122 research outputs found
Potato tuber expression of Arabidopsis <i>WRINKLED1</i> increase triacylglycerol and membrane lipids while affecting central carbohydrate metabolism
No full textTuber and root crops virtually exclusively accumulate storage products in the form of carbohydrates. An exception is yellow nutsedge (Cyperus esculentus) in which tubers have the capacity to store starch and triacylglycerols (TAG) in roughly equal amounts. This suggests that a tuber crop can efficiently handle accumulation of energy dense oil. From a nutritional as well as economic aspect, it would be of interest to utilize the high yield capacity of tuber or root crops for oil accumulation similar to yellow nutsedge. The transcription factor WRINKLED1 from Arabidopsis thaliana, which in seed embryos induce fatty acid synthesis, has been shown to be a major factor for oil accumulation. WRINKLED1 was expressed in potato (Solanum tuberosum) tubers to explore whether this factor could impact tuber metabolism. This study shows that a WRINKLED1 transcription factor could induce triacylglycerol accumulation in tubers of transformed potato plants grown in field (up to 12 nmol TAG/mg dry weight, 1% of dry weight) together with a large increase in polar membrane lipids. The changes in metabolism further affected starch accumulation and composition concomitant with massive increases in sugar content
Production of amylopectin and high-amylose starch in separate potato genotypes
No full textStarch is one of the most important processed products from agriculture. Two main outlets can be identified; starch is either enzymatically processed for the production of sweeteners and as raw material for fermentation or channelled to various applications as dry starch. Native or chemically modified starches are utilized in food as well as non-food applications, where the specific physicochemical properties are main determinants for their respective use. Starch consists of two different molecules, amylose and amylopectin. To be able to take the full benefit of the unique properties of either component it is of interest to divide the production of amylose and amylopectin into separate plant genotypes. In the presented work, potatoes producing either amylopectin or high-amylose starch were achieved using genetic modification. For potato transformation a highly efficient protocol was developed for a herbicide selection gene instead of the commonly used nptII antibiotic selection gene. In order to achieve respective starch qualities, the expression of genes important for amylopectin or amylose synthesis was silenced. Antisense technology as well as the expression of dsRNA was investigated where the expression of dsRNA was determined to be at least ten-fold more efficient for gene silencing. An added benefit of dsRNA expression was that a higher fraction of silenced transgenic lines compared to the use antisense were associated with single copy T-DNA integrations. One amylopectin potato line was furthermore characterized regarding genetic and chemical composition. The T-DNA was found integrated as an inverted repeat with the inverted repeat region extending into potato chromosomal DNA. This transgenic locus was found to be more consistent with integration into a double-stranded chromosomal break than insertion by a mechanism nicking one strand of the locus. The high-amylose trait generally resulted in a higher tuber fresh weight yield, much elevated sugar levels and a decreased starch content. Amylose levels were obtained where very limited amounts of material recognizable as amylopectin could be found. The production of amylopectin and amylose was divided into separate genotypes but additional factors are needed to be able to produce amylose at levels comparable to starch contents of cultivated potatoes
Wax ester profiling of seed oil by nano-electrospray ionization tandem mass spectrometry
No full textBackground: Wax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts. Results: We present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS. Conclusions: We developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.European Commissio
Production of moth sex pheromone precursors in Nicotiana spp.: a worthwhile new approach to pest control [Elektronisk resurs]
No full textPheromones are environmentally friendly alternatives to traditional pesticides for pest control. They are widely applied for insect monitoring, mating disruption and mass trapping.Nicotiana benthamianaandN. tabacumare potential green biomass production platforms of moth sex pheromones. Using these twoNicotianaspecies as plant factories, we expressed biosynthetic genes of plant and insect origin in leaf tissue. Moth sex pheromone precursors (E)-11-tetradecenoic acid, (Z)-11-tetradecenoic acid and (Z)-11-hexadecenoic acid were produced by introducing the acyl-ACP thioesterasesCpuFatB1fromCuphea pulcherrimaorCpaFatB2fromC. palustrisand the fatty acyl desaturasesAve increment 11fromArgyrotaenia velutinana,CpaE11fromChoristoneura parallelaorAtr increment 11fromAmyelois transitella, under the control of CaMV-35S promoter. Among theNicotianaspp. transformants, the best line produced (Z)-11-hexadecenoic acid at 17.6% of total fatty acids in leaves, during flowering stage, corresponding to 335 mu g of (Z)-11-hexadecenoic acid per gram of fresh leaf. The (Z)-11-hexadecenoic acid production lines fromN. benthamianawere selected for further propagation to obtain homozygous lines. In theN. benthamianaT2 generation, the production quantity of (Z)-11-hexadecenoic acid was stable. Our study demonstrates the feasibility of stable transformation ofN. benthamianafor production of moth pheromone precursors in vegetative tissue
Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
No full textClustered regularly interspaced short palindromic repeats and CRISPR-associated protein-9 (CRISPR-Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA-free genome editing method, using delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr-RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene-free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT–RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2–3% of the regenerated shoots from the RNP-experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.Fil: Andersson, Mariette. Swedish University Of Agricultural Sciences; SueciaFil: Turesson, Helle. Swedish University Of Agricultural Sciences; SueciaFil: Olsson, Niklas. Swedish University Of Agricultural Sciences; SueciaFil: Fält, Ann Sofie. Swedish University Of Agricultural Sciences; SueciaFil: Ohlsson, Pia. Swedish University Of Agricultural Sciences; SueciaFil: Gonzalez, Matías Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Samuelsson, Mathias. Lyckeby Starch AB; SueciaFil: Hofvander, Per. Swedish University Of Agricultural Sciences; Sueci
Tomato protoplasts as cell target for ribonucleoprotein (RNP)-mediated multiplexed genome editing
No full textThe possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)-mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency.Key message RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts
Release of moth pheromone compounds from Nicotiana benthamiana upon transient expression of heterologous biosynthetic genes [Elektronisk resurs]
No full textBackground Using genetically modified plants as natural dispensers of insect pheromones may eventually become part of a novel strategy for integrated pest management. Results In the present study, we first characterized essential functional genes for sex pheromone biosynthesis in the rice stem borer Chilo suppressalis (Walker) by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana, including two desaturase genes CsupYPAQ and CsupKPSE and a reductase gene CsupFAR2. Subsequently, we co-expressed CsupYPAQ and CsupFAR2 together with the previously characterized moth desaturase Atr increment 11 in N. benthamiana. This resulted in the production of (Z)-11-hexadecenol together with (Z)-11-hexadecenal, the major pheromone component of C. suppressalis. Both compounds were collected from the transformed N. benthamiana headspace volatiles using solid-phase microextraction. We finally added the expression of a yeast acetyltransferase gene ATF1 and could then confirm also (Z)-11-hexadecenyl acetate release from the plant. Conclusions Our results pave the way for stable transformation of plants to be used as biological pheromone sources in different pest control strategies
Identification of 1,6-hexadecanediol and its wax diesters in chloroplasts of Nicotiana benthamiana
No full textMain conclusionExpression of the Arabidopsis phytyl ester synthase PES2 in Nicotiana benthamiana chloroplasts resulted in the accumulation of fatty acid phytyl esters and wax diesters containing the novel alkanediol 1,6-hexadecanediol.AbstractDihydric long-chain alcohols carrying two hydroxyl groups are low abundant in plants and are mostly found in the cutin layer of leaves or the suberin of roots. Transient expression of the phytyl ester synthase PES2 from Arabidopsis thaliana in Nicotiana benthamiana resulted in the accumulation of fatty acid phytyl esters (FAPEs) and of a new lipid class that was identified as wax diesters of 1,6-hexadecanediol, carrying mostly lauric acid (12:0) and myristic acid (14:0) residues. The synthesis of FAPE and wax diesters was only observed when PES2 was targeted to the chloroplasts, in agreement with the finding that both FAPE and wax diesters are chloroplast-localized. The accumulation of wax diesters following PES2 expression demonstrates that the dihydric long-chain alcohol, 1,6-hexadecanediol, is an authentic compound produced in N. benthamiana chloroplasts. 1,6-Hexadecanediol in N. benthamiana is likely synthesized by a chloroplast-localized fatty acid reductase (FAR) in combination with a P450 monooxygenase. PES2-mediated acylation might result in chloroplast-trapping of 1,6-hexadecanediol which is possibly an intermediate in the biosynthesis of functional compounds in leaves or other plant organs
Green Chemistry Production of Codlemone, the Sex Pheromone of the Codling Moth (Cydia pomonella), by Metabolic Engineering of the Oilseed Crop Camelina (Camelina sativa) [Elektronisk resurs]
No full textSynthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C-12 chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional increment 9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T-2 generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T-4 generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone
A prokaryotic acyl-CoA reductase performing reduction of fatty acyl-CoA to fatty alcohol
No full textAbstractThe reduction of acyl-CoA or acyl-ACP to fatty alcohol occurs via a fatty aldehyde intermediate. In prokaryotes this reaction is thought to be performed by separate enzymes for each reduction step while in eukaryotes these reactions are performed by a single enzyme without the release of the intermediate fatty aldehyde. However, here we report that a purified fatty acyl reductase from Marinobacter aquaeolei VT8, evolutionarily related to the fatty acyl reductases in eukaryotes, catalysed both reduction steps. Thus, there are at least two pathways existing among prokaryotes for the reduction of activated acyl substrates to fatty alcohol. The Marinobacter fatty acyl reductase studied has a wide substrate range in comparison to what can be found among enzymes so far studied in eukaryotes
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