Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation

The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment.     

Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells

Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp. bacteria to elicit Nod responses on their legume hosts. One of the earliest responses is the formation of phosphatidic acid (PA), a novel second messenger in plant cells. Remarkably, pathogens have also been reported to trigger the formation of PA in nonlegume plants. To investigate how host plants can distinguish between symbionts and pathogens, the effects of Nod factor and elicitors (chitotetraose and xylanase) on the formation of PA were investigated in suspension-cultured alfalfa (Medicago sativa) cells. Theoretically, PA can be synthesized via two signaling pathways, i.e. via phospholipase D (PLD) and via phospholipase C in combination with diacylglycerol (DAG) kinase. Therefore, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used to determine the contribution of each pathway to PA formation. In support, PLD activity was specifically measured by using the ability of the enzyme to transfer the phosphatidyl group of its substrate to a primary alcohol. In practice, Nod factor, chitotetraose, and xylanase induced the formation of PA and its phosphorylated product DAG pyrophosphate within 2 min of treatment. However, whereas phospholipase C and DAG kinase were activated during treatment with all three different compounds, PLD was only activated by Nod factor. No evidence was obtained for the activation of phospholipase A(2).     

Nod signal-induced plasma membrane potential changes in alfalfa root hairs are differentially sensitive to structural modifications of the lipochitooligosaccharide

Lipochitooligosaccharide Nod signals are important determinants of host specificity in the Rhizobium -legume symbiosis. The most rapid response of plant cells to the R. meliloti Nod signal NodRm-IV(C16:2,S) reported so far is the depolarization of the plasma membrane potential in alfalfa root hairs. In order to investigate whether this response may be part of a specific signal transduction cascade involved in the nodulation process, its specificity was studied with respect to host-specific modifications of the lipochitooligosaccharide. Five different Nod factors displaying different degrees of activity in inducing root hair deformation or cortical cell divisions on alfalfa were tested. The ability of the Nod factors to elicit plasma membrane depolarization correlated well with their activity in the bioassays. Removal of the sulfate group (NodRm-IV(C16:2)) led to inactivation of the Nod factor. An increase in the length of the chitooligosaccharide backbone (NodRm-V(C16:2,S)) or saturation of the acyl chain (NodRm-IV(C16:0,S)) resulted in severely reduced activity. In contrast, the O -acetyl group at the non-reducing terminus in NodRm-IV(Ac,C16:2,S), which confers substantially higher activity in long-term bioassays, did not enhance plasma membrane depolarization significantly in comparison with the non- O -acetylated factor. Thus, the rapid plasma membrane response is differentially sensitive to various structural motifs of the lipochitooligosaccharide. These data suggest that the different substituents modifying the basic Nod factor structure may have distinct functions, not all of them contributing to the interaction with a putative receptor in root hair cells. However, the overall specificity of the membrane depolarization for the cognate Nod factors raises the possibility that it is involved in a Nod signal transduction pathway.     

Rhizobium Nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs

Application of Nod factors to growing, responsive root hairs of the bean Phaseolus vulgaris induces marked changes in both the intracellular cytosolic free calcium (Ca²⁺) and in the influx of extracellular [Ca²⁺]. The intracellular [Ca²⁺], which has been measured by ratiometric imaging in cells microinjected with fura-2-dextran (70 kDa), elevates within 5 min from approximately 400 n m to 1500 n m in localised zones in the root hair apex. Of particular note is the observation that the elevated regions of [Ca²⁺] appear to shift position during short time intervals. Increases in and fluctuations of the intracellular [Ca²⁺] are also observed in the perinuclear region after 10–15 min treatment with Nod factors. The extracellular Ca²⁺ flux, detected with the non-invasive, calcium specific vibrating electrode, is inwardly directed and also increases quickly in response to Nod factors from 13 pmol cm^–2 s^–1 to 28 pmol cm^–2 s^–1. Chitin-oligomers, which are structurally similar but biologically inactive when compared to the active Nod factors, fail to elicit changes in either intracellular or extracellular Ca²⁺. The similar timing and location of the intracellular elevations and the increased extracellular influx provide support for the idea that Ca²⁺ participates in secretion and cell wall remodelling, which occur in anticipation of root hair deformation and curling.     

Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur.

Three novel nodulation (Nod) factors were synthesized from chitotetraose and three structurally different fluorescent BODIPY-tagged fatty acids. With fluorescence spectroscopic and microscopic techniques, the following aspects were studied: whether these amphiphilic molecules insert in membranes, whether they transfer between different membranes, and whether they are able to transfer from a membrane to a legume root hair. Fluorescence correlation spectroscopy showed that fluorescent Nod factors are present as monomers in PBS buffer at a concentration of 10 nM, but that when either Triton X-100 micelles or dioleoylphosphatidylcholine (DOPC) vesicles are present, the Nod factors are associated with these particles. With time-correlated single-photon counting fluorescence spectroscopy, it was shown that upon Nod factor insertion in the membrane, the rotation of the fluorescent acyl chain was markedly reduced. A fluorescence resonance energy transfer assay was used to study the transfer of Nod factors from one membrane to the other, or from vesicles to root hairs. Nod factors transfer rapidly between membranes or from vesicles to root hair cell walls. However, they do not flip-flop between membrane leaflets. The results provide novel insights for the mode of secretion and transfer of Nod factors during the early steps of the Rhizobium-legume interaction.     

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     

Rapid alkalinization in alfalfa root hairs in response to rhizobial lipochitooligosaccharide signals

Rhizobial lipochitooligosaccharides (Nod factors) function as symbiotic signals that trigger root hair deformations and cortical cell divisions on the roots of leguminous plants in a host-specific manner. By using pH-sensitive microelectrodes, it is shown that alfalfa root hair cells respond to Rhizobium meliloti Nod factors with a rapid intracellular alkalinization of 0.2–0.3 pH units. This alkalinization remained as long as the Nod factor was present, but slowly reversed after removal of the signal. The response was most sensitive to the sulfated tetrameric Nod factor, NodRm-IV(C16:2,S), which is morphogenic on the host plant alfalfa, suggesting a role in a signal transduction cascade. Non-sulfated Nod factor as well as chitooligosaccharides elicited a pH_c change only at elevated concentrations. The increase of PH_c in response to sulfated Nod factor was concomitant with a depolarization of the plasma membrane potential whereas the PH_c change in response to non-sulfated Nod factor occurred in the absence of membrane depolarization. In addition, whereas a first dose of sulfated Nod factor inhibited the subsequent response to a second dose of the same molecule, it did not significantly repress the activity of non-sulfated Nod factor. These results indicate that sulfated and non-sulfated Nod factors act independently and suggest the existence of two Nod signal perception systems, one transmitting the host-specific signal, the other representing an ancient reception system for a generic Nod factor structure.     

Regulation of electrical coupling between Arabidopsis root hairs

Voltage clamp was used to measure the voltage dependence of cell-to-cell coupling via plasmodesmata between higher-plant cells (root hairs of Arabidopsis thaliana (L.) Heynh.). In addition, ionophoresis was used to introduce a variety of ions [Ca²⁺, inositol-trisphosphate, Li⁺, K⁺, Mg²⁺, ethylene glycol-bis(-aminoethyl ether)-N,N,N, N-tetraacetic acid (EGTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), H⁺, and OH^–] to examine whether they regulate cell-to-cell coupling. Electrical coupling showed high variability in this single cell type at the same developmental stage; the coupling ratio ranged from near 0% to about 90% with a mean value of 32%. It was voltage independent for intracellular voltage gradients (transplasmodesmatal) of -163 to 212 mV. While Ca²⁺ closes the plasmodesmatal connections (at concentrations higher than those causing cessation of cytoplasmic streaming), inositol-trisphosphate and lithium are without effect. Apparently, inositol-trisphosphate may not cause increased cytosolic Ca²⁺ in root hairs. Alkalinization by OH ionophoresis caused a modest decline in cell-to-cell coupling, as did acidification by H⁺ ionophoresis (to an extent causing the cell to become flacid). Increases in cytosolic K⁺, Mg²⁺, and the calcium chelator BAPTA by ionophoresis had no effect on cell-to-cell coupling. The regulation (and lack thereof) reported here for plant plasmodesmata is quite similar to that of gap junctions.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid     

