Search

Efficient plastid transformation in Arabidopsis (Arabidopsis thaliana) requires genetic lines that are hypersensitive to spectinomycin due to the absence of a chloroplast acetyl-coenzyme A carboxylase (ACCase) encoded in the acetyl-coenzyme A carboxylase 2 (ACC2) nuclear gene. To obtain plastid transformation-competent oilseed rape (Brassica napus), we inactivated all nuclear encoded, chloroplast targeted ACCase copies using CRISPR–Cas9. Brassica napus (2n = 38, AACC) is a recent interspecific hybrid of Brassica rapa (2n = 20, AA) and B. oleracea (2n = 18, CC) and is expected to have at least two ACC2 copies, one from each parent. The sequenced genome has two ACC2 copies, one that is B. rapa-like and one that is B. oleracea-like. We designed single-guide RNAs (sgRNAs) that could simultaneously inactivate both nuclear ACC2 copies. We expressed Cas9 from a chimeric egg cell promoter 1.2 (EC1.2p) known to yield homozygous or biallelic mutants in Arabidopsis in the T1 generation. To maximize the probability of functionally inactivating both orthologs in a single step, each of the two vectors carried four sgRNAs. Four T0 transgenic lines were obtained by Agrobacterium tumefaciens-mediated hypocotyl transformation. Amplicon sequencing confirmed mutations in ACC2 genes in 10 T1 progeny, in seven of which no wild-type (WT) copy remained. The B. napus T2 seedlings lacking WT ACC2 gene copies exhibited a spectinomycin hypersensitive phenotype, suggesting that they will be a useful resource for chloroplast genome transformation.

Replacing the native clpP1 gene in the Nicotiana plastid genome with homologs from different donor species showed that the extent of genetic incompatibilities depended on the rate of sequence evolution. The plastid caseinolytic protease (Clp) complex plays essential roles in maintaining protein homeostasis and comprises both plastid-encoded and nuclear-encoded subunits. Despite the Clp complex being retained across green plants with highly conserved protein sequences in most species, examples of extremely accelerated amino acid substitution rates have been identified in numerous angiosperms. The causes of these accelerations have been the subject of extensive speculation but still remain unclear. To distinguish among prevailing hypotheses and begin to understand the functional consequences of rapid sequence divergence in Clp subunits, we used plastome transformation to replace the native clpP1 gene in tobacco (Nicotiana tabacum) with counterparts from another angiosperm genus (Silene) that exhibits a wide range in rates of Clp protein sequence evolution. We found that antibiotic-mediated selection could drive a transgenic clpP1 replacement from a slowly evolving donor species (S. latifolia) to homoplasmy but that clpP1 copies from Silene species with accelerated evolutionary rates remained heteroplasmic, meaning that they could not functionally replace the essential tobacco clpP1 gene. These results suggest that observed cases of rapid Clp sequence evolution are a source of epistatic incompatibilities that must be ameliorated by coevolutionary responses between plastid and nuclear subunits.

Targeting the phiC31 phage integrase for direct export from Agrobacterium to chloroplasts reveals the feasibility of retargeting the Agrobacterium Vir proteins for T-DNA delivery to chloroplasts.

The D1 polypeptide of the photosystem II (PSII) reaction center complex contains domains that regulate primary photochemical yield and charge recombination rate. Many prokaryotic oxygenic phototrophs express two or more D1 isoforms differentially in response to environmental light needs, a capability absent in flowering plants and algae. We report that tobacco (Nicotiana tabacum) plants carrying the Synechococcus (Synechococcus elongatus PCC 7942) low-light mutation (LL-E130Q) in the D1 polypeptide (NtLL) acquire the cyanobacterial photochemical phenotype: faster photodamage in high light and significantly more charge separations in productive linear electron flow in low light. This flux increase produces 16.5% more (dry) biomass under continuous low-light illumination (100 μE m−2 s−1, 24 h). This gain is offset by the predicted lower photoprotection at high light. By contrast, the introduction of the Synechococcus high-light mutation (HL-A152S) into tobacco D1 (NtHL) has slightly increased photoprotection, achieved by photochemical quenching, but no apparent impact on biomass yield compared to wild type under the tested conditions. The universal design principle of all PSII reaction centers trades off energy conversion for photoprotection in different proportions across all phototrophs and provides a useful guidance for testing in crop plants. The observed biomass advantage under continuous low light can be transferred between evolutionarily isolated lineages to benefit growth under artificial lighting conditions. However, removal of the selective marker gene was essential to observe the growth phenotype, indicating growth penalty imposed by use of the particular spectinomycin-resistance gene.

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1-3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.

The plastid caseinolytic protease (Clp) complex plays essential roles in maintaining protein homeostasis and comprises both plastid-encoded and nuclear-encoded subunits. Despite the Clp complex being retained across green plants with highly conserved protein sequences in most species, examples of extremely accelerated amino acid substitution rates have been identified in numerous angiosperms. The causes of these accelerations have been the subject of extensive speculation but still remain unclear. To distinguish among prevailing hypotheses and begin to understand the functional consequences of rapid sequence divergence in Clp subunits, we used plastome transformation to replace the native clpP1 gene in tobacco (Nicotiana tabacum) with counterparts from another angiosperm genus (Silene) that exhibits a wide range in rates of Clp protein sequence evolution. We found that antibiotic-mediated selection could drive a transgenic clpP1 replacement from a slowly evolving donor species (S. latifolia) to homoplasmy but that clpP1 copies from Silene species with accelerated evolutionary rates remained heteroplasmic, meaning that they could not functionally replace the essential tobacco clpP1 gene. These results suggest that observed cases of rapid Clp sequence evolution are a source of epistatic incompatibilities that must be ameliorated by coevolutionary responses between plastid and nuclear subunits.

The protocol we report here is based on biolistic delivery of transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin or kanamycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. The tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar. Plastid transformation in a subset of N. tabacum cultivars and in Nicotiana benthamiana requires adjustment of the tissue culture protocol. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome, vectors suitable for regulated gene expression by the engineered PPR10 RNA binding protein as well as systems for marker gene excision.

Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. The example of the marker gene is the bar

Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. The example of the marker gene is the barau gene flanked by loxP sites in the plastid genome. For marker excision Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the barau gene, confers a visual aurea leaf phenotype, thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The success of in planta plastid marker excision proves that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA and the availability of visual marker genes.

The protocol we report here is based on biolistic delivery of transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin or kanamycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. The tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar. Plastid transformation in a subset of N. tabacum cultivars and in Nicotiana benthamiana requires adjustment of the tissue culture protocol. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome, vectors suitable for regulated gene expression by the engineered PPR10 RNA binding protein as well as systems for marker gene excision.

To develop a deliberately engineered expression and purification system for an active chimeric-recombinant tissue plasminogen activator (crtPA) using co-expression with polyhydroxybutyrate (PHB) operon genes. Fusion of crtPA with PhaC-synthase simplified the purification steps through crtPA sedimentation with PHB particles. Moreover, the covalently immobilized crtPA was biologically active as shown in a chromogenic assay. Upon WELQut-protease activity, the released single-chain crtPA converted to the two-chain form which produced a pattern of bands with approx. MW of 32 and 11 kDa in addition to the full length crtPA. Fusion of crtPA with PhaC-synthase not only simplifies purification from the bacterial host lysate, but also co-expression of PHB operon genes creates an oxidative environment, thereby reducing the inclusion body formation possibility. The isolated crtPA-PHB granules exhibited crtPA serine protease activity. Thus, fusion with the PhaC protein could be used as a scaffold for covalent displaying of functional disulfide-rich proteins.

