Biosynthesis of Storage Proteins in Developing Rice Seeds

Abstract

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10-to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76-and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with I14CIleucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kllodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.The major storage proteins of most cereal grains are glutelin and prolamin. In rice grains, however, glutelin is the major protein of the starchy endosperm, constituting at least 80%o of the total protein, prolamin accounting for less than 5% (12). Rice glutelin has a mol wt of 6 x I05 according to Tecson et al. (27), but Takeda et a!. (25) demonstrated that it has a heterogeneous mol wt. The increase in glutelin in the developing rice grain coincides with the appearance of PB' in the starchy endosperm 7 to 8 DAF (7,29), and glutelin is found exclusively in PB (7,26,30). In a previous paper (26), we reported two types of PB in the starchy endosperm of rice grains and described their isolation. One (type I, PB-I) is spherical with a concentric ring structure, whereas the other (type II, PB-II) is stained homogeneously by osmium tetroxide and does not have this structure. PB-I contains prolamin, and PB-II is rich in glutelin and globulin. The glutelin in PB-II is composed of two principal subunits, the 22-to 23-and 37-to 39-kD complexes, and the prolamin in PB-I is composed mainly of 13-kD polypeptide.

Ultrastructure of protein bodies in mutant rice (Oryza sativa L.) with altered storage protein composition

“…Koshihikari harvested from an experimental field showed a similar pattern to Koshihikari grown in a glasshouse in terms of the SDS-PAGE profile during seed development. In Koshihikari, 37-39 kDa glutelin and 22-23 kDa glutelin were predominant from 5 DAF to 15 DAF during ripening, as reported previously (Yamagata et al 1982). In agreement with previous reports (Li andOkita 1993, Yamagata et al 1982), the ratio of prolamin to total protein increased during the latter stages of seed development (Fig.…”

“…3A). These structures of PB in Koshihikari were similar to those reported previously (Yamagata et al 1982(Yamagata et al , 1986. PB-II occupied the visual field at 15 DAF (Fig.…”

“…1A). The polypeptides were classified into six fractions; 57 kDa pre-glutelin, 37-39 kDa glutelin, 26 kDa globulin, 22-23 kDa glutelin, 10-16 kDa prolamins, and other polypeptides in accordance with previous reports (Ogawa et al 1987, Yamagata et al 1982. The percentages of each fraction were calculated from densitometric analysis of the SDS-PAGE gel and expressed as the relative percentage of the total intensity (Fig.…”

“…Lgc1 and glb1 have deletions of a glutelin gene and the 26 kDa globulin gene, respectively (Kusaba et al 2003, Morita et al 2007). Glutelin and globulin are components of PB-II (Yamagata et al 1982), but the influence of their reduction on PB structure was different. Studies on protein-sorting mutants revealed that deficiency of protein disulfide isomerase or vacuolar processing enzyme affects PB structure (Kumamaru et al 2010, Takemoto et al 2002, Wang et al 2009).…”

“…PB-I consists mainly of prolamins and comprises approximately 20% of milled rice protein. On the other hand, PB-II is composed mainly of deposits of glutelins and globulins and constitutes approximately 60% of milled rice protein (Ogawa et al 1987, Tanaka et al 1980, Yamagata et al 1982. Humans are not able to digest rice seed storage proteins completely (Tanaka et al 1975), and morphological studies have suggested that PB-I is identical to the indigestible remains of rice found in the feces of monogastric animals (Barber et al 1998).…”