Growth, Gravitropism, and Endogenous Ion Currents of Cress Roots (Lepidium sativum L.) : Measurements Using a Novel Three-Dimensional Recording Probe

A novel, three-dimensional recording, vibrating probe was used for measuring the density and direction of the endogenous ionic current of cress roots (Lepidium sativum L.) bathed in low salt media (artificial pond water, APW). Roots submerged in regular APW and growing vertically show the following current pattern. Current of 0.7 microampere/square centimeter density enters or leaves the root cap; the current changes direction frequently. Current of 1.6 microamperes/square centimeter enters the meristem zone most of the time. Maximum current with a density of 2.2 microamperes/square centimeter enters the apical elongating zone, i.e. between 0.8 and 1.2 millimeters behind the root tip. The current density decreases to 1.4 microamperes/square centimeter at 2 millimeters, i.e. in the central elongating zone, and to 1.0 microampere/square centimeter at 3 millimeters, i.e. in the basal elongating zone. The current direction changes from inward to predominantly outward between 1.2 and 3 millimeters behind the tip. Measurements on opposite flanks of the roots indicate that the current pattern is fairly symmetrical. After placing the roots horizontally, the density of the endogenous current remains stable, but the current direction changes at the root cap and in the meristem zone. The current leaves the root on the upper side and enters on the lower side, causing a highly asymmetrical current pattern at the very tip. The current pattern at the upper and lower side further away from the tip remains the same as in vertical roots. Roots submerged in low Ca²⁺ APW show a very different current pattern, no gravitropism, and no change of the current pattern after horizontal orientation. In these roots current enters the root cap and the basal elongating zone and leaves the apical elongating zone. Three conclusions are drawn from these results: First, plant roots elongate by two different modes of growth that are correlated with different current directions. They grow by cytoplasmic enlargement at sites of inward current and by turgor-driven elongation at sites of outward current. Second, a change in the current pattern at the root cap and in the meristem zone is a clear indicator of later gravitropism. Third, Ca²⁺ ions are involved in the gravistimulated change in the current pattern, probably affecting the activity of plasmalemma H⁺-ATPases.     

The spatially variable inhibition by water deficit of maize root growth correlates with altered profiles of proton flux and cell wall pH

Growth of elongating primary roots of maize (Zea mays) seedlings was approximately 50% inhibited after 48 h in aerated nutrient solution under water deficit induced by polyethylene glycol 6000 at -0.5 MPa water potential. Proton flux along the root elongation zone was assayed by high resolution analyses of images of acid diffusion around roots contacted for 5 min with pH indicator gel. Profiles of root segmental elongation correlated qualitatively and quantitatively (r(2) = 0.74) with proton flux along the surface of the elongation zone from water-deficit and control treatments. Proton flux and segmental elongation in roots under water deficit were remarkably well maintained in the region 0 to 3 mm behind the root tip and were inhibited from 3 to 10 mm behind the tip. Associated changes in apoplastic pH inside epidermal cell walls were measured in three defined regions along the root elongation zone by confocal laser scanning microscopy using a ratiometric method. Finally, external acidification of roots was shown to specifically induce a partial reversal of growth inhibition by water deficit in the central region of the elongation zone. These new findings, plus evidence in the literature concerning increases induced by acid pH in wall-extensibility parameters, lead us to propose that the apparently adaptive maintenance of growth 0 to 3 mm behind the tip in maize primary roots under water deficit and the associated inhibition of growth further behind the tip are related to spatially variable changes in proton pumping into expanding cell walls.     

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.     

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.     

Rapid effects of indoleacetic Acid and ethylene on the growth of intact pea roots

Root auxanometers were used to determine the growth rates of individual intact primary roots accurately and quickly. The growth of pea (Pisum sativum L.) roots was inhibited by both indoleacetic acid and ethylene within 20 minutes. A supramaximal concentration of ethylene inhibited root growth less than did 5 to 20 mum indoleacetic acid, indicating that inhibition of root growth by auxin was not due only to indoleacetic acid-induced ethylene production. Inhibition of root growth was largely relieved within 60 minutes of removal of both growth regulators.     

Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

Apoplast acidification associated with growth is well documented in roots, coleoptiles, and internodes but not in leaves. In the present study, advantage was taken of the high cuticle permeability in the elongation zone of barley leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H⁺-ATPase (PM-H⁺-ATPase) and K⁺ in this process was of particular interest. pH microelectrodes and an in vitro gel system with bromocresol purple as pH indicator were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer. Test reagents that blocked (vanadate) or stimulated (fusicoccin) PM-H⁺-ATPase or that reduced (Cs⁺, tetraethylammonium) K⁺ uptake were applied. Apoplast pH was lower in growing than in nongrowing leaf tissue and increased in the elongation zone with increasing apoplast K⁺. Vanadate increased apoplast pH and reduced growth, whereas fusicoccin caused the opposite effects. It is concluded that barley leaves exhibit acid-growth-type mechanisms in that apoplast pH is lower in elongating leaf tissue. Both growth and apoplast pH depend on the activity of the PM-H⁺-ATPase and K⁺ transport processes. However, not all of the growth displayed by leaves is dependent on a lower apoplast pH in the elongation zone; up to 50 % of growth is retained when apoplast pH in the elongation zone increases to a value observed in mature tissue.     

Proton Pumping in Growing Part of Maize Root: Its Correlation with 14-3-3 Protein Content and Changes in Response to Osmotic Stress

The spatial pattern of mitotic activity, cell elongation, rate of H+ fluxes, and 14-3-3 protein content were determined in Zea mays roots. We found that the regions along the apical part of the growing root conversely differ in their proton pumping activity. Higher rate of H+ efflux coincides with higher growth rate and correlates with increased 14-3-3 protein content in membrane preparations. The segment consisting of the root cap and the apical part of the meristem exerts net inward proton pumping, which can be inverted under fusicoccin treatment or osmotic stress. In the latter case, this inversion is accompanied by accumulation of 14-3-3 protein in plasma membranes. The results obtained highlight 14-3-3 protein as an obvious candidate for the fine regulation of plasma membrane H+-ATPase in root apex.     

Growth, membrane potential and endogenous ion currents of willow (Salix viminalis) roots are all affected by abscisic acid and spermine

Growth measurements of hormone-treated roots from willow cuttings were combined with electrophysiological recordings to study hormone-induced changes in membrane potential and in endogenous ion currents. The mean growth rate of roots was 10 ± 2 μm min^?1 in regular nutrient solution. It increased to 13 ± 2 μm min⁺¹ after application of spermine and decreased to 0.07 ± 0.01 μm min^?1 after treatment with abscisic acid (ABA). Transient depolarizations were elicited in root cortex cells by spermine, while ABA caused a transient hyperpolarization. All changes in membrane potential were accompanied by transient responses of the endogenous current. These responses suggest that first anions, then cations leave the root during spermine-induced depolarizations. From the changes of the endogenous current an apparent efflux of anions (presumably Cl^?) and cations (presumably K⁺) of 200 to 700 pmol cm^?2 per depolarization was calculated. To further investigate a possible relation between endogenous ion currents, growth and the growth regulators ABA and spermine, long-lasting extracellular vibrating-probe measurements were performed. Control roots showed an inward current of about 1.5 μA cm^?2 at the apical elongation zone and an outward current with a maximum density of 1.3 μA cm^?2 at the central and basal elongation zone. The addition of ABA and spermine (final concentration 0.1 mM) to the bathing medium affected the endogenous current in opposite ways: ABA caused a reduction of inward and outward current, while spermine stimulated both. Since protons are a major component of the endogenous current, and sucrose can be taken up by root cells from the apoplast via symport with H⁺, a role of the endogenous current in growth regulation is indicated.     

Gradients in maize roots: local elongation and pH

The elongation rate, the gradient of the local elongation rate and the surface pH of maize roots were measured over 12 h. A data bank was constituted by storing these values. By sorting these results on the basis of different elongation rates, different classes of root were obtained. Two classes were chosen: the low-growth roots and the high-growth roots. The mean growth of these two root classes was stable with time and differed significantly from one another. The surface pH of the elongation zone was the same for the roots of these two classes, but the roots selected for their higher growth rate had a larger acid efflux in this zone.     

The effects of electric field on the growth of intact seedlings and coleoptile segments ofZea mays L.

The experiments were carried out with 96-h-old intact maize seedlings and 10 mm long coleoptile segments cut 4 mm below the tip. The electric fields were applied longitudinally along the seedlings. The electric field (15 V) caused inhibition of the elongation growth of intact seedlings which was dependent on both the polarity and the duration of the applied voltage. The growth inhibition was greater when the tip of the shoot was positive relative to the roots. The electric field also caused inhibition of indole-3-acetic acid (IAA) and fusicoccin (FC) induced growth of maize coleoptile segments excised from electrically treated seedlings. IAA-induced growth of coleoptile segments was greater when the tip of the shoot was negative to the roots (not in the case of FC-treated segments and intact seedlings). It was suggested that apart from the changes induced by electric field in transport system of auxin the electric field affected also the activity of plasmalemma proton pump.     

Relationship between iron chlorosis and alkalinity in Zea mays

Mengel, K. and Geurtzen, G. 1988. Relationship between iron chlorosis and alkalinity in Zea mays . - Physiol. Plant. 72: 460–465.
Maize ( Zea mays L. cv. Anjou 21) grown in nutrient solution with Fe-EDTA and with nitrate as the sole nitrogen source showed typical Fe-chlorosis symptoms after a growth period of 14–21 days. Alkalinity in roots, stems and leaves of the chlorotic plants was high. Transferring the chlorotic plants from the nitrate-containing nutrient solution to a solution of (NH₄)₂SO₄ resulted in a regreening of leaves within 2–3 days which was associated with a decrease in solution pH, a decrease in alkalinity of plant parts, a translocation of Fe from roots to tops and a release of Fe into the outer solution. Similar effects were obtained when Fe chlorotic plants were transferred to a dilute HO solution with pH 3.5.
Spraying chlorotic leaves with indoleacetic acid or with fusicoccin led also to a regreening of leaves without having a major effect on leaf alkalinity.
Interpretation of the experimental results is based on the assumption that nitrate as sole N source leads to a high pH level in the apoplast resulting in the precipitation of Fe compounds, probably Fe oxide hydrate. Ammonium nutrition has the reverse effect since it lowers the apoplast pH and this can result in the dissolution of Fe compounds. Application of indoleacetic acid as well as fusicoccin supposedly stimulates the proton pumps in the plasmalemma of the leaf tissue. The resulting decrease in apoplast leaf pH in the microenvironment also leads to a dissolution of Fe compounds in the apoplast and thus promotes the uptake of Fe by the symplasm.     

Characteristics and implications of prolonged fusicoccin-induced growth of Avena coleoptile sections

A study has been made of the prolonged growth of Avena coleoptile sections in response to fusicoccin (FC), a phytotoxin that promotes apoplastic acidification. The final amount of FC-induced growth is a function of the FC concentration. Removal of the epidermis speeds up the initial rate of elongation and shortens the duration of the response, without affecting the total amount of extension. A suboptimal FC concentration (7×10⁻⁸ M ) which induces the same rate of proton excretion as does optimal indoleacetic acid (IAA) (1×10⁻⁵ M ), causes elongation which is 60–75% of that induced by IAA in 4 h or 50–65% in 7 h. This suggests that acid-induced extension could make a major contribution to auxin-induced growth for at least 7 h.     

Differential growth and hormone redistribution in gravireacting maize roots

When growing roots are placed in a horizontal position gravity induces a positive curvature. It is classically considered to be the consequence of a faster elongation rate by the upper side compared to the lower side. A critical examination indicates that the gravireaction is caused by differential cell extension depending on several processes. Some of the endogenous regulators which may control the growth and gravitropism of elongating roots are briefly presented. The growth inhibitors produced or released from the root cap move preferentially in a basipetal direction and accumulate in the lower side of the elongation zone of horizontally maintained roots. The identity of these compounds is far from clear, but one of these inhibitors could be abscisic acid (ABA). However, indol-3y1 acetic acid (IAA) is also important for root growth and gravitropism. ABA may interact with IAA. Two other aspects of root cell extension have also to be carefully considered. An elongation gradient measured from the tip to the base of the root was found to be important for the growth of both vertical and horizontal gravireactive roots. It was changed significantly during the gravipresentation and can be considered as the origin of the differential elongation. Sephadex beads have been used as both growth markers and as monitors of surface pH changes when they contain some pH indicator. This technique has shown that the distribution of cell extension along the main root axis is related to a pH gradient, the proton efflux being larger for faster growing parts of roots. A lateral movement of calcium is obtained when Ca2+ is applied across the tips of horizontally placed roots with a preferential transport towards the lower side. Endogenous calcium, which may accumulate inside the endoplasmic reticulum of some cap cells, may also act in the gravireception. These observations and several others strongly suggest that calcium may play an essential role in controlling root growth and several steps of the root gravireaction.     

Do pH changes in the leaf apoplast contribute to rapid inhibition of leaf elongation rate by water stress? Comparison of stress responses induced by polyethylene glycol and down‐regulation of root hydraulic conductivity

We have dissected the influences of apoplastic pH and cell turgor on short-term responses of leaf growth to plant water status, by using a combination of a double-barrelled pH-selective microelectrodes and a cell pressure probe. These techniques were used, together with continuous measurements of leaf elongation rate (LER), in the (hidden) elongating zone of the leaves of intact maize plants while exposing roots to various treatments. Polyethylene glycol (PEG) reduced water availability to roots, while acid load and anoxia decreased root hydraulic conductivity. During the first 30 min, acid load and anoxia induced moderate reductions in leaf growth and turgor, with no effect on leaf apoplastic pH. PEG stopped leaf growth, while turgor was only partially reduced. Rapid alkalinization of the apoplast, from pH 4.9 ± 0.3 to pH 5.8 ± 0.2 within 30 min, may have participated to this rapid growth reduction. After 60 min, leaf growth inhibition correlated well with turgor reduction across all treatments, supporting a growth limitation by hydraulics. We conclude that apoplastic alkalinization may transiently impair the control of leaf growth by cell turgor upon abrupt water stress, whereas direct hydraulic control of growth predominates under moderate conditions and after a 30-60 min delay following imposition of water stress.     

Does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification?

The reduction in growth of maize (Zea mays L.) seedling primary roots induced by salinization of the nutrient medium with 100 millimolar NaCl was accompanied by reductions in the length of the root tip elongation zone, the length of fully elongated epidermal cells, and the apparent rate of cell production: Each was partially restored when calcium levels in the salinized growth medium were increased from 0.5 to 10.0 millimolar. We investigated the possibility that the inhibition of elongation growth by salinity might be associated with an inhibition of cell wall acidification, such as that which occurs when root growth is inhibited by IAA. A qualitative assay of root surface acidification, using bromocresol purple pH indicator in agar, showed that salinized roots, with and without extra calcium, produced a zone of surface acidification which was similar to that produced by control roots. The zone of acidification began 1 to 2 millimeters behind the tip and coincided with the zone of cell elongation. The remainder of the root alkalinized its surface. Kinetics of surface acidification were assayed quantitatively by placing a flat tipped pH electrode in contact with the elongation zone. The pH at the epidermal surfaces of roots grown either with 100 millimolar NaCl (growth inhibitory), or with 10 millimolar calcium ± NaCl (little growth inhibition), declined from 6.0 to 5.1 over 30 minutes. We conclude that NaCl did not inhibit growth by reducing the capacity of epidermal cells to acidify their walls.     

Changes in IAA responsiveness in the elongation region of graviresponding mung bean roots

IAA responsiveness of sections of root tissue taken from the top and bottom of mung bean roots was assessed prior to and at varying times following gravistimulation. Prior to gravistimulation, root tissue sections from the sides of the elongation zone responded similarly to IAA. After gravistimulation (within 5 min), root sections from the bottom of the elongation zone became more responsive to IAA than sections collected from the upper side of the elongation zone. The change in IAA responsiveness of these tissue sections was transient with root sections from both the top and bottom of the elongation zone again exhibiting similar responsiveness to IAA following 15 minutes of gravistimulation.These studies also examined if the root tip is required for the gravity-induced shift in IAA responsiveness in the tissues of the elongation zone. The IAA responsiveness of top and bottom sections of the elongation zone from decapped mung bean roots was assessed at varying times following gravistimulation. The responsiveness to IAA of top and bottom sections changed rapidly in decapped roots, just as had been previously found for intact roots. Although the alteration in responsiveness was transient in decapped roots (just as intact roots), the time it took for the sections to recover previous responsiveness to IAA was extended.These results suggest that the initial growth response of graviresponding roots may be due to a change in the IAA responsiveness of tissues in the elongation zone and not an asymmetric accumulation of IAA on the lower side of the elongation zone. The results also indicate that the gravity-induced shift in IAA responsiveness in the elongation zone occurs independently of the root cap, suggesting that the cells in the elongation region can perceive and respond to gravity independently of the root cap during the intial phases of the gravity response.     

A Two-Dimensional Vibrating Probe Study of Currents around Lateral Roots of Raphanus sativus Developing in Culture

A computer-assisted, two-dimensional vibrating probe was used to study the ionic currents around developing lateral roots of Raphanus sativus in vitro. This system allowed us to superimpose current vectors on the video image of the roots. In a young lateral root, current entered the cap, meristematic, and elongation zones and exited the primary root surface close to the base of the lateral root. As the lateral root grew, current began to exit from its basal (cell maturation zone) end. The densities of currents entering the apical portion of the faster-growing lateral roots in a medium lacking indole 3-acetic acid were about twice as large as those entering the apical region of the slower-growing lateral roots in indole 3-acetic acid-supplemented medium.     

Acid-induced wall loosening is confined to the accelerating region of the root growing zone

The aims of this study were to quantify developmental differences in acid growth along the root axis and to determine whether these differences were due to alterations in cell turgor or cell wall properties. The apoplast pH of maize roots growing in hydroponics was altered from pH 7.0 to pH 3.4 using 2 mol m^-3 citrate-phosphate buffer or unbuffered solutions. Whole root elongation rate rapidly increased and measurement of the local growth profile indicated that this increase in growth occurred in young cells in the accelerating zone (apical 0-4 mm) while more proximal growing cells were unaffected. Unbuffered solutions of identical pH produced qualitatively similar results. Single cell turgor pressures were unchanged between pH treatments both longitudinally and radially in the root tip. This suggests that the rapid acid-induced changes in growth rate were due to an increase in cell wall loosening. Single cell osmotic pressure and water potential were not significantly different between pH treatments. Acid pH caused net solute import at the root tip to increase 3- to 4-fold, which, coupled with the maintenance of turgor and osmotic pressure, indicated that solute import was not limiting expansion. Thus, acidic solutions cause an increase in growth in accelerating but not decelerating regions. It has been shown for the first time that acid growth in intact, growing roots is not due to differences in turgor, assigning these changes to cell wall properties. Possible cell wall biochemical alterations are discussed.     

Inhibition of Auxin Movement from the Shoot into the Root Inhibits Lateral Root Development in Arabidopsis

In roots two distinct polar movements of auxin have been reported that may control different developmental and growth events. To test the hypothesis that auxin derived from the shoot and transported toward the root controls lateral root development, the two polarities of auxin transport were uncoupled in Arabidopsis. Local application of the auxin-transport inhibitor naphthylphthalamic acid (NPA) at the root-shoot junction decreased the number and density of lateral roots and reduced the free indoleacetic acid (IAA) levels in the root and [³H]IAA transport into the root. Application of NPA to the basal half of or at several positions along the root only reduced lateral root density in regions that were in contact with NPA or in regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root development in Arabidopsis roots was also inhibited by excision of the shoot or dark growth and this inhibition was reversible by IAA. Together, these results are consistent with auxin transport from the shoot into the root controlling lateral root development.     

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.