Anders S. Carlsson

High‐value oils from plants

The seed oils of domesticated oilseed crops are major agricultural commodities that are used primarily for nutritional applications, but in recent years there has been increasing use of these oils for production of biofuels and chemical feedstocks. This is being driven in part by the rapidly rising costs of petroleum, increased concern about the environmental impact of using fossil oil, and the need to develop renewable domestic sources of fuel and industrial raw materials. There is also a need to develop sustainable sources of nutritionally important fatty acids such as those that are typically derived from fish oil. Plant oils can provide renewable sources of high-value fatty acids for both the chemical and health-related industries. The value and application of an oil are determined largely by its fatty acid composition, and while most vegetable oils contain just five basic fatty acid structures, there is a rich diversity of fatty acids present in nature, many of which have potential usage in industry. In this review, we describe several areas where plant oils can have a significant impact on the emerging bioeconomy and the types of fatty acids that are required in these various applications. We also outline the current understanding of the underlying biochemical and molecular mechanisms of seed oil production, and the challenges and potential in translating this knowledge into the rational design and engineering of crop plants to produce high-value oils in plant seeds.

Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis

A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487–6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.

Plant oils as feedstock alternatives to petroleum - a short survey of potential oil crop platforms.

Our society is highly depending on petroleum for its activities. About 90% is used as an energy source for transportation and for generation of heat and electricity and the remaining as feedstocks in the chemical industry. However, petroleum is a finite source as well as causing several environmental problems such as rising carbon dioxide levels in the atmosphere. Petroleum therefore needs to be replaced by alternative and sustainable sources. Plant oils and oleochemicals derived from them represent such alternative sources, which can deliver a substantial part of what is needed to replace the petroleum used as feedstocks. Plant derived feedstock oils can be provided by two types of oil qualities, multi-purpose and technical oils. Multi-purpose oils represent oil qualities that contain common fatty acids and that can be used for both food and feedstock applications. Technical oil qualities contain unusual fatty acids with special properties gained from their unique molecular structure and these types of oils should only be used for feedstock applications. As a risk mitigation strategy in the selection of crops, technical oil qualities should therefore preferably be produced by oil crop platforms dedicated for industrial usage. This review presents a short survey of oil crop platforms to be considered for either multi-purpose or technical oils production. Included among the former platforms are some of the major oil crops in cultivation such as oil palm, soybean and rapeseed. Among the later are those that could be developed into dedicated industrial platforms such as crambe, flax, cotton and Brassica carinata. The survey finishes off by highlighting the potential of substantial increase in plant oil production by developing metabolic flux platforms, which are starch crops converted into oil crops.

Functional expression of five Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli.

Very long chain primary alcohols are significant components in cuticle waxes of plants. Fatty acyl-CoA reductases (FARs) catalyze the formation of a fatty alcohol from an acyl-CoA. The Arabidopsis (Arabidopsis thaliana) genome contains eight genes homologous to FAR genes from jojoba (Simmondsia chinensis), silk moth, wheat and mouse. Expression of six Arabidopsis FAR homologs in Escherichia coli resulted in production of alcohols from endogenous E. coli fatty acids by five of these genes, confirming that they encode for FAR enzymes. Only a truncated splicing version of the sixth gene was found, and this gene yielded a protein with no FAR activity. The five functional FAR enzymes yielded distinctly different compositions of fatty alcohols when expressed in E. coli, indicating that the different enzymes may be involved in the production of different types of alcohols in plant cells.

Replacing fossil oil with fresh oil – with what and for what?

Industrial chemicals and materials are currently derived mainly from fossil-based raw materials, which are declining in availability, increasing in price and are a major source of undesirable greenhouse gas emissions. Plant oils have the potential to provide functionally equivalent, renewable and environmentally friendly replacements for these finite fossil-based raw materials, provided that their composition can be matched to end-use requirