Targeting a heterologous protein to multiple plant organelles via rationally designed 5′ mRNA tags
© Voges et al.; licensee BioMed Central Ltd. 2013
Received: 6 March 2013
Accepted: 17 August 2013
Published: 8 September 2013
Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.
We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in Nicotiana benthamiana leaf tissue. TriTag-1 and TriTag-2 use alternative splicing to generate differentially localized GFP isoforms, localizing it to the chloroplasts, peroxisomes and cytosol. TriTag-1 shows a bias for targeting the chloroplast envelope while TriTag-2 preferentially targets the peroxisomes. TriTag-3 embeds a conserved peroxisomal targeting signal within a chloroplast transit peptide, directing GFP to the chloroplasts and peroxisomes.
Our novel signal sequences can reduce the number of cloning steps and the amount of genetic material required to target a heterologous protein to multiple locations in plant cells. This work harnesses alternative splicing and signal embedding for engineering plants to express multi-functional proteins from single genetic constructs.
KeywordsProtein targeting Alternative splicing Signal embedding Localization Peroxisome Chloroplast Cytosol Organelle Nicotiana benthamiana
Plant cells harbor many distinct compartments that share some overlapping function, or are functionally associated in metabolic pathways and development. To enable complex metabolic engineering, plant engineers will require tools to direct single transgenes to multiple compartments. For example, re-engineering photorespiration [1, 2] and isoprenoid synthesis [3, 4] will involve both the chloroplasts and peroxisomes.
A number of synthetic N-terminal and C-terminal extensions are readily available to target heterologous proteins to desired subcellular compartments, such as the chloroplast, peroxisome, mitochondrion, endoplasmic reticulum or the nucleus. Issues around protein targeting have arisen in (1) studying protein function in a coordinated fashion [5, 6], (2) improving holistic plant metabolic engineering efforts [7–9] and (3) increasing yields attained by molecular farming and other protein factory applications . One approach to target proteins to more than one location involves cloning multiple genetic copies, each containing a different localization peptide. Each copy must be introduced by successive retransformation, or alternatively, by backcrossing single transforms . These procedures are time-intensive and yield transformants with multiple spatially distinct copies of a protein expression cassette. Coordinate expression may not be ensured due to context-dependent regulatory effects and/or homology-based silencing . Although dual targeting to certain organelles may instead be achieved by adding a second localization peptide , this approach is limited to the possible combinations that can be made from available N- and C-terminal extensions.
Here we describe a simple technique for targeting of transgenic proteins to multiple organelles, specifically the chloroplast, peroxisome, and cytosol. This combination of organelles is particularly interesting due to their close functional association in photorespiration, isoprenoid biosynthesis, β-oxidation and other metabolic processes [3, 13, 14].
Design for multiple-compartment localization by alternative splicing: TriTag-1 and TriTag-2
To construct TriTag-1 and TriTag-2, a chloroplast-targeting region (CTPa) was taken from protein-L-isoaspartate methyltransferase (PIMT2, At5g50240). PIMT2 is a ubiquitous repair protein, converting exposed isoaspartate residues to aspartate or asparagine residues in aging polypeptides [15, 16]. Various mRNAs produced from PIMT2 are produced by alternative transcription initiation sites and alternative splicing events . In nature, different isoforms are often produced from an individual gene, via the exclusion or inclusion of coding sequences from its mRNA by alternative splicing [17–19]. The spliceforms produced from the 3′ transcription initiation site target the protein to the chloroplast when the targeting sequence is retained, and to the cytosol when it is not.
A peroxisome targeting sequence, PTS2, containing the RLx5HL nonapeptide , was taken from the transthyretin-like S-allantoin synthase gene (TTL; At5g58220). This synthase catalyzes two steps in the allantoin biosynthesis pathway . At least two spliceforms are produced from TTL from internal alternative acceptor junctions. The translated proteins are targeted to the peroxisome if they retain the internal PTS2 site and to the cytosol if the site is removed .
