Chloroplast Expression Research Articles

High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts

A tobacco chloroplast expression vector, pTRVP1, containing the foot-and-mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene, was constructed and transferred to tobacco by biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. PCR and PCR southern blot analysis revealed that the VP1 gene had integrated into the chloroplast genome. Western blot and quantification ELISA assays indicated that the VP1 gene was expressed in tobacco chloroplasts and accounted for 2-3% of total soluble protein. This suggested that plant chloroplasts were an efficient expression system for the potential production of recombinant antigens in plants.

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2–3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.

Recombination and Heterologous Expression of Allophycocyanin Gene in the Chloroplast of <italic>Chlamydomonas reinhardtii</italic>

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.

Microarray Analysis Confirms the Specificity of a Chlamydomonas reinhardtii Chloroplast RNA Stability Mutant

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687-697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.

Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts

Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser → Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.

New advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen, TetC

Vaccines are a proven method of controlling disease. However there are issues with the delivery and administration of vaccines. A particular problem is that the majority of vaccines currently used are injected, which can be unsafe if needles are reused in areas where blood-borne diseases are prevalent. Vaccines targeting the mucosal immune system avoid many of the problems associated with injections. One potential form of mucosal vaccine is based on the expression of vaccine antigens in plants. Current research in this area has focused on the expression of immunogens from the plant's nuclear genome but low expression levels generally achieved using this system have limited progress. In recent work we have used the model antigen, TetC, which confers resistance to Tetanus infection, to demonstrate the feasibility of expressing vaccine antigens at high levels in the plant chloroplast.

A nucleus-encoded factor, CRR2, is essential for the expression of chloroplast ndhB in Arabidopsis.

The chloroplast NDH complex, NAD(P)H dehydrogenase, reduces the plastoquinone pool non-photochemically and is involved in cyclic electron flow around photosystem I (PSI). A transient increase in chlorophyll fluorescence after turning off actinic light is a result of NDH activity. We focused on this subtle change in chlorophyll fluorescence to isolate nuclear mutants affected in chloroplast NDH activity in Arabidopsis by using chlorophyll fluorescence imaging. crr2-1 and crr2-2 (chlororespiratory reduction) are recessive mutant alleles in which accumulation of the NDH complex is impaired. Except for the defect in NDH activity, photosynthetic electron transport was unaffected. CRR2 encodes a member of the plant combinatorial and modular protein (PCMP) family consisting of more than 200 genes in Arabidopsis. CRR2 functions in the intergenic processing of chloroplast RNA between rps7 and ndhB, which is possibly essential for ndhB translation. We have determined the function of a PCMP family member, indicating that the family is closely related to pentatrico-peptide PPR proteins involved in the maturation steps of organellar RNA.

Light-regulated differential expression of pea chloroplast and cytosolic fructose-1,6-bisphosphatases.

The light-regulated differential expression of pea chloroplast and cytosolic fructose-1,6-bisphosphatases (FBPase) was investigated using enzyme activity assay, immunoblot, and Northern blot analyses. The enzyme activities of both chloroplast and cytosolic FBPases gradually increased under continuous white light illumination, although the increase in chloroplast FBPase was more drastic. Northern and immunoblot analyses also indicated that light stimulated the expression of both enzymes. Enzyme activity and the transcript levels of both enzymes gradually decreased under the dark treatment, although protein levels were unchanged for up to 24 h following the initiation of culture in the dark, indicating that reversible modifications of the enzymes may occur during the transition from light to dark (or the reverse). Light pulse experiments using blue (420 nm) and red/far-red (660/730 nm) light were carried out to analyze the photoreceptors related to the light-mediated expression of both enzymes. Expression of the chloroplast enzyme was very sensitive to red or far-red light pulses-it was induced by red light, but suppressed by far-red light pulses, as determined by enzyme activity, immunoblot, and Northern blot analyses, suggesting that red light signaling is involved in the control of chloroplast FBPase expression. However, cytosolic FBPase was virtually insensitive to blue or red/far-red light pulses in terms of enzyme activity, as determined by protein and transcript levels, indicating that cytosolic enzyme expression is not directly regulated by light signals. Instead, the expression of the cytosolic enzyme may be closely related to photosynthetic energy conversion accompanied by continuous white light irradiation.

Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme

Overproduction of cellulolytic enzymes through conventional nucleartransformation approaches posed a major challenge as they can potentiallydegrade the cell wall components and thereby affect transgenic plant growth anddevelopment. In this study, we have tested the possibility to over produce analkali-thermostable xylanase gene from Bacillus sp. StrainNG-27 in tobacco plants through chloroplast expression. Our results showed thatthe xylanase expression can reach up to 6% of the total soluble protein, avaluecomparable to high level expression reported for several non-cellulolyticproteins in tobacco chloroplasts. The chloroplast-expressed xylanase retainedits activity even when the leaves were dried under sun or at 42°C, offering flexibility in the agricultural system intransport and storage. The recombinant enzyme was purified to homogeneity usingsingle step chromatography with more than 85% recovery. Most importantly,transgenic plants were indistinguishable from the control untransformed plantsin their morphology, growth and in seed setting. These results open up newavenues for large scale production of several other industrially usefulcellulolytic enzymes through chloroplast expression.

Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors

High level expression and efficient recovery of recombinant protein aretwo main critical factors that determine the use of transgenic plants asnaturalbioreactors to produce foreign proteins for industrial applications. Wedemonstrate here the potential of a new strategy involving chloroplasttransformation, GUS-fusions and affinity-tag based chromatography tooverexpressand purify a human therapeutic protein, interferon gamma (IFN-g) in tobaccoplants. Our results show that IFN-g accumulation reaches up to 6% of totalsolubleprotein when expressed as a GUS-fusion protein in tobacco chloroplasts.Additionof His-tag simplified the downstream process and the recombinant protein yieldswere considerably high (∼360 μg/g fresh leaf tissue).Further we demonstrate the use of GUS-fusions to identify recombinant proteincontaining fractions very rapidly (< 5 minutes) through simple GUS assay, animportant consideration for those proteins that are highly labile duringlengthyand harsh downstream processing conditions. The chloroplast-produced IFN-g isbiologically as active as the same protein obtained through E.coli expression without any involvement of refolding procedure. Ourresults demonstrate that the new strategy has tremendous potential for largescale production of proteins from heterologous source, independent of theirphysio-chemical and biological properties, using plants as ‘naturalbioreactors’.

Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast.

A Chlamydomonas reinhardtii chloroplast expression vector, pACTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed and transfered to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by PCR, Southern blot, Western blot and ELISA assays after selection on resistant medium and incubation in the dark. The CTBVP1 fusion protein was expressed in C. reinhardtii chloroplast and accounted for up to 3% of the total soluble protein. The fusion protein also retained both GM1-ganglioside binding affinity and antigenicity of the FMDV VP1 and CTB proteins. These experimental results support the possibility of using transgenic chloroplasts of green alga as a mucosal vaccine source.

Evidence for in vivo modulation of chloroplast RNA stability by 3'-UTR homopolymeric tails in Chlamydomonas reinhardtii.

Polyadenylation of synthetic RNAs stimulates rapid degradation in vitro by using either Chlamydomonas or spinach chloroplast extracts. Here, we used Chlamydomonas chloroplast transformation to test the effects of mRNA homopolymer tails in vivo, with either the endogenous atpB gene or a version of green fluorescent protein developed for chloroplast expression as reporters. Strains were created in which, after transcription of atpB or gfp, RNase P cleavage occurred upstream of an ectopic tRNA(Glu) moiety, thereby exposing A(28), U(25)A(3), [A+U](26), or A(3) tails. Analysis of these strains showed that, as expected, polyadenylated transcripts failed to accumulate, with RNA being undetectable either by filter hybridization or reverse transcriptase-PCR. In accordance, neither the ATPase beta-subunit nor green fluorescent protein could be detected. However, a U(25)A(3) tail also strongly reduced RNA accumulation relative to a control, whereas the [A+U] tail did not, which is suggestive of a degradation mechanism that does not specifically recognize poly(A), or that multiple mechanisms exist. With an A(3) tail, RNA levels decreased relative to a control with no added tail, but some RNA and protein accumulation was observed. We took advantage of the fact that the strain carrying a modified atpB gene producing an A(28) tail is an obligate heterotroph to obtain photoautotrophic revertants. Each revertant exhibited restored atpB mRNA accumulation and translation, and seemed to act by preventing poly(A) tail exposure. This suggests that the poly(A) tail is only recognized as an instability determinant when exposed at the 3' end of a message.

The three genomes of Chlamydomonas.

During the past 50 years, the green unicellular alga Chlamydomonas reinhardtii has played a key role as model system for the study of photosynthesis and chloroplast biogenesis. This is due to its well-established nuclear and chloroplast genetics, its dispensable photosynthetic function in the presence of acetate, and its highly efficient nuclear and chloroplast transformation systems. Considerable progress has been achieved in our understanding of the structure, function, inheritance, and expression of nuclear, chloroplast, and mitochondrial genes and of the molecular cross-talk between the nuclear, chloroplast, and mitochondrial genetic systems.

Expressing PHB synthetic genes through chloroplast genetic engineering

Chloroplast integration and expression vector containing expression cassettes for phbB, phbA, phbC and aadA genes was constructed and bombarded into the tobacco chloroplast genome. Transplastomic plants were analyzed with PCR and Southern blot. Their homoplastomy was also judged. Northern dot and RT-PCR analysis were employed to investigate transgene expression at transcriptional level. The results indicate that the chloroplast transformation system is compatible for poly-3-hydroxybutyrate (PHB) production.

Mobile self-splicing group I introns from the psbA gene of Chlamydomonas reinhardtii: highly efficient homing of an exogenous intron containing its own promoter.

Introns 2 and 4 of the psbA gene of Chlamydomonas reinhardtii chloroplasts (Cr.psbA2 and Cr.psbA4, respectively) contain large free-standing open reading frames (ORFs). We used transformation of an intronless-psbA strain (IL) to test whether these introns undergo homing. Each intron, plus short exon sequences, was cloned into a chloroplast expression vector in both orientations and then cotransformed into IL along with a spectinomycin resistance marker (16S rrn). For Cr.psbA2, the sense construct gave nearly 100% cointegration of the intron whereas the antisense construct gave 0%, consistent with homing. For Cr.psbA4, however, both orientations produced highly efficient cointegration of the intron. Efficient cointegration of Cr.psbA4 also occurred when the intron was introduced as a restriction fragment lacking any known promoter. Deletion of most of the ORF, however, abolished cointegration of the intron, consistent with homing. The Cr.psbA4 constructs also contained a 3-(3,4-dichlorophenyl)-1,1-dimethylurea resistance marker in exon 5, which was always present when the intron integrated, thus demonstrating exon coconversion. Remarkably, primary selection for this marker gave >100-fold more transformants (>10,000/microgram of DNA) than did the spectinomycin resistance marker. A trans homing assay was developed for Cr.psbA4; the ORF-minus intron integrated when the ORF was cotransformed on a separate plasmid. This assay was used to identify an intronic region between bp -88 and -194 (relative to the ORF) that stimulated homing and contained a possible bacterial (-10, -35)-type promoter. Primer extension analysis detected a transcript that could originate from this promoter. Thus, this mobile, self-splicing intron also contains its own promoter for ORF expression. The implications of these results for horizontal intron transfer and organelle transformation are discussed.

Plastid gene expression is not associated with midday depression in CO 2 assimilation and electron transport

To investigate the effect of diurnal variations on chloroplastic electron transport as well as accumulation of gene products associated with it, studies were carried out on Populus deltoides, a tree species. Electron transport studies showed two peak responses as a large diurnal change with pronounced midday depression in whole chain (H 2O→ MV) as well as partial reactions for PSII (H 2O→PBQ) and PSI (DCPIP→MV). The electron transport rates first increased from 05:00 h to a maximum at around 09:00 h and then showed a decrease at 13:00 h followed by recovery and further decrease. The pigments associated with electron transport chain did not show any change during the day. Surprisingly midday depression in the accumulation of transcripts and polypeptides related to electron transport was not observed. This suggests that chloroplastic gene expression is not associated with the midday depression observed for both CO 2 assimilation and electron transport. Studies on the transcripts of psbD/C operon during the day showed that there were differences in the processing pattern although the steady state levels of the processed transcripts of this operon did not show any variation.

An association between chloroplast DNA haplotypeand gender in a plant metapopulation

Theoretical models have shown that metapopulation structure can influence the evolution of the sex ratio in gynodioecious plants when the fitness of females and/or hermaphrodites varies among populations with the local sex ratio. Such frequency-dependent fitness has been demonstrated previously in natural populations of Silene vulgaris, a gynodioecious plant in which sex determination is cytonuclear. We investigated whether there was an association between chloroplast DNA (cpDNA) haplotype and sex expression, with the assumption that cpDNA haplotypes could be associated with specific male sterility variants likely found in the mitochondrial DNA, owing to co-transmission of the two cytoplasmic genomes. Two cpDNA haplotypes were studied in a collection of 111 individuals of known sex taken from 20 natural populations found in Virginia, USA. The set of haplotype 1 individuals consisted of 71.4% hermaphrodites, where as only 7.7% of the haplotype 2 individuals were hermaphroditic; a highly statistically significant difference. By extension of a previous model it was also shown that the differential clustering of these two haplotypes into local populations contributes to among-population sex ratio variation and has the potential to lower the fitness of haplotype 2 by greater than 50%, relative to that expected with no population structure.

