Ionic Current Changes Associated with the Gravity-Induced Bending Response in Roots of Zea mays L

A vibrating probe was used to measure the changes in ionic currents around gravistimulated roots of Zea mays L. in an effort to determine whether these currents are involved in stimulus transduction from the root cap to the elongation zone. We did not observe a migration of the previously reported auxin-insensitive current efflux associated with gravity sensing (T. Björkman, A.C. Leopold [1987] Plant Physiol 84:841-846) back from the root cap. Instead, beginning 10 to 15 min after gravistimulation, an asymmetry in current developed simultaneously along the root around the meristem and apical regions of the elongation zone. This asymmetry comprised a proton efflux from the upper surface, which was superimposed on the symmetrical pattern around the vertical root. The gravity-induced proton efflux was inhibited by the application of the auxin transport inhibitor, 2,3,5-triiodobenzoic acid, whereas the calcium channel blocker, lanthanum, had little effect. Because the onset of the gravity-induced current asymmetry coincided both spatially and temporally with the onset of the differential growth response, we suggest that this current efflux may result from auxin-requiring acid-growth phenomena in the upper root tissue. The implications of this simultaneous onset of both proton efflux and elongation for theories about gravity stimulus transduction are discussed.     

Relation of growth process to spatial patterns of electric potential and enzyme activity in bean roots

The electric spatial pattern and invertase activity distribution in growing roots of azuki bean (Phaseolus chrysanthos) have been studied. The electric potential near the surface along the root showed a banding pattern with a spatial period of about 2 cm. It was found that the enzyme activity has a peak around 3-7 mm from the root tip, in good agreement with the position of the first peak of the electric potential, which is located a little behind the elongation zone. An inhomogeneous distribution of ATP content was also detected along the root. Experiments on the electric isolation of the elongation zone from the mature zone and acidification treatment showed that H+ is transported from the mature-side to elongation-side regions, causing tip elongation through an acid-growth mechanism. Both acidification and electric disturbance on growing roots affected growth significantly. Simultaneous measurements of electric potential and enzyme activity clearly showed a good correlation between these two quantities and growth speed. From an analogy with the Characean banding, the spatio-temporal organization via the cell membrane in electric potential and enzyme activity can be regarded as a dissipative structure arising far from equilibrium. These experimental results can be interpreted with a new mechanism that the dissipative structure is formed spontaneously along the whole root, accompanied by energy metabolism, to make H+ flow into the root tip.     

Effect of cadmium on the distribution of hydroxyl radical, superoxide and hydrogen peroxide in barley root tip

In the present study, we investigated the alteration of reactive oxygen species production along the longitudinal axis of barley root tips during Cd treatment. In unstressed barley root tips, H₂O₂ production decreased from the root apex towards the differentiation zone where again, a slight increase was observed towards the more mature region of root. An opposite pattern was observed for O ₂ ^?? and OH^? generation. The amount of both O ₂ ^?? and OH^? was highest in the elongation zone, decreased in the root apex and at the differentiation zone of root, then increased again towards the more mature region of root. An elevated Cd-induced O ₂ ^?? production started in the elongation zone and increased further along the differentiation zone of barley root tip. In contrast, Cd-induced H₂O₂ production was localised to the root elongation zone and to the beginning of the differentiation zone. In contrast to Cd-induced H₂O₂ and O ₂ ^?? production, Cd reduced OH^? production along the whole barley root tip. Our results suggest that not only an increase but also the spatial distribution of reactive oxygen species production is involved in the Cd-induced stress response of barley root tip.     