TriTag-1 contains the elements in this order: a short sequence of PIMT2 containing the start codon, two alternative donor sites flanking chloroplast transit peptide CTPa, a single acceptor site, a short exon that encodes glycine and serine residues, a single donor site, and two alternative acceptor sites flanking the peroxisome targeting sequence PTS2 of the TTL gene (Figure 1a,c). In TriTag-2 the positions of the sequences taken from genes PIMT2 and TTL are reversed (Figure 1b,d). Both tags are designed so that the two alternative splicing events occur independently of each other. As a result, mRNAs encoding chloroplast, peroxisomal, and cytoplasmically localized proteins are expected (Figure 1e,f).
Design for dual-targeting by signal embedding: TriTag-3
Subcellular localization of GFP controls in transient assays
Subcellular localization of TriTag-1 and TriTag-2 fused GFP
Transiently expressed TriTag-2-GFP (Figure 6) display cytosolic localization, as well as a bright punctate pattern indicating a higher level of peroxisomal targeting and a lower signal in the chloroplasts. Overall, TriTag-1 localized GFP preferentially to the chloroplasts, while TriTag-2 localized this protein to the peroxisomes, with similar targeting to the cytoplasm, as evidenced by GFP localization at the cell periphery and presumably the nucleus.
Subcellular localization of TriTag-3 fused to GFP
In this study, we describe simple strategies for localizing a single transgenic protein to multiple cellular compartments in plants. Variation in N-terminal targeting sequences was presumably encoded by alternative splicing of the encoding mRNA or simultaneous function of multiple-targeting sequences as expressed in a single protein. This greatly economizes the amount of DNA transfected. In addition, dual targeting was achieved by an ambiguous N-terminal signal with elements of chloroplast and peroxisomal targeting sequences. We designed three different short, N-terminal elements for coordinate chloroplast, peroxisome and cytosol targeting, termed ‘TriTags”. TriTag-1 and TriTag-2 (Figure 1) were designed by combining naturally occurring DNAs encoding alternatively spliced mRNAs that direct the encoded proteins to either the chloroplast plus cytoplasm  or the peroxisome plus cytoplasm . TriTag-3 (Figure 2) does not rely on alternative splicing and consists of a chloroplast targeting sequence in which a naturally unstructured portion has been replaced with a peroxisomal targeting sequence .
The TriTags function in vivo to target GFP in Nicotiana benthamiana leaf epidermal cells (summarized in Figure 3). We compared confocal images of the TriTags to controls of untagged GFP, and GFP with N-terminal tags that had previously been shown to target chloroplasts and peroxisomes [15, 16, 20, 21]. Plasmid DNA was delivered into leaf cells by standard biolistic transfection. Untagged GFP was localized to the cytoplasm and nucleus, with some nuclear localization being expected because the nuclear pore has a large, aqueous channel that permits entry of molecules up to about 70 kD. TriTag-1 and TriTag-2 mediated GFP expression in the chloroplast, peroxisome, and cytoplasm (plus nucleus), with TriTag-1 showing a slight preference for the chloroplast over peroxisome and TriTag-2 showing the opposite behavior. TriTag-3 mediated strong localization to both the peroxisome and chloroplast, but not detectably to the cytoplasm. These behaviors suggest that alternatively spliced forms of TriTag-1 and TriTag-2 are being produced (Figures 1 and 3).
The re-engineering of photorespiration pathways  illustrates the potential utility of such multiple-targeting elements. Normally during photorespiration, glycolate is generated in the chloroplast and then transported into the cytoplasm and then into the peroxisome, where it is oxidized to glyoxylate in an O2-dependent reaction. The reduction of oxygen, rather than NAD(P)+ as an oxidizing agent represents a waste of reducing equivalents and energy. Kebeish et al.  engineered plants to express in the chloroplast and NAD+-dependent bacterial glycolate metabolizing pathway and found this enhanced the growth of light-limited Arabidopsis. In this situation, the added bacterial pathway competes with transport of glycolate from the chloroplast into the cytoplasm. Expression of the pathway in the cytoplasm and peroxisome could further enhance the amount of glycolate that is metabolized by this more efficient pathway. However, as the pathway involves five polypeptide coding sequences, expression of all proteins in three compartments could be prohibitive.
Our results also suggest that novel alternative splicing systems can be engineered in a straightforward manner. One risk in designing such systems is that it is difficult to predict the efficiency of a novel alternatively spliced system. Future designs may use different alternatively-spliced base genes, whose splice ratios may be better known for quantitation. Further experiments with a more stable vector delivery system (e.g. Agrobacterium transfection) could give us additional material for quantitative PCR or changes in expression over time, and further inform future designs based on engineered alternative splicing.
Plant metabolic engineering remains a formidable effort in terms of time and resources. The field requires simple and efficient technologies for transforming plants with multi-functional proteins. Our system with engineered alternative splicing could be used to target a single transgene to multiple locations, namely the chloroplast, cytosol, and peroxisome. In addition, we demonstrated that a peroxisomal signal embedded within a chloroplast signal allows dual targeting of the transgene. These devices may reduce time and resources spent on plant metabolic engineering.
Strains and plasmids
Plasmids constructed in this study
pORE binary vector expressing untagged soluble modified GFP (smGFP) controlled by the pENTCUP2 promoter .
pORE vector expressing TriTag-1-fused GFP controlled by the pENTCUP2 promoter.
pORE vector expressing TriTag-2-fused GFP controlled by the pENTCUP2 promoter.
pORE vector expressing TriTag-3-fused GFP controlled by the pENTCUP2 promoter.
TriTag synthesis and cloning
TriTag-1, TriTag-2 and TriTag-3 were synthesized (GeneBlocks, IDT, Coralville, IA), and cloned in-frame 5′ of the soluble modified GFP (smGFP) using Gibson assembly . This modified GFP contains three site-directed mutations that increase the protein’s solubility and fluorescence intensity . Based on splice site prediction with NetPlantGene , we predicted that the processed spliceforms of TriTag-1 and TriTag 2 encodes for GFP variants containing regions for chloroplast targeting, peroxisomal targeting or neither. Spliceforms other than those found using NetPlantGene would either incorporate a stop codon or lack organelle-targeting information, causing premature translation or sole targeting to the cytosol, respectively.
All plants were incubated at 16-20°C in a 16/8 hour light/dark cycle and watered twice weekly. Peat-based soil-free media (Metromix, SunGro Horticulture, Vancouver, Canada) was autoclaved 45 min before use. Leaves from 3–5 month old Nicotiana benthamiana seedlings plants were collected for bombardment.
DNA-gold particle complexes for biolistic delivery were prepared according to manufacturer’s instructions for use with the Helios Gene Gun (Bio-Rad, Hercules, CA) as follows: Plasmid DNA (50 μg) containing the tagged GFP gene was pelleted onto 1 μm gold particles (6–8 mg) in a spermidine (100 μL, 0.05 M) and CaCl2 (100 μL, 1.0 M) mixture and resuspended in a polyvinylpyrrolidone/EtOH solution (5.7 mg/mL). The resulting suspension was deposited onto the inside surface area of Tygon plastic tubing (o.d. = 2 mm) and diced into cartridges facilitated by the Tubing Prep Station (Bio-Rad, Hercules, CA). Cartridges were stable up to 6 months dessicated at 4°C. The underside of Nicotiana benthamiana leaves were transformed biolistically using the Helios Gene Gun (Bio-Rad, Hercules, CA) at 150–250 psi He . The leaves were placed on wet filter paper in Petri dishes and stored on a bench-top under ambient lighting and room temperature for 48 hours before imaging analysis.
Target control proteins
As expected, control proteins showed untagged smGFP was distributed extensively in the cytosol and nucleus (Figure 4a,b,c), but not the vacuole, which makes up the bulk of the plant cell volume. This localization pattern matches previous untagged GFP localization studies . Cytosolic and chloroplast localization controls were determined by transient expression of GFP fused to the PIMT2 chloroplast targeting peptide and the native autofluorescence of chlorophyll (Figure 4d,e,f). Peroxisomal localizations were determined by comparing images to transient expression of GFP fused to the TTL peroxisomal targeting peptide, which matches previous localization studies .
Splice junctions within the TriTag-1 and TriTag-2 sequences were predicted using the NetPlantGene server . Targeting to the chloroplast and peroxisome of the TriTag-1 and TriTag-2 splice variants and TriTag-3 were predicted using TargetP  and PeroxisomeDB 2.0 . Peptide structures of CTPb and TriTag-3 were determined using PROFbval on the ROSTLAB server .
Imaging and processing
Bombarded leaves were diced and placed on glass slides in 0.1% Triton-X100 and imaged by fluorescence confocal microscopy (excitation at 489 nm, detection at 500–569 for GFP and 630–700 for chlorophyll autoflourescence) using a 40x water-based objective (numerical aperture 1.10).
Peroxisome targeting signal 2
Arabidopsis transthyretin-like S-allantoin synthase gene
Chloroplast targeting peptide
Arabidopsis protein-L-isoaspartate methyltransferase gene
Chloroplast targeting peptide from PIMT2
Solanum tuberosum ribulose-1,5-biphosphate carboxylase (RuBisCO) small-subunit gene
Chloroplast targeting peptide from rbc S1
Soluble modified green fluorescent protein.
The authors would like to thank Jen Sheen of Massachusetts General Hospital for the generous donation of Nicotiana benthamiana seedlings. Jen Bush helped us greatly in advice in growing plants and setting up a growth chamber area. Tom Ferrante, Garry Cuneo, and Danny Selgrade of the Wyss Institute offered crucial help with confocal microscopy. We would especially like to thank Don Ingber of Children’s Hospital Boston and the Wyss Institute for the generous donation of the BioRad Helios Gene Gun and associated supplies. Funding for this project was provided through a cooperative agreement with the Department of Energy Advanced Research Projects Agency (ARPA-E Award # DE-000079) for PAS, JCW, and MdM, and through Chimerion Biotechnologies, Inc. for MJV.
- Kebeish R, Niessen M, Thiruveedhi K, Bari R, Hirsch H-JJ, Rosenkranz R, Stäbler N, Schönfeld B, Kreuzaler F, Peterhänsel C: Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana. Nat Biotechnol 2007, 25: 593-599. 10.1038/nbt1299View ArticleGoogle Scholar
- Maier A, Fahnenstich H, von-Caemmerer S, Engqvist MKM, Weber APM, Flügge U-I, Maurino VG: Transgenic introduction of a glycolate oxidative cycle into A. thaliana chloroplasts leads to growth improvement. Front Plant Sci 2012,3(February):38.Google Scholar
- Sapir-Mir M, Mett A, Belausov E, Tal-Meshulam S, Frydman A, Gidoni D, Eyal Y: Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes. Plant Physiol 2008, 148: 1219-1228. 10.1104/pp.108.127951View ArticleGoogle Scholar
- Kumar S, Hahn FM, Baidoo E, Kahlon TS, Wood DF, McMahan CM, Cornish K, Keasling JD, Daniell H, Whalen MC: Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts. Metab Eng 2012, 14: 19-28. 10.1016/j.ymben.2011.11.005View ArticleGoogle Scholar
- Hooks KB, Turner JE, Graham IA, Runions J, Hooks MA: GFP-tagging of Arabidopsis acyl-activating enzymes raises the issue of peroxisome-chloroplast import competition versus dual localization. J Plant Physiol 2012, 169: 1631-1638. 10.1016/j.jplph.2012.05.026View ArticleGoogle Scholar
- Zhang X, Hu J: The Arabidopsis chloroplast division protein DYNAMIN-RELATED PROTEIN5B also mediates peroxisome division. Plant Cell 2010, 22: 431-442. 10.1105/tpc.109.071324View ArticleGoogle Scholar
- Baudisch B, Klösgen RB: Dual targeting of a processing peptidase into both endosymbiotic organelles mediated by a transport signal of unusual architecture. Mol Plant 2012, 5: 494-503. 10.1093/mp/ssr092View ArticleGoogle Scholar
- Brandão MM, Silva-Filho MC: Evolutionary history of Arabidopsis thaliana aminoacyl-tRNA synthetase dual-targeted proteins. Mol Biol Evol 2011, 28: 79-85. 10.1093/molbev/msq176View ArticleGoogle Scholar
- Severing EI, van-Dijk AD, van-Ham RC: Assessing the contribution of alternative splicing to proteome diversity in Arabidopsis thaliana using proteomics data. BMC Plant Biol 2011, 11: 82. 10.1186/1471-2229-11-82View ArticleGoogle Scholar
- Hyunjong B, Lee D-S, Hwang I: Dual targeting of xylanase to chloroplasts and peroxisomes as a means to increase protein accumulation in plant cells. J Exp Bot 2006, 57: 161-169.View ArticleGoogle Scholar
- Que Q, Chilton M-DM, de-Fontes CM, He C, Nuccio M, Zhu T, Wu Y, Chen JS, Shi L: Trait stacking in transgenic crops: challenges and opportunities. GM Crops 2010, 1: 220-229. 10.4161/gmcr.1.4.13439View ArticleGoogle Scholar
- Dafny-Yelin M, Tzfira T: Delivery of multiple transgenes to plant cells. Plant Physiol 2007, 145: 1118-1128. 10.1104/pp.107.106104View ArticleGoogle Scholar
- Baker A, Graham IA, Holdsworth M, Smith SM, Theodoulou FL: Chewing the fat: beta-oxidation in signalling and development. Trends Plant Sci 2006, 11: 124-132. 10.1016/j.tplants.2006.01.005View ArticleGoogle Scholar
- Peterhansel C, Horst I, Niessen M, Blume C, Kebeish R, Kürkcüoglu S, Kreuzaler F: Photorespiration. The Arabidopsis Book 2010, 8: e0130.View ArticleGoogle Scholar
- Lowenson JD, Clarke S: Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis. J Biol Chem 1992, 267: 5985-5995.Google Scholar
- Dinkins RD, Majee SM, Nayak NR, Martin D, Xu Q, Belcastro MP, Houtz RL, Beach CM, Downie AB: Changing transcriptional initiation sites and alternative 5′- and 3′-splice site selection of the first intron deploys Arabidopsis protein isoaspartyl methyltransferase2 variants to different subcellular compartments. Plant J 2008, 55: 1-13. 10.1111/j.1365-313X.2008.03471.xView ArticleGoogle Scholar
- Reddy ASN, Rogers MF, Richardson DN, Hamilton M, Ben-Hur A: Deciphering the plant splicing code: experimental and computational approaches for predicting alternative splicing and splicing regulatory elements. Front Plant Sci 2012,3(February):18.Google Scholar
- Black DL: Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 2003, 72: 291-336. 10.1146/annurev.biochem.72.121801.161720View ArticleGoogle Scholar
- Kalsotra A, Cooper TA: Functional consequences of developmentally regulated alternative splicing. Nat Rev Genet 2011, 12: 715-729.View ArticleGoogle Scholar
- Lanyon-Hogg T, Warriner SL, Baker A: Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes. Biol Cell 2010, 102: 245-263. 10.1042/BC20090159View ArticleGoogle Scholar
- Reumann S, Babujee L, Ma C, Wienkoop S, Siemsen T, Antonicelli GE, Rasche N, Lüder F, Weckwerth W, Jahn O: Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms. Plant Cell 2007, 19: 3170-3193. 10.1105/tpc.107.050989View ArticleGoogle Scholar
- Fritz CC, Herget T, Wolter FP, Schell J, Schreier PH: Reduced steady-state levels of rbcS mRNA in plants kept in the dark are due to differential degradation. Proc Natl Acad Sci U S A 1991, 88: 4458-4462. 10.1073/pnas.88.10.4458View ArticleGoogle Scholar
- Schlessinger A, Yachdav G, Rost B: PROFbval: predict flexible and rigid residues in proteins. Bioinformatics 2006, 22: 891-893. 10.1093/bioinformatics/btl032View ArticleGoogle Scholar
- Sadler I, Chiang A, Kurihara T, Rothblatt J, Way J, Silver P: A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein. J Cell Biol 1989,109(6 Pt 1):2665-2675.View ArticleGoogle Scholar
- Bhushan S, Kuhn C, Berglund A-K, Roth C, Glaser E: The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting. FEBS Lett 2006, 580: 3966-3972. 10.1016/j.febslet.2006.06.018View ArticleGoogle Scholar
- Emanuelsson O, Brunak S, von-Heijne G, Nielsen H: Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2007, 2: 953-971. 10.1038/nprot.2007.131View ArticleGoogle Scholar
- Schlüter A, Real-Chicharro A, Gabaldón T, Sánchez-Jiménez F, Pujol A: PeroxisomeDB 2.0: an integrative view of the global peroxisomal metabolome. Nucleic Acids Res 2010,38(Database issue):D800-D805.View ArticleGoogle Scholar
- Stauffer E, Westermann A, Wagner G, Wachter A: Polypyrimidine tract-binding protein homologues from Arabidopsis underlie regulatory circuits based on alternative splicing and downstream control. Plant J 2010, 64: 243-255. 10.1111/j.1365-313X.2010.04321.xView ArticleGoogle Scholar
- Coutu C, Brandle J, Brown D, Brown K, Miki B, Simmonds J, Hegedus DD: pORE: a modular binary vector series suited for both monocot and dicot plant transformation. Transgenic Res 2007, 16: 771-781. 10.1007/s11248-007-9066-2View ArticleGoogle Scholar
- Li F, Liu W, Tang J, Chen J, Tong H, Hu B, Li C, Fang J, Chen M, Chu C: Rice DENSE AND ERECT PANICLE 2 is essential for determining panicle outgrowth and elongation. Cell Res 2010, 20: 838-849. 10.1038/cr.2010.69View ArticleGoogle Scholar
- Silva-Filho MC: One ticket for multiple destinations: dual targeting of proteins to distinct subcellular locations. Curr Opin Plant Biol 2003, 6: 589-595. 10.1016/j.pbi.2003.09.008View ArticleGoogle Scholar
- Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001.Google Scholar
- Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin AP, Keasling JD: BglBricks: A flexible standard for biological part assembly. J Biol Eng 2010, 4: 1. 10.1186/1754-1611-4-1View ArticleGoogle Scholar
- Shetty RP, Endy D, Knight TF: Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2008, 2: 5. 10.1186/1754-1611-2-5View ArticleGoogle Scholar
- Gibson DG: Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 2011, 498: 349-361.View ArticleGoogle Scholar
- Davis SJ, Vierstra RD: Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol Biol 1998, 36: 521-528. 10.1023/A:1005991617182View ArticleGoogle Scholar
- Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouzé P, Brunak S: Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Res 1996, 24: 3439-3452. 10.1093/nar/24.17.3439View ArticleGoogle Scholar
- Woods G, Zito K: Preparation of gene gun bullets and biolistic transfection of neurons in slice culture. J Vis Exp 2008, 12: 10-13.Google Scholar
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