Plastid transformation is routine in tobacco (Nicotiana tabacum) but 100-fold less frequent in Arabidopsis (Arabidopsis thaliana), preventing its use in plastid biology. A recent study revealed that null mutations in ACC2, encoding a plastid-targeted acetyl-coenzyme A carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC β-carboxylase subunit. We bombarded ACC2-defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance (aadA) gene and gfp, encoding the green fluorescence protein GFP. Spectinomycin-resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic messenger RNA, which would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin-hypersensitive Slavice and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Slavice and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops.

We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5'-untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High-level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.

New transformation-competent Arabidopsis lines, with new plastid transformation vectors and a protocol for measuring plastid transformation efficiency, will advance the engineering of the plastid genome in Arabidopsis.

Significance We report cell-to-cell movement of mitochondria through a graft junction of two tobacco species, Nicotiana tabacum and Nicotiana sylvestris . The flowers of the N. tabacum line we used are male sterile due to a sterility-causing mitochondrial genome, whereas the N. sylvestris flowers are fertile. Grafting created an opportunity for organelle movement during the healing process when cell-to-cell connections at the graft junction were restored. We recognized N. sylvestris mitochondrial DNA transfer by restoration of fertile flower anatomy in plants regenerated from graft junctions. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of mitochondria by DNA acquired from other species.

The engineering of plant genomes presents exciting opportunities to modify agronomic traits and to produce high-value products in plants. Expression of foreign proteins from transgenes in the chloroplast genome offers advantages that include the capacity for prodigious protein output, the lack of transgene silencing and the ability to express multicomponent pathways from polycistronic mRNA. However, there remains a need for robust methods to regulate plastid transgene expression. We designed orthogonal activators that boost the expression of chloroplast transgenes harbouring cognate cis-elements. Our system exploits the programmable RNA sequence specificity of pentatricopeptide repeat proteins and their native functions as activators of chloroplast gene expression. When expressed from nuclear transgenes, the engineered proteins stimulate the expression of plastid transgenes by up to ~40-fold, with maximal protein abundance approaching that of Rubisco. This strategy provides a means to regulate and optimize the expression of foreign genes in chloroplasts and to avoid deleterious effects of their products on plant growth. A study used engineered, nuclear-expressed pentatricopeptide repeat proteins with programmable RNA sequence specificity to boost the expression of chloroplast transgenes harbouring cognate cis-elements, providing a method for regulating plastid transgene expression.

We describe a steroid-inducible BABY BOOM system that improves plant regeneration in Arabidopsis leaf cultures and yields fertile plants. Regeneration of Arabidopsis thaliana plants for extended periods of time in tissue culture may result in sterile plants. We report here a novel approach for A. thaliana regeneration using a regulated system to induce embryogenic cultures from leaf tissue. The system is based on BABY BOOM (BBM), a transcription factor that turns on genes involved in embryogenesis. We transformed the nucleus of A. thaliana plants with BBM:GR, a gene in which the BBM coding region is fused with the glucocorticoid receptor (GR) steroid-binding domain. In the absence of the synthetic steroid dexamethasone (DEX), the BBM:GR fusion protein is localized in the cytoplasm. Only when DEX is included in the culture medium does the BBM transcription factor enter the nucleus and turn on genes involved in embryogenesis. BBM:GR plant lines show prolific shoot regeneration from leaf pieces on media containing DEX. Removal of DEX from the culture media allowed for flowering and seed formation. Therefore, use of BBM:GR leaf tissue for regeneration of plants for extended periods of time in tissue culture will facilitate the recovery of fertile plants.

This article is a revision of the previous edition article by P. Maliga, volume 1, pp. 392–402, © 2003, Elsevier Ltd.

We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25-50 mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 × Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.

Summary Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.

Originating from endosymbiotic cyanobacteria, plastids are highly polyploid and have prokaryotic-like transcription and translation machineries. The majority of plastid functions is carried out not from plastid encoded genes but from ~3,000 nuclear genes that target proteins to plastids. The compact 125-kb to 155-kb plastid genome (ptDNA) encodes less than one hundred proteins. However, both plastid and nuclear-encoded traits can be modified by plastid genome engineering. Furthermore, novel genes and operons may be incorporated from heterologous sources to expand the repertoire of biosynthetic pathways in plants. In this review, background is provided on the plastid genetic system and how to obtain genetically uniform plants from plastid transformation. We focus on the principles of plastome engineering and address transgene design enabling biotechnological applications. We assess the state of plastome engineering in the microalga Chlamydomonas reinhardtiii, the bryophytes Physcomitrella patens and Marchantia polymorpha, and flowering plants. In the flowering plant group, plastid transformation is routine only in tobacco, tomato, petunia, potato, soybean, lettuce and cabbage. Future applications of plastid genome manipulation are emphasized in the discussion of potential contributions to food security, biofuel production and plant-based biopharmaceutical or industrial enzyme manufacturing.

Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar (bar(au)) gene that confers a golden leaf phenotype and a spectinomycin resistance (aadA) gene that is necessary for the introduction of the bar(au) gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadA(au) transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aadA(au) genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome.

In transformed tobacco (Nicotiana tabacum) plastids, we flank the marker genes with recombinase target sites to facilitate their posttransformation excision. The P1 phage loxP sites are identical 34-bp direct repeats, whereas the phiC31 phage attB/attP sites are 54- and 215-bp sequences with partial homology within the 54-bp region. Deletions in the plastid genome are known to occur by recombination between directly repeated sequences. Our objective was to test whether or not the marker genes may be lost by homologous recombination via the directly repeated target sites in the absence of site-specific recombinases. The sequence between the target sites was the bar(au) gene that causes a golden-yellow (aurea) leaf color, so that the loss of the bar(au) gene can be readily detected by the appearance of green sectors. We report here that transplastomes carrying the bar(au) gene marker between recombinase target sites are relatively stable because no green sectors were detected in approximately 36,000 seedlings (Nt-pSS33 lines) carrying attB/attP-flanked bar(au) gene and in approximately 38,000 seedlings (Nt-pSS42 lines) carrying loxP-flanked bar(au) gene. Exceptions were six uniformly green plants in the Nt-pSS42-7A progeny. Sequencing the region of plastid DNA that may derive from the vector indicated that the bar(au) gene in the six green plants was lost by gene conversion using wild-type plastid DNA as template rather than by deletion via directly repeated loxP sites. Thus, the recombinase target sites incorporated in the plastid genome for marker gene excisions are too short to mediate the loss of marker genes by homologous recombination at a measurable frequency.

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites.

Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3′rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3″-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3″-adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs.

Because of its prokaryotic-type gene expression machinery, maternal inheritance and the opportunity to express proteins at a high level, the plastid genome (plastome or ptDNA) is an increasingly popular target for engineering. The ptDNA is present as up to 10,000 copies per cell, making selection for marker genes essential to obtain plants with uniformly transformed ptDNA. However, the marker gene is no longer desirable when homoplastomic plants are obtained. Marker-free transplastomic plants can now be obtained with four recently developed protocols: homology-based excision via directly repeated sequences, excision by phage site-specific recombinanses, transient cointegration of the marker gene, and the cotransformation-segregation approach. Marker excision technology will benefit applications in agriculture and in molecular farming.

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.

To identify genetic markers in the Arabidopsis thaliana plastid genome (ptDNA), we amplified and sequenced the rpl2-psbA and rbcL-accD regions in 26 ecotypes. The two regions contained eight polymorphic sites including five insertions and/or deletions (indels) involving changes in the length of A or T mononucleotide repeats and three base substitutions. The 27 alleles defined 15 plastid haplotypes, providing a practical set of ptDNA markers for the Columbia, Landsberg erecta and Wassilewskija ecotypes that are commonly used in genetic studies and also for the C24 and RLD ecotypes that are the most amenable for cell culture manipulations.

Cytoplasmic male-sterile (CMS) lines in maize (Zea mays) have been classified by their response to specific restorer genes into three categories: cms-C, cms-S, and cms-T. A mitochondrial genome representing each of the CMS cytotypes has been sequenced, and male sterility in the cms-S and cms-T cytotypes is linked to chimeric mitochondrial genes. To identify markers for plastid genotyping, we sequenced the plastid genomes of three fertile maize lines (B37, B73, and A188) and the B37 cms-C, cms-S, and cms-T cytoplasmic substitution lines. We found that the plastid genomes of B37 and B73 lines are identical. Furthermore, the fertile and CMS plastid genomes are conserved, differing only by zero to three single-nucleotide polymorphisms (SNPs) in coding regions and by eight to 22 SNPs and 10 to 21 short insertions/deletions in noncoding regions. To gain insight into the origin and transmission of the cms-T trait, we identified three SNPs unique to the cms-T plastids and tested the three diagnostic SNPs in 27 cms-T lines, representing the HA, I, Q, RS, and T male-sterile cytoplasms. We report that each of the tested 27 cms-T group accessions have the same three diagnostic plastid SNPs, indicating a single origin and maternal cotransmission of the cms-T mitochondria and plastids to the seed progeny. Our data exclude exceptional pollen transmission of organelles or multiple horizontal gene transfer events as the source of the mitochondrial urf13-T (unidentified reading frame encoding 13-kD cms-T protein) gene in the cms-T cytoplasms. Plastid genotyping enables a reassessment of the evolutionary relationships of cytoplasms in cultivated maize.

We report plastid marker-gene excision with a transiently expressed CRE, site-specific recombinase. This is a novel protocol that enables rapid removal of marker genes from the approximately 10,000 plastid genome copies without transformation of the plant nucleus. Plastid marker excision was tested in tobacco plants transformed with a prototype polycistronic plastid vector, pPRV110L, designed to express multiple genes organized in an operon. The pMHB10 and pMHB11 constructs described here are dicistronic and encode genes for herbicide (bar) and spectinomycin (aadA) resistance. In vector pMHB11, expression of herbicide resistance is dependent on conversion of an ACG codon to an AUG translation initiation codon by mRNA editing, a safety feature that prevents translation of the mRNA in prokaryotes and in the plant nucleus. In the vectors, the marker gene (aadA) is flanked by 34-bp loxP sites for excision by CRE. Marker excision by a transiently expressed CRE involves introduction of CRE in transplastomic leaves by agro-infiltration, followed by plant regeneration. In tobacco transformed with vectors pMHB10 and pMHB11, Southern analysis and PCR identified approximately 10% of the regenerated plants as marker-free.

Plant-expressed vaccines may provide a unique opportunity for generating anti-pathogen immunity, especially in countries where cold storage is lacking. In the following study, we show that soluble protein from tobacco leaves expressing fragment C of tetanus toxin protected mice against a lethal tetanus toxin challenge. More importantly, we show that a single intranasal (i.n.) vaccination was as efficient as oral delivery, inducing high levels of activated CD4(+) T cells and anti-toxin antibody. Unlike the oral route, i.n. delivery did not require the presence of adjuvant (cholera toxin). Indeed, addition of cholera toxin induced bystander immune responses to plant proteins as well. This is the first study documenting protective immunity by a single i.n. dose of plant vaccine. Plant-based vaccines are promising because they are more heat stable, are easy to produce, cheap and do not require needles.

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.

