Efficient metabolic pathway engineering in transgenic PNAS PLUS tobacco and tomato plastids with synthetic multigene operons

Yinghong 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 review September 28, 2012) The engineering of complex metabolic pathways requires the con- group in the chromanol ring to a free radical, followed by meso- certed expression of multiple genes. In plastids (chloroplasts) of meric stabilization of the molecule’s own unpaired electron. The plant cells, genes are organized in operons that are coexpressed as current models of tocochromanol action in the body are based polycistronic transcripts and then often are processed further into largely on their radical-scavenging properties, in that protection of monocistronic mRNAs. Here we have used the tocochromanol membranes from lipid peroxidation and protection of active sites pathway (providing tocopherols and tocotrienols, collectively also of metabolic enzymes and nucleic acids from damage by free referred to as “vitamin E”) as an example to establish principles of radicals are believed to be the main beneficial effects of toco- successful multigene engineering by stable transformation of the chromanols at the cellular level. chloroplast genome, a technology not afflicted with epigenetic var- Because of the high nutritional value and the benefits of vi- iation and/or instability of transgene expression. Testing a series of tamin E in human health, increasing the tocochromanol content single-gene constructs (encoding homogentisate phytyltransferase, of major agricultural crops has long been in the focus of breeding tocopherol cyclase, and γ-tocopherol methyltransferase) and ratio- programs and genetic engineering approaches (2, 3). Elevated nally designed synthetic operons in tobacco and tomato plants, we vitamin E contents can be achieved either by directing more (i) confirmed previous results suggesting homogentisate phytyl- metabolites into the tocopherol biosynthetic pathway or, alter- SCIENCES transferase as the limiting enzymatic step in the pathway, (ii)com-

natively, by altering the tocopherol spectrum [e.g., by converting APPLIED BIOLOGICAL paratively characterized the bottlenecks in tocopherol biosynthesis other tocopherol forms into α-tocopherol, the form with the in transplastomic leaves and tomato fruits, and (iii)achievedanup highest vitamin E activity; (4)]. to tenfold increase in total tocochromanol accumulation. In addition, Relatively little is known about the regulation of tocochro- our results uncovered an unexpected light-dependent regulatory manol biosynthesis and its interconnection with other metabolic link between tocochromanol metabolism and the pathways of pho- pathways in the plant cell. Mutant analysis and overexpression tosynthetic pigment biosynthesis. The synthetic operon design de- studies in the model plant Arabidopsis thaliana have shed some veloped here will facilitate future synthetic biology applications in light on limiting enzymatic steps and also have revealed some plastids, especially the design of artificial operons that introduce strategies for manipulating the tocopherol content and altering novel biochemical pathways into plants. the tocopherol spectrum (4–6). Here we used stable trans- formation of the plastid (chloroplast) genome in the model plant metabolic engineering | plastid transformation | fruit development | tobacco and the food crop tomato to engineer the tocopherol fruit ripening metabolic pathway and assess the impact of the three plastid- localized enzymes of the pathway (Fig. 1) on tocochromanol ocochromanols (tocopherols and tocotrienols) are products biosynthesis in chloroplasts and chromoplasts. Our results con- Tof plant metabolism that arise from two biochemical path- firm previous data on the limiting role of HPT in the pathway, ways: the shikimate pathway of aromatic amino acid biosynthesis characterize the limitations in tocopherol biosynthesis in trans- and the isoprenoid biosynthetic pathway (Fig. 1). Tocopherols plastomic leaves and fruits, and reveal a synthetic operon design arise from a condensation reaction of homogentisic acid (HGA) that facilitates efficient multigene engineering in plastids. with phytyl diphosphate (PDP), whereas tocotrienols are pro- duced by geranylgeranyl diphosphate (GGDP) attachment to Results HGA (Fig. 1). Both tocopherols and tocotrienols occur in four Expression of Three Enzymes of the Tocopherol Pathway from the natural forms that differ in their methylation patterns of the ar- Tobacco Plastid Genome. Based on overexpression studies in nu- Arabidopsis omatic head group: α, β, γ,andδ. Starting with the condensation clear-transgenic plants, two enzymes in the tocoph- of HGA to the isoprenoid chain (PDP or GGDP), all steps of tocochromanol biosynthesis take place in the plastid. Drawing on the plastid isoprenoid pool, tocochromanol biosynthesis is highly connected to a number of other metabolic pathways in the plastid Author contributions: Y.L., D.K., S.R., and R.B. designed research; Y.L. and H.R. performed compartment, including the biosynthesis of carotenoids, chlor- research; Y.L., H.R., D.K., S.R., and R.B. analyzed data; and R.B. wrote the paper. ophylls, gibberellins, phylloquinone, and plastoquinone (Fig. 1). The authors declare no conflict of interest. Tocochromanols are best known for their vitamin E activity. This article is a PNAS Direct Submission. J.E.M. is a guest editor invited by the Because animals (including humans) cannot synthesize vitamin E, Editorial Board. they depend on the uptake of plant-derived tocochromanols in Data deposition: The sequences of the synthetic constructs reported in this paper have been deposited in the National Center for Biotechnology Information GenBank Nucleo- their diet. In addition to being an essential micronutrient, toco- tide Sequence Database [accession nos. JX235341 (pAtTMT), JX235342 (pSyHPT), chromanols have been linked to a variety of health-promoting JX235343 (pSyTCY), JX235344 (pSyTMT), JX235345 (pTop1), and JX235346 (pTop2)]. effects [e.g., in preventing chronic disease (1)]. Most of these 1Present address: Molecular Biology Laboratory, BB-Biogen, Indonesian Agency for Agri- beneficial properties generally are explained by tocochromanols cultural Research and Development, Ministry of Agriculture, Bogor 16111, Indonesia. being highly potent antioxidants and efficient quenchers of re- 2To whom correspondence should be addressed. E-mail: [email protected]. active oxygen species. This activity comes from the ability of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tocochromanols to donate a hydrogen atom from the hydroxyl 1073/pnas.1216898110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1216898110 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 However, because of lower plastid number per cell, lower plastid genome 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 flux through tocochromanol biosynthesis. To this end, we overexpressed the 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 the same intergenic region in the plastid genome (Fig. 2). Both the expression cassette and the targeting site in the plastid DNA had been tested extensively in previous experiments and were shown to give highly efficient and stable transgene expression from the plastid genome (14–17). To avoid limitations from plant-specific regulation 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 is unlikely to limit flux through the tocochromanol pathway; (4)], we additionally used the gene from A. thaliana (Fig. 2A). All four constructs were introduced into the tobacco plastid genome by particle gun-mediated transformation followed by selection for spectinomycin resistance conferred by a chimeric aadA gene present in all transformation vectors (Fig. 2A) (19). For each construct, two independently generated transplastomic lines were selected for in-depth analysis. The lines are referred to hereafter as “Nt-SyHPT” lines (for Nicotiana tabacum plants expressing the Synechocystis HPT enzyme, with Nt-SyHPT1 and Nt-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 plastid genome), DNA samples were taken after two additional rounds of regeneration under antibiotic selection (20, 21) and analyzed by restriction fragment-length polymorphism (RFLP) analysis (Fig. S1). These experiments (i) confirmed successful trans- formation of the plastid genome in all tested lines, (ii) verified correct integration of the transgenes into the targeted genomic Fig. 1. Metabolic pathway of tocochromanol (tocopherol and tocotrienol) bio- region by homologous recombination (Fig. 2 A and B), and (iii) synthesis in higher plants and its links to other metabolic processes. Tocochro- provided preliminary evidence for homoplasmy of the trans- manol biosynthesis draws on metabolites from two biochemical pathways: the A shikimate pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway formed plastid genome (Fig. S1 ). Homoplasmy ultimately was of isoprenoid metabolism. The key difference between tocopherol and toco- confirmed by seed assays (20) that yielded a uniform population trienol biosynthesis lies in the nature of the side chain, which comes from PDP in of spectinomycin-resistant seedlings (Fig. S1C), consistent with the case of tocopherols and from GGDP in the case of tocotrienols. In tocopherol maternal inheritance of the plastid genome in tobacco (22). biosynthesis, shikimate-derived HGA is connected to PDP in a reaction catalyzed To verify expression of the transgenes from the plastid genome, by HPT. The analogous reaction in tocotrienol biosynthesis is catalyzed by HGGT a set of Northern blot experiments was conducted. Hybridizations α β γ δ and links GGDP to HGA. -, -, -, and -tocochromanols differ only in the number with probes specific to the four transgenes yielded similar transcript and arrangement of methyl groups on the aromatic ring. Enzymes, compounds, patterns (Fig. 3A), in that two prominent hybridizing bands were and bonds specific to tocotrienol biosynthesis are indicated in green. MSBQ, 2-methyl-6-solanesyl-1,4-benzoquinol; MPBQ, 2-methyl-6-phytylbenzoquinone; detected in all transplastomic lines. The lower band corresponds to MGGBQ, 2-methyl-6-geranylgeranylbenzoquinone; DMPBQ, 2,3-dimethyl- the expected size of the transgene transcript. The additional, 5-phytyl-1,4-benzoquinone; DMGGBQ, 2,3-dimethyl-5-geranylgeranyl-1,4- slightly larger mRNA species originates from read-through tran- benzoquinone; SAM, S-adenosyl methionine. scription and has been observed previously in other studies using the same basic transformation vector pKP9 for transgene expres- sion from the plastid genome (15, 16). erol biosynthetic pathway (Fig. 1) were suggested as rate-limiting: When grown to maturity in soil under standard greenhouse homogentisate phytyltransferase [HPT, encoded by the VTE2 conditions, none of the transplastomic lines displayed a discern- gene (5)] and tocopherol cyclase [TCY, encoded by the VTE1 ible phenotype, indicating that chloroplast expression of the locus (8)]. Recent data have called into question the possible three enzymes of the tocopherol pathway has no obvious nega- limiting role of TCY in tocopherol biosynthesis (9, 10). Expres- tive phenotypic consequences. sion of foreign genes from the plastid genome has the advantages that (i) there is no interference from epigenetic processes, and Tocochromanol Biosynthesis in Transplastomic Tobacco Plants (ii) transgene insertion occurs by homologous recombination into Overexpressing the Three Pathway Enzymes from Single-Gene a preselected region of the plastid genome, typically an intergenic Constructs. We next wanted to examine the effect of transgene spacer region (11–13). Therefore, transgene expression is not expression in the transplastomic tobacco lines on tocochromanol burdened by copy number-dependent or insertion site-dependent metabolism. To this end, tocochromanols were extracted from variation in expression levels and epigenetic transgene silencing. leaf tissue with methanol and analyzed by reversed-phase HPLC

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Fig. 2. Introduction of constructs for metabolic engineering of the tocopherol pathway into the tobacco and tomato plastid genomes. (A) Physical maps of the transgene constructs and their targeting region in the tobacco plastid genome. Genes above the line are transcribed from the left to the right; genes below 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 two intergenic regions from the plastid genome (IGR1 and IGR2 in pTop1) or two identical intercistronic expression elements (IEE in pTop2) (38). The transgene expression 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 used as terminator of the operon, and the 3′ UTRs of the tobacco plastid rbcL and rps16 (Nt-Trps16) genes were used to stabilize the processed monocistronic mRNAs. 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: intergenic region between rps2 and atpI.(B) Physical map of the targeting region in the wild-type tobacco plastid genome (N.t. ptDNA). Expected sizes of restriction fragments in RFLP analyses are indicated. (C) Physical map of the targeting region in the wild-type tomato plastid genome (S.l. ptDNA).

using pure standards for quantitation (Fig. 4). Although no that an even stronger HPT overexpression would be needed to significant changes in tocochromanol accumulation were seen enhance tocopherol synthesis further. Alternatively, HPT ex- in the Nt-SyTCY, Nt-SyTMT, and Nt-AtTMT lines, a strong in- pression from the chloroplast genome may have removed the crease in tocopherol accumulation was observed in the Nt-SyHPT HPT bottleneck completely so that now another enzyme in the lines which express the Synechocystis HPT enzyme. Total tocochro- pathway becomes limiting. The most plausible candidate for such manol levels were nearly fivefold higher in Nt-SyHPT plants than a secondarily limiting enzyme seems to be the tocopherol cyclase in wild-type plants (Fig. 4A), confirming previous data suggesting that acts downstream of HPT in the pathway (Fig. 1) (24). To that HPT has a limiting role in tocopherol biosynthesis (5, 10). Most test this possibility, we decided to combine all three chloroplast of the additional tocochromanol accumulating in the Nt-SyHPT enzymes of the tocopherol biosynthetic pathway (HPT, TCY, lines was α-tocopherol, although some β-/γ-tocopherol (not nor- and TMT) in a synthetic operon. mally accumulating in the wild type) was detectable also. This result We initially constructed a bacterial-type operon, pTop1 (Fig. indicates efficient conversion of γ-tocopherol to α-tocopherol in 2A) in which the expression of the operon genes is driven by the HPT-expressing plants and argues against a serious limitation a single promoter, the individual cistrons are separated by two from TMT activity in leaf α-tocopherol synthesis. Accumulation intergenic regions taken from endogenous operons in the chloro- of small amounts of tocotrienols (which are not detectable in the plast genome (Materials and Methods), and the final cistron is wild type) also was observed in the transplastomic HPT-expressing followed by a 3′ UTR to stabilize the polycistronic mRNA (25). lines (Fig. 4A). This activity could be the result of some side activity The operon construct was transformed into tobacco chloroplasts, of HPT as a geranylgeranyltransferase (Fig. 1), as observed and the resulting transplastomic lines (referred to as “Nt-Top1 previously in vitro for the Synechocystis HPT (23). lines”) were purified to homoplasmy (Fig. S1B). Analysis of RNA accumulation in Nt-Top1 transplastomic lines confirmed the Coexpression of Three Pathway Enzymes from Operon Constructs. We presence of the expected tricistronic transcript of ∼3.8 kb but also next wanted to determine what limits tocopherol biosynthesis in revealed abundant shorter-than-expected RNA species (Fig. 3B). our HPT-expressing plants. One possibility is that, in the Nt- This observation suggests that some RNA cleavage and/or deg- SyHPT lines, HPT activity still limits tocopherol biosynthesis and radation of the polycistronic operon transcript occur.

Lu et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 or oligocistronic RNAs (26, 27), presumably by endonucleolytic cleavage (28). In at least some cases, cutting of the polycistronic precursor RNA into monocistronic units is required to facilitate efficient translation of all cistrons, as is supported by the analysis of nuclear mutants defective in distinct intercistronic processing events and by in vitro translation studies (29–32). Some poly- cistronic transcripts in plastids clearly remain unprocessed and are translated efficiently [e.g., the psbE operon comprising four small genes for polypeptides of photosystem II and the dicis- tronic psaA/B mRNA encoding the two reaction center subunits of photosystem I (33, 34)], but the rules determining the de- pendency of translation on intercistronic RNA processing are currently unknown. Thus which (trans)genes in synthetic operons will be expressed efficiently from unprocessed polycistronic transcripts and which transgenes will require intercistronic pro- cessing for efficient translation remains unpredictable, and pre- vious attempts to stack transgenes in operons have produced mixed results (e.g., 35–37). We recently have identified a small RNA element (dubbed the “intercistronic expression element,” IEE) that, when introduced into a chimeric context, proved to be sufficient to trigger pro- cessing of polycistronic transcripts into stable and translatable monocistronic mRNAs (38). Because we suspected the poor per- formance of our Top1 tocopherol operon in chloroplasts might be caused by inefficient translation of the polycistronic mRNA (which, in addition, was not fully stable; Fig. 3), we decided to redesign the operon and use the IEE element to enable cleavage of the operon transcript into stable monocistronic units. This rede- signed synthetic operon, pTop2, was assembled from the coding regions 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 to confer stability of the processed mRNAs (Fig. 2A). Transplastomic Nt-Top2 plants were generated by chloroplast transformation in tobacco and purified to homoplasmy (as confirmed by Southern blot analysis and inheritance assays; Fig. S1 B and C). Transcript analyses by Northern blot experiments demonstrated that the IEE indeed triggered faithful processing of the polycistronic operon transcript 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 synthetic Fig. 3. Analysis of mRNA accumulation in transplastomic tobacco lines operons from the plastid genome. expressing enzymes of the tocopherol biosynthetic pathway. (A) mRNA ac- Having confirmed efficient processing and stable mRNA cumulation in transplastomic lines expressing monocistronic constructs. The accumulation from the redesigned synthetic tocopherol op- transgene-specific hybridization probes detect two major transcript species eron, we next examined tocochromanol accumulation in the in all lines. The smaller species of ∼1.5 kb corresponds to the monocistronic Nt-Top2 lines. Interestingly, the transplastomic Nt-Top2 plants transcript from the transgene, whereas the larger transcript species origi- showed an increase in tocochromanol accumulation that nates from read-through transcription, as observed previously with similar strongly exceeded the increase seen before in our Nt-SyHPT transgenic constructs (15, 62). Sizes of the RNA marker bands are indicated in lines (Fig. 4A). The Nt-Top2 plants also showed a higher kilobases. (B) mRNAs accumulation in transplastomic lines that express the Nt-Sy A polycistronic constructs Top1 and Top2. Note that only in Nt-Top2 lines are abundance of tocotrienols than the HPT lines (Fig. 4 ). the operon transcripts properly processed into three monocistronic mRNAs These data suggest that tocochromanol synthesis can be that can be detected readily with the individual hybridization probes. In boosted further by coexpressing HPT with the downstream contrast, Nt-Top1 lines accumulate a series of larger transcripts, including enzymes in the pathway and provide evidence that TCY ex- the unprocessed tricistronic precursor RNA of ∼3.8 kb. pression limits tocopherol biosynthesis once the HPT bottle- neck has been removed. Nt HPLC analysis of leaf material from -Top1 transplastomic Pigment Synthesis and Stress Tolerance of Transplastomic Tobacco lines was performed to determine how coexpression of the three Plants with Enhanced Tocochromanol Biosynthesis. Our expression tocopherol biosynthesis genes from the operon construct affects studies of tocopherol biosynthetic enzymes in tobacco chloroplasts tocochromanol synthesis in tobacco. Surprisingly, expression of revealed two sets of transplastomic lines with strongly elevated the tocopherol operon resulted in only a moderate increase tocochromanol accumulation: the Nt-SyHPT lines and the Nt-Top2 (∼1.7-fold) in tocopherol accumulation. We suspected inefficient lines. Tocochromanol biosynthesis draws on precursors from the translation of the polycistronic mRNA to be the cause of the isoprenoid biosynthetic pathway (Fig. 1), which provides precursors limited effect of the operon expression on the flux through to- to a variety of other pathways, including carotenoid, chlorophyll, copherol biosynthesis. Transcription of bacterial operons usually phylloquinone, plastoquinone, and gibberellin biosyntheses (39). gives rise to stable polycistronic mRNAs which are translated To test whether these pathways are affected by the strongly in- directly. In contrast, many polycistronic precursor transcripts in creased flux through the tocopherol pathway in Nt-SyHPT and Nt- plastids are posttranscriptionally processed into monocistronic Top2 plants, we measured photosynthetic pigment accumulation

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Fig. 4. Accumulation of tocochromanol and photosynthetic pigment in transplastomic tobacco plants expressing enzymes of the tocopherol biosynthetic pathway. (A) Accumulation levels of the individual tocochromanols in leaves of wild-type plants (Nt-WT) and the various transplastomic lines generated in this study. The third leaf from the top of a mature plant (with a total of seven leaves) was harvested for the analysis. For each plant line, four different plants were measured. Error bars represent the SD. For each construct, two independently generated transplastomic lines were analyzed. Asterisks indicate values that are significantly different from the wild type (P < 0.05). Note that the β and γ forms (of both tocopherols and tocotrienols) could not be separated by chromatography and therefore are represented inasinglebar.(B) Pigment contents in leaves of wild-type plants, transplastomic lines overexpressing HPT, and lines expressing the Top2 operon construct. Note that − − the 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 and Nt-Top2). Plants were exposed to cold stress at 4 °C for 1 mo and then were transferred back to 25 °C for 1 wk. The first three leaves from the top were harvested and photographed. Although wild-type leaves show severe symptoms of photooxidative damage (evidenced by strong pigment loss starting from the leaf margins), the overproducing lines vitamin E recover without serious damage and show only slightly yellow leaf margins toward the tip.

in the tocochromanol-overproducing transplastomic plants and When plants were grown under unnaturally low light conditions − − tested the transplastomic lines in comparative growth assays under (∼190 μE·m 2·s 1), the transplastomic Nt-Top2 plants, but not the a variety of conditions. Nt-SyHPT plants, displayed a mild phenotype (Fig. S2). Young Nt-SyHPT and Nt-Top2 plants grown under medium- to high- leaves in Nt-Top2 plants were light green, a phenotype that dis- − − intensity light (300–1,000 μE·m 2·s 1), displayed no discernible appeared during development so that older leaves were in- phenotype. Surprisingly, when photosynthetic pigment accumu- distinguishable from wild-type leaves (Fig. S2A). Consistent with lation was analyzed, the transplastomic lines showed slightly the light-green phenotype under low-light conditions, the levels of higher levels of carotenoids and chlorophylls than seen in wild- all photosynthetic pigments were slightly lower in Nt-Top2 plants type plants (Fig. 4B). This result suggests that no competition for (but not in Nt-SyHPT plants) than in the wild type (Fig. S2B). isoprenoid substrates occurs in the tocopherol-overproducing Previous work has implicated tocopherols in the protection of plants, at least not to an extent that affects pigment biosynthesis plants from oxidative stress (40–42). To test whether our tocoph- by precursor limitation. erol-overaccumulating Nt-SyHPT and Nt-Top2 transplastomic