Analysis of Some Barley Chlorophyll Mutants and Their Response to Temperature Stress

Six barley chlorophyll (Chl) mutants, viridis, flavoviridis, chlorina, xanhta, lutea, and albina, differed in the contents of Chl (a+b) and carotenoids (Cars). In accordance with their Chl-deficient phenotype, the Chl a and b and Car contents of mutants decreased from viridis to albina, only xantha had the same or even higher concentration of Cars as the wild type plant. The albina mutant completely lacked and xantha had a significantly reduced photosynthetic activity. We found quantitative differences in protein contents between wild type and mutant plants, with the lowest concentration per fresh mass in the albina mutant. Chl fluorescence analysis revealed that heat-treated barley leaves of both the wild type and Chl mutants had a lower photosystem 2 efficiency than the untreated ones. With 35S-methionine labelling and SDS-PAGE we found that six to nine de novo synthetized proteins appeared after heat shock (2 h, 42 °C) in the wild type and Chl mutants. In albina the expression of heat shock proteins (HSPs) was reduced to 50 % of that in the wild type. Hence mainly albina mutants, with a completely destroyed proteosynthetic apparatus of the chloroplasts, are able to synthesize a small set of HSPs. The albina mutant is a very useful tool for the study of different gene expression of chloroplast and nuclear DNA.

Two different modes of early development and nitrogen assimilation in gymnosperm seedlings†

SummaryLight‐independent chloroplast development and expression of genes encoding chloroplast proteins occur in many but not all species of gymnosperms. Early development in maritime pine (Pinus pinaster) seedlings was strongly light‐independent, whereas Ginkgo biloba seedlings exhibited a typical angiosperm‐like morphogenesis with differentiated patterns in light and dark. In pine, chloroplast polypeptides were undetectable in the seed embryo and accumulated in cotyledons of both light‐ and dark‐grown plants in good correlation with light‐independent chlorophyll synthesis. In contrast, chlorophyll and chloroplast proteins were only detected in light‐grown ginkgo. Pine cytosolic glutamine synthetase (GS) and ferredoxin glutamate synthase (Fd‐GOGAT) were present at low levels in the seeds and accumulated at comparable amounts in light‐ and dark‐grown seedlings. Fd‐GOGAT was also barely detectable in the seeds of ginkgo and only accumulated in green plants with mature chloroplasts. In G. biloba seeds and etiolated plants only cytosolic GS was identified, while in light‐grown seedlings this molecular form was present at low abundance and choroplastic GS was the predominant isoenzyme. The above results have been confirmed by immunolocalization of GS protein in pine and ginkgo plantlets. In pine, GS was present in the peripheral cytoplasm of mesophyll cells and also in the phloem region of the vascular bundle. Immunocytochemical analysis showed that the labelling of mesophyll and phloem cells was only cytoplasmic. In developing ginkgo, GS antigens were present in the chloroplasts of mesophyll parenchyma cells of leaflets and green cotyledons. In contrast, a weak labelling of GS was observed in the parenchyma and phloem cells of non‐green cotyledons enclosed in the seed coat. Taking all this into account, our data indicate the existence of two different modes of GS and GOGAT regulation in gymnosperms in close correlation with the differential response of plants to light. Furthermore, the results suggest that glutamine and glutamate biosynthesis is confined to the chloroplast of mesophyll cells in species with light‐dependent chloroplast, development whereas compartmentation would be required in species with light‐independent plastid development.

Changes in transcript levels of chloroplast psbA and psbD genes during water stress in wheat leaves.

In order to examine whether the decrease in gene expression of chloroplast DNA-encoded polypetides contributes to the inhibition of photosystem II (PSII) function during water stress, changes in transcript and template levels of chloroplast psbA and psbD genes (encoding the D1 and D2 reaction center proteins of PSII, respectively) were investigated in spring wheat leaves (Triticum aestivum L. cv. Longchun No. 10) using northern, Southern and dot blot analyses. The results of northern hybridization indicated that stressing wheat seedlings in polyethylene glycol (PEG) solutions with an osmotic potential of -0.5 MPa for 0, 24, 48 and 72 h, caused marked declines in the steady state levels of the psbA and psbD transcripts but did not alter their transcript processing patterns. RNA dot blot analysis further demonstrated that over the whole range of water stress investigated, the transcript levels of the two genes declined by 2- and 3-fold, respectively, relative to the same amount of total RNA. As total RNA decreased 3-fold during the process of stress, the transcript levels of psbA and psbD genes actually declined by 6- and 9-fold, respectively. These results suggest that water stress affects the expression of the psbA and psbD genes, possibly at the transcriptional level. Southern and DNA dot blot analyses consistently showed that water stress did not affect the template levels of either psbA or psbD genes, suggesting that the decreased abundance of psbA and psbD transcripts under water stress is not due to limited gene templates but likely a result of lowered gene transcriptional activity and/or changed mRNA stability.