Efficient metabolic pathway engineering in transgenictobacco and tomato plastids with syntheticmultigene operonsYinghong Lu, Habib Rijzaani1, Daniel Karcher, Stephanie Ruf, and Ralph Bock2

Max-Planck-Institut für Molekulare Pflanzenphysiologie, D-14476 Potsdam-Golm, Germany

Edited by John E. Mullet, Texas A&M University, College Station, TX, and accepted by the Editorial Board January 2, 2013 (received for reviewSeptember 28, 2012)

The engineering of complex metabolic pathways requires the con-certed expression of multiple genes. In plastids (chloroplasts) ofplant cells, genes are organized in operons that are coexpressed aspolycistronic transcripts and then often are processed further intomonocistronic mRNAs. Here we have used the tocochromanolpathway (providing tocopherols and tocotrienols, collectively alsoreferred to as “vitamin E”) as an example to establish principles ofsuccessful multigene engineering by stable transformation of thechloroplast genome, a technology not afflicted with epigenetic var-iation and/or instability of transgene expression. Testing a series ofsingle-gene constructs (encoding homogentisate phytyltransferase,tocopherol cyclase, and γ-tocopherol methyltransferase) and ratio-nally designed synthetic operons in tobacco and tomato plants, we(i) confirmed previous results suggesting homogentisate phytyl-transferase as the limiting enzymatic step in the pathway, (ii) com-paratively characterized the bottlenecks in tocopherol biosynthesisin transplastomic leaves and tomato fruits, and (iii) achieved an upto tenfold increase in total tocochromanol accumulation. In addition,our results uncovered an unexpected light-dependent regulatorylink between tocochromanol metabolism and the pathways of pho-tosynthetic pigment biosynthesis. The synthetic operon design de-veloped here will facilitate future synthetic biology applications inplastids, especially the design of artificial operons that introducenovel biochemical pathways into plants.

metabolic engineering | plastid transformation | fruit development |fruit ripening

Tocochromanols (tocopherols and tocotrienols) are productsof plant metabolism that arise from two biochemical path-

ways: the shikimate pathway of aromatic amino acid biosynthesisand the isoprenoid biosynthetic pathway (Fig. 1). Tocopherolsarise from a condensation reaction of homogentisic acid (HGA)with phytyl diphosphate (PDP), whereas tocotrienols are pro-duced by geranylgeranyl diphosphate (GGDP) attachment toHGA (Fig. 1). Both tocopherols and tocotrienols occur in fournatural forms that differ in their methylation patterns of the ar-omatic head group: α, β, γ, and δ. Starting with the condensationof HGA to the isoprenoid chain (PDP or GGDP), all steps oftocochromanol biosynthesis take place in the plastid. Drawing onthe plastid isoprenoid pool, tocochromanol biosynthesis is highlyconnected to a number of other metabolic pathways in the plastidcompartment, including the biosynthesis of carotenoids, chlor-ophylls, gibberellins, phylloquinone, and plastoquinone (Fig. 1).Tocochromanols are best known for their vitamin E activity.

Because animals (including humans) cannot synthesize vitamin E,they depend on the uptake of plant-derived tocochromanols intheir diet. In addition to being an essential micronutrient, toco-chromanols have been linked to a variety of health-promotingeffects [e.g., in preventing chronic disease (1)]. Most of thesebeneficial properties generally are explained by tocochromanolsbeing highly potent antioxidants and efficient quenchers of re-active oxygen species. This activity comes from the ability oftocochromanols to donate a hydrogen atom from the hydroxyl

group in the chromanol ring to a free radical, followed by meso-meric stabilization of the molecule’s own unpaired electron. Thecurrent models of tocochromanol action in the body are basedlargely on their radical-scavenging properties, in that protection ofmembranes from lipid peroxidation and protection of active sitesof metabolic enzymes and nucleic acids from damage by freeradicals are believed to be the main beneficial effects of toco-chromanols at the cellular level.Because of the high nutritional value and the benefits of vi-

tamin E in human health, increasing the tocochromanol contentof major agricultural crops has long been in the focus of breedingprograms and genetic engineering approaches (2, 3). Elevatedvitamin E contents can be achieved either by directing moremetabolites into the tocopherol biosynthetic pathway or, alter-natively, by altering the tocopherol spectrum [e.g., by convertingother tocopherol forms into α-tocopherol, the form with thehighest vitamin E activity; (4)].Relatively little is known about the regulation of tocochro-

manol biosynthesis and its interconnection with other metabolicpathways in the plant cell. Mutant analysis and overexpressionstudies in the model plant Arabidopsis thaliana have shed somelight on limiting enzymatic steps and also have revealed somestrategies for manipulating the tocopherol content and alteringthe tocopherol spectrum (4–6). Here we used stable trans-formation of the plastid (chloroplast) genome in the model planttobacco and the food crop tomato to engineer the tocopherolmetabolic pathway and assess the impact of the three plastid-localized enzymes of the pathway (Fig. 1) on tocochromanolbiosynthesis in chloroplasts and chromoplasts. Our results con-firm previous data on the limiting role of HPT in the pathway,characterize the limitations in tocopherol biosynthesis in trans-plastomic leaves and fruits, and reveal a synthetic operon designthat facilitates efficient multigene engineering in plastids.

ResultsExpression of Three Enzymes of the Tocopherol Pathway from theTobacco Plastid Genome. Based on overexpression studies in nu-clear-transgenic Arabidopsis plants, two enzymes in the tocoph-

Author contributions: Y.L., D.K., S.R., and R.B. designed research; Y.L. and H.R. performedresearch; Y.L., H.R., D.K., S.R., and R.B. analyzed data; and R.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.E.M. is a guest editor invited by theEditorial Board.

Data deposition: The sequences of the synthetic constructs reported in this paper havebeen deposited in the National Center for Biotechnology Information GenBank Nucleo-tide Sequence Database [accession nos. JX235341 (pAtTMT), JX235342 (pSyHPT),JX235343 (pSyTCY), JX235344 (pSyTMT), JX235345 (pTop1), and JX235346 (pTop2)].1Present address: Molecular Biology Laboratory, BB-Biogen, Indonesian Agency for Agri-cultural Research and Development, Ministry of Agriculture, Bogor 16111, Indonesia.

2To whom correspondence should be addressed. E-mail: rbock@mpimp-golm.mpg.de.

See Author Summary on page 2703 (volume 110, number 8).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1216898110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1216898110 PNAS | Published online February 4, 2013 | E623–E632

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erol biosynthetic pathway (Fig. 1) were suggested as rate-limiting:homogentisate phytyltransferase [HPT, encoded by the VTE2gene (5)] and tocopherol cyclase [TCY, encoded by the VTE1locus (8)]. Recent data have called into question the possiblelimiting role of TCY in tocopherol biosynthesis (9, 10). Expres-sion of foreign genes from the plastid genome has the advantagesthat (i) there is no interference from epigenetic processes, and(ii) transgene insertion occurs by homologous recombination intoa preselected region of the plastid genome, typically an intergenicspacer region (11–13). Therefore, transgene expression is notburdened by copy number-dependent or insertion site-dependentvariation in expression levels and epigenetic transgene silencing.

However, because of lower plastid number per cell, lower plastidgenome copy numbers per cell, and lower gene expression ac-tivity, transgene expression can be reduced significantly in non-green tissues compared with photosynthetically active tissues.We used plastid transformation in the model plant tobacco to

test single-gene expression constructs for their effects on the fluxthrough tocochromanol biosynthesis. To this end, we overexpressedthe three key plastid-localized enzymes specific to tocopherol bio-synthesis, HPT, TCY, and γ-tocopherol methyltransferase (TMT)(Fig. 1), in identical expression cassettes and targeted them to thesame intergenic region in the plastid genome (Fig. 2). Both theexpression cassette and the targeting site in the plastid DNA hadbeen tested extensively in previous experiments and were shown togive highly efficient and stable transgene expression from theplastid genome (14–17). To avoid limitations from plant-specificregulation of enzyme activities, we used the genes encoding HPT,TCY, and TMT from the cyanobacterium Synechocystis sp.PCC6803 (18). For the final enzyme in the pathway, TMT [which isunlikely to limit flux through the tocochromanol pathway; (4)], weadditionally used the gene from A. thaliana (Fig. 2A).All four constructs were introduced into the tobacco plastid

genome by particle gun-mediated transformation followed byselection for spectinomycin resistance conferred by a chimericaadA gene present in all transformation vectors (Fig. 2A) (19).For each construct, two independently generated transplastomiclines were selected for in-depth analysis. The lines are referredto hereafter as “Nt-SyHPT” lines (for Nicotiana tabacum plantsexpressing the Synechocystis HPT enzyme, with Nt-SyHPT1 andNt-SyHPT2 designating the two independently generated lines),“Nt-SyTCY” lines (expressing the Synechocystis TCY enzyme), “Nt-SyTMT” lines (expressing the Synechocystis TMT enzyme), and“Nt-AtTMT” lines (expressing the Arabidopsis TMT).To test the transplastomic lines for homoplasmy (i.e., the ab-

sence of residual wild-type copies of the highly polyploid plastidgenome), DNA samples were taken after two additional roundsof regeneration under antibiotic selection (20, 21) and analyzedby restriction fragment-length polymorphism (RFLP) analysis(Fig. S1). These experiments (i) confirmed successful trans-formation of the plastid genome in all tested lines, (ii) verifiedcorrect integration of the transgenes into the targeted genomicregion by homologous recombination (Fig. 2 A and B), and (iii)provided preliminary evidence for homoplasmy of the trans-formed plastid genome (Fig. S1A). Homoplasmy ultimately wasconfirmed by seed assays (20) that yielded a uniform populationof spectinomycin-resistant seedlings (Fig. S1C), consistent withmaternal inheritance of the plastid genome in tobacco (22).To verify expression of the transgenes from the plastid genome,

a set of Northern blot experiments was conducted. Hybridizationswith probes specific to the four transgenes yielded similar transcriptpatterns (Fig. 3A), in that two prominent hybridizing bands weredetected in all transplastomic lines. The lower band corresponds tothe expected size of the transgene transcript. The additional,slightly larger mRNA species originates from read-through tran-scription and has been observed previously in other studies usingthe same basic transformation vector pKP9 for transgene expres-sion from the plastid genome (15, 16).When grown to maturity in soil under standard greenhouse

conditions, none of the transplastomic lines displayed a discern-ible phenotype, indicating that chloroplast expression of thethree enzymes of the tocopherol pathway has no obvious nega-tive phenotypic consequences.

Tocochromanol Biosynthesis in Transplastomic Tobacco PlantsOverexpressing the Three Pathway Enzymes from Single-GeneConstructs. We next wanted to examine the effect of transgeneexpression in the transplastomic tobacco lines on tocochromanolmetabolism. To this end, tocochromanols were extracted fromleaf tissue with methanol and analyzed by reversed-phase HPLC

Fig. 1. Metabolic pathway of tocochromanol (tocopherol and tocotrienol) bio-synthesis in higher plants and its links to other metabolic processes. Tocochro-manol biosynthesis draws on metabolites from two biochemical pathways: theshikimate pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathwayof isoprenoid metabolism. The key difference between tocopherol and toco-trienol biosynthesis lies in the nature of the side chain, which comes from PDP inthe case of tocopherols and from GGDP in the case of tocotrienols. In tocopherolbiosynthesis, shikimate-derived HGA is connected to PDP in a reaction catalyzedby HPT. The analogous reaction in tocotrienol biosynthesis is catalyzed by HGGTand links GGDP toHGA. α-, β-, γ-, and δ-tocochromanols differ only in the numberand arrangement of methyl groups on the aromatic ring. Enzymes, compounds,and bonds specific to tocotrienol biosynthesis are indicated in green. MSBQ,2-methyl-6-solanesyl-1,4-benzoquinol; MPBQ, 2-methyl-6-phytylbenzoquinone;MGGBQ, 2-methyl-6-geranylgeranylbenzoquinone; DMPBQ, 2,3-dimethyl-5-phytyl-1,4-benzoquinone; DMGGBQ, 2,3-dimethyl-5-geranylgeranyl-1,4-benzoquinone; SAM, S-adenosyl methionine.

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using pure standards for quantitation (Fig. 4). Although nosignificant changes in tocochromanol accumulation were seenin the Nt-SyTCY, Nt-SyTMT, and Nt-AtTMT lines, a strong in-crease in tocopherol accumulation was observed in the Nt-SyHPTlines which express the Synechocystis HPT enzyme. Total tocochro-manol levels were nearly fivefold higher in Nt-SyHPT plants thanin wild-type plants (Fig. 4A), confirming previous data suggestingthatHPThas a limiting role in tocopherol biosynthesis (5, 10).Mostof the additional tocochromanol accumulating in the Nt-SyHPTlines was α-tocopherol, although some β-/γ-tocopherol (not nor-mally accumulating in the wild type) was detectable also. This resultindicates efficient conversion of γ-tocopherol to α-tocopherol inthe HPT-expressing plants and argues against a serious limitationfrom TMT activity in leaf α-tocopherol synthesis. Accumulationof small amounts of tocotrienols (which are not detectable in thewild type) also was observed in the transplastomic HPT-expressinglines (Fig. 4A). This activity could be the result of some side activityof HPT as a geranylgeranyltransferase (Fig. 1), as observedpreviously in vitro for the Synechocystis HPT (23).

Coexpression of Three Pathway Enzymes from Operon Constructs.Wenext wanted to determine what limits tocopherol biosynthesis inour HPT-expressing plants. One possibility is that, in the Nt-SyHPT lines, HPT activity still limits tocopherol biosynthesis and

that an even stronger HPT overexpression would be needed toenhance tocopherol synthesis further. Alternatively, HPT ex-pression from the chloroplast genome may have removed theHPT bottleneck completely so that now another enzyme in thepathway becomes limiting. The most plausible candidate for sucha secondarily limiting enzyme seems to be the tocopherol cyclasethat acts downstream of HPT in the pathway (Fig. 1) (24). Totest this possibility, we decided to combine all three chloroplastenzymes of the tocopherol biosynthetic pathway (HPT, TCY,and TMT) in a synthetic operon.We initially constructed a bacterial-type operon, pTop1 (Fig.

2A) in which the expression of the operon genes is driven bya single promoter, the individual cistrons are separated by twointergenic regions taken from endogenous operons in the chloro-plast genome (Materials and Methods), and the final cistron isfollowed by a 3′ UTR to stabilize the polycistronic mRNA (25).The operon construct was transformed into tobacco chloroplasts,and the resulting transplastomic lines (referred to as “Nt-Top1lines”) were purified to homoplasmy (Fig. S1B). Analysis of RNAaccumulation in Nt-Top1 transplastomic lines confirmed thepresence of the expected tricistronic transcript of ∼3.8 kb but alsorevealed abundant shorter-than-expected RNA species (Fig. 3B).This observation suggests that some RNA cleavage and/or deg-radation of the polycistronic operon transcript occur.

Fig. 2. Introduction of constructs for metabolic engineering of the tocopherol pathway into the tobacco and tomato plastid genomes. (A) Physical maps ofthe transgene constructs and their targeting region in the tobacco plastid genome. Genes above the line are transcribed from the left to the right; genesbelow the line are transcribed from right to left. Relevant restriction sites and resultant fragments sizes in RFLP analyses of transplastomic lines are indicated.For gene names and encoded gene products, see Fig. 1. Two polycistronic constructs (pTop1 and pTop2) were designed by spacing the genes with either twointergenic regions from the plastid genome (IGR1 and IGR2 in pTop1) or two identical intercistronic expression elements (IEE in pTop2) (38). The transgeneexpression cassettes are driven by the ribosomal RNA operon promoter from tobacco (Nt-Prrn) fused to the 5′ UTR from phage T7 gene 10 (14,16) and the 3′UTR of the plastid rbcL gene (Nt-TrbcL). The only exception is that in pTop2 the 3′ UTR of the Chlamydomonas reinhardtii plastid rbcL gene (Cr-TrbcL) was usedas terminator of the operon, and the 3′ UTRs of the tobacco plastid rbcL and rps16 (Nt-Trps16) genes were used to stabilize the processed monocistronicmRNAs. The selectable marker gene aadA is driven by a chimeric ribosomal RNA operon promoter and is fused to the 3′ UTR from the plastid psbA gene (Nt-TpsbA) (19). Relevant restriction sites and fragment sizes are indicated. GOI: gene of interest; IGR1: intergenic region between psbH and petB; IGR2: intergenicregion between rps2 and atpI. (B) Physical map of the targeting region in the wild-type tobacco plastid genome (N.t. ptDNA). Expected sizes of restrictionfragments in RFLP analyses are indicated. (C) Physical map of the targeting region in the wild-type tomato plastid genome (S.l. ptDNA).

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HPLC analysis of leaf material from Nt-Top1 transplastomiclines was performed to determine how coexpression of the threetocopherol biosynthesis genes from the operon construct affectstocochromanol synthesis in tobacco. Surprisingly, expression ofthe tocopherol operon resulted in only a moderate increase(∼1.7-fold) in tocopherol accumulation. We suspected inefficienttranslation of the polycistronic mRNA to be the cause of thelimited effect of the operon expression on the flux through to-copherol biosynthesis. Transcription of bacterial operons usuallygives rise to stable polycistronic mRNAs which are translateddirectly. In contrast, many polycistronic precursor transcripts inplastids are posttranscriptionally processed into monocistronic

or oligocistronic RNAs (26, 27), presumably by endonucleolyticcleavage (28). In at least some cases, cutting of the polycistronicprecursor RNA into monocistronic units is required to facilitateefficient translation of all cistrons, as is supported by the analysisof nuclear mutants defective in distinct intercistronic processingevents and by in vitro translation studies (29–32). Some poly-cistronic transcripts in plastids clearly remain unprocessed andare translated efficiently [e.g., the psbE operon comprising foursmall genes for polypeptides of photosystem II and the dicis-tronic psaA/B mRNA encoding the two reaction center subunitsof photosystem I (33, 34)], but the rules determining the de-pendency of translation on intercistronic RNA processing arecurrently unknown. Thus which (trans)genes in synthetic operonswill be expressed efficiently from unprocessed polycistronictranscripts and which transgenes will require intercistronic pro-cessing for efficient translation remains unpredictable, and pre-vious attempts to stack transgenes in operons have producedmixed results (e.g., 35–37).We recently have identified a small RNA element (dubbed the

“intercistronic expression element,” IEE) that, when introducedinto a chimeric context, proved to be sufficient to trigger pro-cessing of polycistronic transcripts into stable and translatablemonocistronic mRNAs (38). Because we suspected the poor per-formance of our Top1 tocopherol operon in chloroplasts might becaused by inefficient translation of the polycistronic mRNA(which, in addition, was not fully stable; Fig. 3), we decided toredesign the operon and use the IEE element to enable cleavage ofthe operon transcript into stable monocistronic units. This rede-signed synthetic operon, pTop2, was assembled from the codingregions of the three tocopherol genes, the promoter driving tran-scription of the operon, two copies of the 50-nt IEE element sep-arating the three cistrons, and three different 3′UTR sequences toconfer stability of the processed mRNAs (Fig. 2A). TransplastomicNt-Top2 plants were generated by chloroplast transformation intobacco and purified to homoplasmy (as confirmed by Southernblot analysis and inheritance assays; Fig. S1 B and C). Transcriptanalyses by Northern blot experiments demonstrated that the IEEindeed triggered faithful processing of the polycistronic operontranscript into stable monocistronic mRNAs for all three cistrons(Fig. 3B), confirming that the IEE can serve as a sequence context-independent tool to facilitate efficient expression of syntheticoperons from the plastid genome.Having confirmed efficient processing and stable mRNA

accumulation from the redesigned synthetic tocopherol op-eron, we next examined tocochromanol accumulation in theNt-Top2 lines. Interestingly, the transplastomic Nt-Top2 plantsshowed an increase in tocochromanol accumulation thatstrongly exceeded the increase seen before in our Nt-SyHPTlines (Fig. 4A). The Nt-Top2 plants also showed a higherabundance of tocotrienols than the Nt-SyHPT lines (Fig. 4A).These data suggest that tocochromanol synthesis can beboosted further by coexpressing HPT with the downstreamenzymes in the pathway and provide evidence that TCY ex-pression limits tocopherol biosynthesis once the HPT bottle-neck has been removed.

Pigment Synthesis and Stress Tolerance of Transplastomic TobaccoPlants with Enhanced Tocochromanol Biosynthesis. Our expressionstudies of tocopherol biosynthetic enzymes in tobacco chloroplastsrevealed two sets of transplastomic lines with strongly elevatedtocochromanol accumulation: theNt-SyHPT lines and theNt-Top2lines. Tocochromanol biosynthesis draws on precursors from theisoprenoid biosynthetic pathway (Fig. 1), which provides precursorsto a variety of other pathways, including carotenoid, chlorophyll,phylloquinone, plastoquinone, and gibberellin biosyntheses (39).To test whether these pathways are affected by the strongly in-creased flux through the tocopherol pathway in Nt-SyHPT and Nt-Top2 plants, we measured photosynthetic pigment accumulation

Fig. 3. Analysis of mRNA accumulation in transplastomic tobacco linesexpressing enzymes of the tocopherol biosynthetic pathway. (A) mRNA ac-cumulation in transplastomic lines expressing monocistronic constructs. Thetransgene-specific hybridization probes detect two major transcript speciesin all lines. The smaller species of ∼1.5 kb corresponds to the monocistronictranscript from the transgene, whereas the larger transcript species origi-nates from read-through transcription, as observed previously with similartransgenic constructs (15, 62). Sizes of the RNA marker bands are indicated inkilobases. (B) mRNAs accumulation in transplastomic lines that express thepolycistronic constructs Top1 and Top2. Note that only in Nt-Top2 lines arethe operon transcripts properly processed into three monocistronic mRNAsthat can be detected readily with the individual hybridization probes. Incontrast, Nt-Top1 lines accumulate a series of larger transcripts, includingthe unprocessed tricistronic precursor RNA of ∼3.8 kb.

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in the tocochromanol-overproducing transplastomic plants andtested the transplastomic lines in comparative growth assays undera variety of conditions.Nt-SyHPT and Nt-Top2 plants grown under medium- to high-

intensity light (300–1,000 μE·m−2·s−1), displayed no discerniblephenotype. Surprisingly, when photosynthetic pigment accumu-lation was analyzed, the transplastomic lines showed slightlyhigher levels of carotenoids and chlorophylls than seen in wild-type plants (Fig. 4B). This result suggests that no competition forisoprenoid substrates occurs in the tocopherol-overproducingplants, at least not to an extent that affects pigment biosynthesisby precursor limitation.

When plants were grown under unnaturally low light conditions(∼190 μE·m−2·s−1), the transplastomicNt-Top2 plants, but not theNt-SyHPT plants, displayed a mild phenotype (Fig. S2). Youngleaves in Nt-Top2 plants were light green, a phenotype that dis-appeared during development so that older leaves were in-distinguishable from wild-type leaves (Fig. S2A). Consistent withthe light-green phenotype under low-light conditions, the levels ofall photosynthetic pigments were slightly lower in Nt-Top2 plants(but not in Nt-SyHPT plants) than in the wild type (Fig. S2B).Previous work has implicated tocopherols in the protection of

plants from oxidative stress (40–42). To test whether our tocoph-erol-overaccumulating Nt-SyHPT and Nt-Top2 transplastomic

Fig. 4. Accumulation of tocochromanol andphotosynthetic pigment in transplastomic tobaccoplants expressing enzymes of the tocopherol biosynthetic pathway.(A) Accumulation levels of the individual tocochromanols in leaves ofwild-type plants (Nt-WT) and the various transplastomic lines generated in this study. The thirdleaf from the top of amature plant (with a total of seven leaves) was harvested for the analysis. For each plant line, four different plants weremeasured. Error barsrepresent the SD. For each construct, two independently generated transplastomic lineswere analyzed. Asterisks indicate values that are significantly different fromthewild type (P< 0.05).Note that the β and γ forms (of both tocopherols and tocotrienols) could notbe separatedby chromatographyand therefore are representedin a single bar. (B) Pigment contents in leaves ofwild-type plants, transplastomic lines overexpressingHPT, and lines expressing the Top2operon construct. Note thatthe levels of all pigments (carotenoids and chlorophylls) are slightly elevated in the transplastomic lines. Plants were grown under a light intensity of 500 μE·m−2·s−1.(C) Cold stress recovery assay to compare the tolerance to oxidative stress in wild-type plants and transplastomic lines overaccumulating tocopherols (Nt-SyHPT andNt-Top2). Plantswere exposed to cold stress at 4 °C for 1moand thenwere transferred back to 25 °C for 1wk. Thefirst three leaves from the topwereharvested andphotographed. Althoughwild-type leaves show severe symptoms of photooxidative damage (evidenced by strong pigment loss starting from the leafmargins), theoverproducing lines vitamin E recover without serious damage and show only slightly yellow leaf margins toward the tip.