Lu et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 tobacco plants show increased tolerance to photooxidative stress, lines (Fig. 3). Importantly, the tricistronic operon transcripts in we performed cold-stress recovery assays under conditions known the Sl-IPA-Top2, Sl-DG-Top2, and Sl-GP-Top2 lines underwent to promote greatly the production of reactive oxygen species in correct processing into stable monocistronic mRNAs for all chloroplasts (43–45). Although wild-type plants showed severe three genes of the operon, indicating that the IEE, originally symptoms of photooxidative damage (evidenced by leaf bleaching) identified in tobacco (38), also serves as a faithful RNA-pro- under these conditions, both the Nt-SyHPT and the Nt-Top2 cessing element in other species. transplastomic plants were much less affected (Fig. 4C), suggesting that their high tocochromanol content confers strong protection Tocochromanol Biosynthesis in Leaves and Fruits of Transplastomic from oxidative stress. Tomato Plants. Tocochromanol biosynthesis in the six sets of transplastomic tomato plants was analyzed in leaves and fruits Expression of Tocopherol Biosynthetic Enzymes from the Plastid (Fig. 5). To assess the dependence of tocopherol accumulation Genomes of Red-Fruited and Green-Fruited Tomato Varieties. Toco- on leaf age and plant development as well, young leaves (the chromanols of plant origin provide vitamin E to animals and second or third leaf from the top) and mature (fully expanded humans and therefore represent essential components of the but not yet senescent) leaves were analyzed comparatively. In human diet. We previously reported the development of a plastid line with the results obtained with HPT expression in trans- transformation protocol for tomato, a food crop with an edible plastomic tobacco plants, the Sl-SyHPT lines showed a strong fruit (46). To be able to compare the regulation of tocopherol increase in leaf tocochromanol synthesis that was very similar in biosynthesis and the rate-limiting steps in leaves versus fruits and, all three tomato genotypes (Fig. 5 A, C, and E). The increase in at the same time, to test if the results obtained in tobacco are tocochromanol accumulation was more pronounced in mature transferable to an important food crop, we attempted to in- leaves, where tocochromanol levels were approximately sixfold troduce the pSyHPT and pTop2 constructs that had led to greatly higher in the transplastomic lines than in the wild type. As in elevated tocopherol synthesis in transplastomic tobacco into the tobacco leaves, expression of the cyanobacterial HPT enzyme in tomato plastid genome. (Because plastid transformation is con- transplastomic tomato plants induced the biosynthesis of toco- siderably more laborious and time-consuming in tomato than in trienols, which normally do not accumulate in leaves of di- tobacco, initial construct testing and optimization was done in the cotyledonous plants (6). In all three tomato varieties, expression tobacco system.) of the Top2 tocopherol operon led to an even stronger increase Our previous work has shown that, during chloroplast-to-chro- in leaf tocochromanol accumulation than seen with HPT ex- moplast conversion in tomato fruit ripening, plastid gene and pression alone, reaching levels up to tenfold higher than in the transgene expression are strongly down-regulated at both the wild type (Fig. 5 A, C, and E). transcriptional and translational levels (47, 15). To avoid limi- When tocochromanols were measured in ripe tomato fruits of tations from inefficient plastid gene expression and to distinguish the red-fruited variety IPA-6, the increase in tocochromanol accu- between effects from tissue-specific regulation (fruit versus leaf) mulation in the transplastomic lines compared with the wild type and effects from plastid differentiation (chloroplast versus chro- was significantly lower than in leaf tissue (Fig. 5B). Also, no in- moplast), we wanted to compare tocopherol metabolism in red- duction of tocotrienol biosynthesis was seen in red fruits. This result fruited and green-fruited tomato varieties expressing the pSyHPT is consistent with a strong decline in plastid gene expression activity and pTop2 constructs. Because the only established tomato plastid occurring upon chloroplast-to-chromoplast conversion, as has been transformation protocol is for two closely related red-fruited to- observed previously (47, 15), but also could be the result of pre- mato varieties bred in Brazil (46, 48), we first needed to develop cursor limitation (i.e., lower GGDP availability in red fruits). In- protocols for green-fruited varieties (see Fig. S5). For two well- terestingly, ripe tomato fruits of the two green-fruited varieties regenerating commercial varieties, “Dorothy’sGreen” and “Green Dorothy’s Green and Green Pineapple showed much higher toco- Pineapple”, we succeeded in obtaining transplastomic plants using chromanol accumulation than tomatoes of the red-fruited variety optimized selection and regeneration protocols (49). Thus we were IPA-6 (Fig. 5 D and F). Total tocochromanol contents in fruits of able to test whether green-fruited, chloroplast-containing tomato HPT-expressing transplastomic Dorothy’s Green plants were twice varieties express plastid transgenes more efficiently than red- as high as in transplastomic IPA-6 fruits, whereas transplastomic fruited, chromoplast-containing varieties and to analyze tocochro- Green Pineapple fruits had threefold higher tocochromanol con- manol biosynthesis comparatively in ripe green fruits and red fruits. tents. These results correlate with Green Pineapple fruits having We introduced the two constructs that led to strongly elevated the highest chlorophyll content and thus presumably the highest tocopherol synthesis in tobacco (the single-gene construct pSyHPT activity of plastid gene expression (Fig. S5A). Unlike HPT expression and the operon construct pTop2; Fig. 2) into the plastid genomes in IPA-6, expression in Dorothy’s Green and Green Pineapple led of the three tomato varieties that now can be transformed: the red- to tocotrienol accumulation in fruits. This accumulation is consis- fruited variety IPA-6 and the green-fruited varieties Dorothy’s tent with higher HPT expression levels in green-fruited varieties Green and Green Pineapple. The fruits of Green Pineapple differ enhancing the side activity of the Synechocystis HPT as a homoge- from those of Dorothy’s Green in that they are smaller, somewhat ntisic acid geranylgeranyl transferase (HGGT) (Fig. 1) (23). darker green, and have less white (non–green plastid-containing) Surprisingly, coexpression of TCY and TMT with HPT from fruit flesh (see Fig. S5A). the Top2 operon did not result in a further increase in toco- Transplastomic tomato lines were generated with both con- chromanol accumulation in tomato fruits (Fig. 5 B, D, and F). structs and for all three genotypes. The lines are designated Sl- This result is in stark contrast to the situation in tobacco and IPA (for Solanum lycopersicum IPA-6), Sl-DG (for S. lycopersi- tomato leaves, where expression of the tocopherol operon cum Dorothy’s Green), and Sl-GP (for S. lycopersicum Green caused an additional boost in tocochromanol synthesis (Figs. 3A Pineapple). Regenerated transplastomic plants were character- and 5 A, C, and E). This finding indicates that, despite its ized by RFLP analyses (compare Fig. S3 A and B with Fig. 2), overexpression, HPT still represents the rate-limiting enzyme of which verified correct integration into the targeted region of the the tocopherol pathway in the Sl-SyHPT and Sl-Top2 tomatoes tomato plastid genome (50) and provided preliminary evidence and suggests that tocochromanol biosynthesis exhibits different of homoplasmy. The homoplasmic state subsequently was con- bottlenecks in different tissues. firmed by inheritance assays (Fig. S3C). To follow the time course of tocochromanol accumulation Northern blot analyses detected very similar transcript accu- during the fruit-ripening process, four different ripening stages mulation patterns in transplastomic tomato plants (Fig. S4), as were investigated in the red-fruited tomato variety IPA-6 and seen previously in the corresponding transplastomic tobacco the green-fruited tomato variety Green Pineapple. Interestingly,

