The Growth of the Primary Roots and Root Hairs of Sinapis alba and Lepidium sativus in Triton X-100

Seeds of Lepidium and Sinapis were germinated and grown for 3 days in different concentrations of Triton X-100 (0.001–0.1 % v/v). The elongation of the primary root was slightly stimulated by low concentrations. In concentrations above 0.01 %, Triton inhibited root growth and forked root hairs developed. The hairs elongated both at the apex and at the base, exhibited protoplasmic streaming and activity of particulate non-specific esterase. In contrast the growth of the hypocotyl of both Lepidium and Sinapis diminished steadily in increasing concentrations of Triton. Triton also affected the percentage germination of Lepidium, which increased or decreased according to the concentration used. The changes in root growth and germination and the appearance of branched root hairs in abundance coincide with a change in the detergent solution from monomer to aggregated molecules.     

Root hair deformation in the white clover/Rhizobium trifolii symbiosis

Rhizobium trifolii most frequently infects its host white clover (Trifolium repens L.) by means of infection threads formed in markedly curled root hairs. Rhizobium infections are classified as either lateral or apical based on whether they originate in the branches or at the apex of the root hairs. A quantitative estimate of lateral and apical infection in the region of the host root (Trifolium repens L. cv. Regal Ladino) that possessed mature and immature root hairs at the time of inoculation with Rhizobium trifolii TAI (CSIRO, Canberra City, Australia) indicated that lateral infection occurred more frequently in the mature root hair region of the root. Apical infections were more common in the immature root hair region. Cell free filtrates collected from R. trifolii cultured in association with the host roots induced branching in white clover root hairs. A partially purified preparation of the branching factor was obtained from freeze-dried filtrates by ethanol extraction and ion exchange chromatography. Preliminary studies on the characteristics of these substances suggest that some are dialyzable and heat stable white others are non-dialyzable and heat labile. The dialyzable, heat-stable compounds contain neutral sugars and range between 1200 to 10000 daltons in size. In roots that were exposed to low concentrations (6–25 μg-ml^?1) of these partially purified deformation factors before inoculation, the developmentally mature root hairs were deformed at the time of inoculation. Nodules appeared in the mature and immature root hair region of these plants at the same time. In plants exposed to water, nodules were observed in the immature root hair region and mature root hair regions 3 and 5 days after inoculation, respectively. Based on these results, we conclude that the nodule development was hastened in the plants exposed to the root hair-deforming substances because the mature root hairs of these plants were made infectible at the time of inoculation by this exposure.     

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

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

Early cytological events in the infection of the root hairs ofTrifolium repens byRhizobium leguminosarum biovartrifolii observed by glass slide culture in living seedlings

This report describes the early cytological events in the infection byRhizobium leguminosarum biovartrifolii of the root hairs ofTrifolium repens seedlings kept alive on agar medium in glass slide culture experiment. The infection threads bearing rhizobia were formed as soon as the epidermal cells began to emerge as root hairs. On the top of some of these infected emerging root hairs, there were smoky, cell-debris-like bodies, which appeared to be derived from the cell wall dug by rhizobia. Similar bodies were also observed in longer root hairs. None of the root hair cells along the length of the roots which contained infection threads were curled or distorted. A substantial number of pink-colored nodules were later formed on the roots with non-curled infected root hairs.     

Lipochito‐oligosaccharides re‐initiate root hair tip growth in Vicia sativa with high calcium and spectrin‐like antigen at the tip

Lipochito-oligosaccharides, Nod factors secreted by Rhizobium bacteria, are signal molecules that induce deformation of root hairs of their host plant. A bioassay was used for deformation, and the cytological changes induced by specific lipochito-oligosaccharides in root hairs of Vicia sativa L. (vetch), grown between glass slides, were examined. In the assay, root hairs of a particular developmental stage, those that were terminating growth, were susceptible to deformation. These hairs obtained characteristics of tip-growing cells again: (i) a polar cytoplasmic organization and reverse fountain streaming, (ii) an accumulation of a spectrin-like antigen at the tip, and (iii) a tip-focused calcium gradient. Calcium gradients were visualized in Indo-1 loaded root hairs with UV confocal microscopy and ratio-imaging. The results show that hairs respond to the bacterial signal by recovering cytoplasmic polarity and exocytosis.     

Role of Bacterial Polysaccharides in the Adsorption Process of the Rhizobium-Pea. Symbiosis

Phase-contrast and fluorescence microscopy observations showed that pea symbiont R. leguminosarum adsorbed to pea root hairs, but non-symbiont rhizobial strains only adsorbed to a small extent. ¹⁴C-labeled cells were used to assay the number of rhizobial cells adsorbed to a pea root. Capsular polysaccharides or lipopolysaccharides obtained from R. leguminosarum specifically inhibited the adsorption of ¹⁴C-R. leguminosarum cells to a pea root and specifically adsorbed to pea root hairs. Also, they reacted specifically with pea seed lectins. These results suggest that capsular polysaccharides or lipopolysaccharides play an important role in host-specific adsorption. The interaction between the polysaccharides and pea lectins could be the key to determining host specificity in the infection process of Rhizobium-pea symbiosis.     

Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability

Low phosphorus availability stimulates root hair elongation in many plants, which may have adaptive significance in soil phosphorus acquisition. We investigated the effect of low phosphorus on the elongation of Arabidopsis thaliana root hairs. Arabidopsis thaliana plants were grown in plant culture containing high (1000 mmol m^?3) or low (1 mmol m^?3) phosphorus concentrations, and root hair elongation was analysed by image analysis. After 15d of growth, low-phosphorus plants developed root hairs averaging 0.9 mm in length while high-phosphorus plants of the same age developed root hairs averaging 0.3 mm in length. Increased root hair length in low-phosphorus plants was a result of both increased growth duration and increased growth rate. Root hair length decreased logarithmically in response to increasing phosphorus concentration. Local changes in phosphorus availability influenced root hair growth regardless of the phosphorus status of the plant. Low phosphorus stimulated root hair elongation in the hairless axr2 mutant, exogenously applied IAA stimulated root hair elongation in wild-type high-phosphorus plants and the auxin antagonist CM PA inhibited root hair elongation in low-phosphorus plants. These results indicate that auxin may be involved in the low-phosphorus response in root hairs.     

Root hair elongation is inhibited by hypaphorine, the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by indole-3-acetic acid

Hypaphorine, the major indolic compound isolated from the ectomycorrhizal fungus Pisolithus tinctorius, controls the elongation rate of root hairs. At inhibitory concentrations (100 μM), hypaphorine induced a transitory swelling of root hair tips of Eucalyptus globulus Labill. ssp. bicostata. When the polar tip growth resumed, a characteristic deformation was still visible on elongating hairs. At higher hypaphorine concentrations (500 μM and greater), root hair elongation stopped, only 15 min after application. However, root hair initiation from trichoblasts was not affected by hypaphorine. Hypaphorine activity could not be mimicked by related molecules such as indole-3-acetic acid (IAA) or tryptophan. While IAA had no activity on root hair elongation, IAA was able to restore the tip growth of root hairs following inhibition by hypaphorine. These results suggest that hypaphorine and endogenous IAA counteract in controlling root hair elongation. During ectomycorrhiza development, the absence of root hairs might be due in part to fungal release of molecules, such as hypaphorine, that inhibit the elongation of root hairs. Received: 27 October 1999 / Accepted: 14 March 2000     

Involvement of Rhizobium leguminosarum nodulation genes in gene expression in pea root hairs

The mRNA population in pea root hairs was characterized by means of in vitro translation of total root hair RNA followed by 2-dimensional gel electrophoresis of the translation products. Root hairs contain several mRNAs not detectable in total RNA preparations from roots. Most of these root hair-specific mRNAs occur in elongating root hairs at higher levels than in mature root hairs. The expression of some genes in pea root hairs is typically affected by inoculation with Rhizobium leguminosarum. One gene, encoding RH-42, is specifically induced while the expression of another gene, encoding RH-44, is markedly enhanced. Using R. leguminosarum mutants it was shown that the nodC gene is required for the induction and enhancement of expression of the RH-42 and RH-44 genes, respectively, while the Rhizobium chromosomal gene pss1, involved in exopolysaccharide synthesis, is not essential. After induction of the nod genes with apigenin the bacteria excrete into the culture medium a factor that causes root hair deformation. This deformation factor stimulates the expression of the RH-44 gene but does not induce the expression of the gene encoding RH-42.     

Ion channels of intact young root hairs from Medicago sativa

Root hairs are a primary site for nutrient absorption and for initiation of signalling processes linked to variations of the root environment:plant-microbe interactions or abiotic changes. In many of these cases, the earliest detectable response is the modification of plasma membrane transports, detected through alteration of the electrical membrane potential. In spite of this, root hairs have not been extensively used in electrophysiological research so far. Problems with cell shape and current coupling are often prohibitive for microelectrode voltage-clamp on intact root hairs. In the present study, these difficulties have been overcome and the ion channel currents are described for young root hairs from alfalfa seedlings (Medicago sativa cv Sitel). Electrophysiological and pharmacological studies indicated an inward rectifying K⁺ time-dependent current. This current was sensitive to tetraethylammonium and Cs⁺ (10 mM each). Two other currents never shown in root hairs were described: an outward rectifying time-dependent K⁺ current, inhibited by tetraethylammonium and Cs⁺ (10 mM each) allowing K⁺ efflux under strong depolarizations and an instantaneous inward current identified as an anion current, inhibited by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and anthracene-9-carboxylic acid (100 μM each). These results should contribute to the understanding of root hair development and of signalling processes in M. sativa root hairs.     

Colonization of barley roots by Fusarium culmorum and influence of Pseudomonas fluorescens on the process

The stages of barley root colonization by Fusarium culmorum were studied in sterile vermiculite by the method of fluorescent antibodies. The influence of the antagonistic bacterium Pseudomonas fluorescens on the process of root colonization by F. culmorum was demonstrated. In vermiculite inoculated with F. culmorum, the fungus density on the roots increased gradually. In the case of joint inoculation of vermiculite with the fungus and the bacterium, the F. culmorum density on the roots changed abruptly. It was shown that the site of primary colonization of the roots by the fungus was mainly the zone of root hairs. When Pseudomonas fluorescens was present on the roots, F. culmorum colonized not only root hairs, but also the elongation zone, during the first two days. Introduction of Pseudomonas fluorescens into vermiculite resulted in lower intensity of barley root rot.     

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 effects 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-type strain C58 and mutants LBA2525 (virB2::lacZ) and LBA288 (without the 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°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 pilus production in agrobacteria was considered.     

A root hairless barley mutant for elucidating genetic of root hairs and phosphorus uptake

This paper reports a new barley mutant missing root hairs. The mutant was spontaneously discovered among the population of wild type (Pallas, a spring barley cultivar), producing normal, 0.8 mm long root hairs. We have called the mutant bald root barley (brb). Root anatomical studies confirmed the lack of root hairs on mutant roots. Amplified Fragment Length Polymorphism (AFLP) analyses of the genomes of the mutant and Pallas supported that the brb mutant has its genetic background in Pallas. The segregation ratio of selfed F₂ plants, resulting from mutant and Pallas outcross, was 1:3 (–root hairs:+root hairs), suggesting a monogenic recessive mode of inheritance.In rhizosphere studies, Pallas absorbed nearly two times more phosphorus (P) than the mutant. Most of available inorganic P in the root hair zone (0.8 mm) of Pallas was depleted, as indicated by the uniform P depletion profile near its roots. The acid phosphatase (Apase) activity near the roots of Pallas was higher and Pallas mobilised more organic P in the rhizosphere than the mutant. The higher Apase activity near Pallas roots also suggests a link between root hair formation and rhizosphere Apase activity. Hence, root hairs are important for increasing plant P uptake of inorganic as well as mobilisation of organic P in soils.Laboratory, pot and field studies showed that barley cultivars with longer root hairs (1.10 mm), extracted more P from rhizosphere soil, absorbed more P in low-P field (Olsen P=14 mg P kg^–1 soil), and produced more shoot biomass than shorter root hair cultivars (0.63 mm). Especially in low-P soil, the differences in root hair length and P uptake among the cultivars were significantly larger. Based on the results, the perspectives of genetic analysis of root hairs and their importance in P uptake and field performance of cereals are discussed.     

Root Hairs Play a Key Role in the Endophytic Colonization of Olive Roots by <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. with Biocontrol Activity

The use of indigenous bacterial root endophytes with biocontrol activity against soil-borne phytopathogens is an environmentally-friendly and ecologically-efficient action within an integrated disease management framework. The earliest steps of olive root colonization by Pseudomonas fluorescens PICF7 and Pseudomonas putida PICP2, effective biocontrol agents (BCAs) against Verticillium wilt of olive (Olea europaea L.) caused by the fungus Verticillium dahliae Kleb., are here described. A gnotobiotic study system using in vitro propagated olive plants, differential fluorescent-protein tagging of bacteria, and confocal laser scanning microscopy analysis have been successfully used to examine olive roots–Pseudomonas spp. interactions at the single-cell level. In vivo simultaneous visualization of PICF7 and PICP2 cells on/in root tissues enabled to discard competition between the two bacterial strains during root colonization. Results demonstrated that both BCAs are able to endophytically colonized olive root tissues. Moreover, results suggest a pivotal role of root hairs in root colonization by both biocontrol Pseudomonas spp. However, colonization of root hairs appeared to be a highly specific event, and only a very low number of root hairs were effectively colonized by introduced bacteria. Strains PICF7 and PICP2 can simultaneously colonize the same root hair, demonstrating that early colonization of a given root hair by one strain did not hinder subsequent attachment and penetration by the other. Since many environmental factors can affect the number, anatomy, development, and physiology of root hairs, colonization competence and biocontrol effectiveness of BCAs may be greatly influenced by root hair’s fitness. Finally, the in vitro study system here reported has shown to be a suitable tool to investigate colonization processes of woody plant roots by microorganisms with biocontrol potential.     

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.     

SCANNING ELECTRON MICROSCOPY OF RHIZOSHEATHS OF ORYZOPSIS HYMENOIDES

Structural features and microorganisms associated with rhizosheaths (sand grain root sheaths) of Oryzopsis hymenoides were examined by scanning electron microscopy. Rhizosheath structure appears to depend primarily on the extent of root hair growth and the bonding between root hairs and sand grains. Several sources of bonding agents are suggested, but plant products may be the most important. Microorganisms, usually rod forms, were frequently observed in association with coatings, resembling mucilage, on root surfaces and root hairs. The fungus Olpidium and unusual bacterial forms resembling Ancalomicrobium and Hyphomicrobium were observed on or near root surfaces. Mycorrhizae were not observed nor could the presence of actinomycetes be attested to.     

Growth and ultrastructure ofArabidopsis root hairs: therhd3 mutation alters vacuole enlargement and tip growth

The root hairs of plants are tubular projections of root epidermal cells and are suitable for investigating the control of cellular morphogenesis. In wild-typeArabidopsis thaliana (L.) Heynh, growing root hairs were found to exhibit cellular expansion limited to the apical end of the cell, a polarized distribution of organelles in the cytoplasm, and vesicles of several types located near the growing tip. Therhd3 mutant produces short and wavy root hairs with an average volume less than one-third of the wild-type hairs, indicating abnormal cell expansion. The mutant hairs display a striking reduction in vacuole size and a corresponding increase in the relative proportion of cytoplasm throughout hair development. Bead-labeling experiments and ultrastructural analyses indicate that the wavy-hair phenotype of the mutant is caused by asymmetric tip growth, possibly due to abnormally distributed vesicles in cortical areas flanking the hair tips. It is suggested that a major effect of therhd3 mutation is to inhibit vacuole enlargement which normally accompanies root hair cell expansion.     

Root hair-specific disruption of cellulose and xyloglucan in <Emphasis Type="Italic">AtCSLD3</Emphasis> mutants,and factors affecting the post-rupture resumption of mutant root hair growth

The glycosyl transferase encoded by the cellulose synthase-like gene CSLD3/KJK/RHD7 (At3g03050) is required for cell wall integrity during root hair formation in Arabidopsis thaliana but it remains unclear whether it contributes to the synthesis of cellulose or hemicellulose. We identified two new alleles, root hair-defective (rhd) 7-1 and rhd7-4, which affect the C-terminal end of the encoded protein. Like root hairs in the previously characterized kjk-2 putative null mutant, rhd7-1 and rhd7-4 hairs rupture before tip growth but, depending on the growth medium and temperature, hairs are able to survive rupture and initiate tip growth, indicating that these alleles retain some function. At 21°C, the rhd7 tip-growing root hairs continued to rupture but at 5oC, rupture was inhibited, resulting in long, wild type-like root hairs. At both temperatures, the expression of another root hair-specific CSLD gene, CSLD2, was increased in the rhd7-4 mutant but reduced in the kjk-2 mutant, suggesting that CSLD2 expression is CSLD3-dependent, and that CSLD2 could partially compensate for CSLD3 defects to prevent rupture at 5°C. Using a fluorescent brightener (FB 28) to detect cell wall (1 → 4)-β-glucans (primarily cellulose) and CCRC-M1 antibody to detect fucosylated xyloglucans revealed a patchy distribution of both in the mutant root hair cell walls. Cell wall thickness varied, and immunogold electron microscopy indicated that xyloglucan distribution was altered throughout the root hair cell walls. These cell wall defects indicate that CSLD3 is required for the normal organization of both cellulose and xyloglucan in root hair cell walls.