Gibberellic acid-induced microtubule reorientation in dwarf peas is accompanied by rapid modification of an α-tubulin isotype

To test whether shifts induced in microtubuie orientation by gibberellic acid (GA₃) involved changes in tubulin isotypes, pea stem cells were examined when elongation had been enhanced by GA₃. The behaviour of a dwarf recessive mutation (le), with very low endogenous levels of gibberellin, was compared with the tall (Le) plant. Two hours after adding GA₃, cells were measurably longer than controls and this coincided with a net shift of microtubule orientation from longitudinal and oblique to transverse — an effect that was more pronounced in the dwarf. There were always more cells with net-transverse microtubules in GA₃-treated tissue than in controls, but as growth ceased, the major orientation of the microtubule arrays became oblique in both samples. Microtubule reorientation was rapid and was closely correlated with the growth of the cells. Although changes in orientation and isotype were monitored over a 40 h period, immunoblotting 2D gels with the well characterized antibodies YL1/2 and YOL1/34 confirmed that alterations to the α-tubulin constellation could be detected as early as the 2 h time point. Again the effect was especially pronounced in dwarf plants. In the presence of added GA₃, one α-tubulin isotype (designated α₁) retained its position in the α-tubulin constellation (as determined by total protein staining and with YOL1/34 that recognizes detyrosinated as well as tyrosinated tubulin). It was no longer recognized, however, by the anti-tyrosinated α-tubulin antibody YL1/2. This indicates that as GA₃ begins to cause a reorientation of the cortical microtubules (and to enhance the rate of cell elongation) the α₁ isotype is rapidly changed, probably by post-translational modification.