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.     

Inhibition of Low pH-induced Elongation in Avena Coleoptiles by Abscisic Acid

An angular position-sensing transducer was used to make continuous measurements of acid-induced elongation of Avena sativa coleoptile segments. Elongation rates at pH 4.5 (5 mm succinate buffer) were about 5-fold greater than those at pH 6.0. Buffered 0.1 mm abscisic acid produced a partial decrease of the growth rate. Pretreatments with abscisic acid buffered at pH 6.0 usually caused a further reduction of the elongation response when the coleoptile segments were subsequently placed in buffer at pH 4.5 containing abscisic acid. Abscisic acid did not completely prevent the pH effect in any of these experiments, and the brief latent period of the pH response was not affected by abscisic acid treatments. At pH 4.5, where the inhibitory effect of ABA was maximum, low pH-induced elongation was also inhibited by KCN and HgCl₂. These results suggest that pH-(4.5) induced elongation in this system may be dependent on some metabolic processes and that abscisic acid-induced inhibition of this elongation may involve an interaction with these processes.     

Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima

Although rapid auxin-induced growth of coleoptile sections can persist for at least 18 hours, acid-induced growth lasts for a much shorter period of time. Three theories have been proposed to explain this difference in persistence. To distinguish between these theories, the pH dependence for auxin-induced growth of oat (Avena sativa L.) coleoptiles has been determined early and late in the elongation process. Coleoptile sections from which the outer epidermis was removed to facilitate buffer entry were incubated, with or without 10 micromolar indoleacetic acid, in 20 millimolar buffers at pH 4.5 to 7.0 to maintain a fixed wall pH. During the first 1 to 2 hours after addition of auxin, elongation occurs by acid-induced extension (i.e. the pH optimum is <5 and the elongation varies inversely with the solution pH). Auxin causes no additional elongation because the buffers prevent further changes in wall pH. After 60 to 90 minutes, a second mechanism of auxin-induced growth, whose pH optimum is 5.5 to 6.0, predominates. It is proposed that rapid growth responses to changes in auxin concentration are mediated by auxin-induced changes in wall pH, whereas the prolonged, steady-state growth rate is controlled by a second, auxin-mediated process whose pH optimum is less acidic.     

The “acid growth effect” and geotropism

Summary The curvature developed by segments of sunflower hypocotyl exposed to gravitational stimulus was enhanced in buffer solutions between pH 3.4 and 4.0 in the absence of added auxin. This effect was observed both when the segments were submerged during the stimulus and when they floated near the surface of the solution. 5–10 min in a horizontal position was sufficient to induce subsequent curvature.Straight growth of the segments was also promoted in buffers of this pH range.The acid effect on curvature was insensitive to KAsO₂, HgCl₂ and cycloheximide, inhibitors which drastically reduced auxin-induced curvature. Furthermore, acid buffer, but not auxin, restored the ability of segments taken from etiolated and starved plants to respond to gravity. These results suggest that the polarisation following gravistimulus may not be resticted to the asymmetric distribution of auxin and auxin co-factors but may involve a general physiological asymmetry.     

Comparative studies on auxin and fusicoccin actions on plant growth

Mode of action of FC was compared with that of auxin in differentexperimental systems and the following results were obtained.

1. FC, as well as auxin, primarily induced elongation of the epidermisof pea epicotyl segments, but it also promoted elongation ofthe inner tissue, as judged by its action in split stem tests,elongation of hollow-cylinder segments and elongation of unpeeledand peeled segments.
2. FC decreased the minimum stress relaxationtime (T₀) and increasedthe extensibility (mm/gr) of the epidermalcell wall of peaepicotyl segments, as did auxin.
3. FC failedto induce expansion growth of Jerusalem artichoketuber sliceswhen given alone or in combination with kinetinor gibberellicacid.
4. FC at concentrations lower than 10^–6 M, when givenwithauxin at concentrations lower than 0.03 mg/liter, promotedelongationof Avena coleoptile segments in an additive manner,to achievethe maximum elongation at higher concentrations.
5. An antiauxin, 2,4,6-trichlorophenoxyacetic acid, inhibitedtheelongation of Avena coleoptile segments due to auxin butnotthat due to FC.
6. Nojirimycin, an inhibitor of ß-glycosidases,inhibitedelongation of pea internode segments due not onlyto auxin butalso to FC.
7. At concentrations more than 10^–5MFC promoted root elongationof intact lettuce seedlings, whichwas inhibited by exogenousauxin.

