Effect of temperature on growth,proton extrusion and membrane potential in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) coleoptile segments

The effects of temperature (5–45°C) on endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC), and proton extrusion in maize coleoptile segments were studied. In addition, membrane potential changes at some temperatures were also determined. It was found that in this model system endogenous growth exhibits a clear maximum at 30°C, whereas growth in the presence of IAA and FC shows the maximum value in the range 30–35°C and 35–40°C, respectively. Simultaneous measurements of growth and external medium pH indicated that FC at stressful temperatures was not only much more active in the stimulation of growth, but was also more effective in acidifying the external medium than IAA. Also the addition of either IAA or FC to the bathing medium at 30 and 40°C did not change the kinetic characteristic of membrane potential changes observed for both substances at 25°C. However, the increased temperature significantly decreased IAA and FC-induced membrane hyperpolarization. IAA in the incubation medium, at 10°C, brought about additional membrane depolarization (apart from the one induced by low temperature). In contrast to IAA, FC at 10°C caused gradual repolarization of membrane potential, which correlated with both FC-induced growth and FC-induced proton extrusion. A plausible interpretation for temperature-induced changes in growth of maize coleoptile segments is that, at least in part, these changes were mediated via a PM H⁺-ATPase activity.     

pH-Dependent accumulation of indoleacetic acid by corn coleoptile sections

The uptake of auxin by 1-mm slices of corn (Zea mays L.) coleoptiles, a tissue known to transport auxin polarly, depends on the pH of the medium. Short-term uptake of indole-3-acetic acid (IAA) in coleoptiles increases with decreasing pH of the buffer as would be expected if the undissociated weak acid, IAA·H, were more permeable than the auxin anion, IAA^-, and IAA^- accumulates in the tissues because of the higher pH of the cytoplasm. Although uptake of [³H]IAA is reduced in neutral buffers, it is greater than expected if it were limited to just the extracellular space of the tissue. The radioactivity accumulated by the tissue can be quantitatively extracted by organic solvents and identified as IAA by thin-layer chromatography. The tissue radioactivity is freely mobile and can efflux from the tissue. Thus these cells in pH 5 buffer are able to retain an average internal concentration of mobile IAA that is at least several times greater than the external concentration. A prominent feature of auxin uptake from acidic buffers is enhanced accumulation at high auxin concentration. This indicates that, in addition to fluxes of IAA·H, a saturable site is involved in auxin uptake. Whenever the auxin-anion gradient is directed outward, saturating the efflux of auxin anions increases accumulation. Furthermore, the observed slowing of short-term uptake of radioactive IAA by increasing concentrations of IAA or K⁺ indicates either an activation of the presumptive auxin leak or saturation of another carrier-mediated uptake system such as a symport of auxin anions with protons. By contrast in neutral buffers, effects of concentration on uptake rates disappear. This implies that at neutral pH the anion leak is decreased and influx depends on the symport.     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.     

Effects of peeling on the surface structure of the Avena coleoptile: Implications for hormone research

Coleoptiles of oats (Avena sativa L.) are often peeled in order to observe hormone-enhanced acidification of the external medium. It is shown by means of the scanning electron microscope that peeling largely removes a single layer of cells, the epidermis with its cuticle. Strips of intact and damaged epidermal cells remain, but most of the newly exposed surface is composed of cortical cells. The cortical face is relatively intact, except that some cells appear punctured and some are broken when a vascular bundle is pulled out with the epidermis. The surface of the cortex is covered by a thin film which is partially digested by 2% pectinase. The pectinase pretreatment also inhibits indoleacetic-acid- and fusicoccin-enhanced acidification. Thus, although peeling could be involved in proton extrusion, physical damage to the coleoptile cells per se does not seem to be the major stimulus leading to hormone-enhanced acidification.Abbreviations FC fusicoccin - IAA indole-3-acetic acid - SEM scanning electron microscope     

Cell-wall tension of the inner tissues of the maize coleoptile and its potential contribution to auxin-mediated organ growth

Plant organs such as maize (Zea mays L.) coleoptiles are characterized by longitudinal tissue tension, i.e. bulk turgor pressure produces unequal amounts of cell-wall tension in the epidermis (essentially the outer epidermal wall) and in the inner tissues. The fractional amount of turgor borne by the epidermal wall of turgid maize coleoptile segments was indirectly estimated by determining the water potential ^* of an external medium which is needed to replace quantitatively the compressive force of the epidermal wall on the inner tissues. The fractional amount of turgor borne by the walls of the inner tissues was estimated from the difference between -^* and the osmotic pressure of the cell sap (_i) which was assumed to represent the turgor of the fully turgid tissue. In segments incubated in water for 1 h, -^* was 6.1–6.5 bar at a _i of 6.7 bar. Both -^* and _i decreased during auxin-induced growth because of water uptake, but did not deviate significantly from each other. It is concluded that the turgor fraction utilized for the elastic extension of the inner tissue walls is less than 1 bar, i.e. less than 15% of bulk turgor, and that more than 85% of bulk turgor is utilized for counteracting the high compressive force of the outer epidermal wall which, in this way, is enabled to mechanically control elongation growth of the organ. This situation is maintained during auxin-induced growth.Abbreviations and Symbols _i osmotic pressure of the tissue - ₀ external water potential - ^* water potential at which segment length does not change - IAA indole-3-acetic acid - ITW longitudinal inner tissue walls - OEW outer epidermal wall - P turgor Supported by Deutsche Forschungsgemeinschaft (SFB 206).     

Galactose prevents proton excretion but not IAA-induced growth in segments of azuki bean epicotyls

We investigated the effect of galactose on IAA-induced elongation and proton excretion in azuki bean (Vigna angularis Ohwi et Ohashi) segments in order to confirm whether or not protons were involved in auxin-induced growth. Galactose inhibited the IAA-induced decrease in the solution pH but had no inhibitory effect on IAA-induced growth in segments of azuki bean epicotyls. On the other hand, galactose inhibited both IAA-induced growth and proton excretion in oat (Avena sativa L.) coleoptile segments. From these results it is unlikely that IAA-induced growth is mediated by proton excretion at least in azuki bean epicotyls.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

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     

Evidence against the acid-growth theory of auxin action

Four experimental predictions of the acid-growth theory of auxin (indole-3-acetic acid, IAA) 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 reveals that these IAA-induced processes are neither correlated in time nor responding coordinately to cations present in the medium. ii) Exogenous protons are not able to substitute for IAA in causing segment elongation at the predicted pH of 4.5–5.0. Instead, external buffers induce significant segment elongation only below pH 4.5, reaching a maximal response at pH 1.75–2.5. Acid and IAA coact additively, and therefore independently, in the whole range of feasible pH values. iii) Neutral or alkaline buffers (pH 6–10) are unable to abolish the IAA-mediated growth response and have no effect on its lag-phase. iv) Fusicoccin, at a concentration producing the same H⁺ excretion as high concentrations of IAA, is ineffective in inducing segment elongation. Moreover, sucrose and other sugars can quantiatively substritute for IAA in inducing H⁺ excretion but are likewise ineffective in inducing elongation. It is concluded that these results are incompatible with the acid-growth theory of auxin action.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

Auxin-induced changes in the population of translatable messenger RNA in elongating maize coleoptile sections

In-vitro translation products of polyadenylated RNA from untreated and indole-3-acetic acid (IAA)-treated elongating sections of maize (Zea mays L.) coleoptiles were analyzed by twodimensional polyacrylamide gel electrophoresis. Treatment with IAA results in an increased amount of at least four in-vitro translation products. The amounts of two of these translation products are increased within 10 min of IAA treatment.Abbreviation IAA indole-3-acetic acid     

Distribution and redistribution of extension growth along vertical and horizontal gravireacting maize roots

Horizontal primary roots of Zea mays L. were photographed during the course of their gravireaction and during a preceding growth period in the vertical orientation. The displacement, by root elongation, of marker particles on the root surface was recorded. The particle-displacement rates were used to estimate the distribution of elemental elongation rates along opposite sides of the growing root apex. In the temperature range 21–25°C there was a stimulation of local elongation rates along the upper side of a gravireacting root and a reduction (and sometimes a cessation) of elongation along the lower side. Elemental elongation rates have been related to the development of root curvature, and the magnitude of the differential growth between upper and lower sides required for a particular rate of bending has also been estimated. The results complement, and are compatible with, findings relating to the distribution of certain endogenous growth regulators believed to participate in the gravireaction.Abbreviation RELEL relative elemental rate of elongation     

The relationship between diffusible,extractable and conjugated (base-labile) forms of indole-3-acetic acid in isolated coleoptile tips of Zea mays L.

