Transplastomic Tobacco Research Articles

Features of the eGFP Gene Expression in Transplastomic Tobacco Plants

Transplastomic tobacco plants expressing a reporter gene egfp and selectable marker gene aadA as part of a bicistronic operon were obtained using the bioballistics method. Features of egfp gene expression were studied in two groups of transplastomic plants: seed progeny obtained from self-pollination and plants obtained as a result of two successive regeneration cycles from the leaves of the original transformants. A comparative fluorimetric analysis of the recombinant protein accumulation in groups of transplastomic plants and nuclear transformants was carried out. It was found that the amount of recombinant eGFP protein accumulated in the leaves of transplastomic plants was unexpectedly low and did not exceed the level established for nuclear transformants. Real-time PCR results showed that the low level of recombinant eGFP accumulation was not associated with low levels of transgene expression or the presence of nontransgenic copies of the plastid genome in chloroplasts. This is most likely due to limitations imposed at the level of translation of recombinant proteins in chloroplasts.

Membrane protein provision controls prothylakoid biogenesis in tobacco etioplasts.

The cytochrome b559 heterodimer is a conserved component of photosystem II whose physiological role in photosynthetic electron transfer is enigmatic. A particularly puzzling aspect of cytochrome b559 has been its presence in etiolated seedlings, where photosystem II is absent. Whether or not the cytochrome has a specific function in etioplasts is unknown. Here, we have attempted to address the function of cytochrome b559 by generating transplastomic tobacco (Nicotiana tabacum) plants that overexpress psbE and psbF, the plastid genes encoding the two cytochrome b559 apoproteins. We show that strong overaccumulation of the PsbE apoprotein can be achieved in etioplasts by suitable manipulations of the promoter and the translation signals, while the cytochrome b559 level is only moderately elevated. The surplus PsbE protein causes striking ultrastructural alterations in etioplasts; most notably, it causes a condensed prolamellar body and a massive proliferation of prothylakoids, with multiple membrane layers coiled into spiral-like structures. Analysis of plastid lipids revealed that increased PsbE biosynthesis strongly stimulated plastid lipid biosynthesis, suggesting that membrane protein abundance controls prothylakoid membrane biogenesis. Our data provide evidence for a structural role of PsbE in prolamellar body formation and prothylakoid biogenesis, and indicate that thylakoid membrane protein abundance regulates lipid biosynthesis in etioplasts.

Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Transplastomic plants — new approaches to solving “old” problems

Transplastomic plants are capable to accumulate the significant amounts (up to 70% of TSP) of target recombinant proteins in tissues. However, the production of such forms is severely limited by the low yield of initial transformants. This problem requires the development and optimization of new approaches to the delivery of transgenes into chloroplasts and an increase in the frequency of their integration into the plastome. Transplastomic tobacco plants expressing thegfpreporter gene and theaadAselectable marker under the control of thePrrnG10Lpromoter and theTpsbAterminator were obtained in the laboratory of plant bioengineering. It is known that the selected promoter and insertion region (between the tRNA genes of isoleucine and alanine) are capable to provide a high yield of recombinant proteins in the leaves of transplastomic plants [1]. However, the content of recombinant GFP in the leaves of the obtained transplastomic plants was determined at the level of 0.12%, and the variability for this trait was minimal and ranged from 0.09 to 0.16% of TSP. Insufficient accumulation of the target protein in transformants is not associated with transcription disorders or the presence of non-transgenic copies of the plastome. Probably, the low frequency of transformation and the lack of variability between the transformants are the reasons that make it difficult to select highly productive forms. It is proposed to increase the efficiency of targeted delivery of genetic constructs to plastids using single-walled carbon nanotubes loaded with recombinant DNA. This process can also be facilitated by our proposed approach to increase the frequency of DNA double-strand breaks in target regions of the plastome through the use of the CRISPR-Cas9 genome editing system.
 This work was supported by the Russian Science Foundation grant No. 23-24-00545.

