Russell-Like Bodies in Plant Seeds Share Common Features With Prolamin Bodies and Occur Upon Recombinant Protein Production

Abstract

Although many recombinant proteins have been produced in seeds at high yields without adverse effects on the plant, endoplasmic reticulum (ER) stress and aberrant localization of endogenous or recombinant proteins have also been reported. The production of murine interleukin-10 (mIL-10) in Arabidopsis thaliana seeds resulted in the de novo formation of ER-derived structures containing a large fraction of the recombinant protein in an insoluble form. These bodies containing mIL-10 were morphologically similar to Russell bodies found in mammalian cells. We confirmed that the compartment containing mIL-10 was enclosed by ER membranes, and 3D electron microscopy revealed that these structures have a spheroidal shape. Another feature shared with Russell bodies is the continued viability of the cells that generate these organelles. To investigate similarities in the formation of Russell-like bodies and the plant-specific protein bodies formed by prolamins in cereal seeds, we crossed plants containing ectopic ER-derived prolamin protein bodies with a line accumulating mIL-10 in Russell-like bodies. This resulted in seeds containing only one population of protein bodies in which mIL-10 inclusions formed a central core surrounded by the prolamin-containing matrix, suggesting that both types of protein aggregates are together removed from the secretory pathway by a common mechanism. We propose that, like mammalian cells, plant cells are able to form Russell-like bodies as a self-protection mechanism, when they are overloaded with a partially transport-incompetent protein, and we discuss the resulting challenges for recombinant protein production.

Keywords: electron tomography; molecular farming; protein bodies; recombinant protein; subcellular targeting.

Figure 1

Localization of mIL-10 and GAD 67/65 in A. thaliana seed cotyledon cells. Representative electron micrographs showing the best-performing lines for expression of mIL-10 (A–C), GAD 67/65 (D), and (E) wild-type seed. Gold particles are visible in protein storage vacuoles (PSV), the apoplast (apo), and especially in membrane-delimited structures (arrows), surrounded by ribosomes (arrowhead) and forming clusters within the cytoplasm (*). No comparable ER-derived vesicular structures were observed in wild-type seeds. Cell wall (cw), nucleus (n), and oil bodies (ob) are also indicated. Bars = 0.25 μm. Electron micrographs showing the lines producing mIL-10 at lower levels are provided in Supplementary Figure S3.

Figure 2

A large fraction of recombinant mIL-10 is insoluble under physiological conditions and its expression induces ER stress. (A) Sequential extraction of mIL-10, GAD67/65, and wild-type (Col wt) seeds followed by immunoblot analysis. Murine IL-10, GAD67/65, and wild-type (Col wt) seed extracts with saline buffer (lane 1), extraction of the pellet under reducing and denaturing conditions after three washes (lane 2), third saline wash before the re-extraction (lane 3). (B) Relative quantification of mIL-10 amounts in the soluble fraction (S) and in the pellet (P) shown in (A). Quantification is based on five replicates (independent extractions of homozygous seed samples). Error bar depicts ± standard deviation of the mean. (C) Quantitative RT-PCR analysis of BiP3 was performed using mature seeds of wild-type (Col) and transgenic lines expressing mIL-10 (at 0.3 or 0.05 mg/g, respectively) or GAD67/65. Transcript levels of BiP3 were normalized to LSM4 as internal control gene. Expression values are provided as E-ct BIP3 / E-ct LSM4. Error bars depict ± standard deviation of the mean based on three replicates (measurement of three cDNAs derived from separate homozygous seed samples for each line). Plants compared in this experiment were grown at the same time under the same experimental conditions.

Figure 3

ER morphology in transgenic cotyledons of mature seeds. (A,B) In vivo confocal microscopy of cotyledon cells from (A) plants expressing ER-GFP, showing a clear ER network comprising the nuclear envelope (green channel, arrowhead) and (B) an F2 generation ER-GFP × mIL-10 cross showing an altered ER network (green channel, arrows). The blue channel reveals the autofluorescence of PSVs. (C–E) Immuno-localization of mIL-10 in fixed transgenic cotyledons showing clusters (arrows) in the lines with high (C) and moderate (D) production of mIL-10. The low expressing line shows labeling in the PSVs (E, arrowheads) but also in ER-derived structures (F, arrows). Bars = 10 μm (A–E), 0.25 μm (F).

Figure 4

Electron tomography of Russell-like bodies containing mIL-10. Semi-thick sections (300 nm) from freeze-substituted, embedded transgenic seed embryos were imaged at 200 kV. (A,B) Cotyledon cell serial sections of transgenic A. thaliana seeds expressing mIL-10, showing the vesicles containing mIL-10 used for 3D modeling. (C,G) 3D model of the mIL-10-containing vesicles with the vesicles shown from different angles. Vesicles marked with an arrow (C,D) are shown from alternative angles (E–G) revealing their spheroidal shape and lack of connections. Bars = 0.25 μm.

Figure 5

Analysis of transgenic mIL-10 seeds during germination. (A) Immunoblot analysis of mIL-10 seed extracts from mature dry seeds and from seeds 3 days after start of germination (3 DAG) Saline seed extract (lane 1), third pellet wash (lane 2), re-extraction of the pellet under reducing and denaturing conditions (lane 3). (B) Localization of mIL-10 in seeds at 3 DAG by immunoelectron microscopy. The ER-derived bodies containing mIL-10 remain unaltered (arrows), while the electrolucent areas within PSVs indicate that degradation of protein accumulated in this compartment has started. Bar = 1 μm. (C) Representative wild-type seed (Col), 3 DAG. (D) Representative transgenic seed (mIL-10), 3 DAG.

Figure 6

Ectopic DsRed-prolamin bodies in A. thaliana seed cotyledon cells. (A,B) DsRed bodies in the line expressing DsRed-zein alone. (C,D) DsRed bodies in the DsRed-zein × mIL-10 hybrid. (A) Confocal microscopy of A. thaliana cotyledons producing DsRed-zein bodies. Red channel: DsRed fluorescence. See the dark areas within some protein bodies (arrowhead). Blue channel: PSV autofluorescence. Right picture = overlay. (B) Electron microscopy of A. thaliana cotyledons producing DsRed-zein bodies: Localization of DsRed, see the abundant gold probes within the protein body and also the non-labeled areas (*). (C,D) Localization of mIL-10 in cotyledons of DsRed-zein × mIL-10 hybrid seeds. (C) Cotyledon cell overview, oil bodies (ob), protein storage vacuole (PSV). (D) Enlargement of the area outlined in (C). See one labeled mIL-10 body (arrow) and the gold particles decorating the electron-dense material within the DsRed body (white arrow) delimited by a ribosome-studded membrane (double arrow). Bars = 5 μm (A), 0.5 μm (B–D).

Figure 7

Soluble and insoluble antibody fractions from mature seeds of different A. thaliana lines expressing scFv-Fc antibodies MBP-10, HA78, or EHF34, respectively (Van Droogenbroeck et al., 2007). Bands corresponding to the scFv-Fc antibodies with a molecular mass around 55 kD are indicated by an arrow. After extracting A. thaliana seeds in saline buffer containing non-ionic detergent (A), the pellets were re-extracted with the same buffer supplemented with 8 M urea and 5% 2-mercaptoethanol as denaturing and reducing agents (B). About 30 μg of total soluble protein was loaded on SDS-PAGE (reducing conditions, 4–12% Bis-Tris polyacrylamide gel), followed by Coomassie staining.