Bacterial Form II Rubisco can support wild-type growth and productivity in Solanum tuberosum cv. Desiree (potato) under elevated CO2.

The last decade has seen significant advances in the development of approaches for improving both the light harvesting and carbon fixation pathways of photosynthesis by nuclear transformation, many involving multigene synthetic biology approaches. As efforts to replicate these accomplishments from tobacco into crops gather momentum, similar diversification is needed in the range of transgenic options available, including capabilities to modify crop photosynthesis by chloroplast transformation. To address this need, here we describe the first transplastomic modification of photosynthesis in a crop by replacing the native Rubisco in potato with the faster, but lower CO2-affinity and poorer CO2/O2 specificity Rubisco from the bacterium Rhodospirillum rubrum. High level production of R. rubrum Rubisco in the potRr genotype (8 to 10 µmol catalytic sites m2) allowed it to attain wild-type levels of productivity, including tuber yield, in air containing 0.5% (v/v) CO2. Under controlled environment growth at 25°C and 350 µmol photons m2 PAR, the productivity and leaf biochemistry of wild-type potato at 0.06%, 0.5%, or 1.5% (v/v) CO2 and potRr at 0.5% or 1.5% (v/v) CO2 were largely indistinguishable. These findings suggest that increasing the scope for enhancing productivity gains in potato by improving photosynthate production will necessitate improvement to its sink-potential, consistent with current evidence productivity gains by eCO2 fertilization for this crop hit a ceiling around 560 to 600 ppm CO2.

LC-MS untargeted metabolomics assesses the delayed response of glufosinate treatment of transgenic glufosinate resistant (GR) buffalo grasses (Stenotaphrum secundatum L.).

INTRODUCTION: Glufosinate resistant (GR) buffalo grasses were genetically modified to resist the broad-spectrum herbicide, glufosinate by inserting a novel pat gene into its genome. This modification results in a production of additional phosphinothricin acetyltransferase (PAT) to detoxify the deleterious effects of glufosinate. The GR grasses and its associated herbicide form a modern, weeding program, to eradicate obnoxious weeds in turf lawn without damaging the grasses at relatively low costs and labor. As with several principal crops which are genetically modified to improve agricultural traits, biosafety of the GR buffalo grasses is inevitably expected to become a public concern. For the first time, we had previously examined the metabolome of glufosinate-resistant buffalo grasses, using a GC-MS untargeted approach to assess the risk of GR as well as identify any pleotropic effects arising from the genetically modification process. In this paper, an untargeted high-resolution LC-MS (LC-HRMS) untargeted metabolomics approach was carried out to complement our previous findings with respect to GR and wild type (WT) buffalo grasses. OBJECTIVE: One of the major aims of this present work was to compare GR to WT buffalo grasses by including the detection of the secondary metabolome and determine any unprecedented metabolic changes. METHODS: Eight-week old plants of 4 GR buffalo grasses, (93-1A, 93-2B, 93-3 C and 93-5A) and 3 wild type varieties (WT 8-4A, WT 9-1B and WT 9-1B) were submerged in either 5 % v/v of glufosinate or distilled water 3 days prior to a LC-HRMS based untargeted metabolomics analysis (glufosinate-treated or control, samples, respectively). An Ultra-High-Performance Liquid Chromatography (UHPLC) system coupled to a Velos Pro Orbitrap mass spectrometer system was employed to holistically measure the primary and secondary metabolome of both GR and WT buffalo grasses either treated with or without glufosinate and subsequently apply several bioinformatic tools including the automated pathway analysis algorithm, mummichog. RESULTS: LC-HRMS untargeted based metabolomics clearly identified that the global metabolite pools of both GR and WT cultivars were highly similar, providing strong, supporting evidence of substantial equivalence between the GR and WT varieties. These findings indicate that if any associated risks to these GR grasses were somehow present, the risk would be within those acceptable ranges present in the WT. Additionally, mummichog-based pathway analysis indicated that phenylalanine metabolism and the TCA cycle were significantly impacted by glufosinate treatment in the WT cultivar. It was possible that alterations in the relative concentrations of several intermediates in these pathways were likely due to glufosinate-induced production of secondary metabolites to enhance plant defense mechanisms against herbicidal stress at the expense of primary metabolism. CONCLUSIONS: GR buffalo grasses were found to be near identical to its WT comparator based on this complementary LC-HRMS based untargeted metabolomics. Therefore, these results further support the safe use of these GR buffalo grasses with substantial evidence. Interestingly, despite protected by PAT, GR buffalo grasses still demonstrated the response to glufosinate treatment by up-regulating some secondary metabolite-related pathways.

Utilization of GC-MS untargeted metabolomics to assess the delayed response of glufosinate treatment of transgenic herbicide resistant (HR) buffalo grasses (Stenotaphrum secundatum L.).

INTRODUCTION: Herbicide resistant (HR) buffalo grasses were genetically engineered to resist the non-selective herbicide, glufosinate in order to facilitate a modern, 'weeding program' which is highly effective in terms of minimizing costs and labor. The resistant trait was conferred by an insertion of the pat gene to allow for the production of the enzyme phosphinothricin acetyltransferase (PAT) to detoxify the glufosinate inhibitive effect. To date, there are only a few reports using metabolomics as well as molecular characterizations published for glufosinate-resistant crops with no reports on HR turfgrass. Therefore, for the first time, this study examines the metabolome of glufosinate-resistant buffalo grasses which not only will be useful to future growers but also the scientific community. OBJECTIVE: A major aim of this present work is to characterize and evaluate the metabolic alterations which may arise from a genetic transformation of HR buffalo grasses by comprehensively using gas chromatography-mass spectrometry (GC-MS) based untargeted metabolomics. METHODS: Eight-week old plants of 4 HR buffalo grasses, (93-1A, 93-2B, 93-3C and 93-5A) and 3 wild type varieties (WT 8-4A, WT 9-1B and WT 9-1B) were selected for physiological, molecular and metabolomics experiments. Plants were either sprayed with 1, 5, 10 and 15% v/v of glufosinate to evaluate the visual injuries or submerged in 5% v/v of glufosinate 3 days prior to a GC-MS based untargeted metabolomics analysis. In contrast, the control group was treated with distilled water. Leaves were extracted in 1:1 methanol:water and then analysed, using an in-house GC-MS untargeted workflow. RESULTS: Results identified 199 metabolites with only 6 of them (cis-aconitic acid, allantoin, cellobiose, glyceric acid, maltose and octadecanoic acid) found to be statistically significant (p < 0.05) between the HR and wild type buffalo grass varieties compared to the control experiment. Among these metabolites, unusual accumulation of allantoin was prominent and was an unanticipated effect of the pat gene insertion. As expected, glufosinate treatment caused significant metabolic alterations in the sensitive wild type, with the up-regulation of several amino acids (e.g. phenylalanine and isoleucine) which was likely due to glufosinate-induced senescence. The aminoacyl-tRNA biosynthetic pathway was identified as the most significant enriched pathway as a result of glufosinate effects because a number of its intermediates were amino acids. CONCLUSION: HR buffalo grasses were very similar to its wild type comparator based on a comprehensive GC-MS based untargeted metabolomics and therefore, should guarantee the safe use of these HR buffalo grasses. The current metabolomics analyses not only confirmed the effects of glufosinate to up-regulate free amino acid pools in the sensitive wild type but also several alterations in sugar, sugar phosphate and organic acid metabolism have been reported.

Recent developments in metabolomics-based research in understanding transgenic grass metabolism.

BACKGROUND: Transgenic herbicide-resistant (HR) turfgrass together with its associated, broad spectrum herbicides promise cheap, selective and efficient weed control by excluding infested weeds resulting in turf lawn with high uniformity and aesthetic value. The concept of this "weeding program" initiated from modern biotechnology has been widely implemented in several principal crops including maize, soybean, canola and cotton as early as the 1990s. Transgenic HR turfgrass classified as a genetically modified organism (GMO) has undoubtedly caused public concern with respect to its biosafety and legalities similar to well-established HR crops. Nevertheless, applying metabolomics-based approaches which focuses on the identification of the global metabolic state of a biological system in response to either internal or external stimuli can also provide a comprehensive characterization of transgenic grass metabolism and its involvement in biosecurity and public perception. AIM OF REVIEW: This review summaries the recent applications of metabolomics applied to HR crops to predict the molecular and physiological phenotypes of HR turfgrass species, glyphosate-resistant Kentucky bluegrass (Poa pratensis L.) and glufosinate-resistant creeping bentgrass (Agrotis stonifera L.). Additionally, this review also presents background knowledge with respect to the application of metabolomics, transformation of HR crops and its biosafety concerns, turfgrass botanical knowledge and its economic and aesthetic value. KEY SCIENTIFIC CONCEPTS OF REVIEW: The purpose of this review is to demonstrate the molecular and physiological phenotypes of HR turfgrass based on several lines of evidence primarily derived from metabolomics data applied to HR crops to identify alterations on HR turfgrass metabolism as a result of genetic modification that confers resistant traits.

