Some New Methodological and Conceptual Aspects of the "Acid Growth Theory" for the Auxin Action in Maize (Zea mays L.) Coleoptile Segments: Do Acid- and Auxin-Induced Rapid Growth Differ in Their Mechanisms?

Abstract

Two arguments against the "acid growth theory" of auxin-induced growth were re-examined. First, the lack of a correlation between the IAA-induced growth and medium acidification, which is mainly due to the cuticle, which is a barrier for proton diffusion. Second, acid- and the IAA-induced growth are additive processes, which means that acid and the IAA act via different mechanisms. Here, growth, medium pH, and membrane potential (in some experiments) were simultaneously measured using non-abraded and non-peeled segments but with the incubation medium having access to their lumen. Using such an approach significantly enhances both the IAA-induced growth and proton extrusion (similar to that of abraded segments). Staining the cuticle on the outer and inner epidermis of the coleoptile segments showed that the cuticle architecture differs on both sides of the segments. The dose-response curves for the IAA-induced growth and proton extrusion were bell-shaped with the maximum at 10^-4 M over 10 h. The kinetics of the IAA-induced hyperpolarisation was similar to that of the rapid phase of the IAA-induced growth. It is also proposed that the K⁺/H⁺ co-transporters are involved in acid-induced growth and that the combined effect of the K⁺ channels and K⁺/ H⁺ co-transporters is responsible for the IAA-induced growth. These findings support the "acid growth theory" of auxin action.

Keywords: auxin; coleoptile segments; growth; membrane potential; proton extrusion.

Figure 1

Effect of the IAA (10⁻⁴ M) on the growth rate (µm s⁻¹ cm⁻¹) and medium pH of the maize coleoptile segments. The IAA-induced growth (A) and medium pH (B) of the coleoptile segments: with (a) or without (b) the first leaf removed, with a partially abraded cuticle (c) and segments cut to half of their length (5-mm-long segments) (d). The control represents the growth rate of the coleoptile segments in the auxin-free medium (endogenous growth). The coleoptile segments were first preincubated (over 2 h) in an auxin-free medium to which the IAA at a final concentration of 10⁻⁴ M was added (arrow). The inset shows the total IAA-induced elongation growth, which was calculated as the sum of the extension from 3 min interval measurements over 10 h. All of the curves are the means of at least eight independent experiments. Bars indicate ± SE.

Figure 2

Light microscopy photographs of the cross-sections of the maize coleoptile segments (Zea mays L.) that had been stained with the lipophilic dyes in order to visualise the cuticle. (a,c) and (b,d) The cuticle on the outer and inner epidermis of the coleoptile segments that had been stained with Sudan Red 7B under a bright field microscope, respectively. (e,f) After Sudan IV and Nile Red staining under an epifluorescence microscope. The cuticle was observed as a continuous bright red layer or orange-gold, respectively, on both the outer and inner epidermal surfaces. (g) Sudan IV rapid routine staining to identify the cuticle on the outer epidermal surface. Scale bars: (a–g) 20 µm.

Figure 3

Growth rate (µm s⁻¹ cm⁻¹) of the maize coleoptile segments that had been incubated in the presence of 10⁻⁶, 10⁻⁵ and 10⁻⁴ M of the IAA (A) and the dose-response curves for the IAA (10⁻⁷–10⁻² M)-induced total elongation growth of the coleoptile segments as a function of time (B). The coleoptile segments were first preincubated (over 2 h) in an auxin-free medium to which the IAA was added (arrow). The inset shows the total elongation growth (µm cm⁻¹), which was calculated as the sum of the extensions from 3 min interval measurements over 10 h. All of the curves are the means of at least six independent experiments. Bars indicate ± SE.

Figure 4

Kinetics of the medium pH changes of the coleoptile segments that had been incubated in the presence of 10^–6, 10^–5 and 10^–4 M of the IAA (A) and dose-response curves for the IAA (10^–7–10^–2 M)-induced medium pH changes (expressed as changes in the H⁺ concentration per coleoptile segment) of the coleoptile segments as a function of time (B). The coleoptile segments were first preincubated (over 2 h) in an auxin-free medium to which the IAA was added (arrow). The pH values are the means of at least six independent experiments that were performed simultaneously with growth (shown in Figure 3) using the same tissue samples. Bars indicate ± SE.

