27. Glutelin mutants induced by MNU treatment in rice

H. SATOH1, L.Q. QU1, T. KUMAMARU1 and M. OGAWA2

1) Faculty of Agriculture, Kyushu University. Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812 Japan
2) Home Economy, Yamaguchi Prefecture University, Sakurabatake, Yamaguchi, 753 Japan

We have reported several mutants for endosperm storage proteins in rice (Kumamaru et al. 1988). Some mutants seem to be mutations for the deposition process to protein bodies (PB) rather than the biosynthesis (Kumamaru et al. 1987 and Takemoto et al. 1996). We treated the fertilized egg cells of rice cultivars Kinmaze and Taichung 65 (T 65) with MNU (N-methyl-N-nitrosourea) (Satoh et al. 1979) and induced several new kinds of glutelin mutants. This report deals with characterization of seven glutelin mutants and one globulin mutant using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel elec-trophoresis) and isoelectricfocusing gel electrophoresis (IEF) using polyacrylamide gel.

About 3,000 mutant lines, derived from the fertilized egg cell treatment with N-nitoroso-N-methyl urea (MNU) of rice cultivars, Kinmaze and T 65, were screened with SDS-PAGE using a gradient gel with 15% to 25% acrylamide and 0.06% to 0.45% BIS concentration linear. Rice glutelins are composed of at least three acidic subunit bands (α-subunits, MW 37-39kD) and two or three subunit bands (β-subunits, MW 22-23kD). Seven glutelin mutant lines and one globulin deletion mutant line were found among them when compared with their original cultivars, Kinmaze and T 65 (Fig. 1). These glutelin mutants were classified into four groups based on the individual SDS-PAGE band pattern. EM 278 and EM 413 were characterized by increased amount of α-1 subunit band (39kD, α-1H); EM 659 by increased amount of α-3 subunit band (37kD, α-3 H); CM 1707, TCM 221 and TCM 1170 by increased amount of α-1 and α-3 subunit bands (α-1/α-3H); EM 895 by decreased amount of α-1 subunit band and EM 49 by decreased amount of α-1 subunit band together with no or negligible amount of 26 and 16kD globulins. Out of the eight glutelin mutants, five mutants, EM 278, EM 659, CM 1707, TCM 221 and TCM 1170, showed a greatly decreased amount of α-2 subunit bands (α-2L) in common, though their SDS-PAGE band patterns were quite different from each other. These results indicates that it is possible to induce mutants with diversified changes in glutelin polypeptides by mutation treatments. Furthermore, TCM 1170 has significantly increased amount of 57kD polypeptide (57H), in addition to the unique α subunit bands (α-1, 3H/α-2L). We have reported four different 57H mutants which were controlled by a single gene (Kumamaru et al. 1987: Satoh et al. 1994, 1995). All of the 57H mutants reported before were characterized by high amount of 57kD polypeptides together with low amount of acidic and basic subunit polypeptides and little change of SDS-PAGE and IEF band patterns of glutelins. Based on these results and the transmission electron microscopic observation of endosperm cells of these mutants, Takemoto et al. (1996) considered that these 57H mutant genes were concerned with the regulation of processing of glutelins or their transportation to PB-II. The fact that TCM 1170 has significantly increased amount of 57kD polypeptide (57H) in addition to α-1, 3H/α-2L indicates that there are genes responsible for the processing of individual glutelin polypeptides as well as the non-specific genes for the processing of individual glutelin polypeptides. Different from other 57H mutants, TCM 1170 mutant could be expected as a useful materials for the research on the genetic control mechanism of biosynthesis and processing of glutelin in rice endosperm.

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After extraction by 1.0% lactic acid, glutelin fractions were analyzed by horizontal slab IEF system. The results of IEF showed that the glutelin acidic and basic subunits were separated into 12 and 9 bands, respectively, similar to the results of Wen and Luthe (1985). The IFF band patterns differed significantly among mutants (Fig. 2). The pI (isoelectric point) 6.71 band of CM 1707,TCM 221,TCM 1170, EM 278 and EM 659 mutants decreased greatly in common, while the pI 6.50 and pI 6.82 bands of EM 278, pI 6.82 and pI 6.90 bands of CM 1707, TCM 221 and TCM 1170, pI 6.90 and pI 7.38 bands of EM659 increased significantly. These indicated that the pI 6.50 and pI 6.82 bands were the main polypeptide components of α-1 subunit, the pI 6.71 band was that of α-2 subunit, pI 6.90 and pI 7.38 bands were that of α-3 subunit. A new band of pI 6.30 seen in EM278 indicated that this band might be also one of the components of α-1 subunit. It can be deduced from these results that the mutated subunits were controlled by structural genes. Meanwhile the pI 8.74 band, the basic subunit, of CM 1707 and TCM 1170 decreased to-gether with decreased pI 6.71 band. Similarly, the pI 8.58 band decreased simultaneously with pI 6.82 band, the main polypeptides of Eφ-1 subunit, decreased in EM 49 which has no 16 and 26 kD globulins. Little or no changes of basic subunit polypeptides were found in EM 278 and EM 659. It had been reported that the glutelin acidic and basic subunits were first synthesized on rEr as 57kD precursor polypeptide encoded by a structural gene and were formed by proteolysis of the precursor through post-translational cleavage in PB II (Yamagata et al. 1982; Sarker et al. 1986). But the pairing relationships between acidic and basic polypeptides are still unknown. Our studies suggested that pI 6.71 and pI 8.78 bands, pI 6.82 and pI 8.58 bands might have pairing relationships, respectively. The pairing relationships between acidic and basic polypeptides are expected to be clear by using other glutelin subunit mutants derived from the treatment of fertilized egg cells with MNU.

Unlike other cereals, rice storage proteins are characterized by a high content (about 75% of total proteins) of alkali/dilute acid soluble proteins, glutelins, in addition to low content of alcohol soluble proteins, prolamin (about 15% of total proteins) which is the major protein in most of the other cereals, and salt soluble proteins, globulin (about 10% of total proteins). Furthermore, glutelins containing comparatively high amount of essential amino acid, lysine, play a major role in rice storage proteins (Huebner et al. 1990). This is one of the reasons why the quality of rice is much better than that of the proteins of other cereal crops. The glutelin mutants we report here are expeted to be useful materials for studying the genetic regulation of glutelin biosynthesis and improving rice protein.

References

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