Gaining deeper insights with fluorescence and electron microscopy
The natural ability of cereal seeds to store proteins in a stable environment has successfully been exploited to use cereals as platforms for the production of recombinant proteins of pharmaceutical interest [3, 4, 5]. For this reason, the investigation of the different trafficking routes for proteins has been subject of several studies, since the final storage site as well as the route followed by the recombinant protein have an influence in the final yield and quality of such product.
The use of fluorescence and electron microscopy in combination with the analysis of glycan structures on recombinant glycoproteins produced in different systems (monocots and dicots) has allowed to gain a deeper insight into the topic. Indeed, we have proved the presence of protein storage vacuoles in maize  and we have demonstrated crucial differences in the trafficking of proteins in cereal endosperm and dicot seeds. Thus, in cereal endosperm cells the protein storage vacuole is the preferred destination for recombinant proteins that enter the endomembrane system and do not carry further targeting signals [7, 8]. The same proteins are efficiently secreted in dicot seeds like tobacco or Arabidopsis, reflecting the high specialization of the cereal endosperm, a moribund tissue which does not survive beyond germination and whose only purpose is the storage of proteins and starch .
Deposition of recombinant fungal phytase in maize endosperm cells
Figure 1 shows the deposition of recombinant fungal phytase in maize endosperm cells. Recombinant fungal phytase carrying a signal peptide and no further targeting signal was expressed in maize endosperm. Slices of endosperm corresponding to a mid developmental stage were cut with a razor blade and fixed in 4 % paraformaldehyde and 0,5 % glutaraldehyde.
After dehydration through ethanol series, samples were infiltrated and embedded in LRWhite resin and the blocks were allowed to polymerise at 60 °C. Semithin sections (1 µm) were obtained with a Leica Ultracut UCT and stained with toluidine blue (A) or used for immunofluorescence studies (B).
Ultrathin sections (80 nm) were also obtained and stained with uranyl acetate 1 % and lead citrate 3 % in a Leica EM AC20 after immunogold localization of recombinant phytase. Light and immunofluorescence pictures were taken with a Leica DM5500. Ultrathin sections were observed with a Philips EM-400 electron microscope.
See the different protein storage organelles in a mid developmental stage endosperm cell: protein storage vacuoles (arrows) and zein bodies (arrowheads). Phytase is unexpectedly deposited in the protein storage vacuole (B, arrow; C, PSV, arrow) as well as around the zein bodies (C, zb). Starch (s). Bars 10 µm (A, B), 1 µm (C).
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