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tobacco plants show increased tolerance to photooxidative stress,we performed cold-stress recovery assays under conditions knownto promote greatly the production of reactive oxygen species inchloroplasts (43–45). Although wild-type plants showed severesymptoms of photooxidative damage (evidenced by leaf bleaching)under these conditions, both the Nt-SyHPT and the Nt-Top2transplastomic plants weremuch less affected (Fig. 4C), suggestingthat their high tocochromanol content confers strong protectionfrom oxidative stress.

Expression of Tocopherol Biosynthetic Enzymes from the PlastidGenomes of Red-Fruited and Green-Fruited Tomato Varieties. Toco-chromanols of plant origin provide vitamin E to animals andhumans and therefore represent essential components of thehuman diet. We previously reported the development of a plastidtransformation protocol for tomato, a food crop with an ediblefruit (46). To be able to compare the regulation of tocopherolbiosynthesis and the rate-limiting steps in leaves versus fruits and,at the same time, to test if the results obtained in tobacco aretransferable to an important food crop, we attempted to in-troduce the pSyHPT and pTop2 constructs that had led to greatlyelevated tocopherol synthesis in transplastomic tobacco into thetomato plastid genome. (Because plastid transformation is con-siderably more laborious and time-consuming in tomato than intobacco, initial construct testing and optimization was done in thetobacco system.)Our previous work has shown that, during chloroplast-to-chro-

moplast conversion in tomato fruit ripening, plastid gene andtransgene expression are strongly down-regulated at both thetranscriptional and translational levels (47, 15). To avoid limi-tations from inefficient plastid gene expression and to distinguishbetween effects from tissue-specific regulation (fruit versus leaf)and effects from plastid differentiation (chloroplast versus chro-moplast), we wanted to compare tocopherol metabolism in red-fruited and green-fruited tomato varieties expressing the pSyHPTand pTop2 constructs. Because the only established tomato plastidtransformation protocol is for two closely related red-fruited to-mato varieties bred in Brazil (46, 48), we first needed to developprotocols for green-fruited varieties (see Fig. S5). For two well-regenerating commercial varieties, “Dorothy’s Green” and “GreenPineapple”, we succeeded in obtaining transplastomic plants usingoptimized selection and regeneration protocols (49). Thus we wereable to test whether green-fruited, chloroplast-containing tomatovarieties express plastid transgenes more efficiently than red-fruited, chromoplast-containing varieties and to analyze tocochro-manol biosynthesis comparatively in ripe green fruits and red fruits.We introduced the two constructs that led to strongly elevated

tocopherol synthesis in tobacco (the single-gene construct pSyHPTand the operon construct pTop2; Fig. 2) into the plastid genomesof the three tomato varieties that now can be transformed: the red-fruited variety IPA-6 and the green-fruited varieties Dorothy’sGreen and Green Pineapple. The fruits of Green Pineapple differfrom those of Dorothy’s Green in that they are smaller, somewhatdarker green, and have less white (non–green plastid-containing)fruit flesh (see Fig. S5A).Transplastomic tomato lines were generated with both con-

structs and for all three genotypes. The lines are designated Sl-IPA (for Solanum lycopersicum IPA-6), Sl-DG (for S. lycopersi-cum Dorothy’s Green), and Sl-GP (for S. lycopersicum GreenPineapple). Regenerated transplastomic plants were character-ized by RFLP analyses (compare Fig. S3 A and B with Fig. 2),which verified correct integration into the targeted region of thetomato plastid genome (50) and provided preliminary evidenceof homoplasmy. The homoplasmic state subsequently was con-firmed by inheritance assays (Fig. S3C).Northern blot analyses detected very similar transcript accu-

mulation patterns in transplastomic tomato plants (Fig. S4), asseen previously in the corresponding transplastomic tobacco

lines (Fig. 3). Importantly, the tricistronic operon transcripts inthe Sl-IPA-Top2, Sl-DG-Top2, and Sl-GP-Top2 lines underwentcorrect processing into stable monocistronic mRNAs for allthree genes of the operon, indicating that the IEE, originallyidentified in tobacco (38), also serves as a faithful RNA-pro-cessing element in other species.

Tocochromanol Biosynthesis in Leaves and Fruits of TransplastomicTomato Plants. Tocochromanol biosynthesis in the six sets oftransplastomic tomato plants was analyzed in leaves and fruits(Fig. 5). To assess the dependence of tocopherol accumulationon leaf age and plant development as well, young leaves (thesecond or third leaf from the top) and mature (fully expandedbut not yet senescent) leaves were analyzed comparatively. Inline with the results obtained with HPT expression in trans-plastomic tobacco plants, the Sl-SyHPT lines showed a strongincrease in leaf tocochromanol synthesis that was very similar inall three tomato genotypes (Fig. 5 A, C, and E). The increase intocochromanol accumulation was more pronounced in matureleaves, where tocochromanol levels were approximately sixfoldhigher in the transplastomic lines than in the wild type. As intobacco leaves, expression of the cyanobacterial HPT enzyme intransplastomic tomato plants induced the biosynthesis of toco-trienols, which normally do not accumulate in leaves of di-cotyledonous plants (6). In all three tomato varieties, expressionof the Top2 tocopherol operon led to an even stronger increasein leaf tocochromanol accumulation than seen with HPT ex-pression alone, reaching levels up to tenfold higher than in thewild type (Fig. 5 A, C, and E).When tocochromanols were measured in ripe tomato fruits of