From these results it is concluded that FC and auxin have acommon mechanism, which may involve hydrogen ion extrusion,leading to cell wall loosening and thus cell elongation. Thisgrowth is limited to the extent that the cells are capable ofelongating in response to hydrogen ions. Otherwise there isa definite difference in the mode of actions between FC andauxin, including the nature of cellular receptors for thesetwo compounds. (Received August 29, 1974; )     

Rapid Auxin-induced Decrease in Free Space pH and Its Relationship to Auxin-induced Growth in Maize and Pea

A pH microelectrode has been used to investigate the auxin effect on free space pH and its correlation with auxin-stimulated elongation in segments of pea (Pisum sativum) stem and maize (Zea mays var. Bear Hybrid) coleoptile tissue. Auxin induces a decrease in free space pH in both tissues. In maize coleoptiles, free space pH begins to fall within about 12 minutes of exposure to auxin and decreases by about 1 pH unit by approximately 30 minutes. In pea, pH begins to decrease within an average of 15 to 18 minutes of exposure to auxin and falls by about 0.9 pH unit by approximately 40 minutes. Auxin-stimulated elongation, measured in the same two tissues similarly prepared, appears in maize at the earliest 18 minutes after auxin application, while in pea it appears at the earliest 21 to 24 minutes after auxin application. The auxin analogs p-chlorophenoxyisobutyric acid and phenylacetic acid do not stimulate elongation above control levels in maize or pea tissue segments and do not cause a decrease in free space pH in either tissue. These findings are consistent with the acid secretion theory of auxin action.     

Polarity and rate of transport of cyclic adenosine 3,5'-monophosphate in the coleoptile

Transport of tritiated cyclic AMP in the coleoptile of oats (Avena sativa) and corn (Zea mays) is polar, with basipetal to acropetal ratios of 4.0 and 3.2, respectively. The rate of transport is approximately that of indoleacetic acid. The linear velocity of transport, however, is at least five times that of auxin. A loss in transport polarity of the nucleotide occurs in subapical tissues within several hours after decapitation of the coleoptile, accompanied by a decrease in transport rate. The loss in polarity is not reversed by exogenous auxin, but the reduction in transport is. Auxin also inhibits the uptake of cyclic AMP. Exogenous cyclic AMP is metabolized rapidly by coleoptile tissues. If cyclic AMP does have a cellular function in the coleoptile, its transport behavior is compatible with that of a hormone.     

Auxin-induced extension, cell wall loosening and changes in the wall polysaccharide content of barley coleoptile segments

Contents of the cell wall and sugar pool and the response toexogenously applied auxin (cell extension and cell wall loosening)were investigated with barley coleoptile segments excised from4-, 5- and 6-day-old seedlings. The first two groups exhibiteda high capacity to grow in terms of the intact growth rate andwere responsive to auxin, while those excised from 6-day-oldseedlings had a low growth capacity. The cell wall of 4- and5-day-old coleoptile segments contained almost the same amountof noncellulosic wall components per unit length while the 6-day-oldones had a lesser amount. The sugar pool and -cellulose contentper unit length decreased as the coleoptile aged. Auxin-stimulatedextension was most marked in the 4-day-old coleoptile segments.Auxin caused quantitative changes in the cell wall componentsof 4-day-old coleoptiles and, to a lesser extent, of 5-day-oldcoleoptiles, i.e., an increase in the contents of xylose andarabinose, both of which are constituents of noncellulosic polysaccharidesof the cell wall, and of -cellulose and a decrease in the noncellulosicglucose content. Auxin caused very little change in the noncellulosicsugar content and -cellulose content of the cell wall from 6-day-oldcoleoptile segments. The auxin-induced change in mechanicalproperties of the cell wall was significant in 4- and 5-day-oldcoleoptiles but very small in 6-day-old ones. The results suggestedthat the content of noncellulosic wall components is closelyrelated to the intact growth and auxin responsiveness of barleycoleoptiles. (Received April 20, 1978; )     