Isolated, 2.5-mm-long coleoptile tips of Zea mays L. cv. Anjou 210 were analyzed for diffusible and tissue-extractable indole-3-acetic acid (IAA) in comparison with the level of base-labile conjugates at various times after excision. The results indicate that base-labile conjugates of IAA do not serve as major sources of free IAA in maize coleoptile tips.Abbreviations IAA indole-3-acetic acid - TLC thin-layer chromatography     

Growth at reduced turgor: irreversible and reversible cell-wall extension of maize coleoptiles and its implications for the theory of cell growth

The relationship between steady-state elongation rate (G) and turgor pressure (P; G/P curve) was investigated using isolated segments of maize (Zea mays L.) coleoptiles incubated in osmotic solutions of a water potential range of 0 to -10 bar (polyethylene glycol 6000 as osmoticum). Short-term elongation measurements revealed curvilinear G/P curves with a steep slope at high turgor and a shallow slope at low turgor. Owing to a decrease of osmotic pressure and turgor, there was a tendency for straightening of the G/P curves during long-term elongation. An elongation rate of zero was adjusted by lowering the turgor by 4.5 bar at a constant osmotic pressure of 6.7 bar. Auxin increased — whereas abscisic acid decreased — the slope of the G/P curve but these hormones had no effect on the threshold turgor of growth (Y = 2.2 bar). It is concluded that extensibility of the growing cell walls represented by the yielding coefficient of Lockhart's growth equation is turgor-dependent and therefore decreases to a very low value as the turgor approaches Y. When the turgor was kept at Y, a constant segment length was maintained over at least 6 h. However, separation of reversible (l_rev) and irreversible (l_irr) components of total (in vivo) length (l_tot = l_rev + l_irr) W measuring segment length before and after freezing/thawing revealed that l_irr increased continuously and l_rev decreased continuously at constant l_tot. After a step-down in turgor the segments grew in l_irr although they shrank in l_tot over the whole turgor range of 0irr irreversible length - l_rev reversible length - l_tot total length (= l_irr + l_rev) - _i osmotic pressure of cell sap - _i water potential of tissue - _o water potential of incubation medium - ABA abscisic acid - G growth rate - m yielding coefficient - P turgor pressure - PEG polyethylene glycol 6000 - Y yield threshold Supported by Deutsche Forschungsgemeinschaft (SFB 206). We thank R. Hertel for helpful comments.     

Effect of applied and endogenous indol-3-yl-acetic acid on maize root growth

The level of endogenous Indol-3-yl-acetic acid (IAA) measured by gas chromatography-mass spectrometry in the elongating zone of intact primary roots of Zea mays showed a good linear correlation with the growth rate of these roots. When they were treated with IAA, their relative elongation decreased; this indicates a supraoptimal content of endogenous IAA. However, the growth of some of the relatively rapidly extending roots was enhanced by such treatment. Interactions between endogenous and applied IAA in the control of root growth are discussed.Abbreviations GC-MS gas chromatography-mass spectrometry - IAA Indol-3-yl-acetic acid     

Stimulatory effects of potassium iodide on auxin action in the coleoptile segments of maize (Zea mays)

Potassium iodide (KI) was found to stimulate IAA-induced elongation of coleoptile segments in maize (Zea mays L.). The promoting effects of KI on coleoptile elongation, which were optimal at 1 mM in the presence of IAA, did not occur as a result of better conservation of IAA in the incubation medium. In addition, KI did not affect fusicoccin- or epibrassinolide-induced elongation. Additionally, sodium iodide (NaI) induced similar stimulatory effects on IAA-induced elongation, however, potassium chloride (KCl) showed no effect, suggesting that iodide is the active component. KI also enhanced IAA-induced ethylene biosynthesis in maize coleoptile segments. Taken together, these results suggest the involvement of KI-sensitive step(s) in auxin action before effectors of the signal transduction pathway split to elongation growth and ethylene biosynthesis. In-yong Hwang and Soo Chul Chang contributed equally to this work.     

Acid growth effects in maize roots: Evidence for a link between auxin-economy and proton extrusion in the control of root growth

The role of proton excretion in the growth of apical segments of maize roots has been examined. Growth is stimulated by acidic buffers and inhibited by neutral buffers. Organic buffers such as 2[N-morpholino] ethane sulphonic acid (MES) — 2-amino-2-(hydroxymethyl)propane-1,3 diol (Tris) are more effective than phosphate buffers in inhibiting growth. Fusicoccin(FC)-induced growth is also inhibited by neutral buffers. The antiauxins 4-chlorophenoxyisobutyric acid (PCIB) and 2-(naphthylmethylthio) propionic acid (NMSP) promote growth and H⁺-excretion over short time periods; this growth is also inhibited by neutral buffers. We conclude that growth of maize roots requires proton extrusion and that regulation of root growth by indol-3yl-acetic acid (IAA) may be mediated by control of this proton extrusion.Abbreviations IAA indol-3yl-acetic acid - ABA abscisic acid - FC fusicoccin - PCIB 4-chlorophenoxy-isobutyric acid - MES 2(N-morpholino)ethane sulphonic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3-diol - NMSP 2-(naphthylmethylthio)propionic acid     

Cooperation of epidermis and inner tissues in auxin-mediated growth of maize coleoptiles

The function of the epidermis in auxinmediated elongation growth of maize (Zea mays L.) coleoptile segments was investigated. The following results were obtained: i) In the intact organ, there is a strong tissue tension produced by the expanding force of the inner tissues which is balanced by the contracting force of the outer epidermal wall. The compression imposed by the stretched outer epidermal wall upon the inner tissues gives rise to a wall-pressure difference which can be transformed into a water-potential difference between inner tissues and external medium (water) by removal of the outer epidermal wall. ii) Peeled segments fail to respond to auxin with normal growth. The plastic extensibility of the inner-tissue cell walls (measured with a constant-load extensiometer using living segments) is not influenced by auxin (or abscisic acid) in peeled or nonpeeled segments. It is concluded that auxin induces (and abscisic acid inhibits) elongation of the intact segment by increasing (decreasing) the extensibility specifically in the outer epidermal wall. In addition, tissue tension (and therewith the pressure acting on the outer epidermal wall) is maintained at a constant level over several hours of auxin-mediated growth, indicating that the inner cells also contribute actively to organ elongation. However, this contribution does not involve an increase of cell-wall extensibility, but a continuous shifting of the potential extension threshold (i.e., the length to which the inner tissues would extend by water uptake after peeling) ahead of the actual segment length. Thus, steady growth involves the coordinated action of wall loosening in the epidermis and regeneration of tissue tension by the inner tissues. iii) Electron micrographs show the accumulation of striking osmiophilic material (particles of approx. 0.3 m diameter) specifically at the plasma membrane/cell-wall interface of the outer epidermal wall of auxin-treated segments. iv) Peeled segments fail to respond to auxin with proton excretion. This is in contrast to fusicoccin-induced proton excretion and growth which can also be readily demonstrated in the absence of the epidermis. However, peeled and nonpeeled segments show the same sensitivity to protons with regard to the induction of acid-mediated in-vivo elongation and cell-wall extensibility. The observed threshold at pH 4.5–5.0 is too low to be compatible with a second messenger function of protons also in the growth response of the inner tissues. Organ growth is described in terms of a physical model which takes into account tissue tension and extensibility of the outer epidermal wall as the decisive growth parameters. This model states that the wall pressure increment, produced by tissue tension in the outer epidermal wall, rather than the pressure acting on the inner-tissue walls, is the driving force of growth.Abbreviations and symbols E _el, E _pl elastic and plastic in-vitro cell-wall extensibility, respectively - E _tot E _el+E _pl - FC fusicoccin - IAA indole-3-acetic acid - IT inner tissue - ITW inner-tissue walls - OEW outer epidermal wall - osmotic pressure - P wall pressure - water potential     

Physical extensibility of maize coleoptile cell walls: apparent plastic extensibility is due to elastic hysteresis

Segments of maize (Zea mays L.) coleoptiles demonstrate plastic cell-wall extensibility (E_pl) as operationally defined by the amount of irreversible strain elicited by stretching living or frozen-thawed tissue under constant load in an extensiometer (creep test). Changes of E_pl are correlated with auxin- and abscisic-acid-dependent growth responses and have therefore been causally related to hormone-controlled cell-wall loosening. Auxin induces an increase of E_pl specifically in the outer epidermal wall of maize coleoptiles which is considered as the growth-limiting wall of the organ. However, detailed kinetic measurements of load-induced extension of frozen-thawed coleoptile segments necessitates a revision of the view that E_pl represents a true plastic (irreversible) wall deformation. Segments demonstrate no significant irreversible extension when completely unloaded between loading cycles. Moreover, E_pl can be demonstrated repeatedly if the same segment is subjected to repeated loading cycles in the extensiometer. It is shown that these phenomena result from the hysteresis behaviour of the cell wall. Stress-strain curves for loading and unloading form a closed hysteresis loop, the width of which represents E_pl at a particular load. Auxin-treatment of segments leads to a deformation of the hysteresis loop, thereby giving rise to an increase of E_pl. These results show that the creep test estimates the viscoelastic (retarded elastic) properties rather than the plastic properties of the wall.Abbreviations E_tot, E_el, E_pl total, elastic, and plastic cell-wall extensibility as defined by the standard creep test - L load Supported by Deutsche Forschungsgemeinschaft (SFB 206).     

The outer epidermis of Avena and maize coleoptiles is not a unique target for auxin in elongation growth

A controversy exists as to whether or not the outer epidermis in coleoptiles is a unique target for auxin in elongation growth. The following evidence indicates that the outer epidermis is not the only auxin-responsive cell layer in either Avena sativa L. or Zea mays L. coleoptiles. Coleoptile sections from which the epidermis has been removed by peeling elongate in response to auxin. The magnitude of the response is similar to that of intact sections provided the incubation solution contains both auxin and sucrose. The amount of elongation is independent of the amount of epidermis removed. Sections of oat coleoptiles from which the epidermis has been removed from one side are nearly straight after 22 h in auxin and sucrose, despite extensive growth of the sections. These data indicate that the outer epidermis is not a unique target for auxin in elongation growth, at least in Avena and maize coleoptiles.Abbreviations IAA indole-3-acetic acid - PCIB p-chlorophenoxyiso-butyric This research was supported by grants from the National Aeronautics and Space Administration and from the U.S. Department of Energy. The help of S. Ann Dreyer is gratefully acknowledged.     

Auxin action on proton influx in corn roots and its correlation with growth

At concentrations inhibitory to the elongation of corn (Zea mays L.) roots, the auxins, indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (α-NAA), cause an increase in the pH of the bathing medium; this increase occurs with an average latent period shorter than the latent period for the inhibitory effect of these auxins on elongation. Indole-2-carboxylic acid, an inactive structural analogue of IAA, and β-naphthaleneacetic acid, an inactive analogue of α-NAA, affect neither growth nor the pH of the medium. Since acid pH is known to promote and basic pH to inhibit root elongation, the data are consistent with the hypothesis that hormone-induced modification of cell-wall pH plays a role in the control of elongation of roots, as has been proposed for elongation of stems and coleoptiles.     

Effect of light and gibberellic acid on coleoptile and first-foliage-leaf growth in durum wheat (Triticum durum Desf.)

A comparison has been made of the relative effectiveness of light quality and quantity and gibberellic acid (GA₃) treatment on the elongation growth of the coleoptile and the first foliage leaf in durum wheat (Triticum durum Desf. cvs. Cappelli and Creso). The cultivar Creso is a shortstrawed variety carrying the Gai 1 gene on chromosome 4A, which influences both plant height and insensitivity to applied gibberellins. The main conclusions are as follows: 1) coleoptile elongation growth appears to be modulated via the fluencerate-dependent action of a blue-light receptor and via a low energy response of phytochrome; 2) the inhibition of first-foliage-leaf growth depends on the operation of a single blue-light-responsive photoreceptor; 3) high energy blue light produces the same inhibitory effect on the two wheat cultivars, whereas at relatively low fluences of white and blue light, the cultivar Creso is more sensitive; 4) the insensitivity to applied GA₃ exerted by the gene Gai 1 in Creso is independent of light; 5) in Cappelli, the action of light on coleoptiles appears to be independent of the applied GA₃, whereas the hormone is able to change the pattern of growth inhibition of the first-foliage-leaf.Abbreviations BL blue light - FR far-red light - GA gibberellin - GA₃ gibberellic acid - R red light - WL white light