Apoplastic alkalinization is instrumental for the inhibition of cell elongation in the Arabidopsis root by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid

Abstract

In Arabidopsis (Arabidopsis thaliana; Columbia-0) roots, the so-called zone of cell elongation comprises two clearly different domains: the transition zone, a postmeristematic region (approximately 200-450 μm proximal of the root tip) with a low rate of elongation, and a fast elongation zone, the adjacent proximal region (450 μm away from the root tip up to the first root hair) with a high rate of elongation. In this study, the surface pH was measured in both zones using the microelectrode ion flux estimation technique. The surface pH is highest in the apical part of the transition zone and is lowest at the basal part of the fast elongation zone. Fast cell elongation is inhibited within minutes by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid; concomitantly, apoplastic alkalinization occurs in the affected root zone. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, can partially rescue this inhibition of cell elongation, whereas the inhibitor N,N'-dicyclohexylcarbodiimide does not further reduce the maximal cell length. Microelectrode ion flux estimation experiments with auxin mutants lead to the final conclusion that control of the activity state of plasma membrane H(+)-ATPases is one of the mechanisms by which ethylene, via auxin, affects the final cell length in the root.

Figure 1.

A, Plot of the surface pH along the Arabidopsis root. The surface pH along the Arabidopsis root is recorded using the MIFE system and sampled every 50 μm from 125 μm of the root tip off to the root hair zone (at 1,050 μm). The extracellular pH reaches a maximum at a distance of 225 μm from the root tip and decreases toward the elongation zone. The pH values in one point are quite stable as seen in the inset of the figure. For each single point the average was taken from a MIFE recording lasting at least 50 s. B, Plot of the H⁺-flux (mean ± sd, n = 4) along the Arabidopsis root. The flux was measured with the MIFE system as described in A.

Figure 2.

A, Changes in pH (ΔpH, mean ± sd, n = 3) at a distance between 400 and 500 μm from the Arabidopsis root tip measured during 120 min after the addition of ACC (+ACC, final concentration 5 μm) and after the addition of KCl as a control. The traces were recorded with a time interval of 10 s between data points. For clarity symbols indicating the mean and sd bars were placed at 5-min intervals only. ACC addition causes an alkalinization in the zone of the root that marks the transition from slow to fast elongating cells. The addition of 5 μm ACC did not significantly alter the pH of the medium in the experimental chamber (results not shown). Before ACC addition, the sampling points at this position displayed a steady influx and remained at a constant pH (results not shown). B, Effect of addition of ACC or KCl (control) on the H⁺-flux (mean ± sd, n = 3) measured with the MIFE system at a distance of 400 to 500 μm from the root tip. Treatments were as described in A.

Figure 3.

Modulation of the H⁺-ATPase activity by the activator FC and the inhibitor DCCD and its effect on the Arabidopsis root growth extent (in percent) in control roots and in roots in which the ethylene content is increased by ACC. FC can partially rescue the inhibition of cell elongation imposed by ACC, whereas DCCD reduces cell lengths only in control conditions, but not in the presence of ACC. Treatments with different lowercase characters above the bars are statistically significant different at the P < 0.01 level.

Figure 4.

Changes in pH (ΔpH, mean ± sd, n = 3) at a distance between 400 and 500 μm from the Arabidopsis root tip measured during 120 min in control conditions (circles) and after the addition of ACC (black symbols) and/or FC (triangles) and after the addition of KCl as a control (white symbols). ACC addition causes an alkalinization in this point along the root, whereas FC reduces the changes in pH to a minimum, regardless of the presence of ACC.

Figure 5.

Changes in pH (ΔpH, mean, n = 3) at a distance between 400 and 500 μm from the root tip in Arabidopsis auxin transport mutants measured during 120 min in control conditions (white symbols) and after the addition of ACC (black symbols). ACC has no clear effect on the pH changes in aux1-22 (circles), whereas the effect on pH in axr2-1 (triangles) restores the behavior of a wild-type plant under normal conditions. Axr3-1 (squares) exhibits an hyperalkalinization response after the addition of ACC. For clarity, the sd bars are omitted (in general the sds were comparable with those found in Fig. 4).

Figure 6.

Model of interactions of ethylene with different auxin-response mutations (axr3-1 and axr2-1), the auxin transporter mutant (aux1-22), the H⁺-pumping ATPase inhibitor DCCD, and the H⁺-pumping ATPase activator FC. In the cartoon on the left of the root tip the disruption of auxin transport from the vascular bundle into the root tip and from there to the cortical cells in the elongation zone by the mutation in the aux1-22 gene, is depicted. The box on the right shows the regulation of the proton-pumping ATPase in the cells in the elongation zone. The mutation in the aux1-22 gene prevents the accumulation of auxin in the cell to ATPase-inhibiting levels. The gain-of-function mutation axr3-1 increases the auxin responses while axr2-1 induces auxin insensitivity. In this model ethylene is assumed to interfere with auxin transport and biosynthesis, increasing the cellular auxin concentration to a level that is inhibitory to the H⁺-pumping ATPase activity. From a previous study it is known that addition of ethylene increases ROS production and leads to hydroxyproline-rich glycoprotein cross-linking in the cell wall.