The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.This work was mainly supported by ERA Biotech (www.erabiotech.com). Additional support was supplied by grant SGR 2009/703 funded by the Generalitat de Catalunya (www10.gencat.net) and grants CDS2007/00036 of Consolider Ingenio program and TRA 2009/0124 of TRACE program funded by Ministerio de Ciencia e Inovación (MICINN, www.micinn.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Two Structural Domains Mediate Two Sequential Events in [gamma]-Zein Targeting: Protein Endoplasmic Reticulum Retention and Protein Body Formation.

[gamma]-Zein is a maize storage protein synthesized by endosperm cells and stored together with [alpha]- and [beta]-zeins in specialized organelles called protein bodies. Previous studies have shown that in maize there is only one type of protein body and it is derived directly from the endoplasmic reticulum (ER). In this article, we describe the domains of [gamma]-zein involved in ER retention and the domains involved in protein body formation. To identify the signal responsible for [gamma]-zein retention in ER-derived protein bodies, DNAs encoding various deletion mutants of [gamma]-zein were constructed and introduced into Arabidopsis as a heterologous system. By using pulse-chase experiments and immunoelectron microscopy, we demonstrated that the deletion of a proline-rich domain at the N terminus of [gamma]-zein puts an end to its retention in the ER; this resulted in the secretion of the mutated protein. The amino acid sequence of [gamma]-zein necessary for ER retention is the repeat domain composed of eight units of the hexapeptide PPPVHL. In addition, we observed that only those [gamma]-zein mutants that contained both the proline-rich repeat domain and the C-terminal cysteine-rich domain were able to form ER-derived protein bodies. We suggest that the retention of [gamma]-zein in the ER could be a result of a protein-protein association or a transient interaction of the repeat domain with ER membranes

The expression pattern of the tonoplast intrinsic protein gamma-TIP in Arabidopsis thaliana is correlated with cell enlargement

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SYNTHESIS AND DEPOSITION OF COIXIN IN SEEDS OF COIX-LACRYMA-JOBI

The synthesis and assembly of prolamins into protein bodies of Coix lacryma-jobi seeds were investigated. Coixins, the Coix prolamins, are grouped into two distinct classes namely alpha- and gamma-coixins. Alpha-coixins are constituted by four size classes, while gamma-coixins comprise only one molecular weight class, SDS-PAGE and western blot analysis of prolamins extracted from endosperm during seed development showed that alpha-coixins are synthesized at earlier developmental stages than gamma-coixin. In vitro translation of polysomal RNA attached to protein bodies isolated from mid-maturation endosperm showed that these polyribosomes are highly enriched in coixin messages. Polysomal RNAs isolated from all developmental stages were electrophoresed and probed with cDNA clones representing alpha- and gamma-coixins. The results confirmed the earlier expression of alpha-coixins and also demonstrated that coixin RNA accumulation in the endosperm occurs mainly at seed mid maturation. Protein bodies isolated from immature endosperm contained all coixin components as determined by SDS-PAGE and western blot analyses. Immunocytochemical analysis by electron and light microscopy revealed that the coixin components are spread all over the protein bodies. The protein bodies are localized in the starchy endosperm cells filling the spaces left by the starch granules. They are surrounded by continuous membranes and are larger than the protein bodies described for maize.83216918

Synthesis And Deposition Of Coixin In Seeds Of Coix Lacryma-jobi

The synthesis and assembly of prolamins into protein bodies of Coix lacryma-jobi seeds were investigated. Coixins, the Coix prolamins, are grouped into two distinct classes namely α- and γ-coixins. Alpha-coixins are constituted by four size classes, while γ-coixins comprise only one molecular weight class. SDS-PAGE and western blot analysis of prolamins extracted from endosperm during seed development showed that α-coixins are synthesized at earlier developmental stages than γ-coixin. In vitro translation of polysomal RNA attached to protein bodies isolated from mid-maturation endosperm showed that these polyribosomes are highly enriched in coixin messages. Polysomal RNAs isolated from all developmental stages were electrophoresed and probed with cDNA clones representing α- and γ-coixins. The results confirmed the earlier expression of α-coixins and also demonstrated that coixin RNA accumulation in the endosperm occurs mainly at seed mid maturation. Protein bodies isolated from immature endosperm contained all coixin components as determined by SDS-PAGE and western blot analyses. Immunocytochemical analysis by electron and light microscopy revealed that the coixin components are spread all over the protein bodies. The protein bodies are localized in the starchy endosperm cells filling the spaces left by the starch granules. They are surrounded by continuous membranes and are larger than the protein bodies described for maize. © 1992.832169180Osborne, Our present knowledge of plant proteins (1908) Science, 28, pp. 417-427Miflin, Shewry, The biology and biochemistry of cereal seed prolamins (1979) Seed Protein Improvement in Cereals and Grain Legumes, 1, pp. 137-158. , 3rd edn, IAEA, ViennaLarkins, Seed storage proteins: characterization and biosynthesis (1981) The Biochemistry of Plants, 6, pp. 449-489. , 3rd edn, P.K. Stumpf, E.E. Conn, Academic, New YorkKhoo, Wolf, Origin and development of protein granules in maize endosperm (1970) American Journal of Botany, 57, pp. 1042-1050Burr, Burr, Zein synthesis in maize endosperm by polyribosomes attached to protein bodies (1976) Proc. Natl. Acad. Sci. U.S.A., 73, pp. 515-519. , 3rd ednLarkins, Hurkman, Synthesis and deposition of zein in protein bodies of maize endosperm (1978) PLANT PHYSIOLOGY, 62, pp. 256-263Pernollet, The protein bodies of seeds: ultrastructure, biochemistry, biosynthesis and degradation (1978) Phytochemistry, 17, pp. 1473-1480Marks, Lindell, Larkins, Quantitative analysis of the accumulation of zein mRNA during maize endosperm development (1985) J. Biol. Chem., 260, pp. 16445-16450Clayton, The awnless species of Andropogoneae (1973) Kew Bulletin, 28, pp. 49-58Clayton, Notes on tribe Andropogoneae (Gramineae) (1983) Kew Bulletin, 35, pp. 813-818Jain, Banerjee, Preliminary observations on the ethnobotany of the genus Coix (1974) Econ. Bot., 28, pp. 38-42Arora, Job's tears (Coix lacryma-jobi) — a minor food and fooder crop from North Eastern India (1977) Econ. Bot., 31, pp. 358-366Shaaffhausen, Adlay or Job's tears — a cereal of potentially greater economic importance (1952) Economic Botany, 6, pp. 216-227Raimo, Leme, Contribuiçao para o estudo dos substitutos do farelo de trigo na alimentaçao das aves: I cereal Adlay (1950) Bot. Ind. Anim., 11, pp. 85-89Torres, Bergamin, O cereal do Adlay Estudo de seu valor agricola e nutricional para aves (1951) Anais da Escola Superior de Agricultura Luiz de Queiroz, 8, pp. 669-685Venkateswarlu, Chaganti, Job's Tears (Coix lacryma-jobi L.) (1973) ICAR Tech. Bull. (Agric.), 43-44, pp. 1-54Ottoboni, Leite, Targon, Crozier, Arruda, Characterization of the storage protein in seed of Coix lacryma-jobi var Adlay Journal of Agricultural and Food Chemistry, 38, pp. 631-635Leite, Ottoboni, Targon, Silva, Turcinelli, Arruda, Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis (1990) Plant Molecular Biology, 14, pp. 734-751Ottoboni, Leite, Targon, Silva, Arruda, Heterogeneity of Coix, maize and teosinte prolamins detected by isoelectric focusing (1990) Rev. Bras. Genet., 13, pp. 313-322Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (1970) Nature, 227, pp. 680-684Naito, Dubé, Beachy, Differential expression of conglycinin α- and β-subunit genes in transgenic plants (1988) Plant Mol. Biol., 11, pp. 109-123Maniatis, Fritsch, Sambrook, (1982) Molecular Cloning: A Laboratory Manual, , 3rd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYMechler, Isolation of messenger RNA from membrane bound polysomes (1987) Methods Enzymol., 152, pp. 241-248Ludevid, Torrent, Martinez-Izquierdo, Puigdomenech, Palau, Subcellular localization of glutenin-2 in maize (Zea mays L.) endosperm (1984) Plant Mol. Biol., 3, pp. 227-234Benner, Phillips, Messing, Genetic analysis of methionine-rich storage protein accumulation in maize (1989) Theor. Appl. Genet., 78, pp. 761-767Burr, Burr, Rubenstein, Simon, Purification and translation of zein messenger RNA from maize endosperm protein bodies (1978) Proc. Natl. Acad. Sci. U.S.A., 75, pp. 696-700. , 3rd ednDonovan, Lee, Longhurst, Cell-free synthesis of wheat prolamins (1982) Australian Journal of Plant Physiology, 9, pp. 59-68Matthews, Miflin, In vitro synthesis of barley storage proteins (1980) Planta, 149, pp. 262-268Torrent, Poca, Campos, Ludevid, Palau, In maize, glutelin-2 and low molecular weight zeins are synthesized by membrane-bound polyribosomes and translocated into microsomal membranes (1986) Plant Mol. Biol., 7, pp. 393-403Brineagar, Paterson, Synthesis of oat globulin precursor Analogy to legume 11S storage protein synthesis (1982) PLANT PHYSIOLOGY, 70, pp. 1767-1769Viotti, Sala, Marotta, Alberi, Balducci, Soave, Genes and mRNAs coding for zein polypeptides in Zea mays (1979) Eur. J. Biochem., 102, pp. 211-222Park, Lewis, Rubenstein, Heterogeneity of zein mRNA and protein in maize (1980) PLANT PHYSIOLOGY, 65, pp. 98-106Pérez-Grau, Cortadas, Puigdomenech, Palau, Accumulation and subcellular localization of glutelin-2 transcripts during maturation of maize endosperm (1986) FEBS Lett., 202, pp. 145-148Taylor, Schussler, Liebonberg, Protein body formation in the starchy endosperm of developing Sorghum bicolor (L.) Moench seeds (1985) S. Afr. J. Bot., 51, pp. 35-40Pernollet, Moose, Structure and location of legume and cereal seed storage proteins (1983) Seed Proteins, pp. 155-191. , J. Daussant, J. Mosse, J. Vaughan, Academic Press, LondonLending, Larkins, Changes in the zein composition of protein bodies during maize endosperm development (1989) Plant Cell, 1, pp. 1011-1023Lending, Chesnut, Shaw, Larkins, Structure of maize protein bodies and immunocytochemical localization of zeins (1988) Protoplasma, 143, pp. 51-62Bechtel, Juliano, Formation of protein bodies in the starchy endosperm of rice (Oryza sativa L.): a reinvestigation (1980) Ann. Bot., 45, pp. 503-509Oparka, Harris, Rice protein body formation: all types are initiated by dilation of the endosplasmic reticulum (1982) Planta, 154, pp. 184-18

Vegetative and seed-specific forms of tonoplast intrinsic protein in the vacuolar membrane of Arabidopsis thaliana

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Accumulation of cell wall hydroxyproline-rich glycoprotein mRNA is an early event in maize embryo cell differentiation.

The accumulation of the mRNA coding for a hydroxyproline-rich glycoprotein (HRGP), an abundant component of the wall from the cells of vegetative tissues, has been observed in maize embryo by in situ hybridization. The HRGP mRNA accumulates in the embryo axis and not in the scutellum and preferentially in dividing and provascular cells. The histone H4 mRNA is distributed in similar tissues but is restricted to defined groups of cells, indicating that these two gene products have a different steady-state level of accumulation during the cell cycle. The HRGP mRNA appears to be a useful marker for early formation of the vascular systems. The mRNA accumulation correlates in space and time with cells having a low content of cellulose in their walls, suggesting that the mRNA is produced in the early stages of cell wall formation before complete deposition of cellulose