Plant Growth Regul (2007) 52:141–150 DOI 10.1007/s10725-007-9184-0
ORIGINAL PAPER
Effect of temperature on growth, proton extrusion and membrane potential in maize (Zea mays L.) coleoptile segments Waldemar Karcz Æ Zbigniew Burdach
Received: 28 June 2006 / Accepted: 29 March 2007 / Published online: 17 April 2007 Springer Science+Business Media B.V. 2007
Abstract The effects of temperature (5–458C) 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 308C, whereas growth in the presence of IAA and FC shows the maximum value in the range 30–358C and 35–408C, 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 408C did not change the kinetic characteristic of membrane potential changes observed for both substances at 258C. However, the increased temperature significantly decreased IAA and FC-induced membrane hyperpolarization. IAA in the incubation medium, at 108C, brought about additional membrane depolarization (apart from the one induced by low temperature). In contrast to IAA, FC at 108C caused gradual repolarization of membrane potential, which correlated with both FC-induced growth and
W. Karcz (&) Z. Burdach Faculty of Biology, Department of Plant Physiology, University of Silesia, ul. Jagiellonska 28, Katowice 40032, Poland e-mail:
[email protected]
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. Keywords Auxin Coleoptile segments Elongation growth Fusicoccin Medium pH Membrane potential Abbreviations IAA Indole-3-acetic acid FC Fusicoccin Em Membrane potential PM Plasma membrane Q10 Temperature coefficient SGR Spontaneous growth response
Introduction It is well known that plants possess numerous mechanisms which enable them to perceive, transduce and respond to a variety of environmental stresses. Temperature extremes are major environmental factors which limit plant growth and development. On the other hand, these two processes are tightly regulated by plant growth substances among which indole-3-acetic acid (IAA) plays a key role (Davies 2004). Over the last few years progress has
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been made towards the elucidation of IAA-induced signal transduction pathways (reviewed in Hagen and Guilfoyle 2002; Scherer 2002; Christian et al. 2006; Fuchs et al. 2006), whereas our knowledge concerning the regulation of plant growth at low or high temperature is still limited. Taking into account the above, it is reasonable to point out that temperature-dependent changes in growth can be mediated, in part, by auxin (IAA). Little research has been published concerning the mechanisms of IAA-induced growth at various temperatures. Initial studies on the temperature dependence of IAA-induced growth from Ray and Ruesink (1962) and Rayle and Cleland (1972), showed that the rate of auxin-induced cell elongation was strongly dependent upon temperature; the Q10 for the rate of cell elongation was higher at lower temperatures than at elevated temperatures. In turn, Nissl and Zenk (1969), in short-term experiments, found that the lag-phase of the growth response to auxin was gradually shortened with increasing medium temperature from 21 to 408C. It has also been shown that the binding of auxin to its receptor was temperature-dependent (Ray and Dohrmann 1977; Lo¨bler and Kla¨mbt 1985). It has recently been proposed that temperature-induced hypocotyl elongation in Arabidopsis correlates with an increase in free IAA concentration and depends on auxin transport (Gray et al. 1998). In turn, Rapparini et al. (2002) showed that IAA metabolism in Lemna gibba undergoes dynamic changes in response to growth temperature. A well studied aspect of auxin action, in maize coleoptile segments, is its effect on cell elongation and proton extrusion (Kutschera and Schopfer 1985a, b; Lu¨then et al. 1990; Claussen et al. 1996; Karcz et al. 1990, 1995; Karcz and Burdach 2002). According to the ‘‘acid-growth theory’’ (Rayle and Cleland 1970, 1992; Hager et al. 1971, 1991), auxin-induced cell wall acidification provides favorable conditions for cell wall loosening, a requirement for cell elongation. The ability of protons to cause cell wall loosening is mediated by a family of proteins called expansins (for review see Cosgrove et al. 2002). At least in maize coleoptile segments, auxin-induced medium acidification was mediated by increased activity and/or amount of the PM H+-ATPase (Hager et al. 1991; Frias et al. 1996).
