Lipochito-oligosaccharide nodulation factors stimulate cytoplasmic polarity with longitudinal endoplasmic reticulum and vesicles at the tip in vetch root hairs

Vetch root hair development has four stages: bulge, growing, growth terminating, and full-grown hair. In the assay we used, the nodulation factor induced swellings and outgrowths in growth-terminating hairs. Bulges, swellings, and full-grown hairs have transverse endoplasmic reticulum (ER) and no tip-accumulated vesicles. Growing hairs and outgrowths show vesicle accumulation in the tip and longitudinal subapical ER. Bulge walls and walls of swellings appear mottled.     

Endogenous ion currents traverse growing roots and root hairs of Trifolium repens

Abstract The patterns of naturally growing ion currents associated with horizontally growing roots of Trifolium repens L. seedlings were measured in a simple low-salt bathing medium using a highly sensitive vibrating electrode. Current consistently enters the main elongation zone of the root and leaves from mature, elongated tissue. This current enters and leaves with densities of ca. 4.0–11.0 mA m^?2 and 4.0 mA m^?2, respectively. Current was also delected entering the zone of emerging root hairs and also the root-hair tips themselves. These results are a further example of the involvement of self-generated electrical fields in plant developmental processes. Possible, secondary rhizosphere-associated effects of the extracellular loop of the developmental current are also suggested.     

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.     

Redistribution of actin, profilin and phosphatidylinositol-4,5-bisphosphate in growing and maturing root hairs

The continuously changing polar cytoplasmic organization during initiation and tip growth of root hairs is reflected by a dynamic redistribution of cytoskeletal elements. The small G-actin binding protein, profilin, which is known to be a widely expressed, potent regulator of actin dynamics, was specifically localized at the tip of root hairs and co-distributed with a diffusely fluorescing apical cap of actin, but not with subapical actin microfilament (MF) bundles. Profilin and actin caps were present exclusively in the bulge of outgrowing root hairs and at the apex of elongating root hairs; both disappeared when tip growth terminated, indicating a tip-growth mechanism that involves profilin-actin interactions for the delivery and localized exocytosis of secretory vesicles. Phosphatidylinositol-4,5-bisphosphate (PIP₂), a ligand of profilin, was localized almost exclusively in the bulge and, subsequently, formed a weak tip-to-base gradient in the elongating root hairs. When tip growth was eliminated by the MF-disrupting inhibitor cytochalasin D, the apical profilin and the actin fluorescence were lost. Mastoparan, which is known to affect the PIP₂ cycle, probably by stimulating phospholipases, caused the formation of a meshwork of distinct actin MFs replacing the diffuse apical actin cap and, concomittantly, tip growth stopped. This suggests that mastoparan interferes with the PIP₂-regulated profilin-actin interactions and hence disturbs conditions indispensable for the maintenance of tip growth in root hairs. Received: 11 March 1999 / Accepted: 27 May 1999     

The plasma membrane of growing root hairs is composed of zones of local differentiation

Growing root hairs of cress (Lepidium sativum L.) were investigated using freeze-fracture and electron-microscopic techniques. Three zones of differentiation could be detected: the tip zone, the zone of vacuolation and the foot zone. Corresponding to these zones, the plasmatic fracture face of the plasma membrane showed areas of pronounced differentiation with respect to the distribution and frequency of intramembranous particles (IMPs). The tip zone was characterized by an irregular fracture plane caused by a large number of blisters which were more or less free of IMPs. These blisters coincided in size and shape with Golgi vesicles accumulated in the ground cytoplasm near the very tip. Outside these blisters, IMPs were randomly distributed. The surrounding cell wall was very thin and mainly composed of amorphous material. The plasma membrane of the vacuolation zone often revealed areas of hexagonally ordered particles (HOPS). Such patterns of particles were observed in chemically fixed and unfixed root hairs with a maximum surface density of 1200 HOPS per area. Mostly, however, 15–50 HOPS per area were found. The number of such areas increased with increasing distance from the tip up to five areas per m². Additionally, imprints of large cellulose microfibrils could be detected in unfixed material; they were mainly parallel to the root-hair axis and sometimes ended in areas of HOPS. However, HOPS were observed only in approximately 60% of the root hairs. Otherwise, large areas free of IMPs were interspersed between areas of randomly distributed IMPs. The particle frequency was relatively low and varied greatly in the tip as well as in the vacuolation zone, that is, from 1200 to 2000 IMPs m^-2. Finally, the plasma membrane of the foot zone showed a very constant number of approx. 2000 IMPs m^-2. These particles were mainly distinct and randomly distributed. In this zone, HOPS were never observed in spite of the fact that the cell wall was composed of numerous parallel-running cellulose microfibrils. Since membrane material is mainly incorporated in the tip zone where IMPs are statistically distributed, the results indicate that the plasma membrane of the outgrowing part of the root-hair cells is characterized by a high lateral mobility of its components. Furthermore, they indicate that specifically arranged particles are involved in the synthesis of cellulose microfibrils. These areas of HOPS seem to be locally restricted and — or limited with respect to their lifetime.Abbreviations cmf(s) cellulose microfibril(s) - EF extraplasmatic fracture face - HOPS hexagonally ordered particles - IMP intramembranous particle - PF plasmatic fracture face - pm plasma membrane Dedicated to Professor Dr. Kurt Mühlethaler, Zürich, on the occasion of his 65th birthday     

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.     

Pressure regulation of the electrical properties of growing Arabidopsis thaliana L. root hairs.

Actively growing Arabidopsis thaliana L. (Columbia wild type) root hairs were used to examine the interplay between cell turgor pressure and electrical properties of the cell: membrane potential, conductance, cell-to-cell coupling, and input resistance. Pressure was directly modulated using a pressure probe or indirectly by changing the extracellular osmolarity. Direct modulation of pressure in the range of 0 to about 15 x 10(5) Pa (normal turgor pressure was 6.8 +/- 2.0 x 10(5) Pa, n = 29) did not affect the membrane potential, conductance, coupling, or input resistance. Indirect modulation of turgor pressure by adding (hyperosmotic) or removing (hypo-osmotic) 200 mM mannitol/sorbitol affected the potential and conductance but not cell-to-cell coupling. Hypo-osmotic treatment depolarized the potential about 40 mV from an initial potential of about -190 mV and increased membrane conductance, consistent with an increase in anion efflux from the cell. Hyperosmotic treatment hyperpolarized the cell about 25 mV from the same initial potential and decreased conductance, consistent with a decline in cation influx. The results are likely due to the presence of an "osmo-sensor," rather than a "turgor-sensor," regulating the cell's response to osmotic stress.     

Cytoarchitecture and pattern of cytoplasmic streaming in root hairs ofMedicago truncatula during development and deformation by nodulation factors

Summary The cytoarchitecture and the pattern of cytoplasmic streaming change during the development of root hairs ofMedicago truncatula and after a challenge with nodulation (Nod) factors. We measured the speed and orientation of movement of 1–2 μm long organelles. The speed of organelle movement in cytoplasmic strands in the basal part of growing root hairs is 8–14 μm/s and is of the circulation type like in trichoblasts, bulges before tip-growth initiation, and full-grown hairs. In the subapical area of growing hairs, reverse-fountain streaming occurs discontinuously at a slower net speed. The reason for the slower speed is the fact that organelles often stop and jump. Reverse-fountain streaming is a pattern in which the main direction of organelle transport reverses 180 degrees before the cell tip is reached. Within minutes after their application to roots,Rhizobium leguminosarum-derived Nod factors, cause an increase and divergence in the subapical cytoplasmic strands. This phenomenon can best be observed in the growth-terminating hairs, since in hairs of this developmental stage, subapical cytoplasmic strands are transvacuolar. First, the tips of these hairs swell. The organelle movement in the swelling tip increases up to the level normal for circulation streaming, and the number of strands with moving organelles increases. When a new polar outgrowth emerges, reverse-fountain streaming is set up again, with all its characteristics like those seen in growing hairs. This outgrowth may obtain a new full root hair length, by which these hairs may become twice as long as nonchallenged hairs. Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

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.     

Electrogenic transport properties of growing Arabidopsis root hairs : the plasma membrane proton pump and potassium channels

Ion transport, measured using double-barreled micropipettes to obtain current-voltage relations, was examined in Arabidopsis thaliana root hairs that continued tip growth and cytoplasmic streaming after impalement with the micropipette. To do this required in situ measurements with no handling of the seedlings to avoid wounding responses, and conditions allowing good resolution microscopy in tandem with the electrophysiological measurements. Two ion transport processes were demonstrated. One was a tetraethylammonium-sensitive potassium ion current, inward at hyperpolarized potentials and outward at depolarized potentials. The addition of tetraethylammonium (a potassium channel blocker) caused the potential to hyperpolarize, indicating the presence of a net inward potassium current through the ion channels at the resting potential. The potassium influx was sufficient to “drive” cellular expansion based upon growth rates. Indeed, tetraethylammonium caused transient inhibition of tip growth. The other electrogenic process was the plasma membrane proton pump, measured by indirect inhibition with cyanide or direct inhibition by vanadate. The proton pump was the dominant contribution to the resting potential, with a very high current density of about 250 microamperes per square centimeter (seen only in young growing root hairs). The membrane potential generated by the proton pump presumably drives the potassium influx required for cellular expansion. The pump appears to be a constant current source over the voltage range −200 to 0 millivolts. With this system, it is now possible to study the physiology of a higher plant cell in dynamic living state using a broad range of cell biological and electrophysiological techniques.     

Changes in trace metal species and other components of the rhizosphere during growth of radish

Changes in the properties of soil solution in the rhizosphere of developing radish plants were investigated. Variations in these properties were expected to affect the distribution and speciation of metals in the soil and soil solution. Applications of essential nutrients were linked to plant transpiration rates and prevented excess addition of nutrient ions, so that subtle changes in soil solution composition would not be obscured. Soil solution pH, the concentration of dissolved organic carbon (DOC) and the concentrations of major and trace elements in solution were found to vary over time. Strict control of fertilizer additions led to the maintenance of a relatively low ionic strength in the soil solution, and under such conditions trace metal solubility appeared to be highy influenced by the concentration of DOC. A chemical speciation analysis was performed which showed that, while dissolved Cd and Zn were largely uncomplexed in unplanted soil, Cd and Zn in the rhizosphere existed mainly as complexed forms. It is hypothesized that this is partly a result of Ca-metal-ligand equilibrium in solution, with higher Ca concentrations in unplanted soil leading to more of the Cd and Zn in solution existing in the uncomplexed state. Changes in the concentrations of uncomplexed Cd and Zn with time gave the best correlations with changes in plant uptake of these metals over time, supporting the hypothesis that plants mainly absorb the free metal ion from soil solution.     

Immediate and steady state extracellular ionic fluxes of growing Arabidopsis thaliana root hairs under hyperosmotic and hypoosmotic conditions

Osmotic‐induced shifts in extracellular ionic fluxes at the apex of growing Arabidopsis thaliana L. root hairs were examined using the extracellular self‐referencing (vibrating) ion‐selective probe technique. With either APW 7 (artificial pond water, pH 7) or APW 7 plus 200 m M mannitol/sorbitol, a steady state was reached in which growth rates were the same (0.75 and 0.82 µm min⁻¹) as were the net fluxes of calcium (inward), potassium (outward), and chloride (inward). The outward flux of protons was about 20‐fold larger in APW 7 plus 200 m M mannitol/sorbitol compared to APW 7 ( P = 0.028). Significant changes in ionic fluxes within 5 min of osmotic changes were observed for hyperosmotic treatment: a 7.5‐fold increase in the inward calcium flux ( P = 0.041) and a 1.7‐fold increase in the outward potassium flux ( P = 0.007). Although the change in the calcium flux was consistent with a process of osmotic adjustment (and not a consequence of binding/release from the cell wall), the magnitude of the flux was considerably less than the potassium efflux. Unlike the situation in other organisms where volume regulation explicitly relies upon modulation of ionic fluxes (especially sodium, potassium and chloride), the root hairs may rely on other mechanisms of osmotic regulation, possibly coupled to differences in proton efflux under steady state growth conditions. The root hairs do exhibit an osmotic‐induced electrical signal (R. R. Lew. 1996. Plant Physiol. 112: 1089–1100) which may be a component of initial signal transduction controlling osmotic regulation.     

Effect of aluminum on cytoplasmic Ca2+ homeostasis in root hairs of Arabidopsis thaliana (L.)

Aluminum inhibition of root growth is a major world agricultural problem where the cause of toxicity has been linked to changes in cellular calcium homeostasis. Therefore, the effect of aluminum ions (Al) on changes in cytoplasmic free calcium concentration ([Ca²⁺]_c) was followed in root hairs of wild-type, Al-sensitive and Al-resistant mutants of Arabidopsis thaliana (L.) Heynh. Generally, Al exposure resulted in prolonged elevations in tip-localized [Ca²⁺]_c in both wild-type and Al-sensitive root hairs. However, these Al-induced increases in [Ca²⁺]_c were not tightly correlated with growth inhibition, occurring up to 15 min after Al had induced growth to stop. Also, in 32% of root hairs examined growth stopped without a detectable change in [Ca²⁺]_c. In contrast, Al-resistant mutants showed little growth inhibition in response to AlCl₃ exposure and in no case was a change in [Ca²⁺]_c observed. Of the other externally applied stresses tested (oxidative and mechanical stress), both were found to inhibit root hair growth, but only oxidative stress (H₂O₂, 10 μM) caused a prolonged rise in [Ca²⁺]_c similar to that induced by Al. Again this increase occurred after growth had been inhibited. The lack of a tight correlation between Al exposure, growth inhibition and altered [Ca²⁺]_c dynamics suggests that although exposure of root hairs to toxic levels of Al causes an alteration in cellular Ca²⁺ homeostasis, this may not be a required event for Al toxicity. The elevation in [Ca²⁺]_c induced by Al also strongly suggests that the phytotoxic action of Al in root hairs is not through blockage of Ca²⁺-permeable channels required for Ca²⁺ influx into the cytoplasm. Received: 24 October 1997 / Accepted: 6 March 1998     

Influence of minerals on cytoplasmic streaming in root hairs of intact wheat seedlings (Triticum aestivum L.)

The calcium dependency of the cytoplasmic streaming of wheat root hairs was demonstrated by adding the Ca-Ionophore A 23187. Within three minutes the streaming velocity was decreased dramactically. The influence of ammonium on the cytoplasmic streaming is highly pH-dependent. While at a pH of 9.0 an inhibitory effect was observed even at low ammonium concentrations (0.5 mM) no effect could be measured at a pH of 6.5. Nitrate, independently of medium pH had no effect on the cytoplasmic streaming. The same is true for aluminium. It is suggested that at pH 9 ammonium permiates the plasmalemma as NH₃. Due to higher cytoplasmic pH ( 7.5), NH₃ is protonated leading to an increase in cytoplasmic pH. Ammonium may displace sorbed calcium leading to an increase in the free cytoplasmic calcium responsible for the cessation of the streaming. Alternative explanations are discussed.Abbreviations HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid     

Micro-vesicles,pyriform vesicles and macro-vesicles associated with the plasma membrane in the root hairs ofVicia hirsuta after freeze-substitution

After freeze-substitution, micro-vesicles were found only in close proximity to the plasma membrane. Macro and pyriform vesicles were found throughout the cytosol, but also ‘packaged’ close to the plasma membrane, the package delineated by electron transparent outlines similar to the endoplasmic reticulum. These outlines appeared to be continuous with nearby endoplasmic reticulum and were always associated with Golgi bodies and microtubules. Micro-vesicles were found only in grazing sections of the plasma membrane made between the apical dome and the region of the nucleus, where the cell is the most cytoplasmic, and only in close proximity to the plasma membrane. Micro-vesicles were also found in close proximity to microtubules as well as other vesicle types. From the results it is suggested that pyriform and micro-vesicles may have specialised roles in root hair tip growth.     

Control of cytoplasmic pH under anoxic conditions and its implication for plasma membrane proton transport in Medicago sativa root hairs

In root hairs of Medicago sativa, pH-sensitive microelectrodeshave been applied to study cytoplasmic pH-regulation. To inhibitorslike oligomycin, antimycin A, cyanide and the exchange of O₂for N_2, the root hairs respond with a distinct cytoplasmic acidification.Whereas the cytoplasmic pH under aerobic conditions rests at7.28 0.11 SE (n = 168), under conditions of (chemical) anoxiathe cytoplasmic pH is shifted to a stable, well-regulated 6.78 0.08 SE (n = 81). Once this pH is attained in the presenceof one inhibitor, addition of another has no effect. 2-deoxyglucoseand N-acetylglucosamine, both inhibitors of glycolysis at thehexokinase level, increase cytoplasmic pH by about 0.3 pH units,as do glucogenic amino acids. It is suggested that aerobic energymetabolism does not contribute to acidosis of these cells. SincepH-shift and pump deactivation can be separated by using poorrespiratory inhibitors, it is concluded that the switch from‘aerobic’ to ‘anaerobic’ pH is not correlatedwith proton pump activity. Inversely, since cytoplasmic pH neitherresponds to pump activation by FC with alkalinization, nor topump deactivation by cyanide with acidification, it is alsoconcluded that changes in pump activity do not affect cytoplasmicpH. Key words: Cytoplasmic pH regulation, Medicago, pH-sensitive microelectrodes, proton transport, root hairs     

Inhibitory role of root hairs on transport within root culture bioreactors

An experimental system was developed to produce root cultures of Hyoscyamus muticus with and without the profuse root hairs. Growth in the presence of 7.6 microM pyrene butyric acid (PBA) and 2.2 mM phosphate virtually eliminated root hairs, whereas growth rate, general morphology and nutrient yields remained unchanged in well-mixed flask culture. These root cultures were used to demonstrate decreased flow resistance in a tubular reactor as a result of root hair removal. To assess the impact on bioreactor performance, hairy and hairless root cultures were grown in a highly characterized 15-L bubble column bioreactor. In the absence of root hairs, the mixing was greatly enhanced; mixing times became shorter for the hairless culture at roughly 100 g (fresh weight)/L. By the end of the 3-week culture period, the mixing time of the hairy culture was 29 times longer than that of the hairless culture. The growth rate of the hairless culture in the bioreactor was as much as 2.4 times greater than growth of the hairy culture under the same conditions. The improved reactor performance was reflected in greater biomass accumulation and respiratory activity. These results show that the root hairs-which facilitate nutrient uptake in a static soil environment-are detrimental to growth in a liquid environment as an effect of their stagnating fluid flow and limiting oxygen availability.