Plant cells possess three DNA-containing compartments, the nucleus, the mitochondria and the plastids. Accordingly, plastid gene regulation is fairly complex. Albeit plastids retained their own genome and prokaryotic-type gene expression system by a plastid-encoded RNA polymerase (PEP), they need a second nuclear-encoded plastid transcription activity, NEP. Candidate genes for putative NEP catalytic subunits have been cloned in Arabidopsis thaliana (AtRpoTp) and Nicotiana sylvestris (NsRpoTp). To provide evidence for RpoTp as a gene encoding a NEP catalytic subunit, we introduced the AtRpoTp and NsRpoTp cDNAs into the tobacco nucleus under the control of the strong constitutive CaMV 35S promoter. Analysis of transcription from NEP and PEP promoters in these transgenic plants using primer extension assays revealed enhanced transcription from typical type I NEP promoters as PatpB-289 in comparison with the wild type. These data provide direct evidence that RpoTp is a catalytic subunit of NEP and involved in recognition of a distinct subset of type I NEP promoters.

Plastids of higher plants are semi-autonomous cellular organelles that have their own genome and transcription-translation machinery. Examples of plastid functions are photosynthesis and biosynthesis of starch, amino acids, lipids and pigments. Plastid functions are encoded in approximately 120 plastid genes and approximately 3,000 nuclear genes. Although many embryo and seedling lethal nuclear genes are required for chloroplast biogenesis, until now deletion of plastid genes either had no phenotypic consequence (8 genes), or caused a mutant phenotype but did not affect viability (13 genes). Here we identify an essential plastid gene. By using the CRE-lox site-specific recombination system we have deleted clpP1 (caseinolytic protease P1), one of the three genes (clpP1, ycf1 and ycf2) whose disruption had previously only been possible in a fraction of the 1,000-10,000 plastid genome copies in a cell. Loss of the clpP1 gene product, the ClpP1 protease subunit, results in ablation of the shoot system of tobacco plants, suggesting that ClpP1-mediated protein degradation is essential for shoot development.

Fragment C (TetC) is a non-toxic 47 kDa polypeptide fragment of tetanus toxin that can be used as a subunit vaccine against tetanus. Expression of TetC in Escherichia coli and yeast was dependent on the availability of synthetic genes that were required to improve translation efficiency and stabilize the mRNA. To explore the feasibility of producing TetC in tobacco leaves, we attempted expression of both the bacterial high-AT (72.3% AT) and the synthetic higher-GC genes (52.5% AT) in tobacco chloroplasts. We report here that the bacterial high-AT mRNA is stable in tobacco chloroplasts. Significant TetC accumulation was obtained from both genes, 25 and 10% of total soluble cellular protein, respectively, proving the versatility of plastids for expression of unmodified high-AT and high-GC genes. Mucosal immunization of mice with the plastid- produced TetC induced protective levels of TetC antibodies. Thus, expression of TetC in chloroplasts provides a potential route towards the development of a safe, plant-based tetanus vaccine for nasal and oral applications.

This invention relates to methods and compositions for obtaining Arabidopsis and Brassica plants. Specifically, the method provides culturing protocols and compositions that facilitate the regeneration of transformed plants following delivery of beneficial DNA molecules.

RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.

The plastid ribosomal RNA (rrn) operon promoter was fused with DNA segments encoding the leader sequence (5′-untranslated region [UTR]) of plastid mRNAs to compare their efficiency in mediating translation of a bacterial protein neomycin phosphotransferase (NPTII) in tobacco (Nicotiana tabacum) chloroplasts. In young leaves, NPTII accumulated at 0.26% and 0.8% of the total soluble leaf protein from genes with theclpP and atpB 5′-UTR, respectively. Interestingly, expression of NPTII from the promoter with theclpP 5′-UTR (0.26% NPTII) caused a mutant (chlorotic) phenotype, whereas plants accumulating approximately 0.8% NPTII from the atpB 5′-UTR were normal green, indicating that the mutant phenotype was independent of NPTII accumulation. Low levels of monocistronic clpP mRNA and accumulation of intron-containing clpP transcripts in the chlorotic leaves suggest competition between the clpP 5′-UTR in the chimeric transcript and the native clpP pre-mRNA (ratio 16:1) for an mRNA maturation factor. Because maturation of 11 other intron-containing mRNAs was unaffected in the chlorotic leaves, it appears that the factor is clpP specific. The mutant phenotype is correlated with reduced levels (approximately 2 times) of the ClpP1 protease subunit, supporting an important role for ClpP1 in chloroplast development.

