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

Saito Y, Kishida K, Takata K, Takahashi H, Shimada T, Tanaka K, Morita S, Satoh S, Masumura T - J. Exp. Bot. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan.

ABSTRACT
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.

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Related in: MedlinePlus

Association of an ER-resident molecular chaperone, BiP, with PB-I and PB-like structures of 35S:Pro-GFP plants. Immunoelectron micrographs of PB-Is in starchy endosperm cells of seeds (A) and PB-like structures in leaf cells (B) and root cells (C) of 35S:Pro-GFP plants obtained using anti-pumpkin BiP antibodies. Bar in A = 500 nm; bars in B and C = 200 nm.
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fig8: Association of an ER-resident molecular chaperone, BiP, with PB-I and PB-like structures of 35S:Pro-GFP plants. Immunoelectron micrographs of PB-Is in starchy endosperm cells of seeds (A) and PB-like structures in leaf cells (B) and root cells (C) of 35S:Pro-GFP plants obtained using anti-pumpkin BiP antibodies. Bar in A = 500 nm; bars in B and C = 200 nm.

Mentions: The PB-like structures in the leaf cells were often observed in close proximity to the membrane (Fig. 7D, E). To determine whether the PB-like structures were derived from the ER, an immunoelectron microscopy analysis was performed with antibodies raised against pumpkin BiP, an ER-resident molecular chaperone. The anti-BiP antibodies were localized in the peripheral region of PB-Is in WT rice endosperm (Takahashi et al., 2005). Immunogold labelling of BiP revealed the presence of gold particles within PB-Is in the endosperm of the 35S:Pro-GFP plants (Fig. 8A). In the leaf and root cells, the gold particles were detected in electron-dense structures (Fig. 8B, C), and the size and shape of these structures were highly similar to those of the PB-like structures observed in Fig. 7. Figure 8B shows the presence of electron-dense structures on the membrane, causing the area to appear swollen. Gold particles were also detected within the continuous ER membrane region (Fig. 8B). The electron-dense structures labelled with anti-BiP antibodies were not present in WT and 35S:GFP plants (data not shown). These results showed that the PB-like structures are derived from rough ER.


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

Saito Y, Kishida K, Takata K, Takahashi H, Shimada T, Tanaka K, Morita S, Satoh S, Masumura T - J. Exp. Bot. (2009)

Association of an ER-resident molecular chaperone, BiP, with PB-I and PB-like structures of 35S:Pro-GFP plants. Immunoelectron micrographs of PB-Is in starchy endosperm cells of seeds (A) and PB-like structures in leaf cells (B) and root cells (C) of 35S:Pro-GFP plants obtained using anti-pumpkin BiP antibodies. Bar in A = 500 nm; bars in B and C = 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2651459&req=5

fig8: Association of an ER-resident molecular chaperone, BiP, with PB-I and PB-like structures of 35S:Pro-GFP plants. Immunoelectron micrographs of PB-Is in starchy endosperm cells of seeds (A) and PB-like structures in leaf cells (B) and root cells (C) of 35S:Pro-GFP plants obtained using anti-pumpkin BiP antibodies. Bar in A = 500 nm; bars in B and C = 200 nm.
Mentions: The PB-like structures in the leaf cells were often observed in close proximity to the membrane (Fig. 7D, E). To determine whether the PB-like structures were derived from the ER, an immunoelectron microscopy analysis was performed with antibodies raised against pumpkin BiP, an ER-resident molecular chaperone. The anti-BiP antibodies were localized in the peripheral region of PB-Is in WT rice endosperm (Takahashi et al., 2005). Immunogold labelling of BiP revealed the presence of gold particles within PB-Is in the endosperm of the 35S:Pro-GFP plants (Fig. 8A). In the leaf and root cells, the gold particles were detected in electron-dense structures (Fig. 8B, C), and the size and shape of these structures were highly similar to those of the PB-like structures observed in Fig. 7. Figure 8B shows the presence of electron-dense structures on the membrane, causing the area to appear swollen. Gold particles were also detected within the continuous ER membrane region (Fig. 8B). The electron-dense structures labelled with anti-BiP antibodies were not present in WT and 35S:GFP plants (data not shown). These results showed that the PB-like structures are derived from rough ER.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus