Lysine-rich modified gamma-zeins accumulate in protein bodies of transiently transformed maize endosperms.
During maize seed development, endosperm cells synthesize large amounts of storage proteins, alpha-, beta-, and gamma-zeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize gamma-zein gene through the introduction of lysine-rich (Pro-Lys)n coding sequences at different sites of the gamma-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich gamma-zein constructs under the control of the 1.7 kb gamma-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys)n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the gamma-zein, high levels of protein were observed. In contrast, when (Pro-Lys)n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified gamma-zeins accumulate within these organelles and co-localized with endogenous alpha- and gamma-zeins. The studies reported here show the feasibility of manipulating the gamma-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.
Torrent
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Departament de Genètica Molecular, Centre d'Investigació i Desenvolupament, (CSIC), Barcelona, Spain.
Alvarez
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I
Geli
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MI
Dalcol
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Ludevid
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D
D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D008239:Lysine / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*; D015028:Zein / Q000235:genetics / Q000378:metabolism*
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D015028:Zein; D008239:Lysine