Microelectrode ion and O2 fluxes measurements reveal differential sensitivity of barley root tissues to hypoxia

Hypoxia-induced changes in net H+, K+ and O2 fluxes across the plasma membrane (PM) of epidermal root cells were measured using the non-invasive microelectrode ion flux measurement (MIFE) system in elongation, meristem and mature root zones of two barley (Hordeum vulgare L.) varieties contrasting in their waterlogging (WL) tolerance. The ultimate goal of this study was to shed light on the mechanisms underlying effects of WL on plant nutrient acquisition and mechanisms of WL tolerance in barley. Our measurements revealed that functionally different barley root zones have rather different O2 requirements, with the highest O2 influx being in the elongation zone of the root at about 1 mm from the tip. Oxygen deprivation has qualitatively different effects on the activity of PM ion transporters in mature and elongation zones. In the mature zone, hypoxic treatment caused a very sharp decline in K+ uptake in the WL sensitive variety Naso Nijo, but did not reduce K+ influx in the WL tolerant TX9425 variety. In the elongation zone, onset of hypoxia enhanced K+ uptake from roots of both cultivars. Pharmacological experiments suggested that hypoxia-induced K+ flux responses are likely to be mediated by both K(+) -inward- (KIR) and non-selective cation channels (NSCC) in the elongation zone, while in the mature zone K(+) -outward- (KOR) channels are the key contributors. Overall, our results suggest that oxygen deprivation has an immediate and substantial effect on root ion flux patterns, and that this effect is different in WL-sensitive and WL-tolerant cultivars. To what extent this difference in ion flux response to hypoxia is a factor conferring WL tolerance in barley remains to be answered in future studies.     

Hydrogen Ion Entry as a Controlling Factor in the Acid-growth Response of Green Pea Stem Sections

The ability of green pea (Pisum sativum var. Alaska) stem sections to elongate in response to H⁺ has been reinvestigated. Contrary to the conclusions of Barkley and Leopold, (Plant Physiol. 1973. 52: 76-78) these sections elongate in response to H⁺ whenever H⁺ entry through the cuticle is facilitated by slits, holes, or removal. Both live and frozen-thawed sections can undergo acid-induced elongation. Green pea stems behave as predicted by the acid-growth theory.     

Rapid Changes in the Pattern of Electric Current around the Root Tip of Lepidium sativum L. following Gravistimulation

Using a highly sensitive vibrating electrode, the pattern of naturally occurring electric currents around 1-day-old primary roots of Lepidium sativum L. growing vertically downward and the current pattern following gravistimulation of the root has been examined. A more or less symmetrical pattern of current was found around vertically oriented, downward growing roots. Current entered the root at the root cap, the meristem, and the beginning of the elongation zone and left the root along most of the elongation zone and in the root hair zone. After the root was tilted to a horizontal position, we observed current flowing acropetally at the upper side of the root cap and basipetally at the lower side within about 30 seconds in most cases. After a delay of several minutes, acropetally oriented current was also found flowing along the upper side of the meristematic zone. The apparent density of the acropetal current in the root cap region increased and then decreased with time. Gravitropic curvature was first visible approximately 10 minutes after tilting of the root to the horizontal position. Since the change in the pattern of current in the root cap region precedes bending of the root and is different for the upper and lower side, a close connection is suggested between the current and the transduction of information from the root cap to the elongation zone following graviperception in the cap.     

Effect of trifluralin on the histology and respiration ofAllium cepa root tips

Trifluralin was found to induce isodiametric enlargement ofAllium cepa root tip cells in the elongation zone. It enhanced the endogenous oxygen uptake of the excised root tips. The herbicide was found to have no marked effect on the dry weight of the root tips. It is suggested that the herbicide may exert its effect onAllium cepa root tips through its enhancement of ethylene production.     

Elongation of barley roots in high‐pH nutrient solution is supported by both cell proliferation and differentiation in the root apex

Many crops grow well on neutral or weakly acidic soils. The ability of roots to elongate under high‐external pH would be advantageous for the survival of plants on alkaline soil. We found that root elongation was promoted in some plant species in alkaline‐nutrient solution. Barley, but not tomato, root growth was maintained in pH 8 nutrient solution. Fe and Mn were absorbed well from the pH 8 nutrient solution by both barley and tomato plants, suggesting that the different growth responses of these two species may not be caused by insolubilization of transition metals. The ability of intact barley and tomato plants to acidify external solution was comparable; in both species, this ability decreased in plants exposed to pH 8 nutrient solution for 1 w. Conversely, cell proliferation and elongation in barley root apices were facilitated at pH 8 as shown by microscopy and cell‐cycle‐related gene‐expression data; this was not observed in tomato. We propose that barley adapts to alkaline stress by increasing root development.     

