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

Saito, Yuhi; Kishida, Koichi; Takata, Kazuyuki; Takahashi, Hideyuki; Shimada, Takeaki; Tanaka, Kunisuke; Morita, Shigeto; Satoh, Shigeru; Masumura, Takehiro

doi:10.1093/jxb/ern311

Cited by 46 publications

(63 citation statements)

References 58 publications

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“…When protoplasts expressing zeolin were treated with 2-ME or when a zeolin devoid of the six Cys residues was expressed in the protoplasts, the solubility of the protein was enhanced, allowing its trafficking along the secretory pathway (4). Similarly, a fluorescent protein fused to rice prolamine accumulated in the ER of leaf cells by forming polymers that required reducing agents for complete solubilization (48). The formation of disulfide-linked oligomers or large aggregates does not exclusively occur in plants.…”

Section: Discussionmentioning

confidence: 99%

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

Llop‐Tous

Madurga

Giralt

et al. 2010

Journal of Biological Chemistry

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The N-terminal proline-rich domain of ␥-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL) 8 of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4 -6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys 7 and Cys 9 ) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.The mechanisms by which prolamins, which lack the ER 3 retention (H/K)DEL motif, are retained and assembled in the ER-derived PBs are only partially understood. Different factors act as determinants of PB biogenesis, some of them derived from cis-cargo properties and others with a cellular trans-origin (1). It has been proposed that the physico-chemical properties of prolamins, such as hydrophobicity and disulfide bond formation, promote specific interactions resulting in the formation of large self-assemblies that are responsible for their retention in the ER and PB formation (2-4). These polymers could be excluded because of their size from being carried by the COP II vesicles that transport cargo proteins to the Golgi complex (5). However, the generic COPII vesicles are flexible enough for large cargo loading, including that of procollagen (more than 300 nm in size) (6) and the collagen VII trimer (900 kDa) (7).In maize, four distinct types of prolamins (␣-, ␤-, ␥-, and ␦-zein) coexist in the PBs (8) and play distinct roles in PB formation. Recently, maize storage protein mutants created through RNAi showed that ␥-RNAi maize mutant lines exhibited slightly altered protein body formation and that a more drastic effect was observed in the ␤-␥ combined mutant, where protein bodies showed an irregular shape, particularly in their periphery (9).Various studies on zein accumulation in heterologous expression systems suggest that ␥-zein and ␤-zein mediate t...

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Section: Discussionmentioning

confidence: 99%

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

Llop‐Tous

Madurga

Giralt

et al. 2010

Journal of Biological Chemistry

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“…Goat anti-mouse IgG conjugated with 10-nm colloidal gold particles (Sigma-Aldrich; diluted 1:50) were used as secondary antibody. Grids were stained with 2% uranyl acetate and examined with FEI Tecnai G2 20 transmission electron microscope at 120 kV (Saito et al, 2009;Li et al, 2011).…”

Section: Electron Microscopy and Immunolabelingmentioning

confidence: 99%

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice

et al. 2018

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Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana. Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security.

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“…[5][6][7][8]11,19) In addition, rice prolamin has been shown to form ER-derived PBs in plant tissues such as rice leaves, roots, 12) and calli.…”

Section: Discussionmentioning

confidence: 99%

“…12,13) However, rice prolamins lack the characteristic domains found in maize γ-zein and wheat γ-gliadin, indicating there may be another factor in the retention in ER and the formation of ER-derived PB-like structures. 14) To identify the domain of rice prolamins that is responsible for the formation of ER-derived PB-like structures, we have here expressed a chimeric gene encoding a fusion protein of rice prolamin-deletion mutants and green fluorescent protein (prolamin-GFP) in a heterologous yeast expression system.…”

Section: )mentioning

confidence: 99%

Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system

Masumura

Shigemitsu

Morita

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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Cereal prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat prolamins, rice prolamins can form ERderived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH 2 -terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.

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

Cited by 46 publications

References 58 publications

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice

Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system

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