Nicotiana tabacum: An Update on PEG-Mediated Plastid Transformation.

Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (106 viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed plastids are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of plastids in Nicotiana tabacum using an optimized protoplast culture protocol. PEG-mediated plastid transformation has the potential to be developed into a high-throughput, automated pipeline.

Low frequency paternal transmission of plastid genes in Brassicaceae.

Plastid-encoded genes are maternally inherited in most plant species. Transgenes located on the plastid genome are thus within a natural confinement system, preventing their distribution via pollen. However, a low-frequency leakage of plastids via pollen seems to be universal in plants. Here we report that a very low-level paternal inheritance in Arabidopsis thaliana occurs under field conditions. As pollen donor an Arabidopsis accession (Ler-Ely) was used, which carried a plastid-localized atrazine resistance due to a point mutation in the psbA gene. The frequency of pollen transmission into F1 plants, based on their ability to express the atrazine resistance was 1.9 × 10(-5). We extended our analysis to another cruciferous species, the world-wide cultivated crop Brassica napus. First, we isolated a fertile and stable plastid transformant (T36) in a commercial cultivar of B. napus (cv Drakkar). In T36 the aadA and the bar genes were integrated in the inverted repeat region of the B. napus plastid DNA following particle bombardment of hypocotyl segments. Southern blot analysis confirmed transgene integration and homoplasmy of plastid DNA. Line T36 expressed Basta resistance from the inserted bar gene and this trait was used to estimate the frequency of pollen transmission into F1 plants. A frequency of <2.6 × 10(-5) was determined in the greenhouse. Taken together, our data show a very low rate of paternal plastid transmission in Brassicacea. Moreover, the establishment of plastid transformation in B. napus facilitates a safe use of this important crop plant for plant biotechnology.

Nicotiana tabacum: PEG-mediated plastid transformation.

Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (10(6) viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed chloroplasts are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of chloroplasts in Nicotiana tabacum using an optimized protoplast culture protocol.

Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana.

Antimicrobial peptides are important defense compounds of higher organisms that can be used as therapeutic agents against bacterial and/or viral infections. We designed several antimicrobial peptides containing hydrophobic and positively charged clusters that are active against plant and human pathogens. Especially peptide SP1-1 is highly active with a MIC value of 0.1 µg/ml against Xanthomonas vesicatoria, Pseudomonas corrugata and Pseudomonas syringae pv syringae. However, for commercial applications high amounts of peptide are necessary. The synthetic production of peptides is still quite expensive and, depending on the physico-chemical features, difficult. Therefore we developed a plant/tobacco mosaic virus-based production system following the 'full virus vector strategy' with the viral coat protein as fusion partner for the designed antimicrobial peptide. Infection of Nicotiana benthamiana plants with such recombinant virus resulted in production of huge amounts of virus particles presenting the peptides all over their surface. After extraction of recombinant virions, peptides were released from the coat protein by chemical cleavage. A protocol for purification of the antimicrobial peptides using high resolution chromatographic methods has been established. Finally, we yielded up to 0.025 mg of peptide per g of infected leaf biomass. Mass spectrometric and NMR analysis revealed that the in planta produced peptide differs from the synthetic version only in missing of N-terminal amidation. But its antimicrobial activity was in the range of the synthetic one. Taken together, we developed a protocol for plant-based production and purification of biologically active, hydrophobic and positively charged antimicrobial peptide.

Recombinant Whirly1 translocates from transplastomic chloroplasts to the nucleus.

Whirly1 was shown to be dually located in chloroplasts and nucleus of the same cell. To investigate whether the protein translocates from chloroplasts to the nucleus, we inserted a construct encoding an HA-tagged Whirly1 into the plastid genome of tobacco. Although the tagged protein was synthesized in plastids, it was detected in nuclei. Dual location of the protein was confirmed by immunocytological analyses. These results indicate that the plastidial Whirly1 is translocated from the plastid to the nucleus where it affects expression of target genes such as PR1. Our results support a role of Whirly1 in plastid-nucleus communication.

Targeted inactivation of the tobacco plastome origins of replication A and B.

