37. Appearance of a new band in cell-wall isoperoxidases of radiation- induced dwarf rice mutants

Laboratory of Radiation Genetics and Chemical Mutagenesis, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan

With respect to the role of peroxidase, several properties have been described, such as degradation of indole-3-acetic acid (Febhardt 1982), polymerization of lignin precursors (Harkin and Obst 1973) and formation of covalent bonds in ligno proteins or ligno polysaccharides of cell wall (Whitmore 1978a, b). In the present study, differences in isoperoxidases between semidwarf and dwarf mutant lines and their parental cultivar were investigated by means of isoelectric-focusing gel electrophoresis.

The semidwarf cultivar Reimei, a dwarf mutant line Fukei No. 71 and their original cultivar Fujiminori were used. The former two were derived from Fujiminori after ⁶⁰Co gamma-exposure with 20 kR and 30 kR, respectively (Futsuhara 1968).

After the hulls were removed from each seed, the seeds were soaked in 70% ethanol for 5 minutes and in 2% sodium hypochlorite solution for 30 minutes for sterilization and were then rinsed with sterile water. They were allowed to germinate in light at 32 degrees C for 72 hours in 6 cm Petri-dishes containing 4 ml of sterile distilled water. Protein from rice seedlings was fractionated as described by Cassab et al. (1985) and Tierney et al. (1988). Germinating seeds were ground in 0.1% potassium-acetate and 4mM Na\2\S\2\O\5\ at pH 5.0. The homogenate was centrifuged at 3000 X g to extract soluble proteins. Membrane proteins of the pellet was extracted with 0.5% Nonidet P-40 (Sigma) plus 2mM Na\2\S\2\O\5\ and the pelet was washed extensively with cold 2mM Na\2\S\2\O\5\ solution to remove the detergent (Nonidet P-40). The pellet was then extracted with 0.2M CaCl\2\ plus 4mM Na\2\S\2\O\5\ overnight at 4 degrees C. Each fraction containing 50 ug protein was used for isoelectric-focusing electrophoresis, which was carried out using disk polyacrylamide gel (5.25%) containg Ampholine (pH3.5-10 LKB). Electrophoresis was performed at 200V for the first 14 hours, followed by 400V for the last one hour. Then, the electrofocused gel was stained with the reacting mixture containing 0.1% o- dianisidine, 0.03% hydrogen peroxide and 0.06M acetate buffer (pH 4.9) at 4 degrees C.

Fig. 1,A,B and C show the peroxidase zymograms of soluble, membrane and cell- wall proteins, respectively, each obtained from germinating seeds of Fujiminori (lane 1), Reimei (lane 2) and Fukei No. 71 (lane 3). In soluble protein (A), the three strains, Fujiminori, Reimie and Fukei No. 71, showed an identical pattern of 15 isozyme bands varying in intensity. In membrane protein (B), also, the three strains showed an identical pattern of 13 isozyme bands. In cell-wall protein (C), however, Reimei and Fukei No. 71 had a band a pI 6.5 which was not present in Fujiminori, as indicated by an arrowhead. The intensity of this band was higher in Fukei No. 71 than in Reimei. It was also found that Reimei and Fukei No. 71 had higher peroxidase activity than Fujiminori, as will be reported elsewhere.

The dwarfism of Reimei is controlled by gene sd-1 (Kikuchi and Ikehashi 1984); that of Fukei No. 71 is due to d-50 (RGN 1, p. 34; Futsuhara 1968). Except for these recessive dwarfing genes, Reimei and Fukei No. 71 would have the same genotype as of Fujiminori as the three strains show the same characters (Futsuhara 1968). It may be concluded that the occurrence of a new band of cell-wall peroxidase, with higher activity in Fukei No. 71 than in Reimei, is due to the action of the dwarfing genes induced by gamma-irradiation.

Fig. 1. Peroxidase zymograms of rice seedlings. Lane 1-Fujiminori; 2-Reimei; 3-Fukei No. 71. A: In soluble protein, B: In membrane protein, C: In cell-wall protein.

Cassab, G.J., J. Nieto-Sotelo, J.B. Cooper, G.J. van Holst and J.E. Varner, 1985. A developmentally regulated hydroxyproline-rich glycoprotein from the cell walls of soybean seed coats. Plant Physiol 77: 532-535.

Futsuhara, Y., 1968. Breeding of a new rice variety Reimei by gamma-ray irradiation. Gamma Field Symp. 7: 87-109.

Gebhardt, K., 1982. Activation of indole-3-acetic acid oxidation from horseradish and prunus by phewnols and H\2\O\2\. Plant Growth Reg. 1: 73-84.

Harkin, J.M. and J.R. Obst, 1973. Lignification in trees: indication of exclusive peroxidase participation. Science. 180: 296-297.

Kikuchi, F. and H. Ikehashi, 1984. Semidwarfing genes of high-yielding rice varieties in Japan. RGN 1: 93-94.

Tierney, M.L., J. Wichert and D. Pluymers, 1988. Analysis of the expression of extensin and p33-related cell wall proteins in carrot and soybean. Mol. Gen. Genet. 211: 393-399.

Whitmore, F.W., 1978a. Lignin-proteins complex catalysed by peroxidase. Plant Sci. Lett. 13: 241-245.

Whitmore, F.W., 1978b. Lignin-carbohydrate complex formed in isolated cell walls of callus. Phytochemistry. 17: 421-424.