the red-fruited variety IPA-6, the increase in tocochromanol accu-mulation in the transplastomic lines compared with the wild typewas significantly lower than in leaf tissue (Fig. 5B). Also, no in-duction of tocotrienol biosynthesis was seen in red fruits. This resultis consistent with a strong decline in plastid gene expression activityoccurring upon chloroplast-to-chromoplast conversion, as has beenobserved previously (47, 15), but also could be the result of pre-cursor limitation (i.e., lower GGDP availability in red fruits). In-terestingly, ripe tomato fruits of the two green-fruited varietiesDorothy’s Green and Green Pineapple showed much higher toco-chromanol accumulation than tomatoes of the red-fruited varietyIPA-6 (Fig. 5 D and F). Total tocochromanol contents in fruits ofHPT-expressing transplastomic Dorothy’s Green plants were twiceas high as in transplastomic IPA-6 fruits, whereas transplastomicGreen Pineapple fruits had threefold higher tocochromanol con-tents. These results correlate with Green Pineapple fruits havingthe highest chlorophyll content and thus presumably the highestactivity of plastid gene expression (Fig. S5A). UnlikeHPT expressionin IPA-6, expression in Dorothy’s Green and Green Pineapple ledto tocotrienol accumulation in fruits. This accumulation is consis-tent with higher HPT expression levels in green-fruited varietiesenhancing the side activity of the Synechocystis HPT as a homoge-ntisic acid geranylgeranyl transferase (HGGT) (Fig. 1) (23).Surprisingly, coexpression of TCY and TMT with HPT from

the Top2 operon did not result in a further increase in toco-chromanol accumulation in tomato fruits (Fig. 5 B, D, and F).This result is in stark contrast to the situation in tobacco andtomato leaves, where expression of the tocopherol operoncaused an additional boost in tocochromanol synthesis (Figs. 3Aand 5 A, C, and E). This finding indicates that, despite itsoverexpression, HPT still represents the rate-limiting enzyme ofthe tocopherol pathway in the Sl-SyHPT and Sl-Top2 tomatoesand suggests that tocochromanol biosynthesis exhibits differentbottlenecks in different tissues.To follow the time course of tocochromanol accumulation

during the fruit-ripening process, four different ripening stageswere investigated in the red-fruited tomato variety IPA-6 andthe green-fruited tomato variety Green Pineapple. Interestingly,

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although the tocochromanol levels increased continuously duringthe fruit-ripening process in both varieties (and in the wild-typeas well as in Sl-SyHPT and Sl-Top2 tomatoes), the rate of in-crease was much higher in the transplastomic Green Pineapplefruits (Fig. S6). This result suggests that continued transgene ex-pression in fruit chloroplasts largely accounts for the very hightocochromanol accumulation levels in ripe tomatoes of the green-fruited varieties (Fig. 5).

DiscussionUsing tocochromanol (vitamin E) biosynthesis as an example,our work described here has uncovered efficient strategies bywhich metabolic pathway engineering can be performed by stabletransformation of the plastid genome. Because vitamin E is anessential vitamin that has a diversity of beneficial physiologicaland disease-preventing properties (1), elevating the vitamin Econtent is an important goal of breeding and genetic engineering

Fig. 5. Tocochromanol accumulation in leaves and fruits of red-fruited and green-fruited transplastomic tomato varieties expressing enzymes of the to-copherol biosynthetic pathway. For each cultivar, wild-type plants, transplastomic lines expressing HPT from a monocistronic construct, and transplastomiclines expressing the Top2 operon were analyzed. Two independently generated transplastomic lines per construct and four different plants per line weremeasured. Error bars represent the SD. Asterisks indicate values that are significantly different from the wild type (P < 0.05). Note that the β and γ forms ofboth tocopherols and tocotrienols could not be separated by chromatography and therefore are represented in a single bar. To determine leaf tocopherolcontents, young (not yet fully expanded) leaves from the top of the plant and mature leaves from the bottom of the plant were comparatively analyzed. (A)Tocochromanol accumulation in leaves of the red-fruited variety IPA-6. (B) Tocochromanol accumulation in fruits of the red-fruited variety IPA-6. (C)Tocochromanol accumulation in leaves of the green-fruited variety Green Pineapple. (D) Tocochromanol accumulation in fruits of the green-fruited varietyGreen Pineapple. (E) Tocochromanol accumulation in leaves of the green-fruited variety Dorothy’s Green. (F) Tocochromanol accumulation in fruits of thegreen-fruited variety Dorothy’s Green. Note that both Dorothy’s Green and Green Pineapple are commercial green-fruited varieties that do not accumulatecarotenoids but ripen normally (so-called “green when ripe” varieties) (63).

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efforts (2, 3, 51–53). In addition to enhancing the nutritionalvalue of crops, increased levels of vitamin E compounds canimprove plant performance under stressful conditions that arelinked to reactive oxygen species (Fig. 3C) (54). Our resultsconfirm that the optimal approaches to maximize vitamin Econtents differ in leafy tissues and nonleafy tissues. In leaves,expression of all three pathway enzymes from a synthetic operonproved to be superior to expression of the rate-limiting enzymeHPT alone (Figs. 3 and 5), perhaps because the efficient con-version of 2-methyl-6-phytylbenzoquinone and 2,3-dimethyl-5-phytyl-1,4-benzoquinone into tocopherols by TCY and TMT(Fig. 1) creates a metabolic sink that ultimately diverts moreprecursors into the pathway. In contrast, in both green and redtomato fruits, HPT expression triggered higher vitamin E syn-thesis than expression of the entire operon (Fig. 5). A possiblereason is that HPT poses a stronger limitation to tocochromanolbiosynthesis in fruits than in leaves, so that coexpression of theother pathway enzymes not only is unable to confer a furtherboost in vitamin E synthesis but even slightly reduces the bene-ficial effects of HPT expression. This effect could be caused bythe two additional transgenes draining away plastid expressioncapacity that is known to be limited in tomato fruits (47, 15). Italso is possible that precursor availability (e.g., phytyl phosphatefrom chlorophyll turnover; ref. 55) limits tocopherol bio-synthesis more strongly in fruits than in leaves and in this waycontributes to the different levels of vitamin E accumulation indifferent tissues (Fig. 5).Previously, impressive results have been obtained by enhancing

the tocopherol pathway via nuclear transformation. In addition,these studies provided interesting insights into limiting toco-chromanol intermediates and rate-limiting biochemical reactions.For example, a comprehensive study in soybean seeds revealedthat, at the metabolite level, the availability of PDP represents themost serious limitation (3). Our data obtained here suggest that,at least in green leaves, plastid engineering can be even moreefficient than nuclear genetic engineering, especially with thesynthetic operon design developed here. Nuclear expression ofHPT in A. thaliana induced an up to 4.4-fold increase in leaf to-copherol (5), whereas in our plastid operon-expressing lines, anup to 10-fold increase was obtained (Fig. 5). However, the mostsignificant advantages of plastid engineering lie in its uniqueprecision (because of transgene integration by homologous re-combination) and the possibility of stackingmultiple transgenes inoperons for coexpression as polycistronic mRNAs, which cur-rently is not feasible in the nuclear genome. The functioning of theIEE (38) in very different sequence contexts, including nptII, yfp,and the three tocopherol biosynthesis genes tested here, suggeststhat the IEE triggers processing of polycistronic transcripts intostable and translatable monocistronic units in a sequence context-independent manner and thus provides a generally usable tool forthe construction of synthetic operons that are expressed effi-ciently. Therefore, the synthetic operon design developed in thisstudy will facilitate future synthetic biology applications in plas-tids, especially the construction of complex artificial operons thatintroduce novel biochemical pathways into plants. Because of thelack of epigenetic gene silencing and position effects in plastids,the transplastomic technology also entails an enormous reductionin the number of transgenic events that need to be generated andanalyzed. For example, although 48 independent HGGT trans-genic events had to be analyzed to identify a strong tocotrienolaccumulation phenotype [in which tocotrienols accounted for 74%of the total content of tocochromanols of maize embryos (6)],generation of one or two transplastomic lines per construct usuallysuffices. The savings become even more significant when multipletransgenes need to be introduced, a process that in conventionalnuclear transformation methodology typically involves cotrans-formation and analysis of even more events or the combination ofindividual transgenic events by crossing (3). Finally, because of

maternal inheritance of the plastid DNA, plastid transformationalso greatly reduces the risk of unwanted pollen transmission, thusimproving the biosafety of transgenic plants. In this respect, webuilt an additional useful feature into our operon vector: two loxPsites that can be used to eliminate the aadA selectablemarker geneby transiently crossing in a nuclear-encoded plastid-targeted Crerecombinase (56, 15).A byproduct of this work has been the development of plastid

transformation for green-fruited tomato varieties. The benefit ofusing green-fruited tomato varieties for the synthesis of nutraceut-icals and pharmaceuticals is twofold. First, the long-term presenceof chloroplasts in the fruit ensures a higher activity of plastid geneexpression than in chromoplast-containing red fruits (47). Lowlevels of gene expression also were described for other nongreenplastid types (e.g., amyloplasts), although recent work has suggestedstrategies to overcome these limitations, at least partially (57–59).Second, green-fruited tomatoes can greatly simplify identity pres-ervation (of transgenic tomatoes, nutritionally enhanced tomatoes,or “pharma” tomatoes), at least in countries where only red-fruitedtomatoes are marketed. The expansion of the genotype range fortomato plastid transformation reported here represents an impor-tant step toward the application of the transplastomic technology inan important food crop on a more routine basis.Analysis of carotenoid and chlorophyll accumulation in

transplastomic plants overaccumulating vitamin E revealed aninteresting regulatory connection between tocochromanol bio-synthesis and the pathways of photosynthetic pigment bio-synthesis. Surprisingly, both carotenoid and chlorophyll levelswere found to be slightly increased in plants overproducing to-copherol, at least when grown under standard conditions (Fig.3B). This observation may suggest that the increased flux throughthe tocochromanol pathway results in a general stimulation ofisoprenoid-using pathways (possibly through positive feedbackregulation). Alternatively, higher levels of protection from pho-tooxidative stress could lead to reduced turnover of photosyn-thetic pigments. Interestingly, the plants expressing the synthetictocopherol operon and showing the strongest overaccumulationof tocochromanols displayed a mild pigment deficiency underlow-light conditions (Fig. S2). This deficiency indicates that thelevel of tocochromanol synthesis we have achieved with ourtransplastomic approach probably is close the maximum that ispossible without negatively affecting metabolism and, hence,plant fitness. It is noteworthy in this respect that spinach, one ofthe vegetable plants with the highest vitamin E content, has ap-proximately four- to fivefold higher tocochromanol levels thantomato fruits (60). Thus, the level in spinach is similar to thelevels reached in our best-performing transplastomic tomatoesbut is surpassed significantly by the levels reached in the leaves oftransplastomic plants expressing the synthetic tocopherol operon.In sum, our work presented here provides efficient strategies

for enhancing the vitamin E content in leaves and fruits throughplastid genome engineering and pinpoints principles of syntheticoperon design for efficient metabolic pathway engineering andsynthetic biology in plastids.

Materials and MethodsPlant Material and Growth Conditions. Tobacco (N. tabacum cv. Petite Havana)plants were grown under aseptic conditions on agar-solidified medium con-taining 30 g/L sucrose (19). The tomato (S. lycopersicum) variety IPA-6 is a com-mercial red-fruited cultivar bred in Brazil. The green-fruited varieties GreenPineapple and Dorothy’s Green are presumably unrelated heirloom tomatoes(of unclear genetic origin) that were first described by tomato growers inthe United States (http://t.tatianastomatobase.com:88/wiki/Green_Pineapple;http://t.tatianastomatobase.com:88/wiki/Dorothy%27s_Green). Both are com-mercial varieties that do not accumulate carotenoids but ripen normally (so-called “green when ripe” varieties). Tomato plants were raised from surface-sterilized seeds on the same medium with 20 g/L sucrose. Regenerated shootsfrom transplastomic lines were rooted and propagated on the same medium.Rooted homoplasmic plants were transferred to soil and grown to maturity

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under standard greenhouse conditions (relative humidity: 55%; day tempera-ture: 25 °C; night temperature: 20 °C; diurnal cycle: 16 h light/8 h darkness; lightintensity: 190–600 μE·m−2·s−1). The homoplasmic state of the T1 generationwastested by germinating seeds from selfed transplastomic plants onmediumwithspectinomycin (500 mg/L for tobacco, 100 mg/L for tomato).

Construction of Plastid Transformation Vectors. Monocistronic expressioncassettes for enzymes of the tocopherol pathway were assembled in theplastid expression vector pHK20 (14). The operon constructs pTOP1 andpTOP2 were assembled from the genes cloned individually into the pHK20backbone. The cloning procedures and the sources of genes and expressionelements are described in detail in SI Materials and Methods. Synthetic oli-gonucleotides used are listed in Table S1. The sequences of the syntheticconstructs were submitted to the GenBank Nucleotide Sequence Database atthe National Center for Biotechnology Information and can be obtainedunder the accession nos. JX235342 (pSyHPT), JX235343 (pSyTCY), JX235344(pSyTMT), JX235341 (pAtTMT), JX235345 (pTop1), and JX235346 (pTop2).

Plastid Transformation and Selection of Transplastomic Lines. Young leavesfrom aseptically grown tobacco and tomato plants were bombarded withplasmid-coated 0.6-μm gold particles using a helium-driven biolistic gun(PDS1000He; BioRad) with the Hepta Adapter setup. Primary spectinomycin-resistant lines were selected on plant-regeneration medium containing 500mg/L spectinomycin (19, 46, 48).

To develop a plastid transformation protocol for green-fruited tomatovarieties, we tested a number of commercially grown green-fruited varietiesfor their tissue culture and regeneration properties. In these analyses, weidentified two varieties (Dorothy’s Green and Green Pineapple), that dis-played good regeneration rates from leaf explants and also showed accept-able rates of rooting on synthetic medium as well as good fruit and seed set inthe greenhouse (Fig. S5). Regeneration medium and selection conditions forplastid transformation of green-fruited tomato varieties were as describedpreviously for red-fruited varieties (46, 48). The plastid transformation effi-ciencies expressed as number of confirmed transplastomic lines (pSyHPT +pTop2) per number of selection plates (Petri dishes) with bombarded leafpieces were five pSyHPT + three pTop2 events per 19 plates for IPA-6, ninepSyHPT + eight pTop2 events per 469 plates for Dorothy’s Green, and threepSyHPT + 10 pTop2 events per 674 plates for Green Pineapple.

Spontaneous spectinomycin-resistant tobacco and tomato lines wereidentified by double-selection tests on regeneration medium containing 500mg/L spectinomycin and 500 mg/L streptomycin (19, 20) and subsequentlywere eliminated. Several independent transplastomic lines were generatedfor each construct and were subjected to one to three additional rounds ofregeneration on spectinomycin-containing regeneration medium to enrichthe transplastome and select for homoplasmic cell lines.

Isolation of Nucleic Acids and Hybridization Procedures. Total plant DNA wasextracted from fresh leaf samples by a cetyltrimethylammonium bromide-based method (15). For RFLP analysis by Southern blotting, 5-μg samples of

total DNA were treated with restriction enzymes, separated in 1% agarosegels, and transferred onto Hybond XL nylon membranes (GE Healthcare) bycapillary blotting. For hybridization, [α32P]dCTP-labeled probes were pro-duced by random priming (Multiprime DNA labeling kit; GE Healthcare).

Hybridizations were performed at 65 °C using standard protocols.Total plant RNA was isolated by a guanidine isothiocyanate/phenol-basedmethod (peqGOLD TriFast; Peqlab). RNA samples (3 μg total RNA) weredenatured, separated in denaturing formaldehyde-containing agarosegels (1%), and blotted onto Hybond XL nylon membranes (GE Healthcare)by capillary blotting.

A 550-bp PCR product generated by amplification of a portion of the psaBcoding region using primers P7247 and P7244 (48) and a 283-bp PCR productgenerated by amplification of the psbZ coding region using Pycf9a and Pycf9b(38) were used as RFLP probes to verify plastid transformation and assesshomoplasmy. Transgene-specific probes for Northern blot analyses weregenerated by excising the complete coding regions from plasmid clones.

HPLC Analysis of Pigments and Tocochromanols. Tocochromanols wereextracted with methanol from lyophilized leaf powder or lyophilized tomatofruit powder. Separation, identification, and quantification of tocochroma-nols were carried out with an YMC ODS-A 250 × 4.6 mm column using theAgilent 1100 Series HPLC system. Separation was performed according topublished procedures (42). Reversed-phase HPLC was used to avoid coelutionof γ-tocopherol with plastochromanol-8 (9). Carotenoids and chlorophyllswere isolated from lyophilized leaf powder samples by extraction with 80%(vol/vol) acetone followed by two extractions with 100% acetone. The threeextracts subsequently were combined and used for HPLC analysis. Separation,identification, and quantification of pigments were performed as describedpreviously (61) using the HPLC system specified above. All tocochromanol andpigment species were identified and quantified by comparison with knownamounts of pure standards.

Cold Stress and Recovery Assay. To assess and compare the tolerance oftransplastomic and wild-type plants to oxidative stress, greenhouse-grownplantswere transferred to a growth chamber adjusted to cold stress conditions(temperature: 4 °C; day length: 16 h; light intensity: 70 μE·m−2·s−1). Followingexposure to cold stress for 1 mo, plants were transferred back to normalgrowth conditions (temperature: 25 °C; light intensity: 250μE·m−2·s−1) for 1wk.The top three leaves then were phenotypically compared and photographed.

ACKNOWLEDGMENTS.We thank Stefanie Seeger and Claudia Hasse for helpwith chloroplast transformation, Dr. Eugenia Maximova for microscopy, theMax Planck Institute for Molecular Plant Physiology (MPI-MP) Green Teamfor plant care and cultivation, and Dr. Peter Dörmann of the MPI-MP/Uni-versity of Bonn and the EU-FP7 METAPRO consortium (www.isoprenoid.com)for helpful discussions. This work was supported by a Grant European Union-Seventh Framework Programme METAPRO 244348 from the EuropeanUnion (to R.B.).

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