Differential Responsiveness of Cortical Microtubule Orientation to Suppression of Cell Expansion among the Developmental Zones of Arabidopsis thaliana Root Apex

Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone.     

Short-term aluminium-induced changes in barley root tips

The short-term exposure of barley roots to low Al concentration caused significant root growth inhibition and radial swelling of roots. During Al treatment, the radial expansion of root cells occurred in root tissues representing elongation zone and meristem. Both low pH and Al treatments caused significant disruption of cell membranes in swollen roots. In contrast to Evans blue uptake callose formation was observed only at higher Al concentrations and was detected in both swollen and adjacent root areas. Similarly to Al, exogenous short-term application of indole-3-acetic acid, polar transport inhibitor triiodobenzoic acid, ethylene precursor 1-aminocyclopropane-1-carboxylic acid or H₂O₂ evoked root growth inhibition and radial cell expansion in barley root tip too.     

Evidence for the acid-growth theory of fusicoccin action

Three predictions of the acid-growth theory of fusicoccin (FC) action in inducing cell elongation were reinvestigated using abraded segments of maize (Zea mays L.) coleoptiles. i) Quantitative comparison of segment elongation and medium-acidification kinetics measured in the same sample of tissue shows that these FC-induced processes are strictly correlated in time and respond coordinately to cations present in the medium. ii) Fusicoccin (1 mol l^-1) induces a rapid acidification of the cell-wall solution, reaching a final level of pH 3.8–4.0. Exogenous protons are able to substitute quantitatively for FC in causing segment elongation at pH 3.8–4.0. At pH 4, FC has no additional effect on cell elongation. iii) Neutral buffers (pH 7) completely abolish the FC-mediated growth response. iv) Cycloheximide (10 mg l^-1) inhibits both FC-induced and acid-buffer(pH 4)-induced elongation after a lag of 40–45 min, and FC-induced H⁺ excretion after a lag of 2 h. Under the same conditions, indole-3-acetic acid-induced elongation and H⁺ excretion are inhibited without detectable lag. It is concluded that these results are fully compatible with the acid-growth theory of FC action.Abbreviations IAA indole-3-acetic acid - CHI cycloheximide - FC fusicoccin     

Spatial distribution and speciation of copper in root tips of cucumber revealed by μ-XRF and μ-XANES

The localization, biotransformation, and chemical speciation of copper in root tips of cucumber (Cucumis sativus) were investigated using synchrotron-based micro X-ray fluorescence (μ-XRF) and micro X-ray absorption near edge structure (μ-XANES). The highest content of Cu was found in root cap and meristematic zone whereas low Cu content in elongation and maturation zone. There was a dramatic increase of Cu content in root cap and meristematic zone after treatment with 100 μM CuSO₄ for 72 h. The μ-XANES analysis revealed that most of Cu in root tip was bound with alginate, citrate, and cysteine-like ligands whereas rarely deposited in form of CuO. From root cap to maturation zone, the proportion of Cu bound with alginate-like ligands increased whereas that bound with citrate-like ligands decreased. The proportion of Cu bound with cysteine-like ligands increased from root cap to elongation zone but sharply declined in maturation zone. The results suggested that Cu was chelated by S ligands in the cell walls which protect protoplasm against possible damage caused by Cu excess.     

The Root Apex of Arabidopsis thaliana Consists of Four Distinct Zones of Growth Activities: Meristematic Zone,Transition Zone,Fast Elongation Zone and Growth Terminating Zone

In the growing apex of Arabidopsis thaliana primary roots, cells proceed through four distinct phases of cellular activities. These zones and their boundaries can be well defined based on their characteristic cellular activities. The meristematic zone comprises, and is limited to, all cells that undergo mitotic divisions. Detailed in vivo analysis of transgenic lines reveals that, in the Columbia-0 ecotype, the meristem stretches up to 200 µm away from the junction between root and root cap (RCJ). In the transition zone, 200 to about 520 µm away from the RCJ, cells undergo physiological changes as they prepare for their fast elongation. Upon entering the transition zone, they progressively develop a central vacuole, polarize the cytoskeleton and remodel their cell walls. Cells grow slowly during this transition: it takes ten hours to triplicate cell length from 8.5 to about 35 µm in the trichoblast cell files. In the fast elongation zone, which covers the zone from 520 to about 850 µm from the RCJ, cell length quadruplicates to about 140 µm in only two hours. This is accompanied by drastic and specific cell wall alterations. Finally, root hairs fully develop in the growth terminating zone, where root cells undergo a minor elongation to reach their mature lengths.Key words: Arabidopsis, cytoskeleton, development, differentiation zone, elongation zone, growth, growth terminating zone, meristem, root apex, transition zone     