According to the Kolodner and Tewari model [Kolodner, R.D. and Tewari, K.K. (1975) Nature, 256, 708.], plastid DNA replication involves displacement-loop and rolling-circle modes of replication, which are initiated on a pair of origins of replication (ori). In accordance with the model, such a pair of oris -oriA and oriB- was described in Nicotiana tabacum [Kunnimalaiyaan, M. and Nielsen B.L. (1997b) Nucl. Acids Res. 25, 3681.]. However, as reported previously, both copies of oriA can be deleted without abolishing replication. Deletion of both oriBs was not found [Mühlbauer, S.K. et al. (2002) Plant J. 32, 175.]. Here we describe new ori inactivation lines, in which one oriB is deleted and the other copy is strongly mutated. In addition, lines oriA and oriB were deleted from the same inverted repeat. In contrast to the expectations of the model, neither oriA nor oriB is essential. Some of the deletions led to reduced growth of plants and reduced plastid DNA copy number in later stages of leaf development. The gross structure of plastid DNA was unchanged; however, the location of the ends of branched plastid DNA complexes was different in the inactivation mutants. Taken together, the results indicate that there are additional mechanisms of plastid DNA replication and/or additional origins of replication. These mechanisms seem to be different from those found in eubacteria, which, according to the endosymbiont theory, are the progenitors of plastids.

Linear molecules of tobacco ptDNA end at known replication origins and additional loci.

Higher plant plastid DNA (ptDNA) is generally described as a double-stranded circular molecule of the size of the monomer of the plastid genome. Also, the substrates and products of ptDNA replication are generally assumed to be circular molecules. Linear or partly linear ptDNA molecules were detected in our present study using pulsed-field gel electrophoresis and Southern blotting of ptDNA restricted with 'single cutter' restriction enzymes. These linear DNA molecules show discrete end points which were mapped using appropriate probes. One possible explanation of discrete ends would be that they represent origins of replication. Indeed, some of the mapped ends correlate well with the known origins of replication of tobacco plastids, i.e. both of the oriA sequences and--less pronouncedly--with the oriB elements. Other ends correspond to replication origins that were described for Oenothera hookeri, Zea mays, Glycine max and Chlamydomonas reinhardtii, respectively, while some of the mapped ends were not described previously and might therefore represent additional origins of replication.

Development of novel types of plastid transformation vectors and evaluation of factors controlling expression.

Two new vector types for plastid transformation were developed and uidA reporter gene expression was compared to standard transformation vectors. The first vector type does not contain any plastid promoter, instead it relies on extension of existing plastid operons and was therefore named "operon-extension" vector. When a strongly expressed plastid operon like psbA was extended by the reporter gene with this vector type, the expression level was superior to that of a standard vector under control of the 16S rRNA promoter. Different insertion sites, promoters and 5'-UTRs were analysed for their effect on reporter gene expression with standard and operon-extension vectors. The 5'-UTR of phage 7 gene 10 in combination with a modified N-terminus was found to yield the highest expression levels. Expression levels were also strongly dependent on external factors like plant or leaf age or light intensity. In the second vector type, named "split" plastid transformation vector, modules of the expression cassette were distributed on two separate vectors. Upon co-transformation of plastids with these vectors, the complete expression cassette became inserted into the plastome. This result can be explained by successive co-integration of the split vectors and final loop-out recombination of the duplicated sequences. The split vector concept was validated with different vector pairs.

External control of transgene expression in tobacco plastids using the bacterial lac repressor.

Although several induction systems have been described for plants containing transgenes in the nucleus, to date there is only one method for controlling transgene expression in plastids. This consists of chemical induction of a nuclear gene and import of the gene product into plastids, so that transformation of two cellular compartments is required. Here we describe a system for external control of plastid gene expression which is based entirely on plastid components and can therefore be established in a single transformation step. Our system uses modified promoters containing binding sites for the bacterial lac repressor. Chemical induction can be made with intact plants or after harvesting, which provides ecological and economic benefits.

Inducible trans-activation of plastid transgenes: expression of the R. eutropha phb operon in transplastomic tobacco.

Deleterious effects of constitutive transgene expression can occur if gene products are harmful to the transformed plant. Constraints such as growth inhibition and male sterility have been observed in plastid transformants containing the phb operon encoding the genes required for the production of the polyester polyhydroxybutyric acid (PHB). In order to induce PHB synthesis in tobacco in a well-timed manner, we have constructed a trans-activation system to regulate transcription of the phb operon in plastids. This system consists of a nuclear-located, ethanol-inducible T7RNA polymerase (T7RNAP) which is targeted to plastids harboring the phb operon under control of T7 regulatory elements. Following treatment with 5% ethanol, moderate induction of PHB synthesis was found. PHB amounts reached 1,383 ppm in dry weight, and an overall background activity of 171 ppm was measured in uninduced tissues. On the transcriptional level, T7RNAP induction was proven and we found that the phb operon is transcribed into at least two mRNAs. Without ethanol induction, development of flowers and fertile seeds was possible. Thus, the main problem of inhibitory transgene expression was solved. Our results show that this inducible trans-activation system could serve as an alternative to constitutive expression of transgenes in the plastome.

Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants.

Tobacco plants grown in vitro were supplied with a mixture of [U-13C6]glucose and unlabelled sucrose via the root system. After 20 days, leaves were harvested and extracted with water. Glucose was isolated from the extract and was analysed by 13C NMR spectroscopy. All 13C signals appeared as complex multiplets due to 13C-13C coupling. The abundance of 21 isotopologous glucose species was determined from the 13C NMR signal integrals by numerical deconvolution using a genetic algorithm. The relative fractions of specific isotopologs in the overall excess of 13C-labelled specimens establish flux contributions via glycolysis/glucogenesis, pentose phosphate pathway, citric acid cycle and Calvin cycle including 13CO2 refixation. The fluxes were modelled and reconstructed in silico by a novel rule-based approach yielding the contributions of circular pathways and the degree of multiple cycling events. The data indicate that the vast majority of the proffered [U-13C6]glucose molecules had been modified by catabolism and subsequent glucogenesis from catabolic fragments, predominantly via passage through the citric acid cycle and the pentose phosphate pathway.

Generation of marker-free plastid transformants using a transiently cointegrated selection gene.

Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops, the possibility of marker transfer to wild relatives or microorganisms cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.

Sugarbeet (Beta vulgaris L.): shoot regeneration from callus and callus protoplasts.

The successful application of recombinant DNA technology for crop plants requires efficient regeneration systems. A detailed study on the regeneration potential of callus and callus-derived protoplasts of a recalcitrant species, sugarbeet, was performed. A reproducible and highly efficient method for induction of regenerable friable callus was established from etiolated hypocotyl explants. A reduced sucrose concentration proved beneficial. Successful shoot regeneration could be demonstrated in 10 out of 12 tested lines. Seed germination, followed by callus induction and shoot regeneration required only a single culture medium. Additionally, the regeneration capacity of roots and root-derived callus was demonstrated. Highly efficient plant regeneration was also achieved when using protoplasts isolated from regenerable friable callus induced on etiolated hypocotyls explants. To our knowledge this represents the first report on callus protoplast to plant regeneration in sugarbeet.

Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis.

Tobacco chloroplast transformation is typically achieved using dominant, selectable antibiotic resistance genes such as aadA, nptII and aphA-6. An improvement would be the combination of such a marker with a visual screening system for the early and conclusive detection of plastid transformants. As such, we investigated the use of three photosynthesis-deficient plastid mutants, DeltapetA, Deltaycf3 and DeltarpoA, for the development of a phenotypic selection system. Mutant plants were used as an alternative to the wild-type as source tissue for transformation, re-introducing deleted plastid sequences and using the aphA-6 gene as a selection marker. The reconstitution of the deleted genes in transformed regenerants resulted in shoots with a visually distinct phenotype comparable to the wild-type. This transformation/selection system overcomes the common problems associated with plastid transformation, e.g. the recovery of spontaneous mutants or nuclear insertions. In addition to the benefits offered by phenotypic selection, phenotype reconstitution leads to restoration of photosynthesis, which we assume drives reconstituted plants rapidly towards homoplasmy. As such, repeated cycles of regeneration in the presence of an antibiotic selection agent are no longer required.

PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants.

The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.

Functional analysis of plastid DNA replication origins in tobacco by targeted inactivation.

Sequences described as chloroplast DNA replication origins were analysed in vivo by creating deletion and insertion mutants via plastid transformation in tobacco. Deletion of the described oriA sequence, which is located within the intron of the trnI gene, resulted in heteroplastomic transformants, when the selection marker was inserted within the intron. Removal of the complete intron sequence together with the oriA sequence, however, yielded homoplastomic transformants of normal phenotype, in which wild-type signals were no longer detectable through Southern analysis, thus bringing the role of the described oriA sequence for plastome replication into question. Similarly, deletion of sequence elements upstream of trnI, which have a possible ori function in Oenothera, did not show any effect in tobacco. The two copies of oriB, which are located at the very end of the plastome Inverted Repeats, were targeted with two different transformation vectors in a cotransformation approach. While in initial transformants integration of the selection marker could be detected at both sites, the transgene was found exclusively at one site or the other after additional rounds of regeneration. Whereas the copy of oriB in Inverted Repeat B could be completely deleted, targeting of the copy in Inverted Repeat A resulted in heteroplastomic lines, as the essential ycf1 gene was also affected. Due to the strong selection against cotransformants we conclude that at least one copy of the oriB sequence is essential for plastome replication, whereas replication appears possible without oriA elements.