Recognition of Leguminous Hosts by a Promiscuous Rhizobium Strain

The lima bean (Phaseolus lunatus L.) and the pole bean (Phaseolus vulgaris L.) are nodulated by rhizobia of two different cross-inoculation groups. Rhizobium sp. 127E15, a cowpea-type Rhizobium, can induce effective nodules on the lima bean and partially effective nodules on the pole bean. Rhizobium phaseoli 127K14 can induce effective nodules on the pole bean but does not reciprocally nodulate the lima bean. Root hairs of the lima bean when inoculated with Rhizobium sp. 127E15 showed tip curling and swelling and infection thread formation as observed by light microscopy and scanning electron microscopy. When lima bean root hairs were inoculated with R. phaseoli 127K14, no host-specific responses were observed. Pole bean root hairs that had been inoculated with R. phaseoli 127K14 or Rhizobium sp. 127E15 also showed tip curling and swelling and infection thread formation. Colonization of lima bean root hairs by Rhizobium sp. 127E15 and pole bean root hairs by R. phaseoli 127K14 or Rhizobium sp. 127E15 appeared to involve the elaboration of microfibrils. This study showed that when Rhizobium sp. 127E15 nodulates a host of a different cross-inoculation group, it elicits the same specific host responses as it does from a host of the same cross-inoculation group.     

Role of Lectins in the Specific Recognition of Rhizobium by Lotononis bainesii

Fluorescein isothiocyanate (FITC)-labeled lectin purified from the root of Lotononis bainesii Baker was bound by cells of five out of seven L. bainesii-nodulating strains of Rhizobium under culture conditions. With the exception of a strain of Rhizobium leguminosarum, strains of noninfective rhizobia failed to bind the root lectin under these conditions. The two nonlectin binding L. bainesii-specific strains did not bind root lectin on the L. bainesii rhizoplane although this was observed with three other L. bainesii-nodulating strains. A single Rhizobium japonicum strain bound root lectin on the L. bainesii rhizoplane. There was no evidence of an interaction between the L. bainesii seed lectin and the Rhizobium strains tested.

Root lectin-specific FITC-labeled antibodies were bound to the tips of developing root hairs and lateral growth points of more mature root hairs of L. bainesii seedlings. The damaged edges of severed root hairs always bound FITC-labeled root lectin antibody. Seed lectin-specific FITC-labeled antibodies were not bound to the roots of L. bainesii. The preemergent root hair region of L. bainesii was most susceptible to infection by rhizobia but nodules also emerged in the developing and mature root hair regions. Lectin exposed at growth points on L. bainesii root hairs may provide a favorable site for host plant recognition of infective strains of Rhizobium.

     

Association of Azospirillum with Grass Roots

The association between grass roots and Azospirillum brasilense Sp 7 was investigated by the Fahraeus slide technique, using nitrogen-free medium. Young inoculated roots of pearl millet and guinea grass produced more mucilaginous sheath (mucigel), root hairs, and lateral roots than did uninoculated sterile controls. The bacteria were found within the mucigel that accumulated on the root cap and along the root axes. Adherent bacteria were associated with granular material on root hairs and fibrillar material on undifferentiated epidermal cells. Significantly fewer numbers of azospirilla attached to millet root hairs when the roots were grown in culture medium supplemented with 5 mM potassium nitrate. Under these growth conditions, bacterial attachment to undifferentiated epidermal cells was unaffected. Aseptically collected root exudate from pearl millet contained substances which bound to azospirilla and promoted their adsorption to the root hairs. This activity was associated with nondialyzable and proteasesensitive substances in root exudate. Millet root hairs adsorbed azospirilla in significantly higher numbers than cells of Rhizobium, Pseudomonas, Azotobacter, Klebsiella, or Escherichia. Pectolytic activities, including pectin transeliminase and endopolygalacturonase, were detected in pure cultures of A. brasilense when this species was grown in a medium containing pectin. These studies describe colonization of grass root surfaces by A. brasilense and provide a possible explanation for the limited colonization of intercellular spaces of the outer root cortex.     

The Effect of Nutrient Solution Composition on the Length of Root Hairs of Wheat (Triticum aestivum L.)