Bacterial endophytes and root hairs

Aims

This study aimed to measure the effect of plant diversity on N uptake in grasslands and to assess the mechanisms contributing to diversity effects.

Methods

Annual N uptake into above- and belowground organs and soil nitrate pools were measured in the Jena experiment on a floodplain soil with mixtures of 2–16 species and 1–4 functional groups, and monocultures. In mixtures, the deviation of measured data from data expected from monoculture performance was calculated to assess the contribution of complementarity/facilitation and selection.

Results

N uptake varied from <1 to 45 g?N m^?2 yr^?1, and was higher in grasslands with than without legumes. On average, N uptake was higher in mixtures (21?±?1 g?N m^?2 yr^?1) than monocultures (13?±?1 g?N m^?2 yr^?1), and increased with species richness in mixtures. However, compared to N uptake expected from biomass proportions of species in mixtures, N uptake of mixtures was only slightly higher and a significant surplus N uptake was confined to mixtures containing legumes and non-legumes.

Conclusions

In our study, high N uptake of species rich mixtures was mainly due to dominance of productive species and facilitation by legumes whereas complementarity among non-legumes was of minor relevance.     

Ion currents involved in early Nod factor response in Medicago sativa root hairs: a discontinuous single-electrode voltage-clamp study

Nod factor [NodRm-IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well-known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single-electrode voltage-clamp technique (dSEVC) shows that anion channel, K+ channel and H+-ATPase pump currents are involved in young growing root hairs. The early Nod-factor-induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time-dependent inward K+ current (IRKC). These two ion currents are then down-regulated while the H+ pump is stimulated, allowing long-term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod-factor-induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.     

亚热带典型树种根毛特征及其与共生真菌的关系

根毛和共生真菌增加了吸收面积,提高了植物获取磷等土壤资源的能力。由于野外原位观测根表微观结构较为困难,吸收细根、根毛、共生真菌如何相互作用并适应土壤资源供应,缺乏相应的数据和理论。该研究以受磷限制的亚热带森林为对象,选取了21种典型树种,定量了根毛存在情况、属性变异,分析了根毛形态特征与共生真菌侵染率、吸收细根功能属性之间的关系,探讨了根表结构对低磷土壤的响应和适应格局。结果表明:1)在亚热带森林根毛不是普遍存在的, 21个树种中仅发现7个树种存有根毛, 4个为丛枝菌根(AM)树种, 3个为外生菌根(ECM)树种。其中,马尾松(Pinus massoniana)根毛出现率最高,为86%;2)菌根类型是理解根-根毛-共生真菌关系的关键,AM树种根毛密度与共生真菌侵染率正相关,但ECM树种根毛直径与共生真菌侵染率负相关; 3) AM树种根毛长度和根毛直径、ECM树种根毛出现率与土壤有效磷含量呈负相关关系。该研究揭示了不同菌根类型树种根毛-共生真菌-根属性的格局及相互作用,为精细理解养分获取策略奠定了基础。     

The evolution of root hairs and rhizoids

Background

Almost all land plants develop tip-growing filamentous cells at the interface between the plant and substrate (the soil). Root hairs form on the surface of roots of sporophytes (the multicellular diploid phase of the life cycle) in vascular plants. Rhizoids develop on the free-living gametophytes of vascular and non-vascular plants and on both gametophytes and sporophytes of the extinct rhyniophytes. Extant lycophytes (clubmosses and quillworts) and monilophytes (ferns and horsetails) develop both free-living gametophytes and free-living sporophytes. These gametophytes and sporophytes grow in close contact with the soil and develop rhizoids and root hairs, respectively.

Scope

Here we review the development and function of rhizoids and root hairs in extant groups of land plants. Root hairs are important for the uptake of nutrients with limited mobility in the soil such as phosphate. Rhizoids have a variety of functions including water transport and adhesion to surfaces in some mosses and liverworts.

Conclusions

A similar gene regulatory network controls the development of rhizoids in moss gametophytes and root hairs on the roots of vascular plant sporophytes. It is likely that this gene regulatory network first operated in the gametophyte of the earliest land plants. We propose that later it functioned in sporophytes as the diploid phase evolved a free-living habit and developed an interface with the soil. This transference of gene function from gametophyte to sporophyte could provide a mechanism that, at least in part, explains the increase in morphological diversity of sporophytes that occurred during the radiation of land plants in the Devonian Period.     

How to discriminate between vibration syndrome and local fatigue of motorcyclists

Motorcyclists who work in some offices sometimes complained of coldness, pain and numbness of upper limbs. We studied how to discriminate between vibration syndrome and local fatigue of the motorcyclists. Subjects are 42 motorcyclists of an office in Aichi prefecture. 25 of them held several letters in their left hand when they delivered the letters. They complained of coldness, pain and numbness in the left upper limbs more than in the right limbs (p less than 0.01). We think that it is the local fatigue rather than the disorder of vibration syndrome that causes such symptoms. So it is very important to recognize the existence of local fatigue in order to know how to discriminate between vibration syndrome and local fatigue of the motorcyclists.     

Suppression of arbuscular mycorrhizal colonization and nodulation in split-root systems of alfalfa after pre-inoculation and treatment with Nod factors

Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule-inducing rhizobia. The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation. Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system. In this work, split- root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation systemically influences AMF root colonization and vice versa. Nodules on one half of the split-root system suppressed subsequent AMF colonization on the other half. Conversely, root systems pre-colonized on one side by AMF exhibited reduced nodule formation on the other side. An inhibition effect was also observed with Nod factors (lipo-chito-oligosaccharides). NodSm-IV(C16:2, S) purified from S. meliloti systemically suppressed both nodule formation and AMF colonization. The application of Nod factors, however, did not influence the allocation of (14)C within the split-root system, excluding competition for carbohydrates as the regulatory mechanism. These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.     

Perception of Bradyrhizobium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions

Suboptimal growth conditions, such as low rhizosphere temperature, high salinity, and low pH can negatively affect the rhizobia-legume symbioses, resulting in poor nodulation and lower amounts of nitrogen fixed. Early stages of the Bradyrhizobium japonicum-soybean [Glycine max (L.) Merr.] symbiosis, such as excretion of genistein (the plant-to-bacteria signal) and infection initiation can be inhibited by abiotic stresses; however, the effect on early events modulated by Nod factors (bacteria-to-plant signalling), particularly root hair deformations is unknown. Thus, the objective of this study was to evaluate the perception of Nod factor by soybean root hairs under three stress conditions: low temperature, low pH, and high salinity. Three experiments were conducted using a 1:1 ratio of Nod Bj-V (C(18:1), MeFuc) and Nod Bj-V (Ac, C(16:0), MeFuc). Nod factor induced four types of root hair deformation (HAD), wiggling, bulging, curling, and branching. Under optimal experimental conditions root hair response to the three levels of Nod factor tested (10(-6), 10(-8), and 10(-10) M) was dose-dependent. The highest frequency of root hair deformations was elicited by the 10(-6) M level. Root hair deformation decreased with temperature (25, 17, and 15 degrees C), low pH, and high salinity. Nod factor concentration did not interact with either low temperature or pH. However, salinity strongly inhibited HAD responses to increases in Nod factor concentration. Thus, the addition of higher levels of Nod factor is able to overcome the effects of low pH and temperature stress, but not salinity.