A biotechnological approach for the production of new protein bioplastics.

The future of biomaterial production will leverage biotechnology based on the domestication of cells as biological factories. Plants, algae, and bacteria can produce low-environmental impact biopolymers. Here, two strategies were developed to produce a biopolymer derived from a bioengineered vacuolar storage protein of the common bean (phaseolin; PHSL). The cys-added PHSL* forms linear-structured biopolymers when expressed in the thylakoids of transplastomic tobacco leaves by exploiting the formation of inter-chain disulfide bridges. The same protein without signal peptide (ΔPHSL*) accumulates in Escherichia coli inclusion bodies as high-molar-mass species polymers that can subsequently be oxidized to form disulfide crosslinking bridges in order to increase the stiffness of the biomaterial, a valid alternative to the use of chemical crosslinkers. The E. coli cells produced 300 times more engineered PHSL, measured as percentage of total soluble proteins, than transplastomic tobacco plants. Moreover, the thiol groups of cysteine allow the site-specific PEGylation of ΔPHSL*, which is a desirable functionality in the design of a protein-based drug carrier. In conclusion, ΔPHSL* expressed in E. coli has the potential to become an innovative biopolymer.

The lyophilized chloroplasts store synthetic DARPin G3 as bioactive encapsulated organelles

BackgroundThe high cost of fermentation, purification, cold storage and transportation, short shelf life, and sterile delivery methods of biopharmaceuticals, is a matter for producers and consumers as well. Since the FDA has now approved plant cells for large-scale, cost-effective biopharmaceutical production, the isolation and lyophilization of transplastomic chloroplasts can cover concerns about limitations. DARPins are engineered small single-domain proteins that have been selected to bind to HER2 with high affinity and specificity. HER2 is an oncogene involved in abnormal cell growth in some cancers and the target molecule for cancer immunotherapy.ResultsIn this study, we reported the prolonged stability and functionality of DARPin G3 in lyophilized transplastomic tobacco leaves and chloroplasts. Western blot analysis of lyophilized leaves and chloroplasts stored at room temperature for up to nine months showed that the DARPin G3 protein was stable and preserved proper folding. Lyophilization of leaves and isolated chloroplasts increased DARPin G3 protein concentrations by 16 and 32-fold, respectively. The HER2-binding assay demonstrated that the chloroplast-made DARPin G3 can maintain its stability and binding activity without any affinity drop in lyophilized leaf materials throughout this study for more than nine months at room temperature.ConclusionLyophilization of chloroplasts expressing DARPin G3 would further reduce costs and simplify downstream processing, purification, and storage. Compressed packages of lyophilized chloroplasts were much more effective than lyophilized transplastomic leaves considering occupied space and downstream extraction and purification of DARPin G3 after nine months. These methods facilitate any relevant formulation practices for these compounds to meet any demand-oriented needs.

Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon.

The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design-Build-Test-Learn (DBTL) approach. This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites.This figure was drawn using BioRender ( https://www.biorender.com ).

Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels.

In plant biotechnology and basic research, chloroplasts have been used as chassis for the expression of various transgenes. However, potential unintended side effects of transgene insertion and high-level transgene expression on the expression of native chloroplast genes are often ignored and have not been studied comprehensively. Here, we examined expression of the chloroplast genome at both the transcriptional and translational levels in five transplastomic tobacco (Nicotiana tabacum) lines carrying the identical aadA resistance marker cassette in diverse genomic positions. Although none of the lines exhibits a pronounced visible phenotype, the analysis of three lines that contain the aadA insertion in different locations within the petL-petG-psaJ-rpl33-rps18 transcription unit demonstrates that transcriptional read-through from the aadA resistance marker is unavoidable, and regularly causes overexpression of downstream sense-oriented chloroplast genes at the transcriptional and translational levels. Investigation of additional lines that harbour the aadA intergenically and outside of chloroplast transcription units revealed that expression of the resistance marker can also cause antisense effects by interference with transcription/transcript accumulation and/or translation of downstream antisense-oriented genes. In addition, we provide evidence for a previously suggested role of genomically encoded tRNAs in chloroplast transcription termination and/or transcript processing. Together, our data uncover principles of neighbouring effects of chloroplast transgenes and suggest general strategies for the choice of transgene insertion sites and expression elements to minimize unintended consequences of transgene expression on the transcription and translation of native chloroplast genes.

Chloroplasts evolved an additional layer of translational regulation based on non-AUG start codons for proteins with different turnover rates

Chloroplasts have evolved from photosynthetic cyanobacteria-like progenitors through endosymbiosis. The chloroplasts of present-day land plants have their own transcription and translation systems that show several similarities with prokaryotic organisms. A remarkable feature of the chloroplast translation system is the use of non-AUG start codons in the protein synthesis of certain genes that are evolutionarily conserved from Algae to angiosperms. However, the biological significance of such use of non-AUG codons is not fully understood. The present study was undertaken to unravel the significance of non-AUG start codons in vivo using the chloroplast genetic engineering approach. For this purpose, stable transplastomic tobacco plants expressing a reporter gene i.e. uidA (GUS) under four different start codons (AUG/UUG/GUG/CUG) were generated and β-glucuronidase (GUS) expression was compared. To investigate further the role of promoter sequences proximal to the start codon, uidA was expressed under two different chloroplast gene promoters psbA and psbC that use AUG and a non-AUG (GUG) start codons, respectively, and also showed significant differences in the DNA sequence surrounding the start codon. Further, to delineate the role of RNA editing that creates AUG start codon by editing non-AUG codons, if any, which is another important feature of the chloroplast transcription and translation system, transcripts were sequenced. In addition, a proteomic approach was used to identify the translation initiation site(s) of GUS and the N-terminal amino acid encoded when expressed under different non-AUG start codons. The results showed that chloroplasts use non-AUG start codons in combination with the translation initiation site as an additional layer of gene regulation to over-express proteins that are required at high levels due to their high rates of turnover.

Abatement of microfibre pollution and detoxification of textile dye - Indigo by engineered plant enzymes.

Microfibres (diameter <5 mm) and textile dyes released from textile industries are ubiquitous, cause environmental pollution, and harm aquatic flora, fauna, animals and human life. Therefore, enzymatic abatement of microfibre pollution and textile dye detoxification is essential. Microbial enzymes for such application present major challenges of scale and affordability to clean up large scale pollution. Therefore, enzymes required for the biodegradation of microfibres and indigo dye were expressed in transplastomic tobacco plants through chloroplast genetic engineering. Integration of laccase and lignin peroxidase genes into the tobacco chloroplast genomes and homoplasmy was confirmed by Southern blots. Decolorization (up to 86%) of samples containing indigo dye (100 mg/L) was obtained using cp-laccase (0.5% plant enzyme powder). Significant (8-fold) reduction in commercial microbial cellulase cocktail was achieved in pretreated cotton fibre hydrolysis by supplementing cost effective cellulases (endoglucanases, ß-glucosidases) and accessory enzymes (swollenin, xylanase, lipase) and ligninases (laccase lignin peroxidase) expressed in chloroplasts. Microfibre hydrolysis using cocktail of Cp-cellulases and Cp-accessory enzymes along with minimal dose (0.25% and 0.5%) of commercial cellulase blend (Ctec2) showed 88%-89% of sugar release from pretreated cotton and microfibres. Cp-ligninases, Cp-cellulases and Cp-accessory enzymes were stable in freeze dried leaves up to 15 and 36 months respectively at room temperature, when protected from light. Use of plant powder for decolorization or hydrolysis eliminated the need for preservatives, purification or concentration or cold chain. Evidently, abatement of microfibre pollution and textile dye detoxification using Cp-enzymes is a novel and cost-effective approach to prevent their environmental pollution.

