Beth A. Ahner

High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions

The Thermobifida fusca cel6A gene encoding an endoglucanase was fused to three different downstream box (DB) regions to generate cel6A genes with 14 amino acid fusions. The DB‐Cel6A fusions were inserted into the tobacco (Nicotiana tabacum cv. Samsun) chloroplast genome for protein expression. Accumulation of Cel6A protein in transformed tobacco leaves varied over approximately two orders of magnitude, dependent on the identity of the DB region fused to the cel6A open reading frame (ORF). Additionally, the DB region fused to the cel6A ORF affected the accumulation of Cel6A protein in aging leaves, with the most effective DB regions allowing for high level accumulation of Cel6A protein in young, mature, and old leaves, while Cel6A protein accumulation decreased with leaf age when less effective DB regions were fused to the cel6A ORF. In the most highly expressed DB‐Cel6A construct, enzymatically active Cel6A protein accumulated at up to 10.7% of total soluble leaf protein (%TSP). The strategy used for high‐level endoglucanase expression may be useful for expression of other cellulolytic enzymes in chloroplasts, ultimately leading to cost‐effective heterologous enzyme production for cellulosic ethanol using transplastomic plants. Biotechnol. Bioeng. 2009;102: 1045–1054.

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 β-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 β-glucosidase production for the cellulosic ethanol industry.

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.

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.

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.

Fate of DTPA, EDTA, and EDDS in Hydroponic Media and Effects on Plant Mineral Nutrition

ABSTRACT Synthetic chelators are commonly used in hydroponic media to solubilize iron (Fe); however, the fate of these chelators is unknown. This study examined the persistence of three synthetic chelators, ethylenediaminetetraacetate (EDTA), diethylenetriaminepentaacetate (DTPA), and ethylenediaminedisuccinate (EDDS) in a bench-scale lettuce production system. The EDDS concentration decreased rapidly within 7d, most likely due to biodegradation. The EDTA and DTPA concentrations stayed steady throughout the experiments despite additions to maintain a constant volume and loss of chelator may have been due to either plant uptake or photodegradation of the chelator. Despite large differences in solution chemistry, the final shoot concentrations of iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) were similar among chelator treatments, whereas root concentrations of these same elements were highly variable. The concentration of DTPA in a commercial lettuce production system was measured and highly variable concentrations were found.

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.

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.

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.