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There is more information on the fusicoccin (FC)-induced activation of the PM H+-ATPase. This phytotoxin, produced by the fungus Fusicoccum amygdali, mimics the effects of auxin in many respects (Marre` 1979), although, in the case of medium acidification it stimulates this process by a mechanism entirely different from that of auxin. Recently, it was shown that an FC-binding site arises from interaction of the 14-3-3 protein dimer with the C-terminal autoinhibitory domain of the H+ATPase and that FC stabilizes this complex (Jahn et al. 1997; Baunsgaard et al. 1998; Fuglsang et al. 1999; Oecking and Hagemann 1999; Wu¨rtele et al. 2003). In the past few years, a function of 14-3-3 proteins in the regulation of PM H+-ATPase activity under stress has been proposed (for review see Roberts et al. 2002). The main objective of the present study was to compare the effects of auxin (IAA) and FC on growth, medium pH and membrane potential in maize coleoptile segments incubated at a wide range of temperatures (5–458C).
Materials and methods Plant material The experiments were performed with 10 mm-long segments cut from 4-day-old coleoptiles of Zea mays L. cv. K33 · F2 starting 3 mm below the tip. The primary leaf was removed. Conditions for growing the maize seedlings have been described previously (Karcz and Burdach 2002). Chemicals One-millimolar aqueous stock (ethanolic) solution was prepared from indole-3-acetic acid (Serva, Heidelberg, Germany). Fusicoccin was purchased from Sigma and added from a 0.1 mM stock (ethanolic) solution. Growth and pH measurements Growth experiments were carried out in an apparatus, which allowed simultaneous measurements of elongation growth and pH of the incubation medium
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(Karcz et al. 1990; Karcz and Burdach 2002). Briefly, the optical system used for growth measurements (shadow graph method) permitted the recording of longitudinal extension of a stack of 21 segments (each 10 mm in length). The volume of the incubation medium (1 mM KCl, 0.1 mM NaCl, 0.1 mM CaCl2; initial pH 5.8–6.0) in the elongation and pHmeasuring apparatus was 6.3 ml (0.3 ml segment 1). It is noteworthy that in this apparatus the incubation medium also flowed through the lumen of the coleoptile cylinders (Karcz et al. 1995). This feature permits the treatment solutions to be in direct contact with the interior of the segments, which significantly enhanced both the elongation growth (c. 30%) of the coleoptile segments and acidification (c. 0.6 pH unit) of the medium (Karcz et al. 1995). This experimental set-up enabled coleoptile abrasion to be avoided that inhibits (c. 30%) elongation growth (Kutschera and Schopfer 1985a; Lu¨then et al. 1990; Karcz et al. 1995). Measurements of pH were performed with the pHmeter CP-315 (Elmetron, Poland) and pH electrode OSH 10-10 (Metron, Poland). Growth and pH were read every 15 min under the same conditions. Temperature control was obtained by immersing the elongation and pH-measuring system in a thermostatically controlled water bath. Prior to the addition of growth substances to the incubation medium, the coleoptile segments were equilibrated for 2 h at the desired temperature ±0.58C. All manipulations and growth measurements were carried out under dim green light. Electrophysiology The electrophysiological experiments were performed on intact, 10 mm-long, coleoptile segments. A standard electrophysiological technique was used to determine membrane potential as previously described by Karcz and Burdach (2002). The membrane potential (Em) was measured by recording the voltage between a 3 M KCl-filled glass micropipette inserted into the parenchymal cells and a reference electrode in the bathing medium containing the same composition as used in growth experiments. Before the electrophysiological experiments, the coleoptile segments were preincubated for 2 h at the desired temperature in an intensively
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aerated bathing medium. Subsequently, one coleoptile was transferred into a perfusion Plexiglass chamber, which contained the bathing medium at the desired temperature. Medium flow was driven by a peristaltic pump PP 1B-05A (Zalimp, Poland), which also allowed both the flow of the bathing medium at the desired temperature and the change to the bathing medium in the chamber (usually four chamber volumes within less than 2 min). The microelectrodes were inserted into the cells under microscope by means of a micromanipulator (Hugo Sachs Elektronik, Germany). Micropipettes were prepared as previously described by Karcz and Burdach (2002).
Results Temperature dependence of endogenous growth and growth in the presence of IAA or FC Figure 1 shows the effect of temperature (5–458C) on endogenous growth (growth in medium without growth substances) of maize coleoptile segments determined in the elongation and pH-measuring apparatus (see ‘‘Materials and methods’’). In this set-up the segments, after their excision, were incubated (in an intensively aerated medium) for at least 7 h at the desired temperature. As can be seen in Fig. 1, the growth of maize coleoptile segments increased with increasing medium temperature from 10 to 308C. The extreme temperatures in the range of 5–108C and 40–458C, respectively, strongly inhibited segment elongation. The maximal endogenous growth of maize coleoptile segments was observed at 308C (Fig. 1, inset). At this temperature, the growth of the segments was 60% greater compared with segments grown at 258C. Figure 2 shows the effect of temperature on growth of maize coleoptile segments in the presence of auxin. The segments were first preincubated (for 2 h) at the desired temperature, whereupon IAA, at a final concentration of 10 mM, was added. The maximal growth in the presence of IAA was observed in the range of 30–358C (Fig. 2, inset). FC added to the incubation medium in the same way as IAA, at a final concentration of 1 mM, enhanced the growth of segments to the maximal value at
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Fig. 1 Effect of temperature on endogenous growth (growth in medium without growth substances) of maize coleoptile segments. The growth of a stack of 21 segments (10 mm in length), expressed as elongation (mm cm 1), was measured as described in ‘‘Materials and methods’’. The inset shows the temperature dependence of endogenous growth as a function of time. Values are means of nine independent experiments. The statistical analysis (using software Statistica) showed that the differences between values of elongation growth for 58C and 108C are statistically not significant at 420 min (LSD-test, P < 0.05)
35–408C (Fig. 3, inset). At 458C, the elongation of the segments in the presence of FC was reduced by 40% compared to the maximal value at 35–408C. In contrast to IAA, FC significantly enhanced the elongation growth of segments over the range of lower temperatures (5–108C). Effect of temperature on pH changes of the incubation medium The data in Fig. 4 indicate that coleoptile segments incubated at 258C (reference temperature), without growth substances, characteristically changed the pH of the medium: usually within the first 2 h an increase of pH, to near neutral, was observed, followed by a slow decrease to a value around 5.4–5.6 after 7 h. When coleoptile segments were incubated at 58C (data not shown but similar to 108C) and 108C, this characteristic pattern of change in external medium pH was disturbed (Fig. 4). At these temperatures, a gradual increase of medium pH was observed instead
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Fig. 2 Effect of temperature on growth of maize coleoptile segments in the presence of indoleacetic acid (IAA) (10 mM). The growth of a stack of 21 segments, expressed as elongation (mm cm 1), was measured as described in ‘‘Materials and methods’’. After preincubation (over 2 h at the desired temperature) of the coleoptile segments in control medium, IAA was added (arrow). Values are means of 11 independent experiments. The inset shows the temperature dependence of growth in the presence of IAA as a function of time. The differences between values of elongation growth, in the presence of IAA, for 5 and 108C, and for 30 and 358C are statistically not significant at 420 min (LSD-test, P < 0.05)
of a slow decrease. At 30 and 358C the kinetics of medium pH change were not significantly different compared to that measured at 258C. At higher temperatures (40 and 458C) the acidification of the external medium, expressed as difference between H+ concentration at 7 and 2 h (D [H+]), was significantly lower compared to that at 30–358C (Fig. 4, inset). When IAA or FC was added (after 2 h of preincubation) at 258C to the medium containing coleoptile segments, an additional decrease of medium pH, as compared to medium without growth substances, was observed (Figs. 4, 5). As indicated in Figs. 5 and 6, FC added at 258C was much more effective in acidification of the medium, as compared to IAA. For FC, 5 h after its addition, the pH of the incubation medium dropped to approximately pH 3.9, whereas for IAA the pH was only 5.3. Addition of IAA or FC at 208C (data not shown) and 308C had practically no effect on the characteristic change in medium pH observed at 258C (Fig. 5). At 358C, approximately 3 h after addition of
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Fig. 3 Effect of temperature on growth of maize coleoptile segments in the presence of fusicoccin (FC) (1 mM). The growth of a stack of 21 segments, expressed as elongation (mm cm 1), was measured as described in ‘‘Materials and methods’’. After preincubation (over 2 h at the desired temperature) of the coleoptile segments in control medium, FC was added (arrow). Values are means of 11 independent experiments. The inset shows the temperature dependence of growth in the presence of FC as a function of time. The differences between values of elongation growth, in the presence of FC, for 35 and 408C are statistically not significant at 420 min (LSD-test, P < 0.05)
Fig. 4 Effect of temperature on the medium pH of maize coleoptile segments. Values for pH are means of nine independent experiments performed simultaneously with endogenous growth using the same tissue sample (as described in ‘‘Materials and methods’’). To avoid illegibility of the figure only some curves have been shown. The inset shows temperature dependence of medium pH expressed as D[H+], where D[H+] means difference between H+ concentration ([H+]) at 420 min and 120 min. The differences between values of pH for temperatures showed in figure are statistically significant at 420 min (LSD-test, P < 0.05)
IAA to the incubation medium, a recovery of pH (a net uptake of protons) was observed. It was found, however, that at low temperatures (5 and 108C) the kinetics of pH change differed significantly for both growth substances; the segments treated with IAA at 5 (kinetic not shown) and 108C were virtually not able to acidify the external medium, whereas FC at these temperatures caused slow, but marked acidification of the medium (Figs. 5, 6). Temperature maxima for IAAand FC-induced medium acidification (D[H+]) were observed over the ranges of 30–358C and 35–408C, respectively (Fig. 6).
and 1 mM, respectively. The Em of the parenchymal cells at 258C, before being changed in response to IAA or FC, was 115.8 ± 9.8 mV (mean ± SD, n = 14). The addition of IAA to the incubation medium at 258C (Fig. 7A) characteristically changed the Em of the parenchymal cells: after initial, transient depolarization of Em by 8.1 ± 3.9 mV (mean ± SD, n = 7) a delayed hyperpolarization, during which the membrane potential became 9.9 ± 4.2 mV (mean ± SD, n = 7) more negative than the observed original potential. In contrast to IAA, 1 mM FC caused an immediate hyperpolarization of 25.2 ± 9.8 mV (mean ± SD, n = 9). Preincubation of maize coleoptile segments over 2 h at 308C (the temperature at which maximal growth in the presence of IAA was observed) and 408C (the temperature at which maximal growth in the presence of FC was observed) caused hyperpolarization of the Em by 7.5 and 15.4 mV, as compared to the Em at 258C,
Effect of temperature on IAA and FC-induced membrane potential (Em) changes After insertion of the microelectrode into the cell and stabilization of Em, the bathing medium was refreshed (at the same salt composition and temperature), containing IAA or FC at a final concentration of 10
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Fig. 5 Effect of temperature on indoleacetic acid (IAA) and fusicoccin (FC)-induced medium pH changes of maize coleoptile segments. Growth effectors were added after 2 h of preincubation at the desired temperature (arrow). Values for IAA and FC are means of nine independent experiments performed simultaneously with growth using the same tissue sample (as described in ‘‘Materials and methods’’). To avoid illegibility of the figure only some curves have been shown. The statistical analysis showed that the differences between values of pH in the presence of IAA are statistically not significant for 30 and 358C at 420 min. In the presence of FC the differences between values of pH for 25, 35 and 408C are statistically not significant (LSD-test, P < 0.05)
Fig. 6 Effect of temperature on indoleacetic acid (IAA) and fusicoccin (FC)-induced proton extrusion (expressed as D[H+], where D[H+] means difference between H+ concentration ([H+]) at 420 min and 120 min) in maize coleoptile segments. Growth effectors were added after 2 h of preincubation at the desired temperature. Values for IAA and FC are means of nine independent experiments performed simultaneously with growth using the same tissue sample (as described in ‘‘Materials and methods’’). Bars indicate ± SD
Fig. 7 Effect of temperature on indoleacetic acid (IAA) and fusicoccin (FC)-induced changes in membrane potential (Em) of parenchymal coleoptile cells. At time 0 (arrow) the control medium was changed for a new one, at the same temperature and salt composition, containing in addition IAA or FC.
Representative curves for each substance are shown. Adequate mean value are indicated in Table 1. (A) IAA and FC added at 30 and 408C, respectively. For comparison effect of both substances at 258C was also shown. (B) IAA and FC added at 108C
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respectively (Table 1, Fig. 7A). If either IAA or FC was added to the segments after a 2 h preincubation at 30 or 408C, respectively, the hyperpolarization of the Em was increased and this increase was significantly higher in the case of FC application (Table 1, Fig. 7A). Preincubation of maize coleoptile segments (over 2 h) at 108C brought about depolarization of the Em to a level of 66.9 ± 6.6 mV (mean ± SD, n = 7) (Fig. 7B). The addition of IAA at this low level of Em caused an additional membrane depolarization by 5–7 mV followed by a slight recovery of Em (Fig. 7B). However, when FC was applied under the same experimental conditions, as IAA, hyperpolarization of the Em by 5 mV was observed (Table 1, Fig. 7B).
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Effect of temperature on endogenous growth and simultaneously measured pH of the incubation medium
The major goal of the studies described here was to understand the link between the signal transduction pathways activated in response to extreme temperatures and plant growth substances. 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 known how these substances work at extreme temperatures. As far as we know, the temperature dependence of growth in the presence of IAA and FC, proton extrusion (simultaneously measured with growth) and membrane potential in grass coleoptile cells have never been examined over a wide range of temperatures (5–458C).
The endogenous growth (growth in medium without growth substances) of the coleoptile segments showed a clear maximum at 308C and became smaller as the medium temperature increased or fell from 308C (Fig. 1). At extreme temperatures (5–108C) the growth of maize coleoptile segments were completely abolished, possibly due to the breakdown of calcium homeostasis or loss of turgor. An explanation for the enhancement of growth at 308C could be reconciled by either of the two main hypotheses proposed for interpreting the nature of a spontaneous growth response (SGR). In accordance with the first hypothesis, SGR is the result of a time-dependent increase in tissue sensitivity to a low concentration of endogenous auxin which remains after segment excision (MacDowell and Sirois 1977; Vesper and Evans 1978). The second hypothesis states that SGR is caused by IAA synthesis in the coleoptile segments after excision (Evans and Schmitt 1975; Weiler et al. 1981). Thus, it is possible that increased tissue sensitivity to endogenous IAA or increased synthesis of IAA may occur at 308C. Recently, it has been shown that temperatureinduced hypocotyl elongation in Arabidopsis correlates with an increase in free IAA concentration (Gray et al. 1998). The characteristic pattern of change in external medium pH, simultaneously measured with endogenous growth of maize coleoptile segments, described here at 258C (Fig. 4) is in good agreement with the results obtained by Peters et al. (1998) and Karcz and
Table 1 Effect of temperature on indoleacetic acid (IAA) and fusicoccin (FC)-induced membrane potential (Em) changes in the parenchymal cells of maize coleoptile segments. Prior to
inserting of the microelectrode into the cell the coleoptile segments were first preincubated over 2 h in bathing medium at the desired temperature
Discussion
Temperature of the incubation medium (8C)
Membrane potential, Em (mV) Em after stabilization in the control Em after 35 min treatment medium with IAA
Em after 35 min treatment with FC
10
66.9 ± 6.6
61.2 ± 5.8
71.9 ± 6.2
25
115.8 ± 9.8
125.7 ± 2.4
141.0 ± 13.7
30
123.3 ± 11.9
131.4 ± 12.1
139.0 ± 12.5
40
131.2 ± 11.3
136.6 ± 12.8
145.8 ± 13.6
The data are means of at least seven independent experiments. Error indicate ±SD
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Burdach (2002). These authors showed that there was a close temporal correlation between the characteristic pattern of external medium pH and endogenous growth rate. In the present study it has been demonstrated that at 5 and 108C (Fig. 4) instead of the characteristic pattern of a slow decrease in external medium pH, a gradual increase (net H+ uptake) is observed. A plausible interpretation for this increase in medium pH is that it may result from the overlapping net H + uptake of the plant tissue in response to wounding (Chastain and Hanson 1982; Hush et al. 1992; Peters 1998) and cold stress (Chastain and Hanson 1982; Shabala and Shabala 2002). Effect of temperature on growth in the presence of IAA and FC, and simultaneously with growth measured medium pH The growth and proton extrusion by maize coleoptile segments observed here at 258C in the presence of IAA and FC (Figs. 2, 3, 5) were in good qualitative agreement with the results obtained with this model system by other (Kutschera and Schopfer 1985a, b; Lu¨then et al. 1990; Karcz et al. 1995; Claussen et al. 1996). The growth of maize coleoptile segments in the presence of IAA showed a maximum at 30–358C (Fig. 2). It should be pointed out that in the case of endogenous growth the increase of temperature from 25 to 308C enhanced growth by 60% (Fig. 1), whereas the same temperature interval growth, in the presence of IAA, increased only by 15% (Fig. 2). The Q10 values calculated from the initial (within first 3 h) slope of growth in the presence of IAA at lower (15–258C) and higher (25–358C) temperatures are in line with published data Rayle and Cleland (1972), who found Q10 = 5 over the first interval and Q10 = 1.2 over the second (3.4 and 1.2 in our experiments, respectively). In turn, Fig. 3 shows that the temperature dependence for growth in the presence of FC had a maximum at 35– 408C. The Q10 values calculated for FC at the same temperature protocol as for IAA were 2.3 and 1.7, respectively (the literature lack comparable data). Taking into account that the Q10 values, were significantly higher at lower temperatures, we suggest that temperature optima observed for endogenous growth and growth in the presence of IAA and FC represent a balance between temperature dependent effects on metabolic pathways and respiratory rates. For example, it has been shown (Bravo-F and Uribe 1981) that
