Auxin and hydrogen ion actions on light-grown pea epicotyl segments III. Effect of auxin and hydrogen ions on stress-relaxation properties

The effect of auxin and hydrogen ions on the stress-relaxationproperty of the cell wall was studied using light-grown peaepicotyl segments. Auxin pretreatment caused a decrease in theminimum relaxation time (T_o) of the cell wall of stem segmentsand the epidermis at different pH values, when auxin had beengiven to stem segments. The in vivo effect of hydrogen ionson the cell wall was found to be the same as that of auxin.The stress supported by the frozen-thawed epidermis relaxedfaster at pH 4.5 than at pH 6.5, resulting in a shorter maximumrelaxation time (T_m) at pH 4.5. This effect of low pH was inhibitedby low temperature. The incipient pH range producing this effecton Tm was found to be 5.2–5.8. (Received July 22, 1974; )     

Auxin- and hydrogen ion-induced cell wall loosening and cell extension in Avena coleoptile segments

Hydrogen ions and auxin induce rapid cell extension of Avenacoleoptile segments. Nojirimycin (5-amino-5-deoxy-D-glucopyranose),a potent glucanase inhibitor, inhibits auxin-induced growthbut does not affect hydrogen ion-induced extension. This inhibitorhas little effect on respiration of coleoptile segments butstrongly inhibits the in vitro activity of ß-glucosidase.Hydrogen ions and auxin decreased the minimum stress-relaxationtime of the cell wall, indicating that both enhanced cell extensionthrough cell wall loosening. The hemicellulosic glucose contentof the cell wall which was decreased by auxin after about a2-hr lag, was not affected by hydrogen ions. These results suggestthat cell wall loosening induced by hydrogen ions may not bethe same as that caused by auxin, although both phenomena arerepresented by the decrease in the minimum stress-relaxationtime. (Received November 1, 1976; )     

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; )     

Comparison of Auxin-induced and Acid-induced Elongation in Soybean Hypocotyl

Acid-induced growth was compared to auxin-induced growth. After a transient pH 4-induced increase in the elongation rate was completed, auxin could still induce an enhanced rate of elongation in soybean (Glycine max) hypocotyl segments. This auxin response occurred both when the medium was changed to pH 6 before auxin addition, and when the auxin was added directly to the pH 4 medium. This postacid response to auxin was persistent, and quite unlike a postacid response to acid, which was again shortlived. One mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7) inhibited the first response to auxin (the first response to auxin being similar to the acid response), but not the second response. This did not appear to be simply a hydrogen ion neutralizing effect, however, since a 50-fold increase in buffer concentration at pH 6 did not inhibit the first response. Decrease in the pH of the external medium, previously shown to occur with excised soybean hypocotyl segments, was not affected by auxin. Furthermore, this pH drop, during which the cells appear to be adjusting their external pH to about 5.4, did not result in an increased rate of elongation. Addition of auxin after the equilibrium pH had been attained did not alter the pH, but it did increase the rate of elongation, eliciting a normal auxin response. It was concluded that hydrogen ions do not mediate in long term auxin-induced elongation in soybean hypocotyl.     

Control of Auxin-induced Stem Elongathn by the Epidermis

Segments of the 4th and 5th internodes of light-grown pea seedlings were used for the study of control of stem elongation. With 5th internodes, at low turgor as well as at water saturation auxin primarily appeared to cause a change in cell wall properties of the epidermis but it showed little effect on expansion af the inner tissue. This was confirmed by comparison of expansion between peeled and unpeeled segments, split tests and by measurements of stress-relaxation properties of the epidermal cell wall. Segments with the central part re-moved elongated well in response to auxin, but the isolated epidermis showed neither auxin-induced elongation nor cell wall loosening. A fungal β-1,3-glucanase appeared, at least partly, to have a similar effect as that of auxin on elongation, by changing cell wall properties of the epidermal cell wall. Peeled segments of 4th internodes expanded very little and auxin had little effect on their epidermal cell wall properties.     

On the Relationship between Extracellular pH and the Growth of Excised Pea Stem Segments

Studies with stem segments of peas (Pisum sativum L. var. Alaska) suggest that the pH of the medium bathing elongating tissue does not always reflect intramural (cell wall) conditions or that pH is not a controlling factor in elongation. Peeled, green segments, and peeled or nonpeeled etiolated segments appear to regulate the pH of their bathing medium causing it to become acidified with or without the addition of auxin. The growth rates of segments are greatest during a period before acidification is evident and slow during the time in which the medium becomes acidified. We cannot reproduce the dramatic auxin-induced pH shifts reported in the literature because the control segments are becoming more acid also; but there is some evidence that acidification may occur in response to auxin treatments. K⁺ additions mimic the acidifying tendency of auxin but are without growth-promoting effect. Emergent growth (an extremely rapid burst of growth following anaerobic treatments) is not accompanied by a drop in pH of the bathing medium. Proper aeration of the bathing medium in extracellular pH studies is crucial and may explain differences between our results and other published accounts. The data suggest that the techniques used for most extracellular pH studies may not very closely approximate in vivo conditions or properly reflect intramural H⁺ concentration fluxes.     

Auxin and hydrogen ion actions on light-grown pea epicotyl segments II. Effect of hydrogen ions on extension of the isolated epidermis

Isolated epidermis treated differently, i.e. fresh, frozen-thawedand methanol-treated, was subjected to extension tests undera tension of 2 ? 10⁷ dyne/cm² in buffer solution at differentpH values. (1) Fresh and frozen-thawed epidermis extended in response tobuffer solution at pH values lower than 5.5. Half of the maximumextension was achieved at pH 4.5. (2) Epidermis boiled in methanol or treated with pronase didnot extend in response to pH 4.5. Low temperature reduced therate of extension of fresh or frozen-thawed epidermis inducedby pH 4.5. (3) Pretreatment of the epidermis with 0.1% deoxycholate for30 min did not inhibit acid-induced extension. (4) Nojirimycin, 3 ? 10^-3M, added to the buffer solution inhibitedacid-induced extension. (5) Epidermis peeled off from segments which had been treatedwith cycloheximide for 90 min, extended in response to pH 4.5. (6) Methanol-treated epidermis did not extend at pH 4.5, butextended somewhat at pH 3.0. These results suggest that hydrogen ions induce cell wall loosening,possibly through activation of wall-bound enzymes. (Received May 17, 1974; )     

Fusicoccin-induced growth and hydrogen ion excretion of Avena coleoptiles: Relation to auxin responses

Summary The fungal toxin fusicoccin (FC) induces both rapid cell elongation and H⁺-excretion in Avena coleoptiles. The rates for both responses are greater with FC than with optimal auxin, and in both cases the lag after addition of the hormone is less with FC. This provides additional support for the acid-growth theory. The FC responses resemble the auxin responses in that they are inhibited by a range of metabolic inhibitors, but the responses differ in three ways. First auxin, but not FC, requires continual protein synthesis for its action. The auxin-induced H⁺-excretion is inhibited by water stress or by low external pH, while the FC-induced H⁺-excretion is much less sensitive to either. It is concluded that auxin-induced and FC-induced H⁺-excretion may occur via different mechanisms.Abbreviations FC fusicoccin - DNP dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - CHl cycloheximide - IAA indoleacetic acid     

Enhancement of wall loosening and elongation by Acid solutions

The ability of low pH and CO₂ to induce rapid cell elongation and wall loosening in the Avena coleoptile has been examined with the use of a continuous growth-recording technique and an Instron extensometer, respectively. In particular, the properties of the response to hydrogen ions have been examined in detail and have been compared with the responses initiated by CO₂ and auxin. The optimal pH for growth is about 3.0, and both the maximal growth rate and wall extensibility are similar to that produced by optimal auxin. The timing (initiated in less than 1 minute) and duration (up to 2 hours) of the response to hydrogen ions, as well as certain other aspects of the growth and wall-loosening responses, are described. It is shown that the pH response can be clearly separated from the CO₂ response. Possible mechanisms for the initiation of the growth response to low pH are briefly discussed.     

Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

Osmoregulation in the Avena Coleoptile : CONTROL OF SOLUTE UPTAKE IN PEELED SECTIONS

Peeled Avena sativa coleoptile sections (i.e. sections from which the epidermis has been removed) have been used to study the control of solute uptake under conditions where the uptake is not limited by the cuticular barrier. In the presence of 2% sucrose, auxin enhances the rate at which the total osmotic solutes increase, but this appears to be a response to the increased growth rate, inasmuch as the auxin effect is eliminated when growth is inhibited osmotically. When sections are incubated in sucrose or in 20 millimolar NaCl, the osmotic concentration increases until a plateau is reached after 8 to 24 hours. Auxin has no effect on the initial rate of increase in osmotic concentration but causes the osmotic concentration to reach a plateau earlier and at a lower osmotic conentration value. This difference in steady-state osmotic concentration is, in part, a response to auxin itself, as it persists when auxin-induced growth is inhibited osmotically. The upper limit for osmotic concentration does not appear to be determined by the turgor pressure, inasmuch as a combination of sucrose and NaCl gave a higher plateau osmotic concentration than did either solute alone. We suggest that the rate of solute uptake is determined by the availability of absorbable solutes and by the surface area exposed to the solutes. Each absorbable solute reaches a maximum internal concentration independent of other absorbable solutes; the steady-state osmotic concentration is simply the sum of these individual internal concentrations.     

Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.     

Light interacts with auxin during leaf elongation and leaf angle development in young corn seedlings

Modern corn ( Zea mays L.) varieties have been selected for their ability to maintain productivity in dense plantings. We have tested the possibility that the physiological consequence of the selection of the modern hybrid, 3394, for increased crop yield includes changes in responsiveness to auxin and light. Etiolated seedlings in the modern line are shorter than in an older hybrid, 307, since they produce shorter coleoptile, mesocotyl, and leaves (blade as well as sheath). Etiolated 3394 seedlings, as well as isolated mesocotyl and sheath segments, were less responsive to auxin and an inhibitor of polar auxin transport, N-1-naphthylphthalamic acid (NPA). Reduced response of 3394 to auxin was associated with less reduction of elongation growth by light (white, red, far-red, blue) than in 307, whereas the activity of polar auxin transport (PAT) and its reduction by red or far-red light was similar in both genotypes. NPA reduced PAT in etiolated 3394 seedlings much less than in 307. A characteristic feature of 3394 plants is more erect leaves. In both hybrids, light (white, red, blue) increases leaf declination from the vertical, whereas NPA reduces leaf declination in 307, but not in 3394. Our results support findings that auxin and PAT are involved in elongation growth of corn seedlings, and we show that light interacts with auxin or PAT in regulation of leaf declination. We hypothesize that, relative to 307, more erect leaves in the modern hybrid may be primarily a consequence of a reduced amount of auxin receptor(s) and reduced responsiveness to light in etiolated 3394 plants. The more erect leaves in 3394 may contribute to the tolerance of the modern corn hybrid to dense planting.     

Promotion of growth and hydrogen ion efflux by auxin in roots of maize pretreated with ethylene biosynthesis inhibitors

Low concentrations of auxin (e.g. 10⁻¹⁰m) do not promote the growth of intact seedling roots of maize (Zea mays L. Bear Hybrid WF 9 × 38). Higher concentrations are inhibitory. When the roots are pretreated with the ethylene biosynthesis inhibitors, cobalt and aminoethoxyvinylglycine, auxin (10⁻¹⁰ to 10⁻⁸m) strongly promotes their growth. The promotion of growth by auxin in pretreated roots is preceded by enhanced hydrogen ion secretion from the roots. The data indicate that hormone-enhanced hydrogen ion secretion may play a role in the rapid promotion of root growth by auxin. The ability of auxin to promote the growth of intact roots is discussed in relation to the Cholodny/Went hypothesis of hormonal control of root geotropism.     

Physiological evidence for auxin-induced hydrogen-ion secretion and the epidermal paradox

Summary Peeled Avena coleoptile sections will respond to auxin only if the molarity of the incubation buffer at pH 6.2 is less than 5 mM. This inhibition of auxin-induced growth is not due to toxicity or to a reduction of turgor below the critical value needed for extension but rather appears to be related simply to buffering capacity. These data therefore serve as physiological evidence that H⁺-secretion is an intregal part of auxin-induced cell wall loosening. Other data obtained utilizing peeled plant sections and epidermal strips suggest that the epidermis does not directly control cell extension growth. A model is proposed to explain the curvature response in split-segments tests in terms of a H⁺ gradient across the section. As far as tested this model appears to be an alternative to an older concept which implied that the curvature phenomenon in split sections was mediated by special properties of the epidermal layer. Our results suggest that the curvature response may be more directly attributable to the presence of the cuticle.     

Auxin-induced elongation of short maize coleoptile segments is supported by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one

Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 μM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much smaller in segments shorter than 2–3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially purified methanol extract of maize coleoptiles supported long-term, auxin-controlled elongation. The active material was also found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is not the general auxin cofactor but may specifically “spare” the co-auxin in maize. Received: 27 June 2000 / Accepted: 16 October 2000     

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     

The role of the epidermis in auxin-induced and fusicoccin-induced elongation of Pisum sativum stem segments

The effects of peeling and wounding on the indole-3-acetic acid (IAA) and fusicoccin (FC) growth response of etiolated Pisum sativum L. cv. Alaska stem tissue were examined. Over a 5 h growth period, peeling was found to virtually eliminate the IAA response, but about 30% of the FC response remained. In contrast, unpeeled segments wounded with six vertical slits exhibited significant responses to both IAA and FC, indicating that peeling does not act by damaging the tissue. Microscopy showed that the epidermis was removed intact and that the underlying tissue was essentially undamaged. Neither the addition of 2% sucrose to the incubation medium nor the use of a range of IAA concentrations down to 10^-8 M restored IAA-induced growth in peeled segments, suggesting that lack of osmotic solutes and supra-optimal uptake of IAA were not important factors over this time period. It is concluded that, although the possibility remains that peeling merely allows leakage of hydrogen ions into the medium, it seems more likely that peeling off the epidermis removes the auxin responsive tissue.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

In vitro promotion by auxins of divalent ion release from soybean membranes

Release of divalent ions from membrane pellets of soybean hypocotyls was promoted by the natural auxin, indole-3-acetic acid, and the synthetic auxin, 2,4-dichlorophenoxyacetic acid. The calcium release occurred at auxin concentrations as low as 1 nanomolar, and maximum release was observed at 1 micromolar. Hormone concentrations greater than 1 micromolar showed reduced effectiveness in releasing membrane-associated calcium. 2,3-Dichlorophenoxyacetic acid, a weak-auxin analog of 2,4-dichlorophenoxyacetic acid, did not promote calcium release. In some experiments, the analog actually promoted calcium association with the membranes. Red blood cells treated in a similar manner to soybean hypocotyl membranes did not release calcium in response to indole-3-acetic acid. The release phenomenon was hormone specific but not ion specific. Auxin released manganese from membranes in a manner similar to that of calcium. The calcium release, following auxin treatment, is accompanied by a decrease in membrane-associated sites for calcium binding.     

Auxin-induced H Secretion in Helianthus and Its Implications

We have examined the ability of Helianthus hypocotyl segments as well as segments from a variety of other species to elongate in response to H⁺ and to secrete H⁺ in response to auxin and fusicoccin. In all cases a positive response was obtained when the cuticular barrier was abraded with carborundum. Removal of the cuticular barrier by “peeling” prevented detection of both auxin-induced elongation and H⁺ secretion. Fusicoccin-induced growth and acid secretion are not prevented by peeling. These results suggest considerable tissue selectivity with respect to auxin action but considerably less specificity with respect to fusicoccin. It seems likely that in many dicots auxin-enhanced proton secretion and elongation are controlled by the epidermis and/or closely associated cell layers. The data presented in this paper provide further support for the acid growth theory of auxin action.