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(S178D), 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 righthanded 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 300g 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.

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Seedlings of Arabidopsis α-tubulin 6 mutant (tua6 ) were cultivated under microgravity conditions in the European Modular Cultivation System on the International Space Station, and growth and cell wall properties of their hypocotyls were analyzed (the Resist Wall experiment). Seeds of tua6 mutant were shown to germinate and grow normally until the seedling stage under microgravity conditions, as at 1 G on the ground. The seedlings were naturally air-dried in orbit, which were then recovered and transported to earth. When the mechanical properties of the cell wall of rehydrated hypocotyls were examined with a tensile tester, the hypocotyls showed typical stress-strain and stress-relaxation curves, as normally fixed or frozen materials. Also, no promine nt dif fe r e nc e s we r e d e te c te d in the extensibility or the stress-relaxation parameters of the cell wall between spaceg r ow n hy p o c o t yl s a n d g r o u n d c o n t r o l s , suggesting that tua6 hypocotyls formed the regular cell wall architecture under microgravity conditions. The results and lessons learned from the Resist Wall experiment are expected to provide the basis for the following space experiments to clarify the mechanism of gravity resistance in plants.