42. Protein kinase activity in diverse tissues of two isogenic lines of rice

2) Department of Agronomy, National Chung Hsing University, Taichung, 40227 Taiwan, China

3) Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, 11529 Taiwan, China

The significance of protein phosphorylation in signal transduction has long been realized in animal systems (Hanks et al. 1988; Morrison et al. 1988). In this study, we compared the in vitro phosphorylation of proteins in crude extracts of a monocot plant. We used two isogenic rice lines, differing in heading time of flowers. They are T65 and its early-heading line T65(20)E^a carrying Ef-1^a (Tsai 1973).

Crude extracts were prepared from these two lines, sampled on the same days, 5th and 108th days after seeding. In vitro phosphorylation activity was assayed in samples of the same protein concentration in the following buffer: lOmM Tris-HCI (pH 7.5), 5mM MgCl\2\, 1mM 2-mercaptoethanol, 1OOmM NaCl, 0.015%(v/v) NP-40, 100 uM ATP and 9 uCi of gamma-³²P-ATP (ICN Radiochemicals, 7000 Ci/mmole). Final Ca++ concentration was made 9 uM by the addition of 2mM EDTA and 1.86mM CaCl2 (Perrin and Sayce 1976). The assay was terminated by the addition of an equal volume of 2X SDS-PAGE loading buffer, boiled and loaded onto a 9-15% gradient SDS polyacrylamide gel. After electrophoresis, the gel was dried and exposed to X-ray film.

Fig. 1 shows the extent of in vitro protein phosphorylation from various tissues of the two isogenic lines. The phosphorylated protein patterns varied between the two lines in almost all tissues examined. Most distinct were the differences observed in root and panicles of the 108 day-old plants and grains of the 5 day-old seedlings. Proteins phosphorylated in root from T65 ranged in apparent molecular weights between 10,000 and 30,000, whereas only one protein of apparent molecular weight of 95,000 was phosphorylated in root from T65(20)E^a. At least ten proteins of panicles from T65 were phosphorylated in

Fig. 1. Autoradiogram of in vitro labeled proteins prepared from diverse tissues of rice. Lanes 1-6 represent extracts sampled from 108 day-old plants, and lanes 7-12 from 5 day- old seedlings, respectively. Proteins from the parental T65 line are shown in lanes 1, 3, 5, 7, 9 and 11; the isogenic line, T65(20)E^a in lanes 2, 4, 6, 8, 10 and 12.

vitro. Their apparent molecular weights ranged from 12,000 to 98,000. In contrast, to panicles from T65(20)E^a, a whole array of additional proteins of various molecular weights were phosphorylated in vitro. In grains of the 5 day-old seedlings, the autoradiogram of T65 depicted one major protein of 13,000 and a minor one of 95,000. On the other hand, besides the different phosphorylation extent of the above two proteins, three additional proteins were phosphorylated to various extents in T65(20)E^a. Less distinct, nevertheless clear, differences were observed between the two lines in leaves of the 108 day-old plants and radicles and shoots of the 5 day-old seedlings. Addition of 10 uM cAMP to the incubation mix to replace Ca++ did not alter the phosphorylation patterns (data not shown). The significance of these differences remains unclear until further studies are conducted.

Hanks, S. K., A. M. Quinn and T. Hunter, 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241: 42-52.

Morrison, D. K., D. R. Kaplan, U. Rapp and T. M. Roberts, 1988. Signal transduction from membrane to cytoplasm: growth factor and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity. Proc. Nati. Acad. Sci. USA 85: 8855-8859.

Perrin,D.D. and I. G. Sayce, 1976. Computer calculation of equilibrium calculations in mixtures of metal ions and complexing species. Talanta 14: 833-842.

Tsai, K-H., 1973. Comparison of intraallelic-structure related to the major earliness genes involved in isogenic and radiation-induced early-maturing lines of a rice variety. Taichung 65. J. Agr. Assoc. China, New Series 84: 23-47.