Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes

Abstract

SUMMARYRice prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.

Additional Nitrogen Fertilization at Heading Time of Rice Down-Regulates Cellulose Synthesis in Seed Endosperm

“…Cys-poor prolamins are accumulated at late stages of grain filling [43], and their expression is increased during suppression of glutelins, which are the most abundant storage protein in rice [44]. Both the present data and previous reports suggest that the expression of Cys-poor prolamin is controlled at the mRNA level, and effectively stores excess nitrogen during additional fertilization [43], [44].…”

“…Cys-poor prolamins are accumulated at late stages of grain filling [43], and their expression is increased during suppression of glutelins, which are the most abundant storage protein in rice [44]. Both the present data and previous reports suggest that the expression of Cys-poor prolamin is controlled at the mRNA level, and effectively stores excess nitrogen during additional fertilization [43], [44]. This may be because Cys-poor prolamin has the simple primary structure with the absence of cysteine residues, and is directly accumulated in the endoplasmic reticulum where protein synthesis occurs.…”

The dynamic behavior of storage organelles in developing cereal seeds and its impact on the production of recombinant proteins

“…Protein trafficking and organelle reshaping can also be investigated by looking at the behavior of recombinant proteins, including storage proteins fused to fluorescent tags (Tosi et al, 2009; Saito et al, 2012; Shigemitsu et al, 2013; Wakasa and Takaiwa, 2013) or foreign proteins that have been produced in the context of molecular farming. Endogenous glycoproteins are rarely found in the endosperm (Woo et al, 2001) and are difficult to follow due to the lack of appropriate detection antibodies, so recombinant glycoproteins are particularly useful because they can be traced through intracellular pathways by immunolocalization and by characterizing their glycan modifications (Drakakaki et al, 2006; Arcalis et al, 2010).…”

In Vivo Digestibility of Rice Prolamin/Protein Body-I Particle Is Decreased by Cooking

“…In the case of rice proteins, Bradbury et al (16) reported that the decreased protein digestibility due to cooking does not result from the formation of isopeptide linkages between the e-amino group of lysine (which would make it resistant to trypsin degradation) and the g-carboxyl group of glutamic acid residues, but may instead be due to the formation of a cystine-rich core that is resistant to proteases. In addition, Saito et al (17) reported that the localization of prolamin species in PB-Is was different and 16 and 10 kDa prolamin, sulfur-containing amino acids rich in proteins, were mainly located in the core and in the middle layer of PB-Is, respectively. Based on these reports and our results, we hypothesize that hydrophobic interactions and disulfide linkages in the PB-Is contribute to the decrease in prolamin digestibility that occurs with cooking.…”