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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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Accumulation of prolamin–GFP fusion proteins in different tissues of transgenic plants. Seeds, leaves, and roots of WT (W), 35S:GFP (G), and 35S:Pro-GFP (P) plants were subjected to SDS–PAGE followed by immunoblot with anti-GFP and anti-13 kDa prolamin antibodies. The upper and lower bands correspond to prolamin–GFP and GFP, respectively. The truncated prolamin–GFP was observed in the leaf and root tissues of 35S:Pro-GFP plants (asterisks). The arrowheads indicate the non-specific signal. The molecular masses are given on the left in kDa.
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fig3: Accumulation of prolamin–GFP fusion proteins in different tissues of transgenic plants. Seeds, leaves, and roots of WT (W), 35S:GFP (G), and 35S:Pro-GFP (P) plants were subjected to SDS–PAGE followed by immunoblot with anti-GFP and anti-13 kDa prolamin antibodies. The upper and lower bands correspond to prolamin–GFP and GFP, respectively. The truncated prolamin–GFP was observed in the leaf and root tissues of 35S:Pro-GFP plants (asterisks). The arrowheads indicate the non-specific signal. The molecular masses are given on the left in kDa.

Mentions: To investigate the accumulation of the prolamin–GFP fusion proteins in different tissues of the transgenic rice plant, immunoblot analysis of protein extracts from seeds, leaves, and roots was performed with anti-GFP antibodies (Fig. 3). Bands with apparent molecular masses of ∼27 kDa and ∼40 kDa were detected in the 35S:GFP and 35S:Pro-GFP in all tissues, respectively (Fig. 3). The 40 kDa mass is consistent with the predicted molecular size of prolamin–GFP fusion proteins, because the molecular sizes of prolamin and GFP are 13 kDa and 27 kDa, respectively. The ∼40 kDa polypeptides also reacted with anti-13 kDa prolamin antibodies (Fig. 3). These results suggested that the prolamin–GFP fusion proteins accumulated stably not only in the seeds but also in the leaves and roots. The bands of 13, ∼23, and ∼27 kDa were also detected in the seeds of all plants by anti-13 kDa prolamin antibodies. The 13 kDa proteins are endogenous 13 kDa prolamin. The bands of 23 kDa and 27 kDa are non-specific signals. In addition, the processed form of the prolamin–GFP fusion proteins was detected in the leaves and roots of 35S:Pro-GFP plants (Fig. 3).


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)

Accumulation of prolamin–GFP fusion proteins in different tissues of transgenic plants. Seeds, leaves, and roots of WT (W), 35S:GFP (G), and 35S:Pro-GFP (P) plants were subjected to SDS–PAGE followed by immunoblot with anti-GFP and anti-13 kDa prolamin antibodies. The upper and lower bands correspond to prolamin–GFP and GFP, respectively. The truncated prolamin–GFP was observed in the leaf and root tissues of 35S:Pro-GFP plants (asterisks). The arrowheads indicate the non-specific signal. The molecular masses are given on the left in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Accumulation of prolamin–GFP fusion proteins in different tissues of transgenic plants. Seeds, leaves, and roots of WT (W), 35S:GFP (G), and 35S:Pro-GFP (P) plants were subjected to SDS–PAGE followed by immunoblot with anti-GFP and anti-13 kDa prolamin antibodies. The upper and lower bands correspond to prolamin–GFP and GFP, respectively. The truncated prolamin–GFP was observed in the leaf and root tissues of 35S:Pro-GFP plants (asterisks). The arrowheads indicate the non-specific signal. The molecular masses are given on the left in kDa.
Mentions: To investigate the accumulation of the prolamin–GFP fusion proteins in different tissues of the transgenic rice plant, immunoblot analysis of protein extracts from seeds, leaves, and roots was performed with anti-GFP antibodies (Fig. 3). Bands with apparent molecular masses of ∼27 kDa and ∼40 kDa were detected in the 35S:GFP and 35S:Pro-GFP in all tissues, respectively (Fig. 3). The 40 kDa mass is consistent with the predicted molecular size of prolamin–GFP fusion proteins, because the molecular sizes of prolamin and GFP are 13 kDa and 27 kDa, respectively. The ∼40 kDa polypeptides also reacted with anti-13 kDa prolamin antibodies (Fig. 3). These results suggested that the prolamin–GFP fusion proteins accumulated stably not only in the seeds but also in the leaves and roots. The bands of 13, ∼23, and ∼27 kDa were also detected in the seeds of all plants by anti-13 kDa prolamin antibodies. The 13 kDa proteins are endogenous 13 kDa prolamin. The bands of 23 kDa and 27 kDa are non-specific signals. In addition, the processed form of the prolamin–GFP fusion proteins was detected in the leaves and roots of 35S:Pro-GFP plants (Fig. 3).

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