Expression strategies for the efficient synthesis of antimicrobial peptides in plastids

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides.

Producing fluorescent plants to lure and trap insect pests.

Producing fluorescent plants to lure and trap insect pests.

High-level expression of anti-interleukin-6 single chain variable fragment through plastid transformation technology

Interleukin-6 (IL-6) is a pleiotropic cytokine which participates in the pathogenesis of a variety of clinical disorders, including many kinds of cancers. Anti-IL-6 antibody was proved to be useful for the immunotherapy of various inflammatory diseases. Plants are low-cost platforms for producing specific proteins of therapeutic interest. Two dependent transplastomic tobacco lines expressing murine anti-IL-6 single chain variable fragment (scFv) were generated after bombardment and regeneration, homoplasmy was then verified by Southern blotting analysis. The anti-IL-6 scFv gene was successfully expressed at both transcriptional and translational levels in transplastomic tobacco plants. Functional anti-IL-6 scFv accumulated to 1% of total soluble proteins, namely 41 mg/kg fresh weight. There was no obvious phenotypic difference between the wild-type and the transplastomic tobacco plants, including the growth rate, the height of mature plants and the number of siliques. The high-level expression of anti-IL-6 scFv indicates the potential for cost-effective production of scFV using transplastomic plants.

Control of a sap-sucking insect pest by plastid-mediated RNA interference

Expression of double-stranded RNAs in plastids offers great potential for the efficient control of chewing insects. However, many insect pests do not consume plant tissue but rather feed on the host plant by sucking sap from the vascular system. Whether or not plastid-mediated RNA interference (RNAi) can be employed to control sap-sucking insects is unknown. Here, we show that five species of sap-sucking hemipteran insects acquire plastid RNA upon feeding on plants. We generated both nuclear transgenic and transplastomic tobacco plants expressing double-stranded RNAs targeting the MpDhc64C gene, a newly identified efficient target gene of RNAi whose silencing causes lethality to the green peach aphid Myzus persicae. In a whole-plant bioassay, transplastomic plants exhibited significant resistance to aphids, as evidenced by reduced insect survival, impaired fecundity, and decreased weight of survivors. The protective effect was comparable with that conferred by the best-performing nuclear transgenic plants. We found that the proportion of aphids on mature leaves of transplastomic plants was significantly lower compared with that of nuclear transgenic plants. When aphids were allowed to infest only the mature leaves, transplastomic plants grew significantly faster and were overall better protected from the pest compared with nuclear transgenic plants. When monitored by electrical-penetration-graph analyses and aphid avoidance response experiments, the insects displayed remarkable alterations in feeding behavior, which was different in nuclear transgenic and transplastomic plants, likely reflecting specific avoidance strategies to toxic RNA molecules. Taken together, our study demonstrates that plastid-mediated RNAi provides an efficient strategy for controlling at least some sap-sucking insect pests, even though there is most likely no or only very little chloroplast RNA in the sap.

Resistance to RNA interference by plant‐derived double‐stranded RNAs but not plant‐derived short interfering RNAs in Helicoverpa armigera

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

Efficient control of western flower thrips by plastid-mediated RNA interference

Plastid-mediated RNA interference (PM-RNAi) has emerged as a promising strategy for pest control. Expression from the plastid genome of stable double-stranded RNAs (dsRNAs) targeted against essential insect genes can effectively control some herbivorous beetles, but little is known about the efficacy of the transplastomic approach in other groups of pest insects, especially nonchewing insects that do not consume large amounts of leaf material. Here we have investigated the susceptibility of the western flower thrip (WFT, Frankliniella occidentalis), a notorious pest in greenhouses and open fields, to PM-RNAi. We show that WFTs ingest chloroplasts and take up plastid-expressed dsRNAs. We generated a series of transplastomic tobacco plants expressing dsRNAs and hairpin RNAs (hpRNAs) targeted against four essential WFT genes. Unexpectedly, we discovered plastid genome instability in transplastomic plants expressing hpRNAs, suggesting that dsRNA cassettes are preferable over hpRNA cassettes when designing PM-RNAi strategies. Feeding studies revealed that, unlike nuclear transgenic plants, transplastomic plants induced a potent RNAi response in WFTs, causing efficient suppression of the targeted genes and high insect mortality. Our study extends the application range of PM-RNAi technology to an important group of nonchewing insects, reveals design principles for the construction of dsRNA-expressing transplastomic plants, and provides an efficient approach to control one of the toughest insect pests in agriculture and horticulture.