The length of root hairs of wheat (Triticum aestivum L.) growingin Long Ashton nitrate nutrient solution was affected mainlyby pH and the concentrations of calcium and nitrate. Root hairlength was decreased by high pH (> 7.0) and by low calciumconcentrations but was increased by nitrate. Buffering at pH6.5 was found to affect the relationship between nutrient solutionstrength and the length of root hairs. The interactions betweencalcium, nitrate and buffering were determined. Phosphate concentrationdid not affect the length of root hairs of wheat, although lowconcentrations have been reported to stimulate root hair growthin other plants. The mechanisms by which pH, calcium and nitratemay affect root hair length are discussed. Key words: Root hairs, Triticum aestivum, Wheat, Nutrient solutions     

Ultrastructure,differentiation and cell wall texture of trichoblasts and root hairs of ceratopteris thalictroides (L.) brongn. (Parkeriaceae)

Trichoblasts and root hairs of Ceratopteris thalictroides (L.) Brongn. were studied by different techniques to survey their morphological features. Trichoblasts could be identified at an early stage by an intra-vacuolar precipitate appearing during fixation. Special attention was paid to root-hair initiation. No structures or changes were observed that play a role in the initiation of papilla formation. When the papilla is formed, vesicles and periplasmic membranes can be observed which may play a role in the weakening of the cell wall during the papilla outgrowth.During root-hair growth, the nucleus of the trichoblast moves from the trichoblast to a subapical position in the root hair. The nuclei of all root cells contain 2 types of nuclear inclusions, one of which is proteinaceous.The cell wall of the Ceratopteris root hair has a helicoidal texture and because the cortical microtubules run longitudinally in the root hair, no correlation can be made between the directions of microtubules and microfibrils in these root hairs.     

Interactions between root hair length and arbuscular mycorrhizal colonisation in phosphorus deficient barley (Hordeum vulgare)

Aims

Phosphorus (P) limits crop yield and P-fertilisers are frequently applied to agricultural soils. However, supplies of quality rock phosphate are diminishing. Plants have evolved mechanisms to improve P-acquisition and understanding these could improve the long-term sustainability of agriculture. Here we examined interactions between root hairs and arbuscular mycorrhizal (AM) colonisation in barley (Hordeum vulgare L.).

Methods

Barley mutants exhibiting different root hair phenotypes, wild type barley and narrowleaf plantain (Plantago lanceolata L.) were grown in the glasshouse in P-sufficient and P-deficient treatments and allowed to develop AM colonization from the natural soil community. Plants were harvested after 6 weeks growth and root hair length, AM-fungal colonisation, shoot biomass and P-accumulation measured.

Results

Under P-deficient conditions, root hair length and AM colonisation were negatively related suggesting that resources are allocated to root hairs rather than to AM fungi in response to P-deficiency. There was evidence that barley and narrowleaf plantain employed different strategies to increase P-acquisition under identical conditions, but root hairs were more effective.

Conclusions

This research suggests future barley breeding programmes should focus on maintaining or improving root hair phenotypes and that pursuing enhancements to AM associations under the prevalent agricultural conditions tested here would be ineffectual.     

Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation

The endoplasmic reticulum (ER) of the model legume Lotus japonicus was visualized using green fluorescent protein (GFP) fused with the KDEL sequence to investigate the changes in the root hair cortical ER in the presence or absence of Mesorhizobium loti using live fluorescence imaging. Uninoculated root hairs displayed dynamic forms of ER, ranging from a highly condensed form to an open reticulum. In the presence of M. loti, a highly dynamic condensed form of the ER linked with the nucleus was found in deformed, curled, and infected root hairs, similar to that in uninoculated and inoculated growing zone I and II root hairs. An open reticulum was primarily found in mature inoculated zone III root hairs, similar to that found in inactive deformed/curled root hairs and infected root hairs with aborted infection threads. Co-imaging of GFP-labeled ER with light transmission demonstrated a correlation between the mobility of the ER and other organelles and the directionality of the cytoplasmic streaming in root hairs in the early stages of infection thread formation and growth. ER remodeling in root hair cells is discussed in terms of possible biological significance during root hair growth, deformation/curling, and infection in the Mesorhizobium–L. japonicus symbiosis.     

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.     

Receptor Site on Clover and Alfalfa Roots for Rhizobium

Sites on white clover and alfalfa roots that bind Rhizobium trifolii and R. meliloti capsular polysaccharides, respectively, were examined by fluorescence microscopy. Fluorescein isothiocyanate-labeled capsular material from R. trifolii bound specifically to root hairs of clover but not alfalfa. Binding was most intense at the root hair tips. Treatment of clover roots with 2-deoxyglucose (2-dG) prevented binding of R. trifolii capsular material to the roots. The sugar 2-dG enhanced the elution of clover root protein, which could bind to and specifically agglutinate R. trifolii but not R. meliloti or R. japonicum. The mild elution procedure left the roots intact. Agglutination of R. trifolii and passive hemagglutination of rabbit erythrocytes coated with the capsular material of R. trifolii were specifically inhibited by 2-dG. These results suggest that clover roots contain proteins that cross-link complementary polysaccharides on the surface of clover root hairs and infective R. trifolii through 2-dG-sensitive binding sites. Alfalfa root hairs were shown to specifically bind to a surface polysaccharide from R. meliloti.     

Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.     

Root hairs are the most important root trait for rhizosheath formation of barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)

Background and AimsRhizosheaths are defined as the soil adhering to the root system after it is extracted from the ground. Root hairs and mucilage (root exudates) are key root traits involved in rhizosheath formation, but to better understand the mechanisms involved their relative contributions should be distinguished.MethodsThe ability of three species [barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)] to form a rhizosheath in a sandy loam soil was compared with that of their root-hairless mutants [bald root barley (brb), maize root hairless 3 (rth3) and root hairless 1 (Ljrhl1)]. Root hair traits (length and density) of wild-type (WT) barley and maize were compared along with exudate adhesiveness of both barley and maize genotypes. Furthermore, root hair traits and exudate adhesiveness from different root types (axile versus lateral) were compared within the cereal species.Key ResultsPer unit root length, rhizosheath size diminished in the order of barley > L. japonicus > maize in WT plants. Root hairs significantly increased rhizosheath formation of all species (3.9-, 3.2- and 1.8-fold for barley, L. japonicus and maize, respectively) but there was no consistent genotypic effect on exudate adhesiveness in the cereals. While brb exudates were more and rth3 exudates were less adhesive than their respective WTs, maize rth3 bound more soil than barley brb. Although both maize genotypes produced significantly more adhesive exudate than the barley genotypes, root hair development of WT barley was more extensive than that of WT maize. Thus, the greater density of longer root hairs in WT barley bound more soil than WT maize. Root type did not seem to affect rhizosheath formation, unless these types differed in root length.ConclusionsWhen root hairs were present, greater root hair development better facilitated rhizosheath formation than root exudate adhesiveness. However, when root hairs were absent root exudate adhesiveness was a more dominant trait.     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

Heterogeneous nutrient supply promotes maize growth and phosphorus acquisition: additive and compensatory effects of lateral roots and root hairs

Background and AimsRoot proliferation is a response to a heterogeneous nutrient distribution. However, the growth of root hairs in response to heterogeneous nutrients and the relationship between root hairs and lateral roots remain unclear. This study aims to understand the effects of heterogeneous nutrients on root hair growth and the trade-off between root hairs and lateral roots in phosphorus (P) acquisition.MethodsNear-isogenic maize lines, the B73 wild type (WT) and the rth3 root hairless mutant, were grown in rhizoboxes with uniform or localized supply of 40 (low) or 140 (high) mg P kg⁻¹ soil.ResultsBoth WT and rth3 had nearly two-fold greater shoot biomass and P content under local than uniform treatment at low P. Significant root proliferation was observed in both WT and rth3 in the nutrient patch, with the WT accompanied by an obvious increase (from 0.7 to 1.2 mm) in root hair length. The root response ratio of rth3 was greater than that of WT at low P, but could not completely compensate for the loss of root hairs. This suggests that plants enhanced P acquisition through complementarity between lateral roots and root hairs, and thus regulated nutrient foraging and shoot growth. The disappearance of WT and rth3 root response differences at high P indicated that the P application reduced the dependence of the plants on specific root traits to obtain nutrients.ConclusionsIn addition to root proliferation, the root response to a nutrient-rich patch was also accompanied by root hair elongation. The genotypes without root hairs increased their investment in lateral roots in a nutrient-rich patch to compensate for the absence of root hairs, suggesting that plants enhanced nutrient acquisition by regulating the trade-off of complementary root traits.     

Development and persistence of sandsheaths of Lyginia barbata (Restionaceae): relation to root structural development and longevity

Background and Aims

Strongly coherent sandsheaths that envelop perennial roots of many monocotyledonous species of arid environments have been described for over a century. This study, for the first time, details the roles played by the structural development of the subtending roots in the formation and persistence of the sheaths.

