Gravity-induced modifications to development in hypocotyls of Arabidopsis tubulin mutants

Abstract

We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Delta), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300 g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in modifications to morphology and orientation of microtubule arrays by 1g gravity and hypergravity in tubulin mutants.

Figure 1.

Effects of hypergravity on elongation growth and lateral thickening in hypocotyls of Arabidopsis tubulin mutants. Wild type (WT) and tubulin mutants were grown on agar medium at 1g for 48 h at 25°C. Seedlings were then transferred to 1g or 300g conditions, and grown for a further 24 h at 25°C. A, The length was measured using a scale. B, The diameter of hypocotyls was measured with a digital stereoscopic microscope. Values are means ± se (n = 20). *, Mean values with significant differences between 1g and 300g treatments (P < 0.05).

Figure 2.

Cell elongation and twisting profiles in an epidermal cell file in hypocotyls of Arabidopsis tubulin mutants. Wild type (WT) and tua6 were grown as in Figure 1. A, The length of individual cells from the top (cell 1) to the base (cell 20) was measured with SEM and elongation for 24 h was calculated. B, The alignment angle of individual cells was measured with SEM. Values are means ± se (n = 15).

Figure 3.

Surface view of epidermal cell files in Arabidopsis tubulin mutants grown at 1g or 300g. Wild type (WT) and tubulin mutants were grown as in Figure 1. The outer surface of cells 10 to 12 from the top of hypocotyls was observed with SEM. Supplementary lines denote the longitudinal axis of hypocotyls and the alignment of epidermal cell files, for measuring the alignment angle. The bar denotes 100 μm.

Figure 4.

Effects of hypergravity on alignment angle of epidermal cell files in hypocotyls of Arabidopsis tubulin mutants. Wild type (WT) and tubulin mutants were grown as in Figure 1, and the angle of cells 10 to 12 to the longitudinal axis was measured using a protractor, as in Figure 3. Values are means ± se (n = 20). *, Mean values with significant differences between 1g and 300g treatments (P < 0.05).

Figure 5.

Immunofluorescence images of cortical microtubules in hypocotyls of Arabidopsis tubulin mutants. Wild type (WT) and tubulin mutants were grown as in Figure 1. Epidermal cells 10 to 12 were stained as described in “Materials and Methods.” Typical examples of two adjacent cells with distinct orientation of cortical microtubules are shown. The bar denotes 10 μm.

Figure 6.

Frequency distribution of alignment of cortical microtubules in hypocotyls of Arabidopsis tubulin mutants. Wild type (WT) and tubulin mutants were grown as in Figure 1. All values were taken from immunofluorescence micrographs at cells 10 to 12, as shown in Figure 5. Data were obtained from epidermal cells in 10 different hypocotyls and normalized as percentage.

Figure 7.

Effects of hypergravity on alignment of cortical microtubules in hypocotyls of Arabidopsis tubulin mutants. Wild type (WT) and tubulin mutants were grown as in Figure 1. Values are mean alignment angle of cortical microtubules measured in Figure 6 ± se (n = 114–334). *, Mean values with significant differences between 1g and 300g treatments (P < 0.05).

Figure 8.

Effects of gadolinium ions on alignment of epidermal cell files in hypocotyls of Arabidopsis tubulin mutants. Arabidopsis seedlings were grown at 1g or 300g in the presence (Gd³⁺) or absence (buffer) of 30 μm gadolinium chloride for 72 h at 25°C. A, The outer surface of cells 10 to 12 was observed as in Figure 3. The bar denotes 100 μm. B, The angle of epidermal cell files was measured as in Figure 4. Values are means ± se (n = 20). WT, Wild type.

Figure 9.

Effects of gadolinium ions on alignment of cortical microtubules in hypocotyls of Arabidopsis tubulin mutants. Wild type (WT) and tubulin mutants were grown as in Figure 8. A, Immunofluorescence images of cortical microtubules. Epidermal cells 10 to 12 were stained as described in “Materials and Methods.” B, Frequency distribution of alignment of cortical microtubules. All values were taken from immunofluorescence micrographs at cells 10 to 12. C, Mean alignment angle of cortical microtubules measured in B ± se (n = 111–278).