nod− S. meliloti or after mock-inoculation, a response resembling the phenotype of root hairs, after handling, of the Medicago truncatula mutant, dmi2. Mtdmi2 is Nod−Myc− due to a defect in a gene encoding a Nodule Receptor Kinase (NORK). Like Mtdmi2, the root hair swelling response appears in part to be mediated by touch because Masym3 root hairs not contacted by either bacteria or drops of water or buffer remain elongated and do not exhibit tip swelling.

Nuclear dynamics during the simultaneous and sustained tip growth of multiple root hairs arising from a single root epidermal cell

Nuclear dynamics in root hairs, which depends upon the actin cytoskeleton, appears to be an important factor in root-hair tip growth. Previous evidence suggests that there is an absolute requirement for the nucleus to be a fixed distance from the growing root-hair tip for tip growth to proceed. To test this hypothesis, nuclear dynamics were examined in root-hair cells bearing multiple root hairs. The majority of root-hair cells of transgenic plants overexpressing the ROP2 GTPase (ROP2 OX) bear multiple root hairs. Simultaneous and sustained fast tip growth occurred in multiple root hairs of ROP2 OX, with the continual presence of tip-localized cytoplasm in these growing hairs. Nuclear dynamics were imaged in ROP2 OX by co-expressing a transgene encoding a nuclear localization signal (NLS)-green fluorescent protein (GFP) fusion protein. The nucleus was in continual proximity to one of the growing root-hair tips, whilst the other tip elongated at a similar rate but in the absence of the nucleus from the shank of that root hair. To test whether this phenomenon was an artefact of ROP2 overexpression, nuclear dynamics were examined in wild-type and NLS-GFP transgenic plants. Multiple root hairs on the same cell underwent simultaneous and sustained fast tip growth, with the nucleus lying deep within the shank of only one of these hairs. The nucleus was also moved into the root-hair tip during the severe root-hair tip branching which is characteristic of ROP2 OX transgenic plants. These results suggest that fast tip growth can proceed in some multiple root hairs at extreme distances from the nucleus.

A transient decrease in reactive oxygen species in roots leads to root hair deformation in the legume–rhizobia symbiosis

* A possible role for reactive oxygen species (ROS) in root hair deformation in response to Nod factor (NF) was investigated using Medicago truncatula nodulation mutants, and an inhibitor and precursors of ROS. * In wild-type roots, ROS efflux transiently decreased approximately 1 h after NF treatment. Transcript accumulation of two NADPH oxidase homologs, respiratory burst oxidase homolog 2 (MtRBOH2) and MtRBOH3, also transiently decreased at 1 h. However, in the nonnodulating mutant Nod factor perception (nfp), transcript accumulation did not change. * Exogenous application of ROS prevented root hair swelling and branching induced by NF. When accumulation of ROS was prevented by diphenylene iodonium (DPI), NF did not induce root hair branching. Root treatment with DPI alone reduced ROS efflux and induced root hair tip swelling. Transient treatment of roots with DPI mimicked NF treatment and resulted in root hair branching in the absence of NF. A transient DPI treatment did not induce root hair branching in the nonlegumes Arabidopsis thaliana and tomato (Lycopersicon esculentum). * The results suggest a role for the transient reduction of ROS accumulation in governing NF-induced root hair deformation in legumes.

Extracellular ATP in Plants. Visualization, Localization, and Analysis of Physiological Significance in Growth and Signaling

Extracellular ATP (eATP) in animals is well documented and known to play an important role in cellular signaling (e.g. at the nerve synapse). The existence of eATP has been postulated in plants; however, there is no definitive experimental evidence for its presence or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it may play in plant growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel reporter; i.e. fusing a cellulose-binding domain peptide to the ATP-requiring enzyme luciferase. Application of this reporter to plant roots allowed visualization of eATP in the presence of the substrate luciferin. Luciferase activity could be detected in the interstitial spaces between plant epidermal cells and predominantly at the regions of actively growing cells. The levels of eATP were closely correlated with regions of active growth and cell expansion. Pharmacological compounds known to alter cytoplasmic calcium levels revealed that ATP release is a calcium-dependent process and may occur through vesicular fusion, an important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is not only essential for root hair growth, but also dependent on the cytoplasmic calcium levels. Whereas application of exogenous ATP and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and nonhydrolyzable ATP (betagammameATP). However, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased ROS activity, suggesting that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.

Distribution of G-actin is related to root hair growth of wheat.

