Yuhi Saito

A green fluorescent protein fused to rice prolamin forms protein body-like structures in transgenic rice

Prolamins, a group of rice (Oryza sativa) seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and deposited in ER-derived type I protein bodies (PB-Is) in rice endosperm cells. The accumulation mechanism of prolamins, which do not possess the well-known ER retention signal, remains unclear. In order to elucidate whether the accumulation of prolamin in the ER requires seed-specific factors, the subcellular localization of the constitutively expressed green fluorescent protein fused to prolamin (prolamin–GFP) was examined in seeds, leaves, and roots of transgenic rice plants. The prolamin–GFP fusion proteins accumulated not only in the seeds but also in the leaves and roots. Microscopic observation of GFP fluorescence and immunocytochemical analysis revealed that prolamin–GFP fusion proteins specifically accumulated in PB-Is in the endosperm, whereas they were deposited in the electron-dense structures in the leaves and roots. The ER chaperone BiP was detected in the structures in the leaves and roots. The results show that the aggregation of prolamin–GFP fusion proteins does not depend on the tissues, suggesting that the prolamin–GFP fusion proteins accumulate in the ER by forming into aggregates. The findings bear out the importance of the assembly of prolamin molecules and the interaction of prolamin with BiP in the formation of ER-derived PBs.

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

Rice 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.

Improvement in the in vivo digestibility of rice protein by alkali extraction is due to structural changes in prolamin/protein body-I particle.

Rice prolamin, constituting type-I protein body (PB-I), is indigestible and causes deterioration of rice protein nutritional quality. In this study, the in vivo digestibility of rice protein isolates was investigated by tracing their intraluminal transit in the gastrointestinal (GI) tract of rats by western blotting and by observing the structures excreted in the feces by electron microscopy. Two types of rice protein isolates, produced by alkali extraction (AE-RP) and by starch degradation (SD-RP), were compared. The protein patterns in the isolates were similar, but their digestion in the GI-tract showed striking differences. In the AE-RP group, 13-kDa prolamin (13P) quickly disappeared in the lower GI tract and was not excreted in the feces. By contrast, in the SD-RP group, 13P accumulated massively and nearly intact PB-Is were excreted. These results indicate that the in vivo digestibility of prolamin can be improved by alkali extraction through structural changes to it.

Ultrastructure of mature protein body in the starchy endosperm of dry cereal grain.

The development of the protein body in the late stage of seed maturation is poorly understood, because electron-microscopy of mature cereal endosperm is technically difficult. In this study, we attempted to modify the existing method of embedding rice grain in resin. The modified method revealed the ultrastructures of the mature protein body in dry cereal grains.

Thin Frozen Film Method for Visualization of Storage Proteins in Mature Rice Grains

There are technical difficulties in obtaining intact sections of cereal grains in which mature cells and their subcellular structures are well preserved. Here we describe a simple method for sectioning hard mature rice grains. It makes possible accurate localization of storage proteins in high-quality histological sections of rice endosperm.

Control of foreign polypeptide localization in specific layers of protein body type I in rice seed.

Key messageProlamin–GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin–GFP fusion proteins were gradually digested from outside by pepsin digestion.AbstractIn rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin–GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin–GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin–GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin–GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin–GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion.

Separation and identification of rice prolamins by two-dimensional gel electrophoresis and amino acid sequencing.

There are difficulties in detecting and separating rice prolamin polypeptides by 2D-PAGE analysis because prolamin polypeptides are insoluble, and the amino acid sequences show high homology among them. In this study, we improved the prolamin extraction method and the 2D-PAGE procedure, and succeeded in separating prolamin polypeptide species by 2D-PAGE and in identifying major prolamin polypeptide sequences.

Cyclic electron flow around PSI functions in the photoinhibited rice leaves

We investigated the regulation mechanism of cyclic electron flow around photosystem I (CEF-PSI) in the rice leaves which suffered from photosystem II (PSII) photoinhibition. High-light (HL) treatment [2000 µmol photon m–2 s–1 at 0% carbon dioxide (CO2), 2% oxygen (O2) and 25°C] of rice leaves decreased both the maximum quantum efficiency of PSII (Fv/Fm) and the light-dependent O2-evolution rate [V(O2)]. High-light treatment did not affect the relative electron flux in PSI [Φ(PSI) × PFD]. In non-treated leaves, increasing in the photon flux density (PFD) enhanced V(O2), Φ(PSI) × PFD and the ratio of oxidized P700 to total P700 [(P700+)/(P700)total]. Φ(PSI) × PFD continued to increase even after the saturation of V(O2) against PFD. These results suggested that the electrons not used for the major electron sink, photosynthetic carbon reduction-cycle, did turnover in PSI, that is, CEF-PSI functioned at a higher PFD. High-light treatments did not affect the activity of CEF-PSI and increased (P700+)/(P700)total in the lower PFDs, compared to non-treated leaves. The activity of CEF-PSI depends on the amount of oxidized PQ. Photoinhibition of PSII suppressed electron influx from PSII to photosynthetic linear electron transport. The enhanced (P700+)/(P700)total suggested the increase in the ratio of oxidized plastoquinone (PQ) to total PQ, which supported the activity of CEF-PSI in the photoinhibited leaves.

Analysis of storage protein distribution in rice grain of seed-protein mutant cultivars by immunofluorescence microscopy.

Abstract Localization of storage proteins in rice grains of seed-protein mutant cultivars, low-glutelin cultivars LGC-1, LGCsoft and a 26-kDa-globulin-deficient low-glutelin cultivar LGC-Katsu, was examined by immunofluorescence microscopy using fluorescence-labeled antibodies of 13 kDa prolamin and 23 kDa glutelin. Abundant 13 kDa prolamin and 23 kDa glutelin was observed in the outer regions of rice grains. Image analysis revealed that peaks of fluorescence intensity of both proteins were located at 2–7% of the width or longitudinal length of brown rice distant from the outer surface of brown rice (RDOS) on the dorsal, ventral, and apical sides of brown rice. In these seed-protein mutant cultivars and a normal-type cultivar Nihonmasari, the foot of the distribution peaks of both proteins were located at 20–30% RDOS on the ventral and apical side. In contrast, on the dorsal side of rice grain, varietal differences of RDOS at the foot of the distribution peak of both proteins varied with the cultivar. The RDOS was 20 –40%; and Nihonmasari >LGC-1≒LGCsoft >LGC-Katsu. Although the quantity of 13 kDa prolamin and 23 kDa glutelin greatly varied with the cultivar in the outer region of rice grain, both proteins were distributed uniformly at low levels in the inner region in all cultivars. Furthermore, SDS-PAGE analysis showed that a larger quantity of 13 kDa prolamin localized on the ventral than the dorsal side of rice grains in seed-protein mutant cultivars, especially in LGC-Katsu.

Randomized, double-blind, placebo-controlled, parallel-group study of the effect of Lactobacillus paracasei K71 intake on salivary release of secretory immunoglobulin A

Lactobacillus paracasei K71 was shown to be effective in alleviating the severity of atopic dermatitis in a randomized controlled trial, and a preliminary open-label trial suggested that strain K71 intake enhanced secretory immunoglobulin A (sIgA) release in the saliva. This study investigated the effect of K71 on sIgA release in a randomized, double-blind, placebo-controlled, parallel-group trial. The trial included 62 Japanese subjects aged 20–64 years with relatively low rates of salivary sIgA release. Subjects (n=31 in each group) were randomly given a tablet containing 100 mg (approximately 2 × 1011 bacteria) of K71 or a placebo tablet daily for 12 weeks. After eliminating data for eight subjects (four in each group) who met the exclusion criteria for efficacy analysis, data for 54 subjects were analyzed. The change in the rate of salivary sIgA release 8 weeks after initiation of the study compared with baseline was significantly higher in the K71 tablet group (105.5 ± 119.0 µg/min) than in the placebo group (52.7 ± 62.6 µg/min; p=0.047). There were no adverse events associated with intake of tablets containing K71. The safety of intake of L. paracasei K71 was also confirmed in an independent open-label trial with 20 healthy subjects who consumed excessive amounts of K71-containing food. L. paracasei K71 intake may therefore have some benefits in promoting mucosal immune function.