Figure 5

Effect of the IAA (10^–5 and 10^–4 M) and FC (10^–6 M) on the membrane potential (E_m) of the parenchymal coleoptile cells simultaneously measured with the growth and medium pH. At 5 min (arrow), the IAA or FC was added to the growth medium. The typical results that were selected from at least seven experiments is presented. The adequate mean values are indicated in the text.

Figure 6

Effect of the acid buffer (pH 4) and its combination with the IAA or TEA-Cl on the growth rate (µm s⁻¹ cm⁻¹) and elongation (µm cm⁻¹) of the maize coleoptile segments. The coleoptile segments were first preincubated (over 2 h) in the control, after which this medium was changed for a new one containing the acid buffer or its combination with the IAA (10⁻⁴ M) or TEA-Cl (30 mM). The inset on the right side shows the total elongation over 10 h. The typical results were selected from six experiments. The adequate mean values are indicated in the text.

Figure 7

Effect of the acid buffer (pH 4) and its combination with the IAA on the membrane potential (E_m) of the parenchymal coleoptile cells. At time 5 min (arrow), the control medium was changed for a new one with the same salt composition, but that also contained the acid buffer (pH) or its combination with the IAA (10⁻⁴ M). An arrow pointing down indicates the exchange of the medium for the control medium. The representative curves for each variant are shown. The adequate mean values are indicated in the text.

Figure 8

Hypothetical model for the molecular mechanisms of acid- and auxin-induced rapid growth. This model assumes that the K⁺/H⁺ co-transporters are responsible for the acid-induced rapid growth, while both the K⁺ channels and K⁺/H⁺ co-transporters are involved in the auxin-induced growth. On the basis of the available and our own results, we speculate that at high H⁺ concentration in the medium (pH 4) the proton pump current amplitude decreases and the membrane potential (E_m) shifts to less negative values (here from −110 to ca. −50 mV, Figure 7). At a K⁺ concentration in the cytosol of 100 mM and an extracellular K⁺ concentration of 1 mM (in our experiments) the equilibrium (Nernst) potential for K⁺ (E_K) is equal −118 mV, suggesting that at E_m more positive than E_K (here E_m about −50 mV at pH 4) the K⁺ rectifying inward channels are blocked. Taking into account that an acid buffer (pH 4) caused the rapid and strong growth of the coleoptile segments, it may be suggested, that the K⁺/H⁺ co-transporters are responsible for the acid-induced growth. However, when auxin was added to the incubation medium, it induced (after initial depolarization) a rapid (transient) hyperpolarisation of the E_m (Figure 5) during which the membrane potential was by ca. 20 mV more negative than E_K. This means that in the presence of IAA the inward rectifying K⁺ channels are open and can mediate K⁺ uptake into the cell. Because the kinetics of the IAA-induced hyperpolarisation is similar to that of the first (very rapid) phase of the IAA-induced growth it may be suggested that the inward rectifying K⁺ channels are involved in the phase. The onset of the second phase of IAA-induced growth rate (Figure 3) correlates in time with rapid depolarisation of the E_m observed here after the maximum of the IAA-induced membrane hyperpolarisation was achieved. This finding (transient membrane hyperpolarisation) may indicate that the K⁺/H⁺ co-transporters are responsible for the second phase of the IAA-induced growth.

Figure 9

A schematic drawing of the apparatus that was used to simultaneously measure the elongation growth, medium pH and membrane potential of the coleoptile cells. The longitudinal extension of a stack of the segments was recorded in an intensively aerated solution using an angular transducer. The elongation growth, medium pH and membrane potential were measured with multifunctional computer meters: P, peristaltic pump; M, multifunctional computer meter; E₁ and E₂, reference and internal electrode, respectively.