Lipid production in association of filamentous fungi with genetically modified cyanobacterial cells.

BACKGROUND: Numerous strategies have evolved recently for the generation of genetically modified or synthetic microalgae and cyanobacteria designed for production of ethanol, biodiesel and other fuels. In spite of their obvious attractiveness there are still a number of challenges that can affect their economic viability: the high costs associated with (1) harvesting, which can account for up to 50 % of the total biofuel's cost, (2) nutrients supply and (3) oil extraction. Fungal-assisted bio-flocculation of microalgae is gaining increasing attention due to its high efficiency, no need for added chemicals and low energy inputs. The implementation of renewable alternative carbon, nitrogen and phosphorus sources from agricultural wastes and wastewaters for growing algae and fungi makes this strategy economically attractive. RESULTS: This work demonstrates that the filamentous fungi, Aspergillus fumigatus can efficiently flocculate the unicellular cyanobacteria Synechocystis PCC 6803 and its genetically modified derivatives that have been altered to enable secretion of free fatty acids into growth media. Secreted free fatty acids are potentially used by fungal cells as a carbon source for growth and ex-novo production of lipids. For most of genetically modified strains the total lipid yields extracted from the fungal-cyanobacterial pellets were found to be higher than additive yields of lipids and total free fatty acids produced by fungal and Synechocystis components when grown in mono-cultures. The synergistic effect observed in fungal-Synechocystis associations was also found in bioremediation rates when animal husbandry wastewater was used an alternative source of nitrogen and phosphorus. CONCLUSION: Fungal assisted flocculation can complement and assist in large scale biofuel production from wild-type and genetically modified Synechocystis PCC 6803 strains by (1) efficient harvesting of cyanobacterial cells and (2) producing of high yields of lipids accumulated in fungal-cyanobacterial pellets.

Fungal-assisted algal flocculation: application in wastewater treatment and biofuel production.

BACKGROUND: The microalgal-based industries are facing a number of important challenges that in turn affect their economic viability. Arguably the most important of these are associated with the high costs of harvesting and dewatering of the microalgal cells, the costs and sustainability of nutrient supplies and costly methods for large scale oil extraction. Existing harvesting technologies, which can account for up to 50% of the total cost, are not economically feasible because of either requiring too much energy or the addition of chemicals. Fungal-assisted flocculation is currently receiving increased attention because of its high harvesting efficiency. Moreover, some of fungal and microalgal strains are well known for their ability to treat wastewater, generating biomass which represents a renewable and sustainable feedstock for bioenergy production. RESULTS: We screened 33 fungal strains, isolated from compost, straws and soil for their lipid content and flocculation efficiencies against representatives of microalgae commercially used for biodiesel production, namely the heterotrophic freshwater microalgae Chlorella protothecoides and the marine microalgae Tetraselmis suecica. Lipid levels and composition were analyzed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources from wheat straw and swine wastewater, respectively. The biomass of fungal-algal pellets grown on swine wastewater was used as feedstock for the production of value-added chemicals, biogas, bio-solids and liquid petrochemicals through pyrolysis. Co-cultivation of microalgae and filamentous fungus increased total biomass production, lipid yield and wastewater bioremediation efficiency. CONCLUSION: Fungal-assisted microalgal flocculation shows significant potential for solving the major challenges facing the commercialization of microalgal biotechnology, namely (i) the efficient and cost-effective harvesting of freshwater and seawater algal strains; (ii) enhancement of total oil production and optimization of its composition; (iii) nutrient supply through recovering of the primary nutrients, nitrogen and phosphates and microelements from wastewater. The biomass generated was thermochemically converted into biogas, bio-solids and a range of liquid petrochemicals including straight-chain C12 to C21 alkanes which can be directly used as a glycerine-free component of biodiesel. Pyrolysis represents an efficient alternative strategy for biofuel production from species with tough cell walls such as fungi and fungal-algal pellets.

Co-cultivation of fungal and microalgal cells as an efficient system for harvesting microalgal cells, lipid production and wastewater treatment.

The challenges which the large scale microalgal industry is facing are associated with the high cost of key operations such as harvesting, nutrient supply and oil extraction. The high-energy input for harvesting makes current commercial microalgal biodiesel production economically unfeasible and can account for up to 50% of the total cost of biofuel production. Co-cultivation of fungal and microalgal cells is getting increasing attention because of high efficiency of bio-flocculation of microalgal cells with no requirement for added chemicals and low energy inputs. Moreover, some fungal and microalgal strains are well known for their exceptional ability to purify wastewater, generating biomass that represents a renewable and sustainable feedstock for biofuel production. We have screened the flocculation efficiency of the filamentous fungus A. fumigatus against 11 microalgae representing freshwater, marine, small (5 µm), large (over 300 µm), heterotrophic, photoautotrophic, motile and non-motile strains. Some of the strains are commercially used for biofuel production. Lipid production and composition were analysed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources contained in wheat straw and swine wastewater, respectively. Co-cultivation of algae and A. fumigatus cells showed additive and synergistic effects on biomass production, lipid yield and wastewater bioremediation efficiency. Analysis of fungal-algal pellet's fatty acids composition suggested that it can be tailored and optimised through co-cultivating different algae and fungi without the need for genetic modification.

Dual application of duckweed and azolla plants for wastewater treatment and renewable fuels and petrochemicals production.

BACKGROUND: Shortages in fresh water supplies today affects more than 1 billion people worldwide. Phytoremediation strategies, based on the abilities of aquatic plants to recycle nutrients offer an attractive solution for the bioremediation of water pollution and represents one of the most globally researched issues. The subsequent application of the biomass from the remediation for the production of fuels and petrochemicals offers an ecologically friendly and cost-effective solution for water pollution problems and production of value-added products. RESULTS: In this paper, the feasibility of the dual application of duckweed and azolla aquatic plants for wastewater treatment and production of renewable fuels and petrochemicals is explored. The differences in absorption rates of the key wastewater nutrients, ammonium and phosphorus by these aquatic macrophytes were used as the basis for optimization of the composition of wastewater effluents. Analysis of pyrolysis products showed that azolla and algae produce a similar range of bio-oils that contain a large spectrum of petrochemicals including straight-chain C10-C21 alkanes, which can be directly used as diesel fuel supplement, or a glycerin-free component of biodiesel. Pyrolysis of duckweed produces a different range of bio-oil components that can potentially be used for the production of "green" gasoline and diesel fuel using existing techniques, such as catalytic hydrodeoxygenation. CONCLUSIONS: Differences in absorption rates of the key wastewater nutrients, ammonium and phosphorus by different aquatic macrophytes can be used for optimization of composition of wastewater effluents. The generated data suggest that the composition of the petrochemicals can be modified in a targeted fashion, not only by using different species, but also by changing the source plants' metabolic profile, by exposing them to different abiotic or biotic stresses. This study presents an attractive, ecologically friendly and cost-effective solution for efficient bio-filtration of swine wastewater and petrochemicals production from generated biomass.

Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts.

FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis.

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506-508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4-17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways.

Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis.

The whole genome expression pattern of Arabidopsis in response to the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated using the Affymetrix ATH1-121501 array. Arabidopsis plants were grown in vitro and were exposed to 1 mM 2,4-D for 1 h, after which time gene transcription levels were measured. In response to the treatment 148 genes showed increased levels of transcription and concurrently 85 genes showed decreased levels of transcript. Genes which showed significant change in transcription levels belonged to the following functional categories: transcription, metabolism, cellular communication and signal transduction, subcellular localisation, transport facilitation, protein fate, protein with binding function or cofactor requirement and regulation of/interaction with cellular environment. Interestingly 25.3% of the genes regulated by the treatment could not be classified into a known functional category. The data obtained from these experiments were used to assess the current model of auxinic herbicide action and indicated that 2,4-D not only modulates the expression of auxin, ethylene and abscisic acid (ABA) pathways but also regulates a wide variety of other cellular functions.

GSTT1 null genotype increases risk of premenopausal breast cancer.

The NAT2, GSTM1 and GSTT1 genes are known candidate cancer susceptibility markers and have been investigated in breast cancer susceptibility with conflicting results. We conducted a case-control study to investigate the role of NAT2, GSTM1 and GSTT1 in premenopausal breast cancer. Women with the GSTT1 null genotype were found to have a significant 3.15-fold increased risk of breast cancer (95% CI = 1.7-5.8), while GSTM1 and NAT2 genotypes were not associated with breast cancer risk. Our results suggest that the GSTT1 null genotype may play a role in early onset breast cancer.