The effects of temperature and IAA concentration on the latent period for IAA-induced rapid growth of Avena coleoptile segments

Summary Indoleacetic acid buffered at pH 7.0 induces a high growth rate in Avena coleoptile segments after a latent period, the duration of which is dependent upon both IAA concentration and temperature. A minimum latent period of 7.3 min is observed at 25° C with 10^-3 M IAA in phosphate buffer at pH 7.0.In contrast, 5×10^-3 M IAA made up in 0.01 M KH₂PO₄ alone, promotes elongation almost immediately, regardless of whether the segments have been previously incubated in 0.01 M KH₂PO₄ at pH 4.7, or phosphate buffer at pH 7.0. This immediate response is unaffected by 10^-4 M KCN which abolishes the rapid growth induced by 5×10^-3 M IAA buffered at pH 7.0 but does not affect the immediate appearance of low-pH-induced growth. Since we consistently find solutions of 5×10^-3 M IAA in 0.01 M KH₂PO₄ to have a pH of 3.5, our results indicate that the immediate growth response elicited by this solution is attributable to its low pH rather than to the presence of IAA as previously reported in the literature.     

Polar auxin transport and auxin-induced elongation in the absence of cytoplasmic streaming

Summary When cytoplasmie streaming in oat and maize coleoptile cells is completely inhibited by cytochalasin B (CB), polar transport of auxin (indole-3-acetic acid) continues at a slightly reduced rate. Therefore, cytoplasmic streaming is not required for polar transport. Auxin induces elongation in CB-inhibited coleoptile and pea stem segments, but elongation rate is reduced about 40% by CB. Therefore, stimulation of cytoplasmic streaming cannot be the means by which auxin promotes cell elongation, but streaming may be beneficial to elongation growth although not essential to it. A more severe inhibition of elongation develops after several hours in CB. With coleoptiles this could be due to inhibition of sugar uptake; in pea tissue it may be due to permeability changes and cytoplasmic degeneration. CB does not disorganize or disorient microfilament bundles when it inhibits streaming in maize, but appears instead to cause hypercondensation of microfilament material.     

Auxin-indnced changes in barley coleoptile cell wall composition

Auxin induces extension growth of barley coleoptile segments,causing cell extension and cell wall loosening represented bya change in mechanical properties of the cell wall. This responsedecreased after the segments were starved for more than 12 hrin buffer solution. Auxin decreased the noncellulosic glucosecontent of the cell wall of the segments starved for 0 and 6hr, but very little that of segments starved for 12 and 18 hr.The contents of arabinose, xylose and galactose, among noncellulosicpolysaccharides, and -cellulose of the cell wall increased duringthe starvation, but auxin did not affect them. The auxin-induceddecrease in glucose content was inhibited by nojirimycin, apotent inhibitor of ß-glucanase, which inhibited auxin-inducedextension and changes in mechanical properties of the cell wall,suggesting that cell wall loosening, and thus cell extension,resulted from partial degradation of ß-glucan of thecell wall. (Received April 20, 1978; )     

Mode of dinitroaniline herbicide action I. Analysis of the colchicine-like effects of dinitroaniline herbicides

Colchicine and a variety of dinitroaniline herbicides (DNHs)produce a similar pattern of inhibition of elongation, inductionof swelling in the elongation zone, depolarization of cell enlargement,and induction of multiple nuclei in corn seedling roots. However,a 1000-fold higher concentration of colchicine is needed toproduce effects quantitatively similar to those of oryzalin.Both colchicine- and DNH-inhibition of elongation start at about3 hr. Since these compounds cause swelling and inhibition ofelongation in -seedling roots, segments from the root elongationzone and intact roots in the presence of cell division inhibitors(all growing without cell division), it appears that the inhibitionof root elongation is caused in part by their effect on cellelongation independent of their effect on cell division. Sincethe growth (increase in fresh weight) of -seedling roots andexcised root segments is not inhibited by these compounds, theireffect on the polarity of cell enlargement must be fairly specific.Unlike colchicine, oryzalin applied to the roots did not causeany significant, visible effect on shoot (mesocotyl and coleoptile)growth. These organs are not resistant to oryzalin, for theIAA-induced elongation of coleoptile segments is inhibited whenthey are floated in oryzalin solutions. As expected, when coleoptilesegments are incubated in ¹⁴C-oryzalin, it is taken up rapidlyand not degraded. The failure of root-applied oryzalin to affectseedling shoot growth is due to lack of transport to the shoots. (Received June 14, 1977; )     

Calorimetric studies of the elongation of Avena coleoptile segments

Elongation rate and heat produced by Avena coleoptile segments suspended in sucrose buffer solutions were measured at pH values from 3.5 to 8.5. The caloric efficiency of elongation (CEE) was defined as the ratio of the rate of elongation to the rate of heat production. Elongation and CEE were greatest at intermediate pH values, but heat production (about 1 cal/g.hr) was insensitive to pH within the limits of experimental error (+/-20%). Quantitative agreement was found between the results of previous respiration studies and the rate of heat production in an aerobic atmosphere, which indicates that oxidative metabolism accounts for essentially all energy changes in the cell, so matter flow is a significant component of the bioenergetics of cell function. Indole-3-acetic acid up to 1 mm, produced about a 10-fold increase in elongation rate, a 5-fold increase of the CEE, and a 25% increase in heat production. Above this concentration, sharp drops in both elongation and heat production occurred, without altering the CEE at pH 6.5, but greatly decreasing the CEE at pH 4.5. Elongation and CEE showed marked decreases after 4 hours in an anaerobic atmosphere, but heat production did not exhibit a proportional decrease. These studies indicate that rate of cell elongation in the presence and absence of auxin is not directly proportional to the overall metabolism of the cell.     

Red Light-inhibited Mesocotyl Elongation in Maize Seedlings: I. The Auxin Hypothesis

Red light-inhibited mesocotyl elongation, which occurs in intact Zea mays L. seedlings, was studied in excised segments which included the coleoptile (or parts therefrom) and apical centimeter of the mesocotyl. Experiments took into account, first, the ability of the segments to regenerate auxin supply sites, and, second, that auxin uptake can be greatly reduced if there is no cut surface, apical to the elongating cells, to act as a port of entry. In all cases, auxin completely reversed the inhibition of elongation by light. The results support the hypothesis that light regulates mesocotyl elongation by controlling auxin supply from the coleoptile. Sucrose concentration had no effect on auxin reversal of light-inhibited elongation, but relatively high concentrations of gibberellic acid (10 μm) could substitute for auxin in this system.     

PHYTOCHROME ACTION IN ORYZA SATIVA L.: I. GROWTH RESPONSES OF ETIOLATED COLEOPTILES TO RED, FAR-RED AND BLUE LIGHT

1. Under continuous irradiation, the growth of intact rice coleoptilewas strongly inhibited by red light, and somewhat preventedby blue and far-red light. The inhibitory effect of red lighton coleoptile elongation was caused by a low-energy brief irradiation,and a single exposure of 1.5 kiloergs cm^–2 incidentenergy of red light brought about the 50% inhibition. This photoinhibitionof growth was observed only after the coleoptile had elongatedto about 10 mm or longer. The red light-induced effect was reversedby an immediately following brief exposure to far-red light,and the photoresponses to red and far-red light were repeatedlyreversible. The escape reaction of red lightinduced effect tookplace at a rate so that 50% of the initial reversibility waslost within 9 hr in darkness at 27. The inhibition by bluelight and reversal by far-red irradiation was also achievedrepeatedly with successive treatments of the coleoptiles. Theevidence for a low intensity red far-red reversible controlof coleoptile growth, indicative of control by phytochrome,seems clearly established in etiolated intact seedlings.
2. Incontrast, the elongation of apically excised rice coleoptilesegments was promoted by a brief exposure to red light in 0.02M phosphate buffer, pH 7, and the effect was almost completelynullified by an immediately subsequent exposure to far-red light.It becomes evident that the growth of intact coleoptiles wasinhibited by a exposure to red light, while that of excisedsegments in a buffer was rather promoted by red irradiation.The direction of red light induced responses, either promotiveor inhibitory, depends upon the method of bioassay using intactcoleoptiles or their excised segments.

