Calcium-dependent protein phosphorylation in hairy roots of Daucus carota

In vitro protein phosphorylation in soluble fractions (85,000×g supernatant) prepared from carrot hairy roots, which were induced by inoculation with Agrobacterium rhizogenes harboring the Ri plasmid, was analyzed by SDS-PAGE and autoradiography. The autoradiograms indicated that at least four proteins, with molecular masses of 65, 57.5, 52, and 38 kDa, were phosphorylated. The presence of Ca2+ enhanced the phosphorylation of the 65, 57.5, and 52 kDa proteins, but the presence of both Ca2+ and calmodulin did not enhance phosphorylation as much as Ca2+ alone. W-7 strongly inhibited the phosphorylation of the 65, 57.5, and 52 kDa proteins while W-5 mildly inhibited the phosphorylation of these proteins. The extent of phosphorylation of these proteins and the growth rate of the hairy roots became higher with increasing incubation temperature. W-7 had a considerable inhibitory effect on the growth of hairy roots, W-5 had a weak inhibitory effect on the growth of the roots, and a high concentration of TMB-8 inhibited growth. These results suggest that the 65, 57.5, and 52 kDa proteins are phosphorylated by CDPK and that the phosphorylation is connected with the increase in growth of carrot hairy roots.