Plastidial Expression of 3β-Hydroxysteroid Dehydrogenase and Progesterone 5β-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco.

The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3β-hydroxysteroid dehydrogenase (3β-HSD) and progesterone 5-β-reductases (P5βR1, P5βR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3β-HSD, P5βR1 and P5βR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3β-HSD and P5βR1 are cytoplasmic, and P5βR2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3β-HSD, P5βR1 and P5βR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering.

Mass Production of Virus-Like Particles Using Chloroplast Genetic Engineering for Highly Immunogenic Oral Vaccine Against Fish Disease.

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), which is one of the most serious fish diseases leading to mass mortality in a wide range of fish species worldwide. Although a few injectable inactivated vaccines are commercially available, there is a need for more labor-saving, cost-effective, and fish-friendly immunization methods. The use of transgenic plants expressing pathogen-derived recombinant antigens as edible vaccines is an ideal way to meet these requirements. In this study, chloroplast genetic engineering was successfully utilized to overexpress the red-spotted grouper NNV capsid protein (RGNNV-CP). The RGNNV-CP accumulated at high levels in all young, mature, and old senescent leaves of transplastomic tobacco plants (averaging approximately 3 mg/g leaf fresh weight). The RGNNV-CP efficiently self-assembled into virus-like particles (RGNNV-VLPs) in the chloroplast stroma of the transgenic lines, which could be readily observed by in situ transmission electron microscopy. Furthermore, intraperitoneal injection and oral administration of the crudely purified protein extract containing chloroplast-derived RGNNV-VLPs provided the sevenband grouper fish with sufficient protection against RGNNV challenge, and its immunogenicity was comparable to that of a commercial injectable vaccine. These findings indicate that chloroplast-derived VLP vaccines may play a promising role in the prevention of various diseases, not only in fish but also in other animals, including humans.

Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco

Integration of prokaryotic nitrogen fixation (nif) genes into the plastid genome for expression of functional nitrogenase components could render plants capable of assimilating atmospheric N2 making their crops less dependent of nitrogen fertilizers. The nitrogenase Fe protein component (NifH) has been used as proxy for expression and targeting of Nif proteins within plant and yeast cells. Here we use tobacco plants with the Azotobacter vinelandii nifH and nifM genes integrated into the plastid genome. NifH and its maturase NifM were constitutively produced in leaves, but not roots, during light and dark periods. Nif protein expression in transplastomic plants was stable throughout development. Chloroplast NifH was soluble, but it only showed in vitro activity when isolated from leaves collected at the end of the dark period. Exposing the plant extracts to elevated temperatures precipitated NifM and apo-NifH protein devoid of [Fe4S4] clusters, dramatically increasing the specific activity of remaining NifH protein. Our data indicate that the chloroplast endogenous [Fe-S] cluster biosynthesis was insufficient for complete NifH maturation, albeit a negative effect on NifH maturation due to excess NifM in the chloroplast cannot be excluded. NifH and NifM constitutive expression in transplastomic plants did not affect any of the following traits: seed size, germination time, germination ratio, seedling growth, emergence of the cotyledon and first leaves, chlorophyll content and plant height throughout development.

Inducible expression of human papillomavirus-16 L1 capsomeres in the plastomes of Nicotiana tabacum: Transplastomic plants develop normal flowers and pollen.

Human papillomavirus type-16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for 7 consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3μg/g of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. These data confirm the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology.