Investigating the production of foreign membrane proteins in tobacco chloroplasts: expression of an algal plastid terminal oxidase

Abstract

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5'UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.

Figure 1. Multiple sequence alignment and phylogenetic analysis of PTOX.

(A) Neighbour-joining phylogenetic dendrogram (spider) based upon an alignment of complete amino acid sequences of PTOX molecules. Grouping in different shades based upon structural and functional homology of PTOX polypeptides found in different species. Numbers at nodes indicate bootstrap confidence values (1000 replicates). The PTOX amino acid sequences used were from Arabidopsis thaliana GenBank accession number: CAA06190, tomato (Lycopersicon esculentum) GenBank accession number: AAG02286, rice (Oryza sativa; cultivar japonica) NCBI accession number: NP_001054199, wheat (Triticum aestivum) GenBank accession number: AAG00450, maize (Zea mays) NCBI accession number: NP_001150780, coffee (Coffea canephora) GenBank accession number: ABB70513, pepper (Capsicum annuum) GenBank accession number: AAG02288, Guillardia theta GenBank accession number: CAI77910, Chlamydomonas reinhardtii NCBI accession number: AF494290 (PTOX1) and NCBI accession number: XP_001703466 (PTOX2), Ostreococcus tauri (green alga) GenBank accession number: CAL58090, Bigelowiella natans (chlorarachniophytes) GenBank accession number: AAP79178, Anabaena variabilis ATCC 29413 (cyanobacteria) GenBank accession number: ABA21297, Nostoc sp. PCC 7120 (cyanobacteria) NCBI NP_486136, Prochlorococcus marinus subsp. Pastoris CCMP1986 (cyanobacteria) NCBI accession number: NP_892455, Prochlorococcus marinus Subsp. ASNC729 (cyanobacteria) Genbank accession number: ABE11017, Prochlorococcus marinus str. MIT 9312 (cyanobacteria) NCBI accession number: YP_396838, Prochlorococcus marinus str. NATL2A (cyanobacteria) NCBI accession number: YP_291624, Synechococcus sp. WH 8102 (cyanobacteria) NCBI accession number: NP_896980, Synechococcus sp. CC9902 (cyanobacteria) NCBI accession number: YP_376451, and Synechococcus sp. BL107 (cyanobacteria) NCBI: ZP_01468216. (B) Multiple sequence alignment of PTOX from Chlamydomonas and Arabidopsis. Conserved iron-binding residues are indicated by black arrows, whereas, exon 8 is boxed. Conserved sequences are shaded black. The transit peptide for At-PTOX is underlined.

Figure 2. Generation of homoplastomic transplastomic plants expressing Cr-PTOX1.

Schematic representation of the plastome region of the wildtype (A) and the transplastomic plant line Cr-PTOX1-I (B) analyzed by Southern blot analysis. Cr-PTOX1-I sequences were cloned in vector pHK40 . The positions of the restriction enzyme BglII used to digest the genomic DNA are shown. The dotted lines represent the size of the expected fragments to be released from wildtype as well as Cr-PTOX1-I after restriction. (C) Total genomic DNA from WT as well as Cr-PTOX1-I was digested and hybridized with rrn16/rps12 digoxigenin labelled probe amplified from WT. (D) Maternal inheritance assay of Cr-PTOX1-I transplastomic plant line. Seeds of Cr-PTOX1-I and WT plants were grown on MS plates with or without 500 mg L⁻¹ spectinomycin at room temperature. Abbreviations: Prrn = 16SRNA operon promoter, TrbcL = Rubisco large subunit terminator, PpsbA = psbA promoter, TpsbA = psbA terminator, WT = wild type, T = transplastomic, ptDNA = Plastid DNA.

Figure 3. Detection of Cr-PTOX1 by SDS-PAGE and Western blot analysis.

Total proteins equivalent to 1 µg of chlorophyll were loaded per well from PTOX1-I plant leaves grown in a greenhouse at high light (125 µmol photons m⁻² s⁻¹) and analysed either by running on a 15% (w/v) denaturing polyacrylamide gel and stained by Coomassie Blue (A) or transferring to PVDF for immunodetection carried out using an anti-HA tag antibody (B). Protein samples from Chlamydomonas reinhardtii expressing HA-tagged Light Harvesting Complex b (HA-LHC b) were used as a positive immunoblotting control. (C) Tobacco plants were grown in a growth room at 50 µmol photons m⁻² s⁻¹. 5 µg of thylakoids (based on chlorophyll) from tobacco, wild type (WT) as well Cr-PTOX1 expressing plants and C. reinhardtii (wild type) were loaded per well for SDS-PAGE and immunoblotting, which was carried out using an anti-PTOX1 antibody. Upper panel shows a Coomassie-stained gel, whereas, the bottom panel shows the immunoblot. (D) Differential extraction of thylakoids to determine whether Cr-PTOX1 is being targeted to the membrane. Thylakoids extracted from Cr-PTOX1-I plant leaves were washed with different buffers and centrifuged. The supernatant and pellet fractions (∼1 µg chlorophyll per well) were loaded and immunoblotted using different antibodies against membrane bound proteins.

Figure 4. Immunodetection of Cr-PTOX1 protein by BN and 2D SDS-PAGE.

Thylakoid membranes from Cr-PTOXI-I or WT containing 4 µg chlorophyll were solubilised and separated by BN-PAGE. One of the BN-PAGE gel lanes was stained with Coomassie blue (A) and others were run in the second dimension by denaturing SDS-PAGE. One of the gels obtained was silver-stained (B) while other two were immunoblotted with anti-HA tag (C) or anti-D1 antibodies (D).

Figure 5. Changes in post-illumination Fo rise at various time points during fluorescence induction.

WT and Cr-PTOX1-I plants were grown at low light (50 µmol photons m⁻² s⁻¹). 10-week-old plants were then analysed for post-illumination Fo rise. Dark-adapted WT (A) and Cr-PTOX1-I (B) leaves were illuminated by 96-µmol photons m⁻² s⁻¹ white light (AL) for various time points over a period of 30 min. Post-illumination fluorescence kinetics was monitored by placing leaves in the dark for 5 min after illuminating leaves for 5, 10, 15, 20 and 25 min. Saturating flashes of 1000 µmol photons m⁻² s⁻¹, 600 ms duration each at 1 min interval were applied throughout the period (24 measurements). Abbreviations: AL = Actinic light, MB = Measuring Beam, Fm = Maximum fluorescence from dark-adapted leaves after saturating flash, Fo = minimum fluorescence in dark-adapted leaves before illumination.

Figure 6. Involvement of Cr-PTOX1 in PQ oxidation and effect of propyl gallate on Cr-PTOX1 activity.

WT and Cr-PTOX1-I plants were grown at low light (50 µmol photons m⁻² s⁻¹). 10-week-old plants were then analysed by measuring chlorophyll fluorescence. The fluorescence decay in leaf discs of WT (black trace) and Cr-PTOX1-I plants (red trace) were measured after 1-hour dark adaptation (A). Leaf discs of both WT (B) and Cr-PTOX1-I plants (C) were treated with (broken line) or without (unbroken line) 1 mM propyl gallate (PG) for a period of 3 hours in the dark. Fluorescence values (arbitrary units) are shown on y-axis, whereas, time is shown on x-axis.

Figure 7. Impact of Cr-PTOX1 on plant under high light.

Cr-PTOX1-I plants were grown at 50 µmol photons m⁻² s⁻¹ for a period of 4 weeks (A) and at 125 µmol photons m⁻² s⁻¹ for 10 weeks (B). (C) The plants grown in (B) were placed at 50 µmol photons m⁻² s⁻¹ for 6 weeks. The wildtype plants shown in (B) and (C) are shown for comparison purposes and were not germinated at the same time as the mutant. (D) Phenotypic comparison of two nuclear transformants, At-PTOX-1 and AT-PTOX-2, and transplastomic Cr-PTOX1-I plants at 4-week-old stage grown at 125 µmol photons m⁻² s⁻¹. (E) PSII maximum quantum efficiency was determined by recording Fv/Fm on attached 4–6 week-old healthy leaves of WT and Cr-PTOX1-I plants grown under low light (50 µmol photons m⁻² s⁻¹) and high light conditions (125 µmol photons m⁻² s⁻¹). The leaves were dark adapted for one hour before measuring fluorescence.