Lack of Globulin Synthesis during Seed Development Alters Accumulation of Seed Storage Proteins in Rice

Abstract

The major seed storage proteins (SSPs) in rice seeds have been classified into three types, glutelins, prolamins, and globulin, and the proportion of each SSP varies. It has been shown in rice mutants that when either glutelins or prolamins are defective, the expression of another type of SSP is promoted to counterbalance the deficit. However, we observed reduced abundances of glutelins and prolamins in dry seeds of a globulin-deficient rice mutant (Glb-RNAi), which was generated with RNA interference (RNAi)-induced suppression of globulin expression. The expression of the prolamin and glutelin subfamily genes was reduced in the immature seeds of Glb-RNAi lines compared with those in wild type. A proteomic analysis of Glb-RNAi seeds showed that the reductions in glutelin and prolamin were conserved at the protein level. The decreased pattern in glutelin was also significant in the presence of a reductant, suggesting that the polymerization of the glutelin proteins via intramolecular disulfide bonds could be interrupted in Glb-RNAi seeds. We also observed aberrant and loosely packed structures in the storage organelles of Glb-RNAi seeds, which may be attributable to the reductions in SSPs. In this study, we evaluated the role of rice globulin in seed development, showing that a deficiency in globulin could comprehensively reduce the expression of other SSPs.

RNA Interference-Mediated Simultaneous Suppression of Seed Storage Proteins in Rice Grains

“…The total seed protein was extracted from the powder (100 mg) with 1 ml of sodium dodecyl sulfate (SDS)-urea buffer [250 mM Tris-HCl, pH 6.8, 4% SDS, 8 M urea, 20% glycerol, and 5% β-mercaptoethanol (2-ME)] and quantified using a protein assay kit (Invitrogen) as previously described (Kim et al, 2013; Lee et al, 2015). The total seed protein (10 μg) was separated by gradient SDS (8–17.5%) polyacrylamide gel electrophoresis (Saito et al, 2012).…”

“…Antibodies against GluA, GluB, and globulin were prepared previously in our laboratory (Kim et al, 2013; Lee et al, 2015). Antibodies to prolamin 13a and the BiP and PDI were provided by Dr. B. S. Han (Rural Development Administration, South Korea) and Prof. H. S. Seo (Seoul National University, South Korea), respectively.…”

“…RNA interference (RNAi)-mediated suppression of α-zein resulted in the floury kernel phenotype (Wu and Messing, 2010). To improve the nutrition quality, several groups have performed studies on the molecular and cellular features of SSP-deficient rice and maize seeds (Kawakatsu et al, 2010; Wu and Messing, 2010; Kim et al, 2013; Lee et al, 2015). In the maize prolamin (known as zein)-suppressed mutants, reduction of α-zein (similar to rice 10-kDa prolamin) resulted in an increased level of lysine.…”

“…Moreover, the suppression of α-zein and the simultaneous reduction of both β- and γ-zein led to the formation of abnormal PBs and incomplete embedding of starch granules, resulting in floury kernel phenotype (Wu and Messing, 2010). In SSP-repressed rice transgenic seeds, the suppression of individual SSPs resulted in the increased expression of other SSPs and structural changes in the PBs of endosperm cells without severe seed-phenotypic changes (Kawakatsu et al, 2010; Kim et al, 2012, 2013; Lee et al, 2015). …”

“…Previously, to gain a better understanding of the SSP-regulation system in rice seeds, we studied several transgenic rice plants suppressing respective glutelins, prolamins, and globulin in their seeds (Kim et al, 2012, 2013; Lee et al, 2015). Herein, to gain new insight into the simultaneous suppression of SSPs accumulated in PB-I and PSV of rice seed endosperms, we first generated SSP-suppressed transgenic rice plants that block the simultaneous expression of SSPs grouped in g lutelin A (GluA), p rolamin 13a-I and g lo b ulin using RNA i nterference approach (defined as GPGb-RNAi).…”