Actin distribution in root hair tips is a controversial topic. Although the relationship between Ca2+ gradient and actin dynamics in plant tip-growth has been a focus of study, there is still little direct evidence on the exact relationship in root hair tip-growth. G-actin was labelled by fluorescein isothiocyanate-DNase I. F-actin was labelled by tetramethylrhodamine isothiocyanate-phalloidin. Actin in root hairs of Triticum aestivum (wheat) was investigated using confocal laser-scanning microscopy. Thick F-actin bundles did not extend into a region of approx. 5-10 microm from the tip of the growing root hairs, although they gave off branches of fine actin filaments in the hair tips. A tip-focused G-actin gradient was shown at the extreme apex of growing root hairs. In full-grown wheat root hairs, the tip-focused G-actin gradient disappeared while the thick F-actin bundles extended into the tips. BAPTA-AM, a Ca2+ disruption agent, also caused the tip-focused G-actin gradient to disappear and the diffuse F-actin bundles to appear in the tips of wheat root hairs. These results suggest that the tip-focused gradient of intracellular G-actin concentration at the extreme apex may be essential for root hair growth, and that preserving the tip-focused gradient needs a high Ca2+ concentration in the root hair tips.

Analysis of the root‐hair morphogenesis transcriptome reveals the molecular identity of six genes with roles in root‐hair development in Arabidopsis

Root-hair morphogenesis is a model for studying the genetic regulation of plant cell development, and double-mutant analyses have revealed a complex genetic network underlying the development of this type of cell. Therefore, to increase knowledge of gene expression in root hairs and to identify new genes involved in root-hair morphogenesis, the transcriptomes of the root-hair differentiation zone of wild-type (WT) plants and a tip-growth defective root-hair mutant, rhd2-1, were compared using Affymetrix ATH1 GeneChips. A set of 606 genes with significantly greater expression in WT plants defines the 'root-hair morphogenesis transcriptome'. Compared with the whole genome, this set is highly enriched in genes known to be involved in root-hair morphogenesis. The additional gene families and functional groups enriched in the root-hair morphogenesis transcriptome are cell wall enzymes, hydroxyproline-rich glycoproteins (extensins) and arabinogalactan proteins, peroxidases, receptor-like kinases and proteins with predicted glycosylphosphatidylinositol (GPI) anchors. To discover new root-hair genes, 159 T-DNA insertion lines identified from the root-hair morphogenesis transcriptome were screened for defects in root-hair morphogenesis. This identified knockout mutations in six genes (RHM1-RHM6) that affected root-hair morphogenesis and that had not previously been identified at the molecular level: At2g03720 (similar to Escherichia coli universal stress protein); At3g54870 (armadillo-repeat containing kinesin-related protein); At4g18640 (leucine-rich repeat receptor-like kinase subfamily VI); At4g26690 (glycerophosphoryl diester phosphodiesterase-like GPI-anchored protein); At5g49270 (COBL9 GPI-anchored protein) and At5g65090 (inositol-1,4,5 triphosphate 5-phosphatase-like protein). The mutants were transcript null, their root-hair phenotypes were characterized and complementation testing with uncloned root-hair genes was performed. The results suggest a role for GPI-anchored proteins and lipid rafts in root-hair tip growth because two of these genes (At4g26690 and At5g49270) encode predicted GPI-anchored proteins likely to be associated with lipid rafts, and several other genes previously shown to be required for root-hair development also encode proteins associated with sterol-rich lipid rafts.

Expression of the legume symbiotic lectin genes psl and gs52 promotes rhizobial colonization of roots in rice

Transgenic rice ( Oryza sativa L. cv. Murasaki) carrying genes encoding pea ( Pisum sativum) lectin (PSL) or wild-soybean ( Glycine soja) lectin-nucleotide phosphohydrolase (GS52) were inoculated with Rhizobium leguminosarum bv. v iciae or Bradyrhizobium japonicum USDA110, respectively, as well as with Rhizobium sp. NGR234, and root colonization was assessed in comparison to comparably inoculated control plants. The data showed that expression of PSL and GS52 significantly promoted rhizobial colonization of root epidermal cells including root hairs in rice. In addition, in the case of R. leguminosarum bv. v iciae and B. japonicum USDA110 colonization of the psl and gs52 transgenic rice plants, respectively, the bacterial cells were found to preferentially home towards and aggregate maximally at the root hair tip regions rather than on the root hair “stalks”. The above data suggest that the lectins PSL and GS52, which participate in rhizobial recognition by root epidermal cells in pea and soybean, respectively, are also able to facilitate rhizobial attachment and colonization of the epidermal cells in rice roots. Moreover, aggregation of R. leguminosarum bv. v iciae and B. japonicum USDA110 cells preferentially at root hair tip regions suggest that similar to legumes, the PSL and GS52 lectins are targeted to the root hair tips in transgenic rice, enabling higher bacterial attachment/colonization at the tip region. Rhizobial colonization at root hair tips in the psl and gs52 rice plants frequently led to the localized dissolution of the cell wall creating perforations at the tip region. It is likely that the presence of lectins, such as PSL and GS52 leads to structural modifications in cell wall organization of the root hair/epidermal cells, making them prone to localized dissolution by the hydrolytic activity of compatible rhizobia to permit invasion of the root cells.

Role of Cellulose Fibrils and Exopolysaccharides of Rhizobium leguminosarum in Attachment to and Infection of Vicia sativa Root Hairs

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.

Bringing PIPs to root tips

Tiny hairs on Arabidopsis thaliana roots elongate via tip growth, in which new membrane is added specifically at the root hair tip. Vincent et al. (page 801) now show that a phosphatidylinositol transfer protein (PITP) related to yeast Sec14p is critical for this polarized growth, suggesting that PIP2 may be the start of the polarity cascade in this system.The authors show that Arabidopsis has a large family of these PITPs. One such PITP is AtSfh1p, which, along with its downstream product PIP2, localized to the tip plasma membrane and on post-Golgi vesicles that accumulate at the hair tips.

A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p nullizygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

Dissection of Arabidopsis ADP-RIBOSYLATION FACTOR 1 Function in Epidermal Cell Polarity

Vesicle trafficking is essential for the generation of asymmetries, which are central to multicellular development. Core components of the vesicle transport machinery, such as ADP-ribosylation factor (ARF) GTPases, have been studied primarily at the single-cell level. Here, we analyze developmental functions of the ARF1 subclass of the Arabidopsis thaliana multigene ARF family. Six virtually identical ARF1 genes are ubiquitously expressed, and single loss-of-function mutants in these genes reveal no obvious developmental phenotypes. Fluorescence colocalization studies reveal that ARF1 is localized to the Golgi apparatus and endocytic organelles in both onion (Allium cepa) and Arabidopsis cells. Apical-basal polarity of epidermal cells, reflected by the position of root hair outgrowth, is affected when ARF1 mutants are expressed at early stages of cell differentiation but after they exit mitosis. Genetic interactions during root hair tip growth and localization suggest that the ROP2 protein is a target of ARF1 action, but its localization is slowly affected upon ARF1 manipulation when compared with that of Golgi and endocytic markers. Localization of a second potential target of ARF1 action, PIN2, is also affected with slow kinetics. Although extreme redundancy precludes conventional genetic dissection of ARF1 functions, our approach separates different ARF1 downstream networks involved in local and specific aspects of cell polarity.

Green Fluorescent Protein-mTalin Causes Defects in Actin Organization and Cell Expansion in Arabidopsis and Inhibits Actin Depolymerizing Factor's Actin Depolymerizing Activity in Vitro

Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-mTalin colocalizes with the actin cytoskeleton, its effect on actin dynamics and cell expansion has not been studied in detail. We created Arabidopsis (Arabidopsis thaliana) plants harboring alcohol inducible GFP-mTalin constructs to assess the effect of GFP-mTalin expression in vivo. We focused on the growing root hair as this is a model cell for studying cell expansion and root hair tip growth that requires a highly dynamic and polar actin cytoskeleton. We show that alcohol inducible expression of GFP-mTalin in root hairs causes severe defects in actin organization, resulting in either the termination of growth, cell death, and/or changes in cell shape. Fluorescence recovery after photobleaching experiments demonstrate that the interaction of GFP-mTalin and actin filaments is highly dynamic. To assess how GFP-mTalin affects actin dynamics we performed cosedimentation assays of GFP-mTalin with actin on its own or in the presence of the actin modulating protein, actin depolymerizing factor. We show that that GFP-mTalin does not affect actin polymerization but that it does inhibit the actin depolymerizing activity of actin depolymerizing factor. These observations demonstrate that GFP-mTalin can affect cell expansion, actin organization, and the interaction of actin binding proteins with actin.

The Arabidopsis COW1 gene encodes a phosphatidylinositol transfer protein essential for root hair tip growth

Root hairs are a major site for the uptake of water and nutrients into plants, and they form an increasingly important model system for the study of development in higher plants. We now report on the molecular genetic analysis of the srh1 mutant in Arabidopsis thaliana impaired in root hair tip growth. We show that srh1 is a new allele of cow1 (can of worms1) and we identified the COW1 gene using a positional cloning strategy. The N-terminus of the COW1 protein is 32% identical to an essential phosphatidylinositol transfer protein (PITP), the yeast Sec14 protein (sec14p) while the C-terminus is 34.5% identical to a late nodulin of Lotus japonicus, Nlj16. We show that expression of the COW1 lipid-binding domain complements the growth defect associated with Sec14p dysfunction in yeast. In addition, we show that GFP fused to the COW1 protein specifically accumulates at the site of root hair outgrowth. We conclude that the COW1 protein is a PITP, essential for proper root hair growth.