We fully sequenced four and partially sequenced six additional plastid genomes of the model legume Medicago truncatula. Three accessions, Jemalong 2HA, Borung and Paraggio, belong to ssp. truncatula, and R108 to ssp. tricycla. We report here that the R108 ptDNA has a ~45-kb inversion compared with the ptDNA in ssp. truncatula, mediated by a short, imperfect repeat. DNA gel blot analyses of seven additional ssp. tricycla accessions detected only one of the two alternative genome arrangements, represented by three and four accessions each. Furthermore, we found a variable number of repeats in the essential accD and ycf1 coding regions. The repeats within accD are recombinationally active, yielding variable-length insertions and deletions in the central part of the coding region. The length of ACCD was distinct in each of the 10 sequenced ecotypes, ranging between 650 and 796 amino acids. The repeats in the ycf1 coding region are also recombinationally active, yielding short indels in 10 regions of the reading frames. Thus, the plastid genome variability we report here could be linked to repeat-mediated genome rearrangements. However, the rate of recombination was sufficiently low, so that no heterogeneity of ptDNA could be observed in populations maintained by single-seed descent.

The protocol we report here is based on biolistic delivery of the transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. Also, the tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar, allowing plastid engineering protocols developed for N. tabacum to be directly applied to N. sylvestris. However, the tissue culture protocol is applicable only in a subset of N. tabacum cultivars. Here we highlight differences between the protocols for the two species. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome as well as systems for marker excision. The simpler genetics of the diploid N. sylvestris, as opposed to the allotetraploid N. tabacum, make it an attractive model for plastid transformation.

Uniform transformation of the thousands of plastid genome (ptDNA) copies in a cell is driven by selection for plastid markers. When each of the plastid genome copies is uniformly altered, the marker gene is no longer needed. Plastid markers have been efficiently excised by site-specific recombinases expressed from nuclear genes either by transforming tissue culture cells or introducing the genes by pollination. Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the bar (au) gene, confers a visual aurea leaf phenotype; thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The bar (au) marker-free plastids are transmitted through seed to the progeny. PCR and DNA gel blot (Southern) protocols to confirm transgene integration and plastid marker excision are also provided herein.

RNA editing in the plastids and mitochondria of higher plants involves C to U conversion of specific nucleotides in the mRNA. This leads to the synthesis of proteins that are different from those predicted by the DNA sequence. Editing appears to have arisen at about the same time in both plastids and mitochondria, suggesting a common evolutionary origin. The problem we address here is whether or not there has been co-evolution of the editing systems in the two organelles. Our test system was editing of the Arabidopsis thaliana mRNAs for ndhB and nad2, and for ndhD and nad4, which encode homologous subunits of the plastid and mitochondrial NAD(P)H dehydrogenases, respectively. The editing sites in the Arabidopsis nad2 and nad4 mRNAs have previously been determined and we report here 19 editing sites in eight mRNAs in Arabidopsis plastids. Out of these, eight sites are localized in the ndhB mRNA. In its mitochondrial counterpart, nad2, 31 editing sites are present, none of which are shared with the ndhB gene. The Arabidopsis ndhD mRNA is edited at four positions, only one of which is shared by its mitochondrial homologue, nad4, which contains 32 editing sites. These findings suggest that, although editing in the two organelles may have derived from a single system, there is no significant conservation of editing sites in cognate mRNAs in plastids and mitochondria.

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine-Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5'-untranslated region (5'-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5'-UTR and DB region promotes accumulation of NPTII up to approximately 16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield ( approximately 23% of TSP) was obtained from a construct in which the T7g10 5'-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.