glup4 controlling a 57H character was located on choromosome12 in rice

VI. Gene and genome structure *41. glup4 controlling a 57H character was located on choromosome 12 in rice* H. SATOU¹, W. X. LI¹, Y. TAKEMOTO¹, T. ITO¹, T. KUMAMARU¹, L.Q. QU¹ and M. OGAWA² 1) Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan 2) Home Economy, Yamaguchi Prefecture University, Yamaguchi,753-8502 Japan Glutelin, a major storage protein in rice, is synthesized as 57kD glutelin precursor on the ER, transported to the vacuole via the Golgi appartus, and formed by cleavaging the precursor into a subunit (4OkD) and b subunit (2OkD) glutelins in the vacuole (Yamagata et al.1982; Takaiwa et al. 1986; Masumura et al. l989). Kumamaru et al. (1988) reported a high deposition of 57kD polypeptides (57H) mutant, CM1787, which is from Japonica rice cultivar "Kinmaze" induced by MNU treatment, and revealed that this character was controlled by a recessive gene, esp2, located on chromosome 11. Takemoto et al. (1996) demonstrated that esp2 mutation was caused by the inhibitation of post-translational modifycation of glutelin precursor. 57H mutations are valuable materials for the study on genetic regulation of post-translational modification of glutelin precursor. So far five genes, esp2, Glup1, glup2, glup3 and glup4 have been identified as 57H mutation in rice (Kumamaru et al.1988, Satoh et al. 1994, Satoh et al. 1995). The chromosome location of glup4 was not known. In this report, we showed that the glup4 is non allelic to other four 57H genes and is located on choromosome 12. EM956 and EM960 were characterized by the increased content of 57kD polypeptides with remarkably decreased 26kD globulin in addition to the decreased amount of both 4OkD and 2OkD glutelins in SDS-PAGE. Both the mutants were crossed with their normal parent cultivar T65. The phenotypes of F₁s were normal. The segregation of normal and mutant types in F₂ showed a good fit to a 3:1 ratio. These results indicated that the 57H character of EM956 and EM960 are due to a single recessive gene. All mutant types in F₂ population had decreased 26kD globulin together with increased 57kD polypeptide accumulation. These results suggested that the reduction of 26kD globulin was the pleiotropic effect of 57H gene in EM956 and EM960. These mutants were crossed with each other. The phenotype of F₁ was 57H type and all of the F₂ seeds derived from this cross combination were the mutant type, showing that the 57H character of EM956 and EM960 was controlled by the same gene. Table 1 shows the segregation pattern in F₂ seeds derived from crosses between EM956 and marker lines of esp2, Glup1, glup2 and glup3. F₁ seeds of the cross between esp2 and EM956 showed the normal phenotype, and F₂ seeds were classified into normal, esp2 and EM956 types. Since the double mutant was sterile, segregation of these three types fitted a 9:3:3 ratio. In the cross between Glup1 and EM956, the F₁ seeds were 57H phenotype, and F₂ seeds were classified into Glup1, normal, EM956 and double mutant types, of which the segregation ratio in F₂ fitted to 9:3:3:1. F₁ seeds of the cross between glup2 and EM956, were normal for the 57H character, and F₂ seeds were classified into normal, 57H and double mutant type. As glup2 and EM956 types were not distinguishable from each other, the F₂ segregation ratio fitted 9:6:1. F₁ seeds of the cross between glup3 and EM956 showed the normal phenotype, and F₂ seeds were classified into normal, glup3, EM956 and double mutant types, with the segregation ratio of 9:3:3:1. These results indicate that the 57H gene of EM956, designated as glup4 , is independent of esp2, Glup1, glup2 and glup3, and that glup4 shows the additive effect with these four genes. Previously, we thought that glup4 was located on chromosome 5 (Unpublished). However, in the results of detailed linkage analyses no linkage was found with marker genes located on chromosome 5. Furthermore, trisomc analysis of EM960 revealed that the segregation in the F₂ seeds from the cross between Triplo 12 and the mutant fitted well to the expected ratio of trisomic segregation (Table2). From these results, we concluded that glup4 is located on chromosome 12. References Kumamaru, T., H. Satoh, N. Iwata, T. Omura, M. Ogawa and Z. Kasai, 1988. Mutant for rice storage proteins. I.Screening of mutants for storage proteins of protein bodies in the starchy endosperm. Theor. Appl. Genet.76: 11-16. Masumura, T., K. Kidzu, Y. Sugiyama, N. Mitsukawa, T. Hibino, K. Tanaka and S. Fujii, 1989. Nucleotide sequence of cDNA encoding major rice glutelin. Plant Mol. Biol. 12: 723-725. Satoh, H., T. Kumamaru, S. Yoshimura and M, Ogawa, 1994. New 57kDa glutelin genes on chromosome 9 in rice. RGN 11: 158-161. Satoh, H., T. Kumamaru, M. Ogawa, M. Shiraishi, B.G. Im, M.Y. Son and Y. Takemoto, 1995. Spontaneous "57H" mutants in rice. RON 12: 194-196. Takaiwa, F., S. Kikuchi and K. Oono, 1986. The structure of rice storage protein glutelin precursor deduced from cDNA. FEBS Lett. 206: 33-35. Takemoto. Y., M. Ogawa, T. Kumamaru, M.Y. Son and H. Satoh, 1996. Characterization of the protein bodies and the polypeptides in esp-2 mutant of rice. RON 13: 106-110. Yamagata, H., T. Sugimoto, K. Tanaka and Z. Kasai, 1982. Biosynthesis of storage proteins in developing rice seeds. Plant Physiol. 70: 1094-1100.

VI. Gene and genome structure

41. glup4 controlling a 57H character was located on choromosome 12 in rice

H. SATOU¹, W. X. LI¹, Y. TAKEMOTO¹, T. ITO¹, T. KUMAMARU¹, L.Q. QU¹ and M. OGAWA²
1) Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan
2) Home Economy, Yamaguchi Prefecture University, Yamaguchi,753-8502 Japan

Glutelin, a major storage protein in rice, is synthesized as 57kD glutelin precursor on the ER, transported to the vacuole via the Golgi appartus, and formed by cleavaging the precursor into a subunit (4OkD) and b subunit (2OkD) glutelins in the vacuole (Yamagata et al.1982; Takaiwa et al. 1986; Masumura et al. l989). Kumamaru et al. (1988) reported a high deposition of 57kD polypeptides (57H) mutant, CM1787, which is from Japonica rice cultivar "Kinmaze" induced by MNU treatment, and revealed that this character was controlled by a recessive gene, esp2, located on chromosome 11. Takemoto et al. (1996) demonstrated that esp2 mutation was caused by the inhibitation of post-translational modifycation of glutelin precursor. 57H mutations are valuable materials for the study on genetic regulation of post-translational modification of glutelin precursor.
So far five genes, esp2, Glup1, glup2, glup3 and glup4 have been identified as 57H mutation in rice (Kumamaru et al.1988, Satoh et al. 1994, Satoh et al. 1995). The chromosome location of glup4 was not known. In this report, we showed that the glup4 is non allelic to other four 57H genes and is located on choromosome 12.

EM956 and EM960 were characterized by the increased content of 57kD polypeptides with remarkably decreased 26kD globulin in addition to the decreased amount of both 4OkD and 2OkD glutelins in SDS-PAGE. Both the mutants were crossed with their normal parent cultivar T65. The phenotypes of F₁s were normal. The segregation of normal and mutant types in F₂ showed a good fit to a 3:1 ratio. These results indicated that the 57H character of EM956 and EM960 are due to a single recessive gene. All mutant types in F₂ population had decreased 26kD globulin together with increased 57kD polypeptide accumulation. These results suggested that the reduction of 26kD globulin was the pleiotropic effect of 57H gene in EM956 and EM960. These mutants were crossed with each other. The phenotype of F₁ was 57H type and all of the F₂ seeds derived from this cross combination were the mutant type, showing that the 57H character of EM956 and EM960 was controlled by the same gene.

     Table 1 shows the segregation pattern in F₂ seeds derived from crosses between EM956 and marker lines of esp2, Glup1, glup2 and glup3. F₁ seeds of the cross between esp2 and EM956 showed the normal phenotype, and F₂ seeds were classified into normal, esp2 and EM956 types. Since the double mutant was sterile, segregation of these three types fitted a 9:3:3 ratio.
     In the cross between Glup1 and EM956, the F₁ seeds were 57H phenotype, and F₂ seeds were classified into Glup1, normal, EM956 and double mutant types, of which the segregation ratio in F₂ fitted to 9:3:3:1.
     F₁ seeds of the cross between glup2 and EM956, were normal for the 57H character, and F₂ seeds were classified into normal, 57H and double mutant type. As glup2 and EM956 types were not distinguishable from each other, the F₂ segregation ratio fitted 9:6:1.
     F₁ seeds of the cross between glup3 and EM956 showed the normal phenotype, and F₂ seeds were classified into normal, glup3, EM956 and double mutant types, with the segregation ratio of 9:3:3:1.
    These results indicate that the 57H gene of EM956, designated as glup4 , is independent of esp2, Glup1, glup2 and glup3, and that glup4 shows the additive effect with these four genes.
     Previously, we thought that glup4 was located on chromosome 5 (Unpublished). However, in the results of detailed linkage analyses no linkage was found with marker genes located on chromosome 5. Furthermore, trisomc analysis of EM960 revealed that the segregation in the F₂ seeds from the cross between Triplo 12 and the mutant fitted well to the expected ratio of trisomic segregation (Table2). From these results, we concluded that glup4 is located on chromosome 12.

References

Kumamaru, T., H. Satoh, N. Iwata, T. Omura, M. Ogawa and Z. Kasai, 1988. Mutant for rice storage proteins. I.Screening of mutants for storage proteins of protein bodies in the starchy endosperm. Theor. Appl. Genet.76: 11-16.

Masumura, T., K. Kidzu, Y. Sugiyama, N. Mitsukawa, T. Hibino, K. Tanaka and S. Fujii, 1989. Nucleotide sequence of cDNA encoding major rice glutelin. Plant Mol. Biol. 12: 723-725.

Satoh, H., T. Kumamaru, S. Yoshimura and M, Ogawa, 1994. New 57kDa glutelin genes on chromosome 9 in rice. RGN 11: 158-161.

Satoh, H., T. Kumamaru, M. Ogawa, M. Shiraishi, B.G. Im, M.Y. Son and Y. Takemoto, 1995. Spontaneous "57H" mutants in rice. RON 12: 194-196.

Takaiwa, F., S. Kikuchi and K. Oono, 1986. The structure of rice storage protein glutelin precursor deduced from cDNA. FEBS Lett. 206: 33-35.

Takemoto. Y., M. Ogawa, T. Kumamaru, M.Y. Son and H. Satoh, 1996. Characterization of the protein bodies and the polypeptides in esp-2 mutant of rice. RON 13: 106-110.

Yamagata, H., T. Sugimoto, K. Tanaka and Z. Kasai, 1982. Biosynthesis of storage proteins in developing rice seeds. Plant Physiol. 70: 1094-1100.