Effects of UV-C radiation on extension growth, H+-extrusion and transmembrane electric potential in maize coleoptile segments

The effect of 253.7 nm ultraviolet radiation on elongation growth, medium acidification and changes in electric potential difference between vacuole and external medium in cells of maize ( Zea mays L.) coleoptile segments was investigated. It was found that irradiation with 390, 1170, 3900 and 5 850 J m⁻² UV-C (ultraviolet radiation 253.7 nm) inhibited elongation growth, whereas at 195 J m⁻² stimulation of growth was observed. The administration of IAA (10⁻⁵ M ) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium, measured simultaneously with growth, showed that the exposure of the segments to UV-C caused inhibition of H⁺-extrusion (or stimulation of H⁺ uptake). The presence of IAA (10⁻⁵ M ) in the incubation medium promoted (except after 5850 J m⁻² irradiation) H⁺-extrusion to a level comparable with that produced by IAA in non-irradiated segments. In UV-C irradiated segments the potential difference underwent significant alterations. Irradiation of coleoptile segments with 390 J m⁻² caused a transient depolarization, which was fully reversible within 30 min, while at higher doses depolarization was irreversible. The hyperpolarization of the membrane potential (MP) in cells of maize coleoptile induced by IAA was completely nullified by subsequent irradiation with UV-C. It is suggested that UV-C inhibited IAA-induced growth by a mechanism independent of cell wall acidification.     

Effect of cadmium on growth, proton extrusion and membrane potential in maize coleoptile segments

Cd accumulation, its effects on elongation growth of maize coleoptile segments, pH changes of their incubation medium and the membrane potential of parenchymal cells were studied. The Cd content increased significantly with exposure to increasing cadmium concentrations. Coleoptile segments accumulated the metal more efficiently in the range 10–100 μM Cd, than in the range 100–1000 μM Cd. Cd at concentrations higher than 1.0 μM produced a significant inhibition of both growth and proton extrusion. 100 μM Cd caused depolarization of the plasma membrane (PM) potential in parenchymal cells. The simultaneous treatment of maize coleoptile segments by indole-3-acetic acid (IAA) and Cd, counteracted the toxic effect of Cd on growth. Moreover, our data also showed that 100 μM Cd suppressed the characteristic IAA-induced hyperpolarization of the membrane potential, causing membrane depolarization. These results indicate that the toxic effect of Cd on growth of maize coleoptile segments might be, at least in part, caused via reduced PM H⁺-ATPase activity.     

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.     

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.     

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.     

Role of chloride ions in the promotion of auxin-induced growth of maize coleoptile segments

Background and Aims

The mechanism of auxin action on ion transport in growing cells has not been determined in detail. In particular, little is known about the role of chloride in the auxin-induced growth of coleoptile cells. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were carried out with maize (Zea mays) coleoptile segments, this being a classical model system for studies of plant cell elongation growth.

Methods

Growth kinetics or growth and pH changes were recorded in maize coleoptiles using two independent measuring systems. The growth rate of the segments was measured simultaneously with medium pH changes. Membrane potential changes in parenchymal cells of the segments were also determined for chosen variants. The question of whether anion transport is involved in auxin-induced growth of maize coleoptile segments was primarily studied using anion channel blockers [anthracene-9-carboxylic acid (A-9-C) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS)]. In addition, experiments in which KCl was replaced by KNO₃ were also performed.

Key Results

Both anion channel blockers, added at 0·1 mm, diminished indole-3-acetic acid (IAA)-induced elongation growth by ∼30 %. Medium pH changes measured simultaneously with growth indicated that while DIDS stopped IAA-induced proton extrusion, A-9-C diminished it by only 50 %. Addition of A-9-C to medium containing 1 mm KCl did not affect the characteristic kinetics of IAA-induced membrane potential changes, while in the presence of 10 mm KCl the channel blocker stopped IAA-induced membrane hyperpolarization. Replacement of KCl with KNO₃ significantly decreased IAA-induced growth and inhibited proton extrusion. In contrast to the KCl concentration, the concentration of KNO₃ did not affect the growth-stimulatory effect of IAA. For comparison, the effects of the cation channel blocker tetraethylammonium chloride (TEA-Cl) on IAA-induced growth and proton extrusion were also determined. TEA-Cl, added 1 h before IAA, caused reduction of growth by 49·9 % and inhibition of proton extrusion.

Conclusions

These results suggest that Cl^– plays a role in the IAA-induced growth of maize coleoptile segments. A possible mechanism for Cl^– uptake during IAA-induced growth is proposed in which uptake of K⁺ and Cl^– ions in concert with IAA-induced plasma membrane H⁺-ATPase activity changes the membrane potential to a value needed for turgor adjustment during the growth of maize coleoptile cells.     

Effect of IAA and 4-Cl-IAA on growth rate in maize coleoptile segments

The dose-response curves for IAA and 4-Cl-IAA-induced growth of Zea mays L. coleoptile segments were studied as a function of time. Moreover, some characteristic growth parameters for both auxins were compared. The dose-response curve of growth rate measured after IAA or 4-Cl-IAA application was bell-shaped in all experiments. The optimum concentration was 10⁻⁶ M for 4-Cl-IAA and was found not to depend on the time of the growth measurement. However, in the case of IAA the optimum shifted from 10⁻⁶ M at the time of maximal growth rate to 10⁻⁵ M or even 10⁻⁴ M, when growth measured 3–4 hours after auxin application was analysed. The relative activity of 4-Cl-IAA-induced growth rate (as compared to IAA) increased significantly with increasing time from addition of this auxin to the medium. For both auxins the time needed to reach the maximal growth rate was clearly related to their concentrations. These data provided further evidence that 4-Cl-IAA is much more active auxin than IAA and can also suggest that IAA is more rapidly metabolized in comparison to 4-Cl-IAA.     

A comparison of the effects of 1,4-naphthoquinone and 2-hydroxy-1,4-naphthoquinone (lawsone) on indole-3-acetic acid (IAA)-induced growth of maize coleoptile cells

The effects of 1,4-naphthoquinone (NQ) and 2-hydroxy-1,4-naphthoquinone (NQ-2-OH) on indole-3-acetic acid (IAA)-induced growth, medium pH changes and membrane potential (E_m) in maize (Zea mays L.) coleoptile cells were determined. In addition, the redox cycling properties of both naphthoquinones were also compared. The dose-response curves constructed for the effects of NQ and NQ-2-OH on endogenous and IAA-induced growth differ in shape. It was found that NQ was by 10–50% more effective in inhibiting IAA-induced growth in maize coleoptile segments than NQ-2-OH. Simultaneous measurements of growth and external medium pH indicated that NQ and NQ-2-OH reduced or eliminated proton extrusion at all of the concentrations used, excluding NQ at 1 µM. It was found that both naphthoquinones at concentrations higher than 10 µM caused the depolarisation of the membrane potential (E_m). Additionally, compared to the controls, NQ- and NQ-2-OH-exposure of coleoptile segments, at concentrations higher than 10 µM, caused an elevation of the hydrogen peroxide (H₂O₂) production and plasma membrane redox activity. The highest catalase activity was observed at 10 µM NQ and it was ca. 18-fold greater (at 4 h) than in the control medium. Moreover, it was also found that NQ and NQ-2-OH, at all concentrations studied, increased the malondialdehyde content of coleoptile segments at 4 h of the experiment. The data presented here are discussed taking into account the “acid growth hypothesis” of auxin action and the mechanisms by which naphthoquinones interact with biological systems.     

The effect of auxins (IAA and 4-Cl-IAA) on the redox activity and medium pH of Zea mays L. root segments

Indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA) were tested at different concentrations and times for their capacity to change the redox activity and medium pH of maize root segments. The dose-response surfaces (dose-response curves as a function of time) plotted for redox activity and changes in medium pH (expressed as ΔpH) had a similar shape for both auxins, but differed significantly at the optimal concentrations. With 4-Cl-IAA, the maximal values of redox activity and medium pH changes were observed at 10⁻¹⁰ M, which was a 100-fold lower concentration than with IAA. Correlations were observed between redox activity and medium pH changes at the optimal concentrations of both IAA and 4-Cl-IAA. The results are discussed herein, taking into account both the concentration of the auxins and the effects produced by them.     

Interactive effects of temperature and heavy metals (Cd, Pb) on the elongation growth in maize coleoptiles

The effect of Cd and Pb on endogenous and IAA-induced elongation growth and medium pH of maize coleoptile segments incubated at 20, 25 and 30 °C was studied. It was found that the elongation of coleoptile segments and proton extrusion increased with the temperature and reached its maximum at 30 °C. For Cd, the maximal inhibition of endogenous and IAA-induced growth as well as medium acidification of coleoptile segments was observed at 25 °C. Meanwhile, Pb, irrespective of the temperature, diminished the growth of the segments by ca. 20%, increasing the acidification of the incubation medium. It was also found that in contrast to Cd, Pb accumulation in maize coleoptile segments did not correlate with temperature. The results suggest that the toxic effect of Cd on elongation growth of coleoptile segments is connected with the decrease of the PM H(+)-ATPase activity and probably with Cd-induced high acivity of IAA oxidase, whereas the effect of Pb did not depend on activity of any of the enzymes.     

Regulation of electrogenic proton pumping by auxin and fusicoccin as related to the growth of Avena coleoptiles

The temporal relations between early responses to indoleacetic acid (IAA), proton secretion, hyperpolarization of the membrane potential, and growth change during the incubation of segments of oat (Avena sativa L.) coleoptiles in a low salt medium. When IAA is added after pretreatment of several hours, proton secretion increases after a latency of 7 minutes and reaches its maximum 10 to 15 minutes later. This timing coincides with both the increase in growth of the segments and the hyperpolarization of the membrane potential of parenchyma cells, consistent with the hypothesis that the change in membrane voltage reflects the activity of an electrogenic proton pump. The extent of IAA-induced hyperpolarization is substantially reduced by elevating [KCl]₀, most likely because this increases the passive conductance of the membrane. Neither growth nor proton secretion is affected by high [KCl]₀ (30 millimolar), indicating that neither process is limited by the magnitude of the membrane potential. These results are consistent with the acid growth hypothesis. Following short incubation times, however, IAA-induced hyperpolarization and growth are detected within 10 minutes, while acidification of the medium is delayed for more than 40 minutes. This result is seemingly in conflict with the acid growth hypothesis, but in freshly cut tissue, the pH of the external medium may not reflect the pH of the epidermal cell walls. The temporal coincidence of auxin-induced growth and hyperpolarization suggests that in freshly isolated segments the hyperpolarization is a more sensitive indication of proton secretion than is acidification of the external aqueous environment.     

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.     

Rapid Hormone-induced Hyperpolarization of the Oat Coleoptile Transmembrane Potential

The effects of the plant growth substances indoleacetic acid (IAA) and fusicoccin on the transmembrane potential of Avena coleoptile cells (at 27-29 C) were studied. Fusicoccin caused hyperpolarization of the membrane potential which started after a lag of less than 20 seconds, and which on average reached -49 mv at an external K(+) concentration of 1 mm and -75 mv at 0.1 mm K(+). IAA caused a hyperpolarization of -25 mv starting after a lag of 7 to 8 minutes. These results suggest that fusicoccin and IAA both activate electrogenic H(+) extrusion.     

Effect of temperature on the dose–response curves for auxin-induced elongation growth in maize coleoptile segments

The dose–response curves for IAA-induced growth in maize coleoptile segments were studied as a function of time and temperature. In addition, the kinetics of growth rate responses at some auxin concentrations and temperatures was also compared. It was found that the dose–response curves for IAA-induced elongation growth were, independently of time and temperature, bell-shaped with an optimal concentration at 10⁻⁵ M IAA. The kinetics of IAA-induced growth rate responses depended on IAA concentration and temperature, and could be separated into two phases (biphasic reaction). The first phase (very rapid) was followed by a long lasting one (second phase), which began about 30 min after auxin addition. For coleoptile segments incubated at 30°C, the amplitudes of the first and second phase were significantly higher, when compared with 25°C, at all IAA concentrations studied. However, when coleoptile segments were incubated at 20°C, the elongation growth of coleoptile segments treated with suboptimal IAA concentrations was diminished, mainly as a result of both phases reduction. In conclusion, we propose that the shape of the dose–response curves for IAA-induced growth in maize coleoptile segments is connected with biphasic kinetic of growth rate response.     

The electrical response of maize to auxins

The electrical response of Zea mays coleoptiles and suspension cultured cells to several growth-promoting auxins (IAA, IBA, 2,4-D, 2,4,5-T, 1-NAA) and some of their structural analogues (2,3-D, 2-NAA) has been tested. In coleoptile two typical electrical responses to IAA are observed: an immediated rapid depolarization, and a hyperpolarization following 7-10 minutes after the first external addition of IAA. Of the other tested compounds only 1-NAA significantly depolarized the cells, whereas all auxins as well as the analogues evoked delayed hyperpolarizations. In contrast, the suspension cells were not hyperpolarized by any of the tested compounds, but were strongly depolarized by IAA, 1-NAA, and to a lesser extent by 2-NAA. In these cells IAA and 1-NAA induced inwardly directed currents of positive charge which both saturated around 12 mA/m2. The strong pH-dependence together with the half-maximal currents 0.49 microM IAA and 0.76 microM 1-NAA point to a symport of the anions with at least 2H+. The delayed plasma membrane hyperpolarization is a different response, and seems to be initiated by the protonated auxin species. In accordance with the current literature, it is interpreted as consequence of a stimulated proton extrusion. The finding that all tested compounds evoked a hyperpolarization, makes this response unspecific. It is concluded that a stimulation of proton extrusion is a necessary, but not sufficient step to induce elongation growth.     

Short-term effects of plant hormones on membrane potential and membrane permeability of dwarf maize coleoptile cells (Zea mays L. d 1) in comparison with growth responses

The membrane potential difference of dwarf maize coleoptile cells is increased by both 10^-5moll^-1 gibberellic acid (GA₃) and indoleacetic acid (IAA) a few minutes after application. A final level is reached after 10–20 min. The membrane permeability ratio P _Na:P _K is altered by both hormones during the first 15 min after application, indicating a rapid effect on the membrane. Elongation growth of coleoptile segments, however, is only stimulated by IAA. The auxin-induced growth as well as the auxin effect on membrane permeability depends on the calcium ion concentration of the medium. It is concluded that IAA acts via a proton extrusion pump that is electrically balanced by a potassium ion uptake, driven by the electromotive force of the pump. The mode of action of GA₃ on elongation growth is assumed to involve a process that depends on the physiologic state of the tissue and/or metabolic energy.Abbreviations IAA indoleacetic acid - GA₃ gibberellic acid - FC fusicoccin - PD electric potential difference between the vacuole and the external medium     

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     

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

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

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.     

Evidence of 4-Cl-IAA and IAA Bound to Proteins in Pea Fruit and Seeds

The auxins 4-chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-acetic acid (IAA) occur naturally in pea vegetative and fruit tissues (Pisum sativum L.). Previous work has shown that 4-Cl-IAA can substitute for the seeds in the stimulation of pea pericarp growth, whereas IAA is ineffective. Both auxins are found as free acids and as low-molecular-weight conjugates from organic solvent-soluble extracts from pea fruit. Here we present evidence for an additional conjugated auxin species that was not soluble in organic solvent and yielded 4-Cl-IAA and IAA after strong alkaline hydrolysis, suggestive of auxin attachment to pea seed and pericarp proteins. The solvent-insoluble conjugated 4-Cl-IAA in young pericarp was on average 15-fold greater than solvent-soluble 4-Cl-IAA. The solvent-insoluble conjugated IAA was approximately half the levels reported for the solvent-soluble IAA fraction. To identify putative 4-Cl-IAA-bound proteins, polyclonal antibodies were raised to 4-Cl-IAA linked to bovine serum albumin protein (BSA). Immunoblots probed with anti-4-Cl-IAA-BSA antiserum detected three to four unique bands (32–40 kDa) in primarily maternal tissues, and a different set of protein bands were detected in mainly embryonic tissues (ca. 65–74 kDa in mature seed). 4-Cl-IAA and IAA were also identified from protein fractions separated by polyacrylamide gel electrophoresis using GC-MS. These data show that the majority of 4-Cl-IAA, the growth-active auxin in young pea pericarp, and significant levels of IAA are linked to protein fractions. Auxin-proteins may function in regulation of free bioactive 4-Cl-IAA and IAA levels, and/or 4-Cl-IAA or IAA may be targeted to specific proteins post-translationally to modify protein function or stability.