(Received July 24, 1967; )     

The lack of effect of cyclic adenosine 3':5'-monophosphate on Avena coleoptile growth

The effects of cyclic adenosine 3':5'-monophosphate (cAMP) on the growth of Avena coleoptile segments over 4 to 10 hours were monitored with a position sensing transducer. At pH 6, cAMP (0.1 mm with and without 2.5 mm glucose; or 2 mm alone) or dibutyryl cAMP (0.1 mm) was added at the beginning of the experiment, or after about 1 hour or after about 6 or 7 hours. Under all conditions tested, cAMP compounds had little or no effect on coleoptile segment elongation. Inasmuch as cAMP does not duplicate the rapid and vigorous elongation obtained with 2 mum auxin, the hypothesis that cAMP is a mediator of auxin activity is not supported by experimental evidence in this system. This conclusion is dependent upon the assumption that the cAMP compounds penetrated the tissue.     

Conversion of isatin to isatate as related to growth promotion in Avena coleoptile and pisum stem sections

Isatin, (indole 2,3-dione), which promotes elongation of Pisum stem sections at concentrations exceeding 0.1 mm, promotes elongation of Avena coleoptile sections only at higher concentrations, exceeding 1 mm. Aged isatin solutions are more active than fresh solutions, due to the slow, spontaneous conversion to isatate (o-aminophenylglyoxylate). A concentration of 0.1 mm aged isatin is as active in Avena coleoptile sections as in peas. Isatate has been independently synthesized and its auxin activity in both Avena coleoptile and Pisum stem sections confirmed. The synthetic isatate is more effective than isatin in both systems. This suggests that the auxin activity of isatin is due to its conversion to isatate.     

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.     

Some aspects of geotropism in coleoptiles

Summary Auxin transport was studied in coleoptile sections that were stimulated geotropically. The early time course of auxin-transport asymmetry was measured. An initial phase in which more IAA was delivered into the receptor for the upper half was found after 5 min of horizontal exposure. After about 15 min this was followed by the expected known asymmetry in which more auxin flows in the lower side of the coleoptile. Upon return of the coleoptile to a vertical position, this asymmetry disappeared within 30 min.Earlier correlations of geosensitivity of the auxin transport system with sedimentation of amyloplasts in comparisons of wild type corn and an amylomaize mutant were confirmed and extended. It was also shown that, in contrast to the geotropic effect, phototropically induced lateral auxin asymmetry was not significantly different in wild type and amylomaize. Eleven other single-gene endosperm starch mutants of corn were compared to their corresponding normals. In all pairs, if a difference in geosensitivity of lateral auxin transport was present, it was correlated with a parallel difference in amyloplast sedimentation: e.g., sugary 1 (67) had an amyloplast asymmetry index of 0.32 and a 13% gravity effect on auxin transport; the paired wild-type had both a greater amyloplast asymmetry (0.61) and a greater gravity effect on transport (23%).Correlations between gravity effects on auxin transport and amyloplasts were also shown in comparisons of apical and basal sections of corn, oat and Sorghum coleoptiles.Further results, confirming the increased effect of centrifugal acceleration greater than 1xg on lateral auxin transport and on curvature, are in agreement with the hypothesis that the pressure exerted by amyloplasts, acting as statoliths, locally stimulates the auxin transport system in the individual cells.with participation by Charles steele and Vicky fan