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K+ uptake in corn roots was more temperature sensitive than respiration, especially below 228C. Above this temperature the Q10 of K+ uptake decreased and was similar to that of respiration. Simultaneous measurements of growth and external medium pH of maize coleoptile segments showed that FC at extreme temperatures was not only much more active in the stimulation of growth, but also acidified the external medium more than IAA (Figs. 2, 3, 5, 6). For example, the coleoptile segments treated with IAA at low temperatures (5 and 108C) were practically unable to acidified the external medium (Figs. 5, 6). In contrast to IAA, FC added at 5 and 108C acidified external medium by 0.39 and 0.80 pH-unit (Fig. 5), respectively. The results reported here provide evidence that at extreme temperatures FC was much more effective, as compared to IAA, in stimulating the growth of maize coleoptile segments and in acidification of their external medium. This observation supports the findings that IAA and FC differ in their signal transduction pathway (Hager 2003). Electrophysiology The kinetics of the IAA and FC-induced membrane potential changes of coleoptile cells observed here at 258C (Fig. 7A) are in good qualitatively agreement with the results obtained by Bates and Goldsmith 1983; Felle et al. 1991; Peters et al. 1992; Keller and Volkenburgh 1996. At present, there is no doubt that plasma membrane hyperpolarization in the presence of IAA or FC was a consequence of stimulation of proton extrusion by a H+-ATPase (Ru¨ck et al. 1993; Hedrich et al. 1995). Incubation of maize coleoptile segments over 2 h at 30 and 408C caused hyperpolarization of the Em by 7.5 and 15.4 mV, as compared to 258C, respectively (Table 1, Fig. 7A). Such hyperpolarization of the Em at high temperatures may reflect either an increase in free IAA, followed by stimulation of proton pumping by the H+-ATPase, or the direct activation of the H+-ATPase. The addition of either IAA or FC to the bathing medium at 308 and 408C did not alter the characteristic pattern of the Em changes observed here for both substances at 258C, but significantly decreased the IAA and FC-induced membrane hyperpolarization (Table 1, Fig. 7A). This observation suggests that the hyperpolarization of Em recorded here at high temperatures and in the presence of IAA or FC were not additive processes.
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Preincubation of maize coleoptile segments over 2 h at 108C caused depolarization of the Em to the level of 66.9 mV (Table 1, Fig. 7B). Several mechanisms, including decrease in H+ pump activity or net H+ and Ca2+ uptake by plant tissues, have been suggested as explanations for membrane potential depolarization under cold stress (Minorsky 1989; Ding and Pickard 1993; Yoshida et al. 1999; Shabala and Shabala 2002). However, the fact that net H+ uptake took place at 108C (Fig. 4), it appears likely that an influx of H+ ions, at least in part, was responsible for membrane depolarization. The electrophysiological and pH-measurements performed at 108C in distillated water (data not shown) support this suggestion. Addition of IAA to the incubation medium at 108C brought about additional membrane depolarization, which only slightly recovered during experiment (Fig. 7B). In contrast to IAA, FC added at this temperature caused gradual repolarization of the Em, which correlated with both growth in the presence of FC (Fig. 3) and FC-induced medium acidification (Fig. 5). In conclusion, the results presented in this paper demonstrate that: (1) endogenous growth of maize coleoptile segments had a clear maximum at 308C (Fig. 1); (2) growth in the presence of IAA and FC differed in terms of temperature maximum (Figs. 2, 3); (3) temperature maxima for IAA- and FC-induced growth correlated with IAA- and FC-induced medium acidification, respectively (Figs. 2, 3, 5, 6); (4) IAA and FC-induced electrogenic activity differed qualitatively and quantitatively over the range of temperatures used. A plausible interpretation for temperature-induced changes in growth of maize coleoptile cells is that, at least in part, these are mediated via a PM H+-ATPase activity, which is regulated by auxin and fusiccocin in different ways. Acknowledgments We wish to thank Professor Peter Minorsky (Mercy College, New York, USA) for critical reading of this manuscript and correcting the English text.
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