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1216898110 Lu et al. Downloaded by guest on September 26, 2021 PNAS PLUS SCIENCES APPLIED BIOLOGICAL

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 transplastomic lines expressing the Top2 operon were analyzed. Two independently generated transplastomic lines per construct and four different plants per line were measured. 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 of both tocopherols and tocotrienols could not be separated by chromatography and therefore are represented in a single bar. To determine leaf tocopherol contents, 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 variety Green Pineapple. (E) Tocochromanol accumulation in leaves of the green-fruited variety Dorothy’s Green. (F) Tocochromanol accumulation in fruits of the green-fruited variety Dorothy’s Green. Note that both Dorothy’s Green and Green Pineapple are commercial green-fruited varieties that do not accumulate carotenoids but ripen normally (so-called “green when ripe” varieties) (63).

although the tocochromanol levels increased continuously during Discussion the fruit-ripening process in both varieties (and in the wild-type Using tocochromanol (vitamin E) biosynthesis as an example, as well as in Sl-SyHPT and Sl-Top2 tomatoes), the rate of in- our work described here has uncovered efficient strategies by crease was much higher in the transplastomic Green Pineapple which metabolic pathway engineering can be performed by stable fruits (Fig. S6). This result suggests that continued transgene ex- transformation of the plastid genome. Because vitamin E is an pression in fruit chloroplasts largely accounts for the very high essential vitamin that has a diversity of beneficial physiological tocochromanol accumulation levels in ripe tomatoes of the green- and disease-preventing properties (1), elevating the vitamin E fruited varieties (Fig. 5). content is an important goal of breeding and genetic engineering

