28. Characterization of the protein bodies and the polypeptides in esp-2 mutant of rice 

Y. takemoto, M. ogawa T. kumamarU, M.Y. son and H. satoh

1) Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki. Fukuoka, 812 Japan.

2) Yamaguchi Women's University, Sakurabatake, Yamaguchi 753, Japan.

3) Plant Breeding and Genetics Research Laboratory, Japan Tobacco INC., Iwata, Shizuoka, 438 Japan

 Glutelins, the predominant rice storage protein, are synthesized and sequestered in the rough endoplasmic reticulum (ER) as the 57kD precursor polypeptide (Yamagata et al. 1982). Glutelin precursor is transferred into the protein vacuole (protein body type-II, PB-II) via the Golgi complex (Krishnan et al. 1986), and then cleaved into the 40kD (a subunit) and 20kD (P subunit) polypeptides (Yamagata et al. 1982).

The protein mutants accumulating 57kD polypeptide (57H mutant) were found in mutant lines induced by the MNU treatment (Kumamaru et al. 1988). Three mutant loci, esp-2 (chr.ll), Glup-l (chr.9) and glup-2 (chr.9), were so far identified for the 57H mutation (Satoh et al. 1994). Kumamaru et al. (1987) suggested that a gene esp-2 is a regulatory gene for the glutelin polypeptide of PB-II. This report deals with the electronmicroscopical observation of the protein bodies (PBs) and the characterization of 57kD polypeptides present in the endosperm of esp-2 mutant. An esp-2 mutant,

Fig. 1. SDS-PAGE and Western blot analyses of the rice storage protein A: Total rice storage proteins stained by Coomasie brilliant blue R-250. B: Western blot analysis of the total rice storage proteins using the anti-b subunit serum. 1: Kinmaze (Original variety), 2: CM 1787(esp-2).
Research Notes 107
CM1787, was characterized by the high content of 57kD polypeptide and the content of 20kD and 40kD glutelin subunits were reduced (Fig. 1, A). The 57kD polypeptides were reacted with anti-glutelin b subunit antibody (Fig. 1, B) indicating that this mutant accumulates the 57kD glutelin precursor along with the glutelin subunits reduction.

Glutelins in the endosperm of a normal counterpart "Kinmaze" were extracted by 1 % lactic acid solution (acid solution) and the presence of prolamin did not affect the extraction of glutelin at all (Fig. 2). In esp-2 mutant, however, most of the 57kD glutelin precursor polypeptide was not extracted by the acid solution and remained in the residue. Only when the prolamin was completely removed by 60% n-propanol and 5% 2-mercaptoethanol, the 57kD polypeptides were able to be extracted by the acid solution (Fig. 2). This fact suggests that the 57kD polypeptides of esp-2 mutant are of the nature of glutelin and possibly coexist with the prolamin polypeptides on the occasion of protein accumulation.

Two types of PB, PB-I and PB-II, are observed in the developing endosperm of a normal counterpart "Kinmaze" by transmission electron microscope, as reported by Tanaka et al. (1980) (Fig. 3, A). In the mutant, a large number of small PBs (new type PBs) were observed in addition to PB-II and PB-I as observed in Kinmaze were not found at all. The new type PBs were spherical in shape and 0.5mm in diameter. These PBs were stained weakly by osmium tetroxide and had no lamellar structure as observed in

Fig. 2. SDS-PAGE analysis of the proteins extracted by 1% lactic acid. 1: Total protein. 2: glutelins extracted by 1% lactic acid. 3: residue after the extraction of glutelin. 4: glutelins extracted by 1 % lactic acid after removing prolamins. 5: residue after the extraction of glutelins and prolamins. 108 Rice Genetics Newsletter Vol. 13

Fig. 3. Electron micrographs of the developing endosperm A: Kinmaze (Original variety), B: CM1787 (esp-2), arrow heads: new type protein bodies. Bar = 0.5 microm.
Research Notes 109

Fig 4 Electron micrographs of the developing endosperm of esp 2 mutant showing the specificities of anti b subunit antibody (A) and anti 13b prolarnin polypeptide antibody (B) for a new type protein body (arrows) Bars=0 5 microm

 110 Rice Genetics Newsletter Vol. 13

PB-I. Many ribosomes were observed on the outside of new type PB in developing endosperm, suggesting that these PBs were derived from ER as well as PB-I. (Fig. 3, B). Immunogold labeling experiments using anti-serum raised against glutelin b subunit of PB-II and 13kD-b prolamin polypeptide of PB-I indicated that the 57kD glutelin precursor of the mutant was deposited in the new type PB together with 13kD-b prolamin polypeptide (Fig. 4, A and B), suggesting that the presence of 57kD glutelin precursor polypeptides in PB-I leads to the formation of the new type PB and thus, the 57kD glutelin precursor polypeptides remain to be not cleaved proteolytically into alpha and beta subunits.

From these result, it is suggested that esp-2 mutation results in the deposition of the 57kD glutelin precursor polypeptides in PB-I and Esp-2 is a gene for the post-translational processing of glutelin and controls to direct the glutelin precursors toward the protein vacuole. (Gene symbol: Old system)

References 

Krishnan, H.B., V.R. Franceschi and T.W. Okita, 1986. Immunochemical studies on the role of the Golgi

complex in protein-body formation in rice seeds. Planta 169: 471 -480.

Kumamani, T., H. Satoh, N. Iwata, T. Omura and M. Ogawa, 1987. Mutant for rice storage proteins. III. Genetic analysis of mutants for storage proteins of protein bodies in the starchy endosperm. Jpn. J. Genet. 62:333-339.

Kumamani, T., H. Satoh, N. Iwata, T. Omura, M. Ogawa and K. Tanaka, 1988. Mutant for rice storage proteins. 1. Screening of mutants for storage proteins in the starchy endosperm. Theor Appl Genet 76: 11-16.

 Satoh, H., T. Kumamani, S. Yosimura and M. Ogawa, 1994. New 57kDa glutelin genes on chromosome 9 in rice. RGN 11: 158-161. Tanaka, K., T. Sugimoto, M. Ogawa and Z. Kasai, 1980. Isolation and characterization of two types of protein bodies in the rice endosperm. Agric. Biol. Chem. 44: 1633-1639. Yarnagata, H., T. Sugimoto, K. Tanaka and Z. Kasai, 1982. Biosynthesis of storage protein in developing rice seeds. Plant Physiol. 70: 1094-1100.