Enhancement of auxin sensitivity in Ranunculus sceleratus by ethylene: A mechanism to escape from hypoxia under temporary submergence

We analyzed auxin-induced and ethylene-enhanced elongation of petiole segments in Ranunculus sceleratus L. The early time course of elongation in petiolar segments was monitored with a computer-based video digitizer system. The application of ethylene-releasing ethrel slightly increased the elongation rate in the absence of IAA. When IAA alone was applied, elongation increased after a latent period of approximately 30 min. Maximal elongation rate was attained immediately after the latent period, and then the stabilized steady rate was recorded. During this phase, addition of ethrel strongly increased the elongation rate after a period of approximately 18 min. Although ethrel could acidify the growth medium, only a small part of the enhanced elongation was due to an acid-growth effect. Most of the growth stimulation was auxin-dependent and must be ascribed to the presence of ethylene. In the presence of ethrel, the log-concentration-response curve of IAA appeared to be shifted to the left. This kinetic analysis indicates an increase, due to ethylene, in the sensitivity of the R. sceleratus petiole to auxin, which results in inducing rapid growth to escape from hypoxia under temporary submergence.     

Constitutive autophagy in plant root cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.     

How cobalt facilitates cadmium- and ethylene precursor-induced growth inhibition and radial cell expansion in barley root tips

Significant root growth inhibition was observed during the very short 5 minute exposure time of barley roots to the low 10 μM concentration of cadmium. In addition to the cadmium-induced root growth inhibition, considerable radial expansion of roots was observed as a characteristic symptom of transient short-term exposure of roots to cadmium. The cadmium-induced radial expansion of roots was observed mainly the cortical cells of elongation zone that were twice as large as in control roots. Similarly as in cadmium-treated roots, short-term treatment with ACC significantly inhibited root growth and caused a marked radial expansion of cortical cells. The ethylene synthesis inhibitor cobalt significantly alleviated both the cadmium- and ethylene precursor-induced root growth inhibition and radial root expansion. The results indicate that ethylene probably plays a crucial role in the short-term cadmium-induced inhibition of root growth and radial cell expansion of barley root tips, which are the very early symptoms of cadmium toxicity.     

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL⁻¹ CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL⁻¹, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.     

Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip

Short-term treatment (30min) of barley roots with a low 10μM Cd concentration induced significant H(2)O(2) production in the elongation and differentiation zone of the root tip 3h after treatment. This elevated H(2)O(2) production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60μM Cd concentration induced robust H(2)O(2) production in the elongation zone of the root tip already 1h after short-term treatment. This robust H(2)O(2) generation caused extensive cell death 6h after short-term treatment. Similarly to low Cd concentration, exogenously applied H(2)O(2) caused marked root growth inhibition, which at lower H(2)O(2) concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10μM Cd-induced root growth inhibition, H(2)O(2) production and root swelling, but was ineffective in the alleviation of 60μM Cd-induced root growth inhibition and H(2)O(2) production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death.     

Effect of pH on IAA Uptake by Maize Root Segments

The uptake of [5-³H]indoleacetic acid (IAA) by Zea mays L. root segments involves nonsaturable and saturable processes. The pH optimum of the saturable component was found to be 5.0. The proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone inhibited at 100 micromolar the saturable component of IAA uptake but had no effect on non-saturable uptake. This indicates that the saturable component of IAA uptake is dependent on the proton gradient across the plasmalemma. The high level of proton extrusion in the elongation zone of the root will stimulate nonsaturable and saturable uptake of IAA in that zone.     

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.