Transient increase in the levels of γ-tubulin complex and katanin are responsible for reorientation by ethylene and hypergravity of cortical microtubules

Abstract

The body shape of a plant is primarily regulated by orientation of cortical microtubules. γ-tubulin complex and katanin are required for the nucleation and the severing of microtubules, respectively. Here we discuss the role of γ-tubulin complex and katanin during reorientation of cortical microtubules. 1-Aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, modifies growth anisotropy of azuki bean epicotyls; it inhibits elongation growth and promotes lateral growth. The ACC-induced reorientation of cortical microtubules from transverse to longitudinal directions preceded the modification of growth anisotropy. The transcript level of γ-tubulin complex (VaTUG and VaGCP3) and katanin (VaKTN1) was increased transiently by ACC treatment. During reorientation of cortical microtubules by hypergravity, which also modifies growth anisotropy of shoots, the expression levels of both γ-tubulin complex and katanin genes were increased transiently. The increase in the number of the nucleated microtubule branch as well as the microtubulesevering activity via upregulation of γ-tubulin complex and katanin genes may be involved in the reorientation of cortical microtubules, and contribute to the regulation of the shape of plant body.

Plant hormones or environmental stimuli modify the body shape of a plant. Cortical microtubules are essential for such modification of the body shape because they regulate the direction of cell expansion. For example, ethylene inhibits longitudinal growth and promotes lateral growth of shoots by causing a predominance of longitudinal microtubules.¹ Also, hypergravity, which suppresses elongation growth and promotes lateral growth in shoots, increased the ratio of cells with longitudinal cortical microtubules.^2,3

Protein complexes containing γ-tubulin, GCP2 and GCP3 are required for microtubule nucleation and proper organization of cortical microtubules.^4–6 Also, γ-tubulin-containing complexes are frequently associated with side walls of cortical microtubules in interphase, and nucleate nascent microtubules as branches diverging by approximately 40° from existing microtubules.⁵ The branching of microtubules via γ-tubulin complex may be involved in the reorientation of cortical microtubules. In the branched microtubules, the original microtubules should be severed from the newly synthesized microtubules and then depolymerized during reorientation of cortical microtubules. Katanin has microtubule-severing activity and may be involved in the reorientation of cortical microtubules.^7,8 Both nucleation and severing of microtubules are assumed to be regulated at the transcriptional levels of γ-tubulin complex and katanin genes during reorientation of microtubules. To confirm this point, we investigated the transcript levels of γ-tubulin complex (VaTUG and VaGCP3) and katanin (VaKTN1) genes during reorientation by ethylene of cortical microtubules in azuki bean epicotyls.⁹

The length of epicotyls was decreased, whereas the diameter was increased with increasing concentration of 1-aminocy-clopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. A significant change in elongation and lateral growth was detected within 1 and 1.5 h after the start of 10⁻⁵ M ACC treatment, respectively. Cells having transverse microtubules were predominant in the epidermis of epicotyls in the absence of ACC. The orientation of cortical microtubules did not change during incubation in the absence of ACC. With increasing concentration of ACC, the percentage of cells with transverse microtubules was decreased, while the percentage of cells with longitudinal microtubules was increased. The reorientation of microtubules from transverse to longitudinal directions was detected already at 0.5 h after the start of 10⁻⁵ M ACC treatment. The increase in the longitudinal microtubules and the decrease in transverse microtubules continued for 2 h after the start of ACC treatment. These results indicate that the modification of growth anisotropy in epicotyls by ethylene is accompanied by the reorientation of microtubules.

The time course of changes in the transcript levels of γ-tubulin complex (VaTUG and VaGCP3) and katanin (VaKTN1) in epicotyls grown in the presence or absence of 10⁻⁵ M ACC were examined in detail. In the absence of ACC, the expression level of VaTUG was almost constant during incubation. By contrast, the level of VaTUG significantly increased within 0.5 h after the start of ACC treatment, achieving the maximum at 1 h. Then, the level decreased and returned to control level at 2 h. Expression profiles of VaGCP3 were similar to those of VaTUG. Taken together, the transcript levels of γ-tubulin complex genes were increased transiently during ACC-induced reorientation of cortical microtubules from transverse to longitudinal directions. Recently, we showed that the levels of γ-tubulin complex genes were also increased transiently during hypergravity-induced reorientation of cortical microtubules from transverse to longitudinal directions.¹⁰ These results suggest that the increase in number of the branched microtubules via upregulation of γ-tubulin complex genes facilitates reorientation of cortical microtubules from transverse to longitudinal directions (Fig. 1).

Figure 1.

A model for reorientation of cortical microtubules. The transiently increased γ-tubulin complex binds onto pre-existing cortical microtubules and nucleates microtubules as branch as demonstrated by Murata et al.⁵ Then, transiently increased katanin severs the newly synthesized microtubule branch. Repeat of branching and severing of microtubules may induce the reorientation of cortical microtubules.

Expression of VaKTN1 showed a similar pattern to that of γ-tubulin complex genes. Namely, the transcript level of katanin gene was also increased transiently during ACC-induced reorientation of cortical microtubules from transverse to longitudinal directions. When expression profiles of katanin gene were compared with those of γ-tubulin complex genes, the peak of expression of katanin gene was delayed by 1.0 to 1.5 h. The delay of the peak of expression of katanin gene was also observed during hypergravity-induced reorientation of cortical microtubules from transverse to longitudinal directions.^10,11 These results indicate that microtubule-severing activities are increased by ethylene and hypergravity via upregulation of katanin gene, resulting in the stimulation of separation of the newly synthesized microtubule branch via γ-tubulin complex (Fig. 1). Repeat of branching and severing of microtubules may induce the reorientation of cortical microtubules from transverse to longitudinal directions.

During reorientation by ethylene or hypergravity of cortical microtubules from transverse to longitudinal directions, the expression levels of both γ-tubulin complex and katanin genes were increased transiently. Are such changes in the transcript levels also induced during reorientation of cortical microtubules in the opposite direction? Reorientation of cortical microtubules from longitudinal to transverse directions was induced by removal of the hypergravity stimulus.² By removal of the hypergravity stimulus, the transcript levels of γ-tubulin complex and katanin genes were increased transiently.^10,11 These results indicate the transcription of γ-tubulin complex and katanin genes is stimulated transiently during reorientation of cortical microtubules, irrespective of the direction of reorientation of the microtubules.

As a summary, we propose a model for reorientation of cortical microtubules (Fig. 1). First, the levels of γ-tubulin complex are increased transiently. The transiently increased γ-tubulin complex binds onto pre-existing cortical microtubules and nucleates microtubules as branch. Then, the levels of katanin are increased transiently, which contribute to sever the newly synthesized microtubule branch. Repeat of branching and severing of microtubules may induce the reorientation of cortical microtubules.

Addendum to: Soga K, Yamaguchi A, Kotake T, Wakabayashi K, Hoson T. 1-Aminocyclopropane-1-carboxylic acid (ACC)-induced reorientation of cortical microtubules is accompanied by a transient increase in the transcript levels of γ-tubulin complex and katanin genes in azuki bean epicotyls. J Plant Physiol. 2010;167:1165–1171. doi: 10.1016/j.jplph.2010.04.001.