Methods

The structural development of root tissues associated with persistent sandsheaths was studied in Lyginia barbata, native to the Western Australian sand plains. Cryo-scanning electron microscopy CSEM, optical microscopy and specific staining methods were applied to fresh, field material. The role of root hairs was clarified by monitoring sheath development in roots separated from the sand profile by fine mesh.

Key Results and Conclusions

The formation of the sheaths depends entirely on the numerous living root hairs which extend into the sand and track closely around individual grains enmeshing, by approx. 12 cm from the root tip, a volume of sand more than 14 times that of the subtending root. The longevity of the perennial sheaths depends on the subsequent development of the root hairs and of the epidermis and cortex. Before dying, the root hairs develop cellulosic walls approx. 3 µm thick, incrusted with ferulic acid and lignin, which persist for the life of the sheath. The dead hairs remain in place fused to a persistent platform of sclerified epidermis and outer cortex. The mature cortex comprises this platform, a wide, sclerified inner rim and a lysigenous central region – all dead tissue. We propose that the sandsheath/root hair/epidermis/cortex complex is a structural unit facilitating water and nutrient uptake while the tissues are alive, recycling scarce phosphorus during senescence, and forming, when dead, a persistent essential structure for maintenance of a functional stele in the perennial Lyginia roots.     

Abscisic Acid Elicits the Water-Stress Response in Root Hairs of Arabidopsis thaliana

Water stress has been shown to cause root hairs to become short and bulbous. Because abscisic acid (ABA) mediates a variety of water-stress responses, we investigated the response of Arabidopsis thaliana root hairs to ABA. When wild-type root hairs were treated with ABA, they exhibited the water-stress response. The Arabidopsis mutants abi1 and abi2, which are insensitive to ABA at the seedling stage, did not display the root hair response. These data suggest that ABA may mediate the response of root hairs to water stress. The drought response of root hairs resulting in an inhibition of tip growth will provide an easy screen to select mutations that are insensitive to ABA and/or involved in tip growth.     

SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis

Background and Aims

Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.

Methods

Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.

Key Results

VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.

Conclusions

These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.     

Influence of cold on organization of actin filaments of different types of root cells in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The effect of the low temperature (+4°C) on the organization of actin filaments (microfilaments) of cells from different growth zones has been studied in the roots of Arabidopsis thaliana (L.). It was found that cold treatment inhibited the growth of the primary root and changed its morphology, causing a formation of large number of deformed (ectopic) root hairs in differentiation zone. The temporal relationship between the disorientation and the organization of actin filaments and the detected changes of growth and morphology of roots after cold treatment was shown. It has been found that actin filaments of root hairs, meristematic cells, cells of elongation zone, and epidermal cells of all root zones of A. thaliana are the most sensitive to the cold.     

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.     

Root hair growth in Arabidopsis thaliana is directed by calcium and an endogenous polarity

Tip growth of plant cells has been suggested to be regulated by a tip-focused gradient in cytosolic calcium concentration ([Ca²⁺]_c). However, whether this gradient orients apical growth or follows the driving force for this process remains unknown. Using localized photoactivation of the caged calcium ionophore Br-A23187 we have been able to artificially generate an asymmetrical calcium influx across the root hair tip. This led to a change in the direction of tip growth towards the high point of the new [Ca²⁺]_c gradient. Such reorientation of growth was transient and there was a return to the original direction within 15 min. Root hairs forced to change the direction of their growth by placing a mechanical obstacle in their path stopped, reoriented growth to the side, and grew past the mechanical blockage. However, as soon as the growing tip had cleared the obstacle, growth returned to the original direction. Confocal ratio imaging revealed that a tip-focused [Ca²⁺]_c gradient was always centered at the site of active growth. When the root hair changed direction the gradient also reoriented, and when growth returned to the original direction, so did the [Ca²⁺]_c gradient. This normal direction of apical growth of Arabidopsis thaliana (L.) Heynh. root hairs was found to be at a fixed angle from the root of 85 ± 6.7 degrees. In contrast, Tradescantia virginiana (L.) pollen tubes that were induced to reorient by touch or localized activation of the caged ionophore, did not return to the original growth direction, but continued to elongate in their new orientation. These results suggest that the tip-focused [Ca²⁺]_c gradient is an important factor in localizing growth of the elongating root hair and pollen tube to the apex. However, it is not the primary determinant of the direction of elongation in root hairs, suggesting that other information from the root is acting to continuously reset the growth direction away from the root surface. Received: 22 April 1997 / Accepted: 14 May 1997