Lu et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 efforts (2, 3, 51–53). In addition to enhancing the nutritional maternal inheritance of the plastid DNA, plastid transformation value of crops, increased levels of vitamin E compounds can also greatly reduces the risk of unwanted pollen transmission, thus improve plant performance under stressful conditions that are improving the biosafety of transgenic plants. In this respect, we linked to reactive oxygen species (Fig. 3C) (54). Our results built an additional useful feature into our operon vector: two loxP confirm that the optimal approaches to maximize vitamin E sites that can be used to eliminate the aadA selectable marker gene contents differ in leafy tissues and nonleafy tissues. In leaves, by transiently crossing in a nuclear-encoded plastid-targeted Cre expression of all three pathway enzymes from a synthetic operon recombinase (56, 15). proved to be superior to expression of the rate-limiting enzyme A byproduct of this work has been the development of plastid HPT alone (Figs. 3 and 5), perhaps because the efficient con- transformation for green-fruited tomato varieties. The benefitof version of 2-methyl-6-phytylbenzoquinone and 2,3-dimethyl-5- using green-fruited tomato varieties for the synthesis of nutraceut- phytyl-1,4-benzoquinone into tocopherols by TCY and TMT icals and pharmaceuticals is twofold. First, the long-term presence (Fig. 1) creates a metabolic sink that ultimately diverts more of chloroplasts in the fruit ensures a higher activity of plastid gene precursors into the pathway. In contrast, in both green and red expression than in chromoplast-containing red fruits (47). Low tomato fruits, HPT expression triggered higher vitamin E syn- levels of gene expression also were described for other nongreen thesis than expression of the entire operon (Fig. 5). A possible plastid types (e.g., amyloplasts), although recent work has suggested reason is that HPT poses a stronger limitation to tocochromanol strategies to overcome these limitations, at least partially (57–59). biosynthesis in fruits than in leaves, so that coexpression of the Second, green-fruited tomatoes can greatly simplify identity pres- other pathway enzymes not only is unable to confer a further ervation (of transgenic tomatoes, nutritionally enhanced tomatoes, boost in vitamin E synthesis but even slightly reduces the bene- or “pharma” tomatoes), at least in countries where only red-fruited ficial effects of HPT expression. This effect could be caused by tomatoes are marketed. The expansion of the genotype range for the two additional transgenes draining away plastid expression tomato plastid transformation reported here represents an impor- capacity that is known to be limited in tomato fruits (47, 15). It tant step toward the application of the transplastomic technology in also is possible that precursor availability (e.g., phytyl phosphate an important food crop on a more routine basis. from chlorophyll turnover; ref. 55) limits tocopherol bio- Analysis of carotenoid and chlorophyll accumulation in synthesismorestronglyinfruitsthaninleavesandinthisway transplastomic plants overaccumulating vitamin E revealed an contributes to the different levels of vitamin E accumulation in interesting regulatory connection between tocochromanol bio- different tissues (Fig. 5). synthesis and the pathways of photosynthetic pigment bio- Previously, impressive results have been obtained by enhancing synthesis. Surprisingly, both carotenoid and chlorophyll levels the tocopherol pathway via nuclear transformation. In addition, were found to be slightly increased in plants overproducing to- these studies provided interesting insights into limiting toco- copherol, at least when grown under standard conditions (Fig. chromanol intermediates and rate-limiting biochemical reactions. 3B). This observation may suggest that the increased flux through For example, a comprehensive study in soybean seeds revealed the tocochromanol pathway results in a general stimulation of that, at the metabolite level, the availability of PDP represents the isoprenoid-using pathways (possibly through positive feedback most serious limitation (3). Our data obtained here suggest that, regulation). Alternatively, higher levels of protection from pho- at least in green leaves, plastid engineering can be even more tooxidative stress could lead to reduced turnover of photosyn- efficient than nuclear genetic engineering, especially with the thetic pigments. Interestingly, the plants expressing the synthetic synthetic operon design developed here. Nuclear expression of tocopherol operon and showing the strongest overaccumulation HPT in A. thaliana induced an up to 4.4-fold increase in leaf to- of tocochromanols displayed a mild pigment deficiency under copherol (5), whereas in our plastid operon-expressing lines, an low-light conditions (Fig. S2). This deficiency indicates that the up to 10-fold increase was obtained (Fig. 5). However, the most level of tocochromanol synthesis we have achieved with our significant advantages of plastid engineering lie in its unique transplastomic approach probably is close the maximum that is precision (because of transgene integration by homologous re- possible without negatively affecting metabolism and, hence, combination) and the possibility of stacking multiple transgenes in plant fitness. It is noteworthy in this respect that spinach, one of operons for coexpression as polycistronic mRNAs, which cur- the vegetable plants with the highest vitamin E content, has ap- rently is not feasible in the nuclear genome. The functioning of the proximately four- to fivefold higher tocochromanol levels than IEE (38) in very different sequence contexts, including nptII, yfp, tomato fruits (60). Thus, the level in spinach is similar to the and the three tocopherol biosynthesis genes tested here, suggests levels reached in our best-performing transplastomic tomatoes that the IEE triggers processing of polycistronic transcripts into but is surpassed significantly by the levels reached in the leaves of stable and translatable monocistronic units in a sequence context- transplastomic plants expressing the synthetic tocopherol operon. independent manner and thus provides a generally usable tool for In sum, our work presented here provides efficient strategies the construction of synthetic operons that are expressed effi- for enhancing the vitamin E content in leaves and fruits through ciently. Therefore, the synthetic operon design developed in this plastid genome engineering and pinpoints principles of synthetic study will facilitate future synthetic biology applications in plas- operon design for efficient metabolic pathway engineering and fi tids, especially the construction of complex arti cial operons that synthetic biology in plastids. introduce novel biochemical pathways into plants. Because of the lack of epigenetic gene silencing and position effects in plastids, Materials and Methods the transplastomic technology also entails an enormous reduction Plant Material and Growth Conditions. Tobacco (N. tabacum cv. Petite Havana) in the number of transgenic events that need to be generated and plants were grown under aseptic conditions on agar-solidified medium con- analyzed. For example, although 48 independent HGGT trans- taining 30 g/L sucrose (19). The tomato (S. lycopersicum)varietyIPA-6isacom- genic events had to be analyzed to identify a strong tocotrienol mercial red-fruited cultivar bred in Brazil. The green-fruited varieties Green accumulation phenotype [in which tocotrienols accounted for 74% Pineapple and Dorothy’s Green are presumably unrelated heirloom tomatoes of the total content of tocochromanols of maize embryos (6)], (of unclear genetic origin) that were first described by tomato growers in generation of one or two transplastomic lines per construct usually the United States (http://t.tatianastomatobase.com:88/wiki/Green_Pineapple; fi fi http://t.tatianastomatobase.com:88/wiki/Dorothy%27s_Green). Both are com- suf ces. The savings become even more signi cant when multiple mercial varieties that do not accumulate carotenoids but ripen normally (so- transgenes need to be introduced, a process that in conventional called “green when ripe” varieties). Tomato plants were raised from surface- nuclear transformation methodology typically involves cotrans- sterilized seeds on the same medium with 20 g/L sucrose. Regenerated shoots formation and analysis of even more events or the combination of from transplastomic lines were rooted and propagated on the same medium. individual transgenic events by crossing (3). Finally, because of Rooted homoplasmic plants were transferred to soil and grown to maturity

