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

Abstract

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.

Keywords: Anion channel blockers; Zea mays maize; auxin; cell growth; chloride uptake; coleoptile segments; elongation growth; membrane potential.

Fig. 1.

Effects of 1 mm (A) and 10 mm (B) KCl or KNO₃ on the growth rate of maize coleoptile segments incubated in the presence or absence of 10 μm IAA. The growth rate of a stack of five segments was measured as described in Materials and methods (first measuring system). IAA was added to the incubation medium at 2 h. Inset shows total elongation growth calculated as the sum of extensions measured at 3-min intervals for 10 h. All curves represent means of at least nine independent experiments. Bars indicate ± s.e. The LSD test is included in Supplementary Data Fig. S2.

Fig. 2.

Effects of anion (A-9-C or DIDS) and cation (TEA-Cl or BaCl₂) channel blockers on the growth rate of maize coleoptile segments incubated in the presence of IAA (10 μm). Coleoptile segments were preincubated (1 h) in a control medium and the channel blockers were then added; IAA was added to the incubation medium at 2 h. Curves represent data obtained from the first measuring system. Inset shows total elongation growth as a bar graph, calculated as the sum of extensions between 120 and 600 min of the experiment. Values are means of at least nine independent experiments. Bars indicate s.e. Means followed by the same letter are not significantly different from each other according to the LSD test (P < 0·05).

Fig. 3.

Effects of anion (A-9-C or DIDS) and cation (TEA-Cl or BaCl₂) channel blockers on the growth rate of maize coleoptile segments incubated in the presence of IAA (10 μm). Auxin was added to the incubation medium at 2 h and channel blockers 3 h later. Curves represent data obtained from the first measuring system. All curves are means of at least eight independent experiments.

Fig. 4.

Effect of 10 mm KCl or 10 mm KNO₃ on the growth rate of maize coleoptile segments incubated in the presence or absence of 10 μm IAA, added to the incubation medium at 2 h. The growth rate of a stack of 20 segments was measured as described in Materials and methods (second measuring system). Inset on the right shows total elongation growth, calculated as the sum of extensions measured at 3-min intervals for 10 h. Inset on the left presents medium pH changes when maize coleoptile segments were incubated in the presence of IAA. All curves represent means of at least nine independent experiments. Bars indicate ± s.e. The LSD test for growth is included in Supplementary Data Fig. S2.

Fig. 5.

Effects of 10 mm KCl, 10 mm KNO₃ and 5 mm KCl plus 5 mm KNO₃ on the growth rate of maize coleoptile segments incubated in the presence of 10 μm IAA added to the incubation medium at 2 h. Inset on the left presents medium pH changes for maize coleoptile segments measured simultaneously with growth. All curves represent means of at least nine independent experiments. Bars indicate ± s.e. The LSD test for growth is included in Supplementary Data Fig. S2.

Fig. 6.

Effects of anion (A-9-C or DIDS) and cation (TEA-Cl) channel blockers on the growth rate of maize coleoptile segments incubated in the presence of IAA (10 μm). Coleoptile segments were preincubated (1 h) in control medium, the channel blockers were then added to the incubation medium, and IAA was added at 2 h. Curves represent data obtained using the second measuring system. Inset on the right shows total elongation growth, calculated as the sum of extensions measured at 3-min intervals for 10 h. Inset on the left presents medium pH changes for maize coleoptile segments incubated in the presence of IAA. All curves represent means of at least seven independent experiments. Bars indicate ± s.e. The LSD test for growth is included in Supplementary Data Fig. S2.

Fig. 7.

Effects of A-9-C and TEA-Cl added together or separately (1 h after starting the experiment) on the growth rate of maize coleoptile segments incubated in the presence of 10 μm IAA. IAA was added to the incubation medium at 2 h. Curves represent data obtained using the second measuring system. Inset on the right shows total elongation growth, calculated as the sum of extensions measured at 3-min intervals for 10 h. Inset on the left presents medium pH changes for maize coleoptile segments incubated in the presence of IAA. All curves represent means of at least nine independent experiments. Bars indicate ± s.e. The LSD test for growth is included in Supplementary Data Fig. S2.

Fig. 8.

Comparison of the effect of an anion channel blocker (A-9-C) on the growth rate of maize coleoptile segments incubated in the presence of 10 mm KCl or 10 mm KNO₃ and 10 μm IAA, added to the medium at 2 h. Inset on the right shows total elongation growth, calculated as the sum of extensions measured at 3-min intervals for 10 h. Inset on the left presents medium pH changes measured simultaneously with growth. All curves represent means of at least seven independent experiments. Bars indicate ± s.e. The LSD test for growth is included in Supplementary Data Fig. S2.