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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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PB-like structures containing prolamin–GFP fusion proteins in the vegetative tissues. (A–E) Immunogold labelling of prolamin–GFP with anti-GFP antibodies in the leaf cells of 35S:Pro-GFP plants as determined using a high pressure frozen/freeze substitution technique. Numerous electron-dense structures labelled with anti-GFP antibodies were visible in the leaf cells (A, arrows). These structures had different shapes and sizes (A–E), and some of them were in close proximity to the rough ER (D and E, arrowheads). (F and G) Immunogold labelling of prolamin–GFP in the root cells of 35S:Pro-GFP plants. The PB-like structures were also visible in the root cells (F and G). Cp, chloroplast; LV, lytic vacuole; CW, cell wall. Bars in A–C, E, and F = 500 nm; bar in D = 200 nm.
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fig7: PB-like structures containing prolamin–GFP fusion proteins in the vegetative tissues. (A–E) Immunogold labelling of prolamin–GFP with anti-GFP antibodies in the leaf cells of 35S:Pro-GFP plants as determined using a high pressure frozen/freeze substitution technique. Numerous electron-dense structures labelled with anti-GFP antibodies were visible in the leaf cells (A, arrows). These structures had different shapes and sizes (A–E), and some of them were in close proximity to the rough ER (D and E, arrowheads). (F and G) Immunogold labelling of prolamin–GFP in the root cells of 35S:Pro-GFP plants. The PB-like structures were also visible in the root cells (F and G). Cp, chloroplast; LV, lytic vacuole; CW, cell wall. Bars in A–C, E, and F = 500 nm; bar in D = 200 nm.

Mentions: To characterize these punctate structures of prolamin–GFP in the leaf and root cells, an ultrastructural analysis was performed with an electron microscope using a high pressure frozen/freeze substitution technique for the preparation of the thin sections. Immunogold labelling of prolamin–GFP on these thin-sectioned samples showed that the gold particles were localized in electron-dense structures in the leaf and root cells of 35S:Pro-GFP plants (Fig. 7). Most of the structures containing prolamin–GFP were spherical, with diameters of 50–500 nm in the leaf cells, but some of them were irregular structures (Fig. 7B) or spherical structures with a diameter of 2 μm (Fig. 7C). In addition, these structures in leaf cells were often surrounded by the membrane (Fig. 7D, arrowheads). Some of these structures were also associated with the membrane (Fig. 7E). These structures labelled with anti-GFP antibodies were found in all types of leaf cells, including mesophyll and vascular cells. The novel structures were also found in the root cells (Fig. 7F, G). The characteristics of these structures in the leaves and roots looked similar to those of PB in the endosperms. Examination of several leaf and root sections of WT, 35S:GFP, and 35S:Pro-GFP plants showed that these PB-like structures labelled with anti-GFP antibodies were found only in 35S:Pro-GFP plants and were not present in WT and 35S:GFP plants (data not shown).


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)

PB-like structures containing prolamin–GFP fusion proteins in the vegetative tissues. (A–E) Immunogold labelling of prolamin–GFP with anti-GFP antibodies in the leaf cells of 35S:Pro-GFP plants as determined using a high pressure frozen/freeze substitution technique. Numerous electron-dense structures labelled with anti-GFP antibodies were visible in the leaf cells (A, arrows). These structures had different shapes and sizes (A–E), and some of them were in close proximity to the rough ER (D and E, arrowheads). (F and G) Immunogold labelling of prolamin–GFP in the root cells of 35S:Pro-GFP plants. The PB-like structures were also visible in the root cells (F and G). Cp, chloroplast; LV, lytic vacuole; CW, cell wall. Bars in A–C, E, and F = 500 nm; bar in D = 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: PB-like structures containing prolamin–GFP fusion proteins in the vegetative tissues. (A–E) Immunogold labelling of prolamin–GFP with anti-GFP antibodies in the leaf cells of 35S:Pro-GFP plants as determined using a high pressure frozen/freeze substitution technique. Numerous electron-dense structures labelled with anti-GFP antibodies were visible in the leaf cells (A, arrows). These structures had different shapes and sizes (A–E), and some of them were in close proximity to the rough ER (D and E, arrowheads). (F and G) Immunogold labelling of prolamin–GFP in the root cells of 35S:Pro-GFP plants. The PB-like structures were also visible in the root cells (F and G). Cp, chloroplast; LV, lytic vacuole; CW, cell wall. Bars in A–C, E, and F = 500 nm; bar in D = 200 nm.
Mentions: To characterize these punctate structures of prolamin–GFP in the leaf and root cells, an ultrastructural analysis was performed with an electron microscope using a high pressure frozen/freeze substitution technique for the preparation of the thin sections. Immunogold labelling of prolamin–GFP on these thin-sectioned samples showed that the gold particles were localized in electron-dense structures in the leaf and root cells of 35S:Pro-GFP plants (Fig. 7). Most of the structures containing prolamin–GFP were spherical, with diameters of 50–500 nm in the leaf cells, but some of them were irregular structures (Fig. 7B) or spherical structures with a diameter of 2 μm (Fig. 7C). In addition, these structures in leaf cells were often surrounded by the membrane (Fig. 7D, arrowheads). Some of these structures were also associated with the membrane (Fig. 7E). These structures labelled with anti-GFP antibodies were found in all types of leaf cells, including mesophyll and vascular cells. The novel structures were also found in the root cells (Fig. 7F, G). The characteristics of these structures in the leaves and roots looked similar to those of PB in the endosperms. Examination of several leaf and root sections of WT, 35S:GFP, and 35S:Pro-GFP plants showed that these PB-like structures labelled with anti-GFP antibodies were found only in 35S:Pro-GFP plants and were not present in WT and 35S:GFP plants (data not shown).

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