Stable Accumulation of Aspergillus niger Phytase in Transgenic Tobacco Leaves

Verwoerd, Theo C.; Paridon, P.A. van; Ooyen, A.J.J. van; Lent, J.W.M. van; Hoekema, A.; Pen, J.

doi:10.1104/pp.109.4.1199

Cited by 154 publications

(74 citation statements)

References 31 publications

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“…This was unexpected, since the phytase contained only a leader peptide and should be secreted. Indeed, we, as well as others, have observed efficient secretion in tobacco leaves (Verwoerd et al, 1995). We therefore postulate that this may be a feature specific to cereal endosperm.…”

Section: Discussionmentioning

confidence: 93%

Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm

et al. 2004

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Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals.

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Section: Discussionmentioning

confidence: 93%

Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm

et al. 2004

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“…This molecule was particularly suitable for our investigation as it has been shown to be secreted in other heterologous systems in which it has been expressed, it is very stable within plant cells, and it is highly glycosylated (Verwoerd et al, 1995;Li et al, 1997;Brinch-Pedersen et al, 2000;Lucca et al, 2001;Ullah et al, 2002Ullah et al, , 2003. Therefore, glycosylation analysis could be carried out to complement data from fluorescence and electron microscopy studies, providing further evidence for tissue-specific intracellular localization.…”

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confidence: 99%

The Intracellular Fate of a Recombinant Protein Is Tissue Dependent

Drakakaki

Marcel²,

Arcalís³

et al. 2006

Plant Physiology

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Recombinant proteins directed to the secretory pathway in plants require a signal peptide for entry into the endoplasmic reticulum. In the absence of further targeting information, such proteins are generally secreted via the default pathway to the apoplast. This has been well documented in protoplasts and leaf tissue, but the trafficking of recombinant proteins in seeds and other storage tissues has rarely been investigated. We used Aspergillus niger phytase as a model glycoprotein to compare the intracellular fate of a recombinant protein in the leaves and seeds of rice (Oryza sativa). Using fluorescence and electron microscopy we showed that the recombinant protein was efficiently secreted from leaf cells as expected. In contrast, within endosperm cells it was retained in endoplasmic reticulum-derived prolamin bodies and protein storage vacuoles. Consistent with our immunolocalization data, the phytase produced in endosperm cells possessed oligomannose and vacuolar-type N-glycans [Man 3 (Xyl)(Fuc)GlcNAc 2 ], whereas the phytase produced in leaves contained predominantly secretion-type N-glycans [GlcNAc 2 Man 3 (Xyl)(Fuc)GlcNAc 2 ]. The latter could not be detected in preparations of the endosperm-derived phytase. Our results show that the intracellular deposition and modification of a recombinant protein is tissue dependent.In eukaryotic cells, polypeptides destined for the secretory pathway contain a short N-terminal transit signal peptide, which facilitates cotranslational import into the endoplasmic reticulum (ER). Such proteins may then be secreted to the cell surface or directed to intracellular domains of the endomembrane system. In the latter case, further positive sorting information is deemed necessary. For example, three different categories of signals are known to direct proteins to vacuolar compartments and these may comprise short peptide tags or structural information rather than a peptide sequence (Neuhaus and Rogers, 1998). In the absence of any sorting information, it is generally accepted that proteins in the plant endomembrane system follow the default pathway and are secreted to the apoplast (Denecke et al., 1990).For glycoproteins, the subcellular destination and the path taken by the protein through the endomembrane system determine the final structure of N-glycan moieties. In the first stage of N-glycosylation (that takes place in the ER), the precursor oligosaccharide Glc 3 Man 9 GlcNAc 2 is cotranslationally added to the protein. As the protein moves through the endomembrane system, this core is modified by various glycosidases and glycosyltransferases to form the final glycan structure (Lis and Sharon, 1993;Lerouge et al., 1998). ER-resident glycoproteins contain high Mantype N-glycans whereas proteins passing though the plant Golgi apparatus contain complex-type N-glycans that are mainly characterized by the presence of a(1-3) Fuc and/or b(1-2) Xyl. These residues are linked to the proximal N-acetyl glucosamine and the b-Man residues of the core (Lerouge et al., 1998). While secrete...

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“…An alternative approach for gal enzyme in seeds of transgenic plants (Pen et al, 1993; Phytate (myo-inositol hexakisphosphate) is the major and legumes! producing supplemental phytase is to synthesize the fun- Verwoerd et al, 1995). The feasibility of this method was illustrated by the production of transgenic tobacco (Nicotiform Of phosphorus in mature plant seeds and Po1len (Reddy et phytate accumulates in seeds during maturation and 1989).…”

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Secretion of Active Recombinant Phytase from Soybean Cell-Suspension Cultures

et al. 1997

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Phytase, an enzyme that degrades the phosphorus storage compound phytate, has the potential to enhance phosphorus availability in animal diets when engineered into soybean (Glycine max) seeds. The phytase gene from Aspergillus niger was inserted into soybean transformation plasmids under control of constitutive and seed-specific promoters, with and without a plant signal sequence. Suspension cultures were used to confirm phytase expression in soybean cells. Phytase mRNA was observed in cultures containing constitutively expressed constructs. Phytase activity was detected in the culture medium from transformants that received constructs containing the plant signal sequence, confirming expectations that the protein would follow the default secretory pathway. Secretion also facilitated characterization of the biochemical properties of recombinant phytase. Soybean-synthesized phytase had a lower molecular mass than did the fungal enzyme. However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molecular mass (49 kD). Temperature and pH optima of the recombinant phytase were indistinguishable from the commercially available fungal phytase. Thermal inactivation studies of the recombinant phytase suggested that the additional protein stability would be required to withstand the elevated temperatures involved in soybean processing.

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Stable Accumulation of Aspergillus niger Phytase in Transgenic Tobacco Leaves

Cited by 154 publications

References 31 publications

Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm

Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm

The Intracellular Fate of a Recombinant Protein Is Tissue Dependent

Secretion of Active Recombinant Phytase from Soybean Cell-Suspension Cultures

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