44. Molecular studies on the rice storage proteins

Institute of Biological Chemistry, Washington State University, Pullman, Wa 99164-6340, USA

The major storage proteins of rice endosperm are the glutelins which comprise up to 75% of the rice grain protein (Tecson et al. 1971; Villareal and Juliano 1978). In contrast, the alcohol-water soluble prolamines, the predominant storage protein in most of the major cereals, comprise less than 5 to 10% of the grain protein. When the glutelin protein fraction of rice seeds was fractionated by SDS polyacrylamide gel electrophoresis, three different size proteins, 51 kD, and 21-22 kD as well as a contaminating 14 kD prolamine were detected. Antibodies were raised against purified preparations of these putative glutelin polypeptides and employed in studies to evaluate their immunological relatedness. Antibodies raised against the 34-37 kD and 21-22 kD polypeptides strongly reacted with the 51 kD polypeptide, and conversely, anti- 51 kD polypeptide cross-reacted with the two aforementioned smaller polypeptides as viewed by Western blotting. Irrespective of the antibody preparation, immunoprecipitation of in vitro translation products results in the appearance of only the 51 kD polypeptide. These results together with those from Yamagata et al. (1982) and Luthe (1985) indicate that the 34-37 kD and 21- 22 kD polypeptides are post-translational proteolytic products of the 51 kD protein (Krishnan and Okita 1986). Messenger transcripts for both prolamine and glutelin are asociated with membrane bound polysomes and direct the synthesis of polypeptides 2 kD larger than the mature forms of these proteins. These results are consistent with the view that both storage proteins are initially synthesized with signal peptides which presumably facilitates their transport and packaging into protein bodies.

Glutelin and prolamine cDNA clones were isolated by antibody screening of a lambda-gt 11 rice seed library. Northern blot studies indicate that the mRNAs for glutelin and prolamine are about 1800 and 800 bases in length, respectively. Glutelin transcripts are present in seed as early as 3-5 days after flowering (DAF) and obtain a maximum level at 15-20 DAF. Prolamine transcripts are first detected at 5-8 DAF and reach their maximum level at 25-27 DAF. The results indicate that there are qualitative and quantitative differences in the expression of the rice storage protein genes during development. The DNA sequence of a 1300 base glutelin cDNA shows strong homology at the carboxyl half of the coding segment to the legume 11S storage proteins.

A rice genomic library was prepared by ligating a partial EcoRi digest of rice DNA in Charon 35. Several genomic clones for both the glutelin and prolamine have been isolated and are presently being characterized. Interestingly, one of the prolamine clones contains a highly conserved 2.5 kb EcoRI fragment, repeated in tandem 4 times. Physical mapping data indicates that the prolamine gene resides within a 1.1 kb fragment within this conserved EcoRI fragment. The tight clustering of the prolamine genes is a reflection of the relatively small gnome of rice as compared to other cereals.

Luthe, D.S., 1983. Storage protein accumulation in developing rice seeds. Plant Sci. Lett. 32: 147-158.

Krishnan, H.B. and T.W. Okita, 1986. Structural relationship among the rice glutelins. Plant Physiol. 81: In Press.

Tecson, E.M.S., B.V. Esmana, L.P. Lontok and B.O. Juliano, 1971. Studies on the extraction and composition of rice endosperm: glutelin and prolamine. Cereal Chem. 48: 186-191.

Villareal, R.M. and B.O. Juliano, 1978. Properties of glutelin from maturre and developing rice grain. Phytochem. 17: 177-182.

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