Overexpression of native ORANGE (OR) and OR mutant protein in Chlamydomonas reinhardtii enhances carotenoid and ABA accumulation and increases resistance to abiotic stress.

The carotenoid content of plants can be increased by overexpression of the regulatory protein ORANGE (OR) or a mutant variant known as the 'golden SNP'. In the present study, a strong light-inducible promoter was used to overexpress either wild type CrOR (CrORWT) or a mutated CrOR (CrORHis) containing a single histidine substitution for a conserved arginine in the microalgae Chlamydomonas reinhardtii. Overexpression of CrORWT and CrORHis roughly doubled and tripled, respectively, the accumulation of several different carotenoids, including ß-carotene, α-carotene, lutein and violaxanthin in C. reinhardtii and upregulated the transcript abundance of nearly all relevant carotenoid biosynthetic genes. In addition, microscopic analysis revealed that the OR transgenic cells were larger than control cells and exhibited larger chloroplasts with a disrupted morphology. Moreover, both CrORWT and CrORHis cell lines showed increased tolerance to salt and paraquat stress. The levels of endogenous phytohormone abscisic acid (ABA) were also increased in CrORWT and CrORHis lines, not only in normal growth conditions but also in growth medium supplemented with salt and paraquat. Together these results offer new insights regarding the role of the native OR protein in regulating carotenoid biosynthesis and the accumulation of several carotenoids in microalgae, and establish a new functional role for OR to modulate oxidative stress tolerance potentially mediated by ABA.

Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins.

BACKGROUND: The global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth. RESULTS: Here, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants' metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant-1) by 50-70%. CONCLUSIONS: The work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.

An array microhabitat device with dual gradients revealed synergistic roles of nitrogen and phosphorous in the growth of microalgae.

Harmful algal blooms (HABs) are an emerging environmental problem contaminating water resources and disrupting the balance of the ecosystems. HABs are caused by the sudden growth of photosynthetic algal cells in both fresh and marine water, and have been expanding in extent and appearing more frequently due to the climate change and population growth. Despite the urgency of the problem, the exact environmental conditions that trigger HABs are unknown. This is in part due to the lack of high throughput tools for screening environmental parameters in promoting the growth of photosynthetic microorganisms. In this article, we developed an array microhabitat device with well defined dual nutrient gradients suitable for quantitative studies of multiple environmental parameters in microalgal cell growth. This device enabled an ability to provide 64 different nutrient conditions [nitrogen (N), phosphorous (P), and N : P ratio] at the same time, and the gradient generation took less than 90 min, advancing the current pond and test tube assays in terms of time and cost. Using a photosynthetic algal cell line, Chlamydomonas reinhardtii, preconditioned in co-limited media, we revealed that N and P synergistically promoted cell growth. Interestingly, no discernible response was observed when single P or N gradient was imposed. Our work demonstrated the enabling capability of the microfluidic platform for screening effects of multiple environmental factors in photosynthetic cell growth, and highlighted the importance of the synergistic roles of environmental factors in algal cell growth.

Correction to: A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts.

[This corrects the article DOI: 10.1186/s13068-018-1127-7.].

Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts.

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts.

BACKGROUND: We investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N-terminally fused to the coding region of cel6A, an endoglucanase from Thermobifida fusca. We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5'UTR, previously reported to enhance protein expression, to regulate the expression of the TetC-cel6A gene. We further investigated the accumulation of TetC-Cel6A under N-deplete growth conditions. RESULTS: Both of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII-cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N-deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N-replete medium. CONCLUSIONS: The DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Altered Microbiome Leads to Significant Phenotypic and Transcriptomic Differences in a Lipid Accumulating Chlorophyte.

Given the challenges facing the economically favorable production of products from microalgae, understanding factors that might impact productivity rates including growth rates and accumulation of desired products, for example, triacylglycerols (TAG) for biodiesel feedstock, remains critical. Although operational parameters such as media composition and reactor design can clearly effect growth rates, the role of microbe-microbe interactions is just beginning to be elucidated. In this study an oleaginous marine algae Chlorella spp. C596 culture is shown to be better described as a microbial community. Perturbations to this microbial community showed a significant impact on phenotypes including sustained differences in growth rate and TAG accumulation of 2.4 and 2.5 fold, respectively. Characterization of the associated community using Illumina 16S rRNA amplicon and random shotgun transcriptomic analyses showed that the fast growth rate correlated with two specific bacterial species ( Ruegeria and Rhodobacter spp). The transcriptomic response of the Chlorella species revealed that the slower growing algal consortium C596-S1 upregulated genes associated with photosynthesis and resource scavenging and decreased the expression of genes associated with transcription and translation relative to the initial C596-R1. Our studies advance the appreciation of the effects microbiomes can have on algal growth in bioreactors and suggest that symbiotic interactions are involved in a range of critical processes including nitrogen, carbon cycling, and oxidative stress.

Use of De Novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols.

Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596) of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.

Cysteine Enhances Bioavailability of Copper to Marine Phytoplankton.

Emiliania huxleyi, a ubiquitous marine algae, was cultured under replete and Cu-limiting conditions to investigate Cu uptake strategies involving thiols and associated redox reactions; comparisons to a model diatom, Thalassiosira pseudonana, were also drawn. Cu-limitation increased rates of cell surface reduction of Cu(II) to Cu(I) in E. huxleyi but not in T. pseudonana. Furthermore, Cu-limited E. huxleyi cells took up more Cu when cysteine was present compared to when no ligand was added, although a dependence on cysteine concentration was not observed. In contrast, Cu uptake by replete cells was dependent upon the relative abundance of inorganic species [Cu(I)']. We also show that cysteine can increase the bioavailability of Cu to Cu-limited cells, of both species, through the reductive release of Cu(I) from fairly strong Cu(II) ligands such as EDTA. Finally, support for a mechanism involving uptake of a Cys-Cu complex in E. huxleyi is drawn from the observation that Cu-limitation significantly enhances cysteine uptake by transporters that exhibit Michaelis-Menten kinetics. These Cu uptake strategies help explain the presence and distribution of dissolved thiols in surface seawater and have implications for the biogeochemical cycling of Cu in low Cu environments.

Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

An array microhabitat system for high throughput studies of microalgal growth under controlled nutrient gradients.

Microalgae have been increasingly recognized in the fields of environmental and biomedical engineering because of its use as base materials for biofuels or biomedical products, and also the urgent needs to control harmful algal blooms protecting water resources worldwide. Central to the theme is the growth rate of microalgae under the influences of various environmental cues including nutrients, pH, oxygen tension and light intensity. Current microalgal culture systems, e.g. raceway ponds or chemostats, are not designed for system parameter optimizations of cell growth. In this article, we present the development of an array microfluidic system for high throughput studies of microalgal growth under well defined environmental conditions. The microfluidic platform consists of an array of microhabitats flanked by two parallel side channels, all of which are patterned in a thin agarose gel membrane. The unique feature of the device is that each microhabitat is physically confined suitable for both motile and non-motile cell culture, and at the same time, the device is transparent and can be perfused through the two side channels amendable for precise environmental control of photosynthetic microorganisms. This microfluidic system is used to study the growth kinetics of a model microalgal strain, Chlamydomonas reinhardtii (C. reinhardtii), under ammonium (NH4Cl) concentration gradients. Experimental results show that C. reinhardtii follows Monod growth kinetics with a half-saturation constant of 1.2 ± 0.3 µM. This microfluidic platform provides a fast (~50 fold speed increase), cost effective (less reagents and human intervention) and quantitative technique for microalgal growth studies, in contrast to the current chemostat or batch cell culture system. It can be easily extended to investigate growth kinetics of other microorganisms under either single or co-culture setting.

Determination of stability constants of Cu(I), Cd(II) & Zn(II) complexes with thiols using fluorescent probes.

Fluorometric competing-ligand titrations were used to measure stability constants of Zn(II), Cd(II) and Cu(I) complexes of cysteine and glutathione (GSH). Cu(I)-stability constants were also determined for the dipeptides Arg-Cys and Gln-Cys which are produced by a marine alga under copper stress. The fluorescent ion indicators FluoZin-1 and BTC (Invitrogen) were used as competing ligands in titrations involving Zn(II) and Cd(II). Phen Green SK (Invitrogen) was likewise used in Cu(I) titrations. Conditional and cumulative general stability constants were determined using a least squares fit of the titration data to speciation models. The measured stability constants of Cd(II) and Zn(II) complexes were consistent with previous work, validating our method and assumptions. Our results also include the first general stability constants for Cu(I)-cysteine complexes and an alternative set for Cu(I)-GSH complexes. While these stability constants indicate that Cu(I) forms strong complexes with thiols, they are not strong enough to effectively buffer Cu(I) in seawater.

Detection of algal lipid accumulation due to nitrogen limitation via dielectric spectroscopy of Chlamydomonas reinhardtii suspensions in a coaxial transmission line sample cell.

In this study, dielectric characterization of algae cell suspensions was used to detect lipid accumulation due to nitrogen starvation. Wild-type Chlamydomonas reinhardtii (CC-125) was cultivated in replete and nitrogen-limited conditions in order to achieve a range of lipid contents, as confirmed by Nile Red fluorescence measurements. A vector network analyzer was used to measure the dielectric scattering parameters of a coaxial region of concentrated cell suspension. The critical frequency fc of the normalized transmission coefficient |S21(*)| decreased with increasing lipid content but did not change with cell concentration. These observations were consistent with a decrease in cytoplasmic conductivity due to lipid accumulation in the preliminary transmission line model. This dielectric sensitivity to lipid content will facilitate the development of a rapid, noninvasive method for algal lipid measurement that could be implemented in industrial settings without the need for specialized staff and analytical facilities.

Bacteriophage 5' untranslated regions for control of plastid transgene expression.

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5' untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5' UTRs and the bacteriophage T7 gene 10 (T7g10) 5' UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5' UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3'-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5' UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5' UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5' UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5' UTR. However, transplastomic plants with the bacteriophage 5' UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5' UTR. Furthermore, expression of the T7g1.3 and T4g23 5' UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation.

Chloroplast transformation for engineering of photosynthesis.

Many efforts are underway to engineer improvements in photosynthesis to meet the challenges of increasing demands for food and fuel in rapidly changing environmental conditions. Various transgenes have been introduced into either the nuclear or plastid genomes in attempts to increase photosynthetic efficiency. We examine the current knowledge of the critical features that affect levels of expression of plastid transgenes and protein accumulation in transplastomic plants, such as promoters, 5' and 3' untranslated regions, RNA-processing sites, translation signals and amino acid sequences that affect protein turnover. We review the prior attempts to manipulate the properties of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) through plastid transformation. We illustrate how plastid operons could be created for expression of the multiple genes needed to introduce new pathways or enzymes to enhance photosynthetic rates or reduce photorespiration. We describe here the past accomplishments and future prospects for manipulating plant enzymes and pathways to enhance carbon assimilation through plastid transformation.

Characterization of a High Affinity Phytochelatin Synthase from The Cd-Utilizing Marine Diatom Thalassiosira pseudonana.

Phytochelatin synthase (PC synthase) is the enzyme that catalyzes the production of phytochelatins, peptides of the structure (γ-Glu-Cys)n -Gly, where n = 2-11, from the sulfhydryl-containing tripeptide glutathione, in response to elevated metal exposure. Biochemical utilization of Cd in the marine diatom Thalassiosira weissfloggi, as well as unusually high ratios of PC to Cd in some Thalassiosira species including T. pseudonana Hasle et Heimdal, motivated the characterization of T. pseudonana PC synthase 1 (TpPCS1). This enzyme is the product of one of three genes in the T. pseudonana genome predicted to encode for a PC synthase based on its homology to canonical PC synthases previously examined. TpPCS1 was cloned, expressed in Escherichia coli and purified under both aerobic and anaerobic conditions. TpPCS1 exhibits several characteristics that set it distinctly apart from the well-studied PC synthase, Arabidopsis thaliana PCS1 (AtPCS1). It is extremely sensitive to oxidation, which suppresses activity, and it is readily inhibited by the addition of Cd in the absence of thiolate ligands. TpPCS1 also has significantly greater affinity for one of its key substrates, the bis-glutathionato-Cd complex. TpPCS1 kinetics is best described by a ternary complex model, as opposed to the ping-pong model used to describe AtPCS1 kinetics. The findings indicate that although the function of TpPCS1 is synonymous to that of AtPCS1, its divergent biochemistry suggests adaptation of this enzyme to the distinct trace metal chemistry of the marine environment and the unique physiological needs of T. pseudonana.

An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts.

Production of enzymes for lignocellulose hydrolysis in planta has been proposed as a lower-cost alternative to microbial production, with plastid transformation as a preferred method due to high foreign protein yields. An important regulator of chloroplast protein production is the downstream box (DB) region, located immediately downstream of the start codon. Protein accumulation can vary over several orders of magnitude by altering the DB region. Experiments in bacteria have suggested that these differences in protein accumulation may result from changes in translation efficiency, though the precise mechanism of DB function is not known. In this study, three DB regions were fused to the bglC ORF encoding a ß-glucosidase from the thermophilic bacterium Thermobifida fusca and inserted into the tobacco (Nicotiana tabacum) plastid genome. More than a two order of magnitude of difference in BglC protein accumulation was observed, dependent on the identity of the DB fusion. Differential transcript accumulation explained some the observed differences in protein accumulation, but in addition, less 3' degradation of bglC transcripts was observed in transgenic plants that accumulated the most BglC enzyme. Chloroplast-produced BglC was active against both pure cellobiose and against tobacco lignocellulose. These experiments demonstrate the potential utility of transplastomic plants as a vehicle for heterologous ß-glucosidase production for the cellulosic ethanol industry.

Extraction of lead and cadmium from soils by cysteine and glutathione.

Metal-thiol complexes can enhance metal uptake by plant roots and microorganisms, therefore it is important to determine whether thiols effectively solubilize these metals from the soil matrix. Extractions were conducted by combining 1 g contaminated soil and 10 mL of 10 mmol L(-1) thiol solution and shaking for 1 h. Both cysteine and glutathione extracted between 5 and 45% of Pb and Cd from laboratory and field-contaminated soils at pHs>6 after 1 h. In the presence of oxygen, the half-life of reduced cysteine was on the order of 0.1 h and dissolved metal concentrations decreased to nearly zero over 24 h. In extractions with glutathione, both the metals and thiol were more stable, with a half-life for glutathione of 23 h, and stable dissolved metal concentrations over 24 h in the presence of oxygen. In cysteine extractions, Pb was primarily removed from the Fe/Mn oxide fraction of the amended soil and dissolved Fe concentrations followed dissolved Pb concentrations, whereas this pool of Pb was unavailable to thiol extraction in aged field contaminated soils. Iron is hypothesized to play a role in the oxidation of cysteine via both reductive dissolution of iron oxides and in the photolytic oxidation of the Fe-thiol complex in the aqueous phase. While overall cysteine was more effective than glutathione at extracting Pb from soils, its propensity to oxidize may limit its ability to increase the bioavailability of this metal to plants or microorganisms in oxic environments.

Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots.

This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb-glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation.

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants.

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10-60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, "secondary" recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene.