Identical accumulation and immobilization of sulfated and nonsulfated Nod factors in host and nonhost root hair cell walls

Nod factors are signaling molecules secreted by Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host. How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs. The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM. Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM. When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium. Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M. truncatula and V. sativa, respectively. Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M. truncatula and V. sativa. The implications for the mode of Nod factor perception are discussed.     

Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs

Root hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.     

Fine structure and immunocytochemistry of monotreme hairs, with emphasis on the inner root sheath and trichohyalin-based cornification during hair evolution

The fine structure of hairs in the most ancient extant mammals, the monotremes, is not known. The present study analyzes the ultrastructure and immunocytochemistry for keratins, trichohyalin, and transglutaminase in monotreme hairs and compares their distribution with that present in hairs of the other mammals. The overall ultrastructure of the hair and the distribution of keratins is similar to that of marsupial and placental hairs. Acidic and basic keratins mostly localize in the outer root sheath. The inner root sheath (IRS) comprises 4-8 cell layers in most hairs and forms a tile-like sheath around the hair shaft. No cytological distinction between the Henle and Huxley layers is seen as cells become cornified about at the same time. Externally to the last cornified IRS cells (homologous to the Henle layer), the companion layer contains numerous bundles of keratin. Occasionally, some granules in the companion layer show immunoreactivity for the trichohyalin antibody. This further suggests that the IRS in monotremes is ill-defined, as the companion layer of placental hairs studied so far does not express trichohyalin. A cross-reactivity with an antibody against sheep trichohyalin is present in the IRS of monotremes, suggesting conserved epitopes across mammalian trichohyalin. Trichohyalin granules in the IRS consist of a framework of immunolabeled coarse filaments of 10-12 nm. The latter assume a parallel orientation and lose the immunoreactivity in fully cornified cells. Transglutaminase immunolabeling is diffuse among trichohyalin granules and among the parallel 10-12 nm filaments of maturing inner root cells. Transglutaminase is present where its substrate, trichohyalin, is modified as matrix protein. Cornification of IRS is different from that of hair fiber cuticle and from that of the cornified layer of the epidermis above the follicle. The different consistency among cuticle, IRS, and corneous layer of the epidermis determines separation between hair fiber, IRS, and epidermis. This allows the hair to exit on the epidermal surface after shedding from the IRS and epidermis. Based on comparative studies of reptilian and mammalian skin, a speculative hypothesis on the evolution of the IRS and hairs from the skin of synapsid reptiles is presented.     

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.     

The root epidermis of<Emphasis Type="Italic"> Echium plantagineum</Emphasis> L.: a novel type of pattern based on the distribution of short and long root hairs

The great majority of angiosperm species form a group in which either every cell in the root epidermis produces a root hair, or the cells that produce these hairs are randomly distributed. We describe, for the first time, pattern in the root epidermal cells of a species within this group. The seedling root of Echium plantagineum L. (Boraginaceae) has an epidermis in which almost every cell produces a root hair, but these are of two types, short hairs (up to 200 micro m) and long hairs (>200 micro m), which are in separate cell files, with the cells bearing long hairs usually separated by one or two files of cells bearing short hairs; the epidermal cells with the long root hairs are longer than the epidermal cells with the short root hairs. The long root hairs are initiated and develop earlier than the short root hairs. Transverse sections of the region of the root which contains only developing long root hairs show that the hair cells are located above anticlinal walls between underlying cortical cells. We regard the distribution of root epidermal cells in E. plantagineum as a sub-type of this group. We discuss the possible evolution, from this sub-type, of another group that is characterised by hair cells and non-hair cells occurring in separate files.     

Conserved and unique features of the maize (Zea mays L.) root hair proteome

Root hairs are unicellular extensions of specialized epidermis cells. Under limiting conditions, they significantly increase the water and nutrient uptake capacity of plants by enlarging their root surface. Thus far, little is known about the initiation and growth of root hairs in the monocot model species maize. To gain a first insight into the protein composition of these specialized cells, the 2573 most abundant proteins of maize root hairs attached to four-day-old primary roots of the inbred line B73 were identified by combining 1DE with nanoLC-MS/MS in a shotgun proteomic experiment. Among the identified proteins, homologues of 252 proteins have been previously associated with root hair formation and development in other species. Comparison of the root hair reference proteome of the monocot species maize with the previously published root hair proteome of the dicot species soybean revealed conserved, but also unique, protein functions in root hairs of these two major groups of flowering plants.     

The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling

The symbiotic infection of the model legume Medicago truncatula by Sinorhizobium meliloti involves marked root hair curling, a stage where entrapment of the microsymbiont occurs in a chamber from which infection thread formation is initiated within the root hair. We have genetically dissected these early symbiotic interactions using both plant and rhizobial mutants and have identified a M. truncatula gene, HCL, which controls root hair curling. S. meliloti Nod factors, which are required for the infection process, induced wild-type epidermal nodulin gene expression and root hair deformation in hcl mutants, while Nod factor induction of cortical cell division foci was reduced compared to wild-type plants. Studies of the position of nuclei and of the microtubule cytoskeleton network of hcl mutants revealed that root hair, as well as cortical cells, were activated in response to S. meliloti. However, the asymmetric microtubule network that is typical of curled root hairs, did not form in the mutants, and activated cortical cells did not become polarised and did not exhibit the microtubular cytoplasmic bridges characteristic of the pre-infection threads induced by rhizobia in M. truncatula. These data suggest that hcl mutations alter the formation of signalling centres that normally provide positional information for the reorganisation of the microtubular cytoskeleton in epidermal and cortical cells.     

Nod factor-induced root hair curling: continuous polar growth towards the point of nod factor application

A critical step in establishing a successful nitrogen-fixing symbiosis between rhizobia and legume plants is the entrapment of the bacteria between root hair cell walls, usually in characteristic 180 degrees to 360 degrees curls, shepherd's crooks, which are formed by the host's root hairs. Purified bacterial signal molecules, the nodulation factors (NFs), which are lipochitooligosaccharides, induce root hair deformation in the appropriate host legume and have been proposed to be a key player in eliciting root hair curling. However, for curling to occur, the presence of intact bacteria is thought to be essential. Here, we show that, when spot applied to one side of the growing Medicago truncatula root hair tip, purified NF alone is sufficient to induce reorientation of the root hair growth direction, or a full curl. Using wild-type M. truncatula containing the pMtENOD11::GUS construct, we demonstrate that MtENOD11::GUS is expressed after spot application. The data have been incorporated into a cell biological model, which explains the formation of shepherd's crook curls around NF-secreting rhizobia by continuous tip growth reorientation.     

AKT1 and TRH1 are required during root hair elongation in Arabidopsis

TRH1 is a member of the AtKT/AtKUP/AtHAK family of potassium carriers that is required for root hair elongation and AKT1 is an inward rectifying potassium channel expressed in the root epidermis, endodermis and cortex of Arabidopsis thaliana. Plants homozygous for the trh1-1 mutation form short root hairs. The Trh1(-) phenotype cannot be suppressed by growing plants homozygous for the trh1-1 mutation in the presence of high external KCl concentration. This indicates an absolute requirement for TRH1 in root hair tip growth. Plants homozygous for the akt1-1 mutation develop longer root hairs than the wild type when grown in 0 mM external potassium, but develop shorter hairs than the wild type when grown in higher concentrations [>10 mM] of potassium. These data indicate that both TRH1 and AKT1 are active in the root hair over a wide range of external potassium concentrations, but suggest they have different functions in the growing hair cell.     

Endoplasmic microtubules configure the subapical cytoplasm and are required for fast growth of Medicago truncatula root hairs

To investigate the configuration and function of microtubules (MTs) in tip-growing Medicago truncatula root hairs, we used immunocytochemistry or in vivo decoration by a GFP linked to a MT-binding domain. The two approaches gave similar results and allowed the study of MTs during hair development. Cortical MTs (CMTs) are present in all developmental stages. During the transition from bulge to a tip-growing root hair, endoplasmic MTs (EMTs) appear at the tip of the young hair and remain there until growth arrest. EMTs are a specific feature of tip-growing hairs, forming a three-dimensional array throughout the subapical cytoplasmic dense region. During growth arrest, EMTs, together with the subapical cytoplasmic dense region, progressively disappear, whereas CMTs extend further toward the tip. In full-grown root hairs, CMTs, the only remaining population of MTs, converge at the tip and their density decreases over time. Upon treatment of growing hairs with 1 microM oryzalin, EMTs disappear, but CMTs remain present. The subapical cytoplasmic dense region becomes very short, the distance nucleus tip increases, growth slows down, and the nucleus still follows the advancing tip, though at a much larger distance. Taxol has no effect on the cytoarchitecture of growing hairs; the subapical cytoplasmic dense region remains intact, the nucleus keeps its distance from the tip, but growth rate drops to the same extent as in hairs treated with 1 microM oryzalin. The role of EMTs in growing root hairs is discussed.     

Cell surface expansion in polarly growing root hairs of Medicago truncatula

Fluorescent microspheres were used as material markers to investigate the relative rates of cell surface expansion at the growing tips of Medicago truncatula root hairs. From the analysis of tip shape and microsphere movements, we propose three characteristic zones of expansion in growing root hairs. The center of the apical dome is an area of 1- to 2- microm diameter with relatively constant curvature and high growth rate. Distal to the apex is a more rapidly expanding region 1 to 2 microm in width exhibiting constant surges of off-axis growth. This middle region forms an annulus of maximum growth rate and is visible as an area of accentuated curvature in the tip profile. The remainder of the apical dome is characterized by strong radial expansion anisotropy where the meridional rate of expansion falls below the radial expansion rate. Data also suggest possible meridional contraction at the juncture between the apical dome and the cell body. The cell cylinder distal to the tip expands slightly over time, but only around the circumference. These data for surface expansion in the legume root hair provide new insight into the mechanism of tip growth and the morphogenesis of the root hair.     

NtGNL1基因通过调控囊泡运输影响烟草根毛的极性生长

极性生长是植物生长发育中的常见现象,但囊泡运输与极性生长的关系还未完全明确。花粉管和根毛是植物细胞极性生长的典型模式。早期研究显示NtGNL1(Nicotiana tabacum GNOM-LIKE 1)通过调节囊泡的后高尔基体转运来影响烟草的花粉管生长。本文以NtGNL1 RNAi转基因植株为材料,研究NtGNL1基因在根毛生长中的作用。结果表明,NtGNL1 RNAi转基因植株的根毛生长明显滞后于野生型,且其根毛出现膨大、弯折、扭曲等形态,与NtGNL1 RNAi转基因植株的花粉管异常形态类似。q RT-PCR检测RNAi转基因株系根毛中PIN1、PIN2、GL2、ROP6、RHD6基因的m RNA表达量,显示PIN2和GL2的表达量显著下调,PIN1、ROP6和RHD6的表达量变化不明显。FM4-64染色表明烟草根表皮细胞和根毛的囊泡分布都受到影响,即NtGNL1基因也影响根毛中的囊泡运输。BFA处理加剧了囊泡的聚集程度,提示根毛尖端还存在其它对BFA敏感并调控囊泡运输的基因。以上证据显示,NtGNL1基因通过囊泡运输途径影响烟草根毛的极性生长,NtGNL1基因的表达下调也影响了PIN2和GL2的表达,从而间接影响根毛的极性生长。     

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–Rhizobium Symbiotic Association

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.     

A mutation in MRH2 kinesin enhances the root hair tip growth defect caused by constitutively activated ROP2 small GTPase in Arabidopsis

Root hair tip growth provides a unique model system for the study of plant cell polarity. Transgenic plants expressing constitutively active (CA) forms of ROP (Rho-of-plants) GTPases have been shown to cause the disruption of root hair polarity likely as a result of the alteration of actin filaments (AF) and microtubules (MT) organization. Towards understanding the mechanism by which ROP controls the cytoskeletal organization during root hair tip growth, we have screened for CA-rop2 suppressors or enhancers using CA1-1, a transgenic line that expresses CA-rop2 and shows only mild disruption of tip growth. Here, we report the characterization of a CA-rop2 enhancer (cae1-1 CA1-1) that exhibits bulbous root hairs. The cae1-1 mutation on its own caused a waving and branching root hair phenotype. CAE1 encodes the root hair growth-related, ARM domain-containing kinesin-like protein MRH2 (and thus cae1-1 was renamed to mrh2-3). Cortical MT displayed fragmentation and random orientation in mrh2 root hairs. Consistently, the MT-stabilizing drug taxol could partially rescue the wavy root hair phenotype of mrh2-3, and the MT-depolymerizing drug Oryzalin slightly enhanced the root hair tip growth defect in CA1-1. Interestingly, the addition of the actin-depolymerizing drug Latrunculin B further enhanced the Oryzalin effect. This indicates that the cross-talk of MT and AF organization is important for the mrh2-3 CA1-1 phenotype. Although we did not observe an apparent effect of the MRH2 mutation in AF organization, we found that mrh2-3 root hair growth was more sensitive to Latrunculin B. Moreover, an ARM domain-containing MRH2 fragment could bind to the polymerized actin in vitro. Therefore, our genetic analyses, together with cell biological and pharmacological evidence, suggest that the plant-specific kinesin-related protein MRH2 is an important component that controls MT organization and is likely involved in the ROP2 GTPase-controlled coordination of AF and MT during polarized growth of root hairs.     

Phosphorylation of MtRopGEF2 by LYK3 mediates MtROP activity to regulate rhizobial infection in Medicago truncatula

The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.     

Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges

Plant root hair formation is initiated when specialized elongating root epidermis cells (trichoblasts) assemble distinct domains at the plasma membrane/cell wall cell periphery complexes facing the root surface. These localities show accumulation of expansin and progressively transform into tip-growing root hair apices. Experimentation showed that trichoblasts made devoid of microtubules (MTs) were unaffected in root hair formation, whereas those depleted of F-actin by the G-actin sequestering agent latrunculin B had their root hair formation blocked after the bulge formation stage. In accordance with this, MTs are naturally depleted from early outgrowing bulges in which dense F-actin meshworks accumulate. These F-actin caps remain associated with tips of emerging and growing root hairs. Constitutive expression of the GFP-mouse talin fusion protein in transgenic Arabidopsis, which visualizes all classes of F-actin in a noninvasive mode, allowed in vivo confirmation of the presence of distinct F-actin meshworks within outgrowing bulges and at tips of young root hairs. Profilin accumulates, at both the protein and the mRNA levels, within F-actin-enriched bulges and at tips of emerging hairs. ER-based calreticulin and HDEL proteins also accumulate within outgrowing bulges and remain enriched at tips of emerging hairs. All this suggests that installation of the actin-based tip growth machinery takes place only after expansin-associated bulge formation and requires assembly of profilin-supported dynamic F-actin meshworks.