Colonization of Wheat Root Hairs and Roots by Agrobacteria

Formation of extracellular structures in pure culture and in interaction with wheat root surface was studied by scanning and transmission electron microscopy. The effect of various factors (growth temperature as well as pretreatment of agrobacteria with kalanchoe extract, acetosyringone, and centrifugation) on formation of extracellular structures was tested. The data on Agrobacterium tumefaciens (wild strain C58 and mutants LBA2525 (virB2::lacZ) and LBA288 (without Ti plasmid)) adhesion to wheat root surface and root hairs after pretreatment of agrobacteria with inducer of virulence genes (vir) acetosyringone were obtained. Formation of agrobacterial cell aggregates on wheat root hair tips was demonstrated. The proportion of root hairs with agrobacterial aggregates on the root hair tip insignificantly changed after pretreatment with acetosyringone but considerably increased after treatment of A. tumefaciens C58 and LBA2525 with kalanchoe leaf extract. The most active colonization of root hairs and formation of agrobacterial aggregates on hair root tips was observed at 22 degrees C. The capacity of agrobacteria for adhesion on monocotyledon surface could be changed by pretreatment of bacteria with various surface-active substances. Bacterial cells subjected to centrifugation had a decreased capacity for attachment to both wheat root surface and root hairs. The relationship between the capacity for adhesion and pile production in agrobacteria was considered.

Touchy DMI2 Plants

The roots of certain legumes can respond to the presence of nitrogen-fixing bacteria (rhizobia), by engulfing the microorganisms and forming a symbiotic relation between the plant and the bacteria. Plants carrying the Does Not Make Infections 2 ( DMI2 ) mutations are incapable of forming a symbiotic relationship with rhizobia. Esseling et al. show that three species of plants with DMI2 phenotypes carrying mutations in the genes encoding the nodulation receptor-like kinase (NORK) are hyperresponsive to mechanical stimuli. Even routinely replacing the medium caused growing root hairs to develop a growth-terminating architecture. Application of nodulation factor to the roots of the mutant plants caused changes to cytoarchitecture and root reorientation, and this morphological response was enhanced compared with that in wild-type plants. When mutant plants were inoculated with their symbiotic partner bacteria, the roots curled around the bacteria, forming a simpler curled structure than that formed by a wild-type root. However, the mutant roots could not internalize the bacteria and did not show increased expression of a reporter coupled to a known nodulation-responsive promoter [ Early Nodulin 11 ( ENOD11 )]. The authors suggest that the nodulation pathway bifurcates and that one arm of the pathway controls cytoarchitecture reorganization and the other arm, which includes NORK, governs the responses needed for bacterial internalization and regulation of nodulation-responsive genes, such as ENOD11 . J. J. Esseling, F. G. P. Lhuissier, A. M. C. Emons, A nonsymbiotic root hair tip growth phenotype in NORK -mutated legumes: Implications for nodulation factor-induced signaling and formation of a multifaceted root hair pocket for bacteria. Plant Cell 16 , 933-944 (2004). [Abstract] [Full Text]

New signalling molecules regulating root hair tip growth

Root hairs are tip-growing tubes that emerge from trichoblasts (hair-forming epidermal cells) along the length of the root. Signalling events involved in the formation of root hairs are largely unknown. However, two recent studies have revealed that signalling enzymes such as NADPH oxidase and phospholipase D are crucial for root hair growth and development. Reactive oxygen species (ROS) produced by NADPH oxidase activate calcium ion channels in the apical plasma membrane leading to the tip-focused calcium gradient, an inherent feature of growing root hairs.

A nonsymbiotic root hair tip growth phenotype in NORK-mutated legumes: implications for nodulation factor-induced signaling and formation of a multifaceted root hair pocket for bacteria.

The Medicago truncatula Does not Make Infections (DMI2) mutant is mutated in the nodulation receptor-like kinase, NORK. Here, we report that NORK-mutated legumes of three species show an enhanced touch response to experimental handling, which results in a nonsymbiotic root hair phenotype. When care is taken not to induce this response, DMI2 root hairs respond morphologically like the wild type to nodulation factor (NF). Global NF application results in root hair deformation, and NF spot application induces root hair reorientation or branching, depending on the position of application. In the presence of Sinorhizobium meliloti, DMI2 root hairs make two-dimensional 180 degrees curls but do not entrap bacteria in a three-dimensional pocket because curling stops when the root hair tip touches its own shank. Because DMI2 does not express the promoter of M. truncatula Early Nodulin11 (ENOD11) coupled to beta-glucuronidase upon NF application, we propose a split in NF-induced signaling, with one branch to root hair curling and the other to ENOD11 expression.