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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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Expression of prolamin–GFP mRNA in different tissues of transgenic plants. RT-PCR analysis of the prolamin–GFP transcript in seeds, leaves, and roots of wild type (WT) and 35S:Pro-GFP plants. The bottom panel shows the expression of the actin genes as a control.
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fig2: Expression of prolamin–GFP mRNA in different tissues of transgenic plants. RT-PCR analysis of the prolamin–GFP transcript in seeds, leaves, and roots of wild type (WT) and 35S:Pro-GFP plants. The bottom panel shows the expression of the actin genes as a control.

Mentions: Prolamins are encoded by a multigene family, and separated into three major groups according to their apparent molecular sizes of 10, 13, and 16 kDa (Ogawa et al., 1987). The 13 kDa prolamins are the most abundant group of prolamins in rice (Horikoshi et al., 1991). A chimeric gene encoding a fusion protein of 13 kDa prolamin (λRM1) and GFP was constructed. The prolamin-coding sequence was fused to the region upstream of GFP. GFP is a soluble protein that is secreted efficiently when introduced into plant ER via a signal peptide (Batoko et al., 2000; Frigerio et al., 2001). This chimeric gene was driven by the CaMV 35S promoter (35S:Pro-GFP; Fig. 1). As a control, the GFP gene driven by the CaMV 35S promoter, without the prolamin-coding sequence, was used (35S:GFP; Fig. 1). The first intron of the superoxide dismutase sodCc2 gene (Sakamoto et al., 1995) was inserted between the promoter and the GFP gene to enhance promoter activity. This construct was transferred into rice (O. sativa L. cv. Nipponbare) by an Agrobacterium-mediated transformation method (Hiei et al., 1994). RT-PCR was used to analyse the expression of the prolamin–GFP gene in different tissues of 35S:Pro-GFP plants (Fig. 2). As expected, the transcripts of the prolamin–GFP gene were detected in all tissues analysed, reflecting the activity of the CaMV 35S promoter. The expression of GFP was not detected in tissues of WT plants.


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)

Expression of prolamin–GFP mRNA in different tissues of transgenic plants. RT-PCR analysis of the prolamin–GFP transcript in seeds, leaves, and roots of wild type (WT) and 35S:Pro-GFP plants. The bottom panel shows the expression of the actin genes as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Expression of prolamin–GFP mRNA in different tissues of transgenic plants. RT-PCR analysis of the prolamin–GFP transcript in seeds, leaves, and roots of wild type (WT) and 35S:Pro-GFP plants. The bottom panel shows the expression of the actin genes as a control.
Mentions: Prolamins are encoded by a multigene family, and separated into three major groups according to their apparent molecular sizes of 10, 13, and 16 kDa (Ogawa et al., 1987). The 13 kDa prolamins are the most abundant group of prolamins in rice (Horikoshi et al., 1991). A chimeric gene encoding a fusion protein of 13 kDa prolamin (λRM1) and GFP was constructed. The prolamin-coding sequence was fused to the region upstream of GFP. GFP is a soluble protein that is secreted efficiently when introduced into plant ER via a signal peptide (Batoko et al., 2000; Frigerio et al., 2001). This chimeric gene was driven by the CaMV 35S promoter (35S:Pro-GFP; Fig. 1). As a control, the GFP gene driven by the CaMV 35S promoter, without the prolamin-coding sequence, was used (35S:GFP; Fig. 1). The first intron of the superoxide dismutase sodCc2 gene (Sakamoto et al., 1995) was inserted between the promoter and the GFP gene to enhance promoter activity. This construct was transferred into rice (O. sativa L. cv. Nipponbare) by an Agrobacterium-mediated transformation method (Hiei et al., 1994). RT-PCR was used to analyse the expression of the prolamin–GFP gene in different tissues of 35S:Pro-GFP plants (Fig. 2). As expected, the transcripts of the prolamin–GFP gene were detected in all tissues analysed, reflecting the activity of the CaMV 35S promoter. The expression of GFP was not detected in tissues of WT plants.

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