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1216898110 Lu et al. Downloaded by guest on September 26, 2021 under standard greenhouse conditions (relative humidity: 55%; day tempera- total DNA were treated with restriction enzymes, separated in 1% agarose PNAS PLUS ture: 25 °C; night temperature: 20 °C; diurnal cycle: 16 h light/8 h darkness; light gels, and transferred onto Hybond XL nylon membranes (GE Healthcare) by − − intensity: 190–600 μE·m 2·s 1). The homoplasmic state of the T1 generation was capillary blotting. For hybridization, [α32P]dCTP-labeled probes were pro- tested by germinating seeds from selfed transplastomic plants on medium with duced by random priming (Multiprime DNA labeling kit; GE Healthcare). spectinomycin (500 mg/L for tobacco, 100 mg/L for tomato). Hybridizations were performed at 65 °C using standard protocols. Total plant RNA was isolated by a guanidine isothiocyanate/phenol-based Construction of Plastid Transformation Vectors. Monocistronic expression method (peqGOLD TriFast; Peqlab). RNA samples (3 μg total RNA) were cassettes for enzymes of the tocopherol pathway were assembled in the denatured, separated in denaturing formaldehyde-containing agarose plastid expression vector pHK20 (14). The operon constructs pTOP1 and gels (1%), and blotted onto Hybond XL nylon membranes (GE Healthcare) pTOP2 were assembled from the genes cloned individually into the pHK20 by capillary blotting. backbone. The cloning procedures and the sources of genes and expression A 550-bp PCR product generated by amplification of a portion of the psaB elements are described in detail in SI Materials and Methods. Synthetic oli- coding region using primers P7247 and P7244 (48) and a 283-bp PCR product gonucleotides used are listed in Table S1. The sequences of the synthetic generated by amplification of the psbZ coding region using Pycf9a and Pycf9b constructs were submitted to the GenBank Nucleotide Sequence Database at the National Center for Biotechnology Information and can be obtained (38) were used as RFLP probes to verify plastid transformation and assess fi under the accession nos. JX235342 (pSyHPT), JX235343 (pSyTCY), JX235344 homoplasmy. Transgene-speci c probes for Northern blot analyses were (pSyTMT), JX235341 (pAtTMT), JX235345 (pTop1), and JX235346 (pTop2). generated by excising the complete coding regions from plasmid clones.

Plastid Transformation and Selection of Transplastomic Lines. Young leaves HPLC Analysis of Pigments and Tocochromanols. Tocochromanols were from aseptically grown tobacco and tomato plants were bombarded with extracted with methanol from lyophilized leaf powder or lyophilized tomato plasmid-coated 0.6-μm gold particles using a helium-driven biolistic gun fruit powder. Separation, identification, and quantification of tocochroma- (PDS1000He; BioRad) with the Hepta Adapter setup. Primary spectinomycin- nols were carried out with an YMC ODS-A 250 × 4.6 mm column using the resistant lines were selected on plant-regeneration medium containing 500 Agilent 1100 Series HPLC system. Separation was performed according to mg/L spectinomycin (19, 46, 48). published procedures (42). Reversed-phase HPLC was used to avoid coelution To develop a plastid transformation protocol for green-fruited tomato of γ-tocopherol with plastochromanol-8 (9). Carotenoids and chlorophylls varieties, we tested a number of commercially grown green-fruited varieties were isolated from lyophilized leaf powder samples by extraction with 80% for their tissue culture and regeneration properties. In these analyses, we (vol/vol) acetone followed by two extractions with 100% acetone. The three identified two varieties (Dorothy’s Green and Green Pineapple), that dis- extracts subsequently were combined and used for HPLC analysis. Separation, played good regeneration rates from leaf explants and also showed accept- identification, and quantification of pigments were performed as described able rates of rooting on synthetic medium as well as good fruit and seed set in previously (61) using the HPLC system specified above. All tocochromanol and SCIENCES the greenhouse (Fig. S5). Regeneration medium and selection conditions for pigment species were identified and quantified by comparison with known APPLIED BIOLOGICAL plastid transformation of green-fruited tomato varieties were as described amounts of pure standards. previously for red-fruited varieties (46, 48). The plastid transformation effi- ciencies expressed as number of confirmed transplastomic lines (pSyHPT + Cold Stress and Recovery Assay. To assess and compare the tolerance of pTop2) per number of selection plates (Petri dishes) with bombarded leaf transplastomic and wild-type plants to oxidative stress, greenhouse-grown pieces were five pSyHPT + three pTop2 events per 19 plates for IPA-6, nine plants were transferred to a growth chamber adjusted to cold stress conditions pSyHPT + eight pTop2 events per 469 plates for Dorothy’s Green, and three μ · −2· −1 pSyHPT + 10 pTop2 events per 674 plates for Green Pineapple. (temperature: 4 °C; day length: 16 h; light intensity: 70 E m s ). Following Spontaneous spectinomycin-resistant tobacco and tomato lines were exposure to cold stress for 1 mo, plants were transferred back to normal μ · −2· −1 identified by double-selection tests on regeneration medium containing 500 growth conditions (temperature: 25 °C; light intensity: 250 E m s )for1wk. mg/L spectinomycin and 500 mg/L streptomycin (19, 20) and subsequently The top three leaves then were phenotypically compared and photographed. were eliminated. Several independent transplastomic lines were generated for each construct and were subjected to one to three additional rounds of ACKNOWLEDGMENTS. We thank Stefanie Seeger and Claudia Hasse for help regeneration on spectinomycin-containing regeneration medium to enrich with chloroplast transformation, Dr. Eugenia Maximova for microscopy, the the transplastome and select for homoplasmic cell lines. Max Planck Institute for Molecular Plant Physiology (MPI-MP) Green Team for 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) Isolation of Nucleic Acids and Hybridization Procedures. Total plant DNA was for helpful discussions. This work was supported by a Grant European Union- extracted from fresh leaf samples by a cetyltrimethylammonium bromide- Seventh Framework Programme METAPRO 244348 from the European based method (15). For RFLP analysis by Southern blotting, 5-μg samples of Union (to R.B.).

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