Cell wall pH and auxin transport velocity

According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.     

Determination of Auxin-Dependent pH Changes in Coleoptile Cell Walls by a Null-Point Method

The present debate on the validity of the "acid-growth theory" of auxin (indole-3-acetic acid, IAA) action concentrates on the question of whether IAA-induced proton excretion into the cell wall is quantitatively sufficient to provide the shift in pH that is required to explain IAA-induced growth (see D.L. Rayle, R.E. Cleland [1992] Plant Physiol 99:1271-1274 for a recent apologetic review of the acid-growth theory). In the present paper a null-point method has been employed for determining the growth-effective cell-wall pH in the presence and absence of IAA after 60 min of treatment. Elongation of abraded maize (Zea mays L.) and oat (Avena sativa L.) coleoptile segments was measured with the high resolution of a displacement transducer. The abrasion method employed for rendering the outer epidermal cell wall permeable for buffer ions was checked with a dye-uptake method. Evidence is provided demonstrating that externally applied solutes rapidly and homogeneously penetrate into the epidermal wall, whereas penetration into the inner tissue walls is strongly retarded. "Titration" curves of IAA-induced and basal elongation were determined by measuring the promoting/inhibiting effect of medium pH under iso-osmotic conditions in the range of pH 4.5 to 6.0. In maize, the null point (no pH-dependent change in elongation rate after 5-10 min of treatment with 10 mmol L-1 citrate buffer) was pH 5.00 after 60 min of IAA-induced growth, and the null-point pH determined similarly in IAA-depleted tissue (10 times smaller elongation rate) was 5.25. Corresponding titration curves with Avena segments led to slightly lower null-point pH values both in the presence and absence of IAA-induced growth. After induction of acid-mediated extension by 1 [mu]mol L-1 fusicoccin (FC) in maize, the null-point pH shifted to 3.9. At 0.5 [mu]mol L-1, FC induced the same elongation rate as IAA but a 9-fold larger rate of proton excretion. At 0.033 [mu]mol L-1, FC induced the same rate of proton excretion as IAA but had no appreciable effect on elongation. The implications of these results against the background of recent attempts to revitalize the acid-growth theory of IAA action are discussed.     

Responses of Avena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic Acid

Proton excretion induced by optimal concentrations of indoleacetic acid (IAA) and fusicoccin (FC) differs not only in maximum rate of acidification but also in the lag before onset of H⁺ excretion and in sensitivity to cycloheximide. Because these differences might simply be a consequence of the difference in rate of proton excretion, FC and IAA have now been compared using oat coleoptiles (cv. Victory) under conditions where the rates of acidification are more similar, i.e. suboptimal FC versus optimal IAA. As the concentration of FC is reduced, the rate of H⁺ excretion decreases, the final equilibrium pH increases, and the lag before detectable acidification increases up to 7-fold. This enhanced lag period is not primarily a consequence of wall buffering, inasmuch as it persists when a low concentration of FC is added to sections which were already excreting H⁺ in response to IAA. An extended lag also occurs, upon reduction of FC levels, in the hyperpolarization of the membrane potential, before enhancement of O₂ uptake and before the increased rate of Rb⁺ uptake. The presence or absence of a lag is not a distinguishing feature between FC and IAA actions on H⁺ excretion and cannot be used to discriminate between their sites of action. In contrast, the insensitivity of FC-induced H⁺ excretion to cycloheximide, as compared with the nearly complete inhibition of this auxin effect by cycloheximide, persists even at dilute concentrations of FC. This seems to be a basic difference in H⁺ excretion by IAA and FC.     

Rapid response of the plasma-membrane potential in oat coleoptiles to auxin and other weak acids

We have compared the effects of the auxin, indole-3-acetic acid (IAA) with that of other weak acids on the plasma-membrane potential of oat (Avena sativa L.) coleoptile cells. Cells treated with 1 M IAA at pH 6 depolarize 20–25 mV in 10–12 min, but they then repolarize, until by 20–25 min their potentials are about 25 mV more negative than the initial value. Similar concentrations of benzoic and butyric acids cause the initial depolarization, but not the subsequent hyperpolarization. The hyperpolarization is therefore specific to IAA. All the weak acids, including IAA, evoke a rapid hyperpolarization when their concentrations are raised to 10 mM. This result indicates that at high concentrations, the uptake of undissociated weak acids activates electrogenic proton pumping, most likely by lowering cytoplasmic pH. In contrast, the hyperpolarization observed with concentrations of IAA four orders of magnitude lower appears to be a specific hormonal effect. This specific, auxin-induced hyperpolarization occurs at the same time as the initiation of net proton secretion and supports the hypothesis that auxin initiates extension growth by increasing proton pumping.Abbreviations FC fusicoccin - IAA indole-3-acetic acid     

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     

Use of a pH-response curve for growth to predict apparent wall pH in elongating segments of maize coleoptiles and sunflower hypocotyls

To determine the relationship between apparent pH of the wall solution and shoot segment elongation, curves for the initial growth rates as a function of pH of the external solution were determined for maize (Zea mays L.) coleoptiles and sunflower (Helianthus annuus L.) hypocotyls and used to predict apparent wall pH in segments responding to indole-3-acetic acid (IAA) and fusicoccin (FC). When a solution having a pH predicted for walls of coleoptile segments responding to IAA was applied to the segments in the presence of IAA, this pH was not maintained. However, when the same was done for coleoptile segments responding to FC, the predicted pH was maintained in the external solution. Sunflower hypocotyl tissue did not maintain the external pH at the predicted value in the presence of either IAA or FC. The results indicate that wall loosening in coleoptiles caused by IAA may not be solely controlled by pH in the wall, yet growth (wall loosening) caused by FC apparently is directly related to wall pH. In sunflower the growth response to neither IAA nor FC appears to be directly correlated with wall pH.     

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.     

Fusicoccin counteracts inhibitory effects of high temperature on auxin-induced growth and proton extrusion in maize coleoptile segments

Plant growth and development are tightly regulated by both plant growth substances and environmental factors such as temperature. Taking into account the above, it was reasonable to point out that indole-3-acetic acid (IAA), the most abundant type of auxin in plants, could be involved in temperature- dependent growth of plant cells. We have recently shown that growth of maize coleoptile segments in the presence of auxin (IAA) and fusicoccin (FC) shows the maximum value in the range 30–35°C and 35–40°C, respectively. Furthermore, 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. The aim of this addendum is to determine interrelations between the action of IAA and FC (applied together with IAA) on growth and medium pH of maize coleoptile segments incubated at high temperature (40°C), which was optimal for FC but not for IAA.Key words: auxin, fusicoccin, coleoptile segments, elongation growth, medium pHA well studied aspect of auxin action especially in maize coleoptile, is its effect on cell elongation, proton extrusion and membrane potential.^1–7 It is now generally agreed that indole-3-acetic acid (IAA), as the principal regulator of plant elongation growth, causes (i) acceleration of elongation growth as compared to endogenous growth, (ii) enhancement of proton extrusion as compared to auxin—free medium, and (iii) transient depolarization followed by a slow hyperpolarization of membrane potential. According to the “acid growth theory” of elongation growth,^8–11 auxin induced cell wall acidification provides favorable conditions for cell wall loosening, a requirement for cell elongation. At least in maize coleoptile segments, auxin induced cell wall acidification is mediated by increased activity and/or amount of the PM H⁺-ATPase.^11,12 In the case of fusicoccin, which mimics the effect of auxin in many respects,¹³ it was shown that FC-binding site arises from interaction of the 14-3-3 protein dimmer with the C-terminal autoinhibitory domain of the H⁺-ATPase and that FC stabilizes this complex.^14–18 It should be pointed out that in spite of abundant literature on the mechanism through which IAA or FC control growth of grass coleoptiles, little is know how these substances work at extreme temperatures. Over the past decade, the involvement of 14-3-3 proteins in plant stress responses has often been suggested.¹⁹ For example, work by Chelysheva et al.,²⁰ and Babakov et al.,²¹ demonstrated that under low temperature and high osmolarity conditions, 14-3-3 proteins interact with the C-terminal autoinhibitory domain of the PM H⁺-ATPase activating the proton pump that play a key role in stress responses in higher plants. We have recently shown²² that FC at 40°C induced maximal growth whereas growth observed at the same temperature in the presence of IAA was reduced by 33% compared to the maximal value at 30°C. It was also found²² that at 40°C the kinetics of the pH change differed significantly for both growth substances; the segments treated with IAA at 40°C were virtually not able to acidify the external medium, whereas FC at this temperature caused practically maximal acidification. In this addendum we have shown that application of FC together with IAA conteracted the inhibitory effect of high temperature (40°C) on IAA-induced growth and proton extrusion in maize coleoptile segments (Fig. 1). For example, the total IAA-induced elongation growth of coleoptile segments at 40°C was 1438.1 ± 134.5 µm cm⁻¹ (mean ± SE, n = 11) while elongation of 2747.4 ± 269.7 µm cm⁻¹ (mean ± SE, n = 11) was observed in IAA applied together with FC (Fig. 1A). The data in Figure 1B indicate that coleoptile segments incubated at 40°C (over 2 h), without growth substances (control) characteristically changed the pH of the medium: usually within the first 30–45 min an increase of pH (by ca. 0.5 pH unit) was observed, followed by a slow decrease of pH. When IAA or FC was added (after 2 h of segment''s incubation in control medium), an additional decrease of pH was observed. As can be seen in Figure 1B, FC added at 40°C was much more effective in acidification of the medium, as compared to IAA. For FC, 5h after its addition, the pH of the incubation medium dropped to pH 4.2, whereas for IAA the pH was only 5.4. However, addition of IAA together with FC at 40°C dropped medium pH approximately to the same value as was observed in the presence of FC only.Open in a separate windowFigure 1Effect of high temperature (40°C) on growth (A) and medium pH (B) of maize coleoptile segments incubated in the presence of IAA (10 µM) and FC (1 µM). The growth of a stack of 21 segments, expressed as elongation (µm cm⁻¹), was measured simultaneously with medium pH at 40°C. After preincubation (over 2 h) of the coleoptile segments in control medium, IAA and FC was added (arrow). Values are means of 11 independent experiments. Bars indicate ± SE. In the case of medium pH SE did not exceed 8%.In conclusion, the results presented in this addendum provide further evidence that FC on the receptor level is much more effective than IAA.     

Effect of thiosulphinates contained in garlic extract on growth, proton fluxes and membrane potential in maize (Zea mays L.) coleoptile segments

The effect of thiosulphinates contained in garlic extract (GE) 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 GE dilutions and the protective effect of dithiothreitol (DTT) against GE toxicity were also determined. It was found that GE at almost all dilutions studied, when added to the incubation medium inhibited endogenous growth as well as growth in the presence of either IAA or FC. Simultaneous measurements of growth and external pH indicated that the administration of GE resulted in a complex change in the pH of the external medium; after an initial transient acidification, pH increased and reached the maximal value followed by a gradual decrease of medium pH. When IAA or FC was added after preincubation of the segments in the presence of GE the changes in medium pH were not significantly different from these obtained with GE only. If the coleoptile segments were first preincubated with GE and subsequently GE was removed, the addition of IAA induced strong growth and medium acidification. Dithiothreitol added together with GE neutralized the toxic effect of GE on growth of coleoptile segments incubated in the presence of IAA. The addition of GE to the control medium caused a depolarization of the membrane potential, the value of witch depended on GE dilution. These results indicate that the toxic effect of GE on growth of plant cells might be caused by disruption of the catalytic function of the plasma membrane H⁺-ATPase on formation of the disulfide bonds.     

Wall extensibility, wall pH and tissue osmolality: significance for auxin and ethylene-enhanced petiole growth in semi-aquatic plants

Abstract. Auxin and ethylene both enhance cell elongation in intact petioles of the semi-aquatic plants Regnellidium diphyllum and Nymphoides peltata. The authors now show that auxin but not ethylene increases the in vitro extensibility of cell walls. No response to ethylene occurs in auxin-depleted tissue. Neither hormone regulates cell expansion by direct control of internal osmolality (OS). During growth of segments, OS (and hence turgor) declines rapidly over the first 5–6 h with a net loss of osmotic solutes. Thereafter, an apparent threshold OS is maintained with net gains in osmostic solutes ( Nymphoides ) or further net losses ( Regnellidium ). Although wall extensibility determines initial rates of hormone-induced cell expansion, the primary control of wall loosening appears to differ in the two species. Nymphoides shows typical 'acid growth', and fusicoccin, auxin and ethylene (with auxin) all enhance proton secretion. In Regnellidium , neither low pH nor fusicoccin (FC) alters the rate of cell expansion, although proton secretion is stimulated by FC. Stress relaxation studies using low pH treatment of living or frozen-thawed segments show increases in the extensibility of walls in vitro for Nymphoides but not for Regnellidium. The authors propose that extensibility may be controlled by wall pH in Nymphoides but the availability of effective wall-loosening sites determines extensibility in Regnellidium.     

Cellular responses to naphthoquinones: juglone as a case study

The effects of juglone (JG) on the endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC) and on proton extrusion were studied in maize coleoptile segments. In addition, membrane potential changes were also determined at chosen JG concentrations. It was found that JG, when added to the incubation medium, inhibited endogenous growth as well as growth in the presence of either IAA or FC. Simultaneous measurements of growth and external pH indicated that inhibition of either IAA-induced growth or proton extrusion by JG was a linear function of JG concentration. Addition of JG to the control medium caused depolarization of the membrane potential (E_m), value of which was dependent on JG concentration and time after its administration. Hyperpolarization of E_m induced by IAA was suppressed in the presence of JG. It was also found that for coleoptile segments initially preincubated with JG, although subsequently removed, addition of IAA was not effective in the stimulation of growth and medium acidification. Taken together, these results suggest that the mechanism by which JG inhibits the IAA-induced growth of maize coleoptile segments involves inhibition of PM H⁺-ATPase activity.     

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     

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     

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.     

Auxin and Ethylene Regulation of Petiole Epinasty in Two Developmental Mutants of Tomato, diageotropica and Epinastic

The epinastic growth responses of petioles to auxin and ethylene were quantified in two developmental mutants of tomato (Lycopersicon esculentum Mill.). In the wild type parent line, cultivar VFN8, the epinastic response of excised petiole sections was approximately log-linear between 0.1 and 100 micromolar indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations, with a greater response to 2,4-D at any concentration. When ethylene synthesis was inhibited by aminoethoxyvinylglycine (AVG), epinasty was no longer induced by auxin, but could be restored by the addition of ethylene gas. In the auxin-insensitive mutant, diageotropica (dgt), no epinastic response to IAA was observed at IAA concentrations that effectively induced epinasty in VFN8. In the absence of added IAA, epinastic growth of dgt petioles in 1.3 microliters per liter exogenous ethylene gas was more than double that of VFN8 petioles. IAA had little additional effect in dgt, but promoted epinasty in VFN8. These results confirm that tomato petiole cells respond directly to ethylene and make it unlikely that the differential growth responsible for epinasty results from lateral auxin redistribution. The second mutant, Epinastic (Epi), exhibits constitutively epinasty, cortical swelling, and root branching symptomatic of possible alternation in auxin or ethylene regulation of growth. Only minor quantitative differences were observed between the epinastic responses to auxin and ethylene of VFN8 and Epi. However, in contrast to VFN8, when ethylene synthesis or action was inhibited in Epi, auxin still induced 40 to 50% of the epinastic response observed in the absence of inhibitors. This indicates that the target cells for epinastic growth in Epi are qualitatively different from those of VFN8, having gained the ability to grow differentially in response to auxin alone. The dgt and Epi mutants provide useful systems in which to study the genetic determination of target cell specificity for hormone action.     

Mechanism of the growth-promoting action of fusicoccin

Summary Analogies and differences between the growth-promoting action of fusicoccin (FC), the toxic agent produced by Fusicoccum amygdali, and of indole-3-acetic acid (IAA) in etiolated pea internode segments were further investigated. It was confirmed that the effect of FC, at optimal concentration, on growth by cell enlargement is markedly higher (ca. 70%) than that of IAA. The lack of an inhibitory effect of FC at high concentrations corresponds to a lack of capacity of the toxin to induce ethylene synthesis. When FC and IAA are present together at suboptimal concentrations, the effects of the two substances are clearly additive. Growth stimulation by a mixture of FC and IAA at optimal concentrations is equal to that by FC alone. NaF, 2,4-DNP, actinomycin D, and cycloheximide strongly depress FC-induced stimulation of cell enlargement. These data are in agreement with the hypothesis that FC promotes growth through some effect on cell-wall extensibility probably identical to the one mediating auxin-induced growth, while the primary site of action of FC is different from that of the natural and synthetic auxins.     

Comparison of the effect of indoleacetic Acid and fusicoccin on the breakdown of phosphatidylinositol in maize coleoptiles

The effect of indoleacetic acid (IAA) and fusicoccin (FC) on the breakdown of phosphatidylinositol in maize (Zea mays L.) coleoptiles has been studied. Coleoptiles were able to incorporate [³H] myo-inositol into the phospholipid fraction almost linearly for 8 hours. Thin layer chromatography analysis of total phospholipids showed that [³H]myo-inositol was incorporated only into phosphatidylinositol. Prelabeled coleoptiles treated with IAA showed a loss of the radioactivity incorporated in the phospholipid fraction, whose level decreased by 34% after 1 hour. Treatment with FC, on the contrary, did not modify the content of labelled phosphatidylinositol with respect to the control. The different effects of IAA and FC and a possible mechanism of IAA action on growth are discussed.     

Rapid Effects of IAA on Cell Surface Proteins from Intact Carrot Suspension Culture Cells

Suspension cultures of carrot (Daucus carrota L.) which had an absolute requirement for exogenously supplied auxin were grown in medium containing indoleacetic acid (IAA) as the sole auxin source. Putative cell surface proteins were extracted from the intact cells. Resupply of IAA to cultures partially depleted of auxin resulted in rapidly increased activities of three enzyme activities subsequently extracted. Two of the enzyme activities which increased, peroxidase and pectinesterase, have been implicated in the literature as important to cell wall development, structure, and growth. The other enzyme activity which was increased, IAA oxidase, may be involved in the degradation of IAA In vivo. Polypeptides in the extracts were found to increase equally as rapidly as the enzymes in response to IAA as determined with sodium dodecyl sulfate-polyacrylamide electrophoretic gels stained with silver. It is not known whether the changes in enzyme and polypeptide levels in the protein extracts were due to auxin effects on protein synthesis, transport, or extractability.     

Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site

To locate functionally the primary site of auxin action in growing cells, the pool of auxin relevant to induction of growth in maize (Zea mays L.) coleoptile sections was determined. A positive correlation was consistently noted between growth and intracellular levels of indole-3-acetic acid (IAA), i.e. growth appears to be relatively independent of the external level of IAA. N-1-Naphthylphthalamic acid (NPA), a potent inhibitor of auxin transport, was used to enhance accumulation of IAA in coleoptile cells. From the use of NPA, it is shown that: 1) increasing the accumulation of IAA in cells, while the external concentration is held constant, resulted in a concomitant increase in growth, and 2) blocking the exit of IAA from cells with NPA sustained an IAA-induced growth response in the absence of externally applied IAA. Furthermore, the absence of any alterations in auxin binding to microsomal fractions by NPA indicates that the action of NPA in causing enhancement of auxin-induced growth is based upon its inhibition of efflux of IAA from the cells. This research was supported by National Science Foundation grant No. DMB 8515925. The careful assistance of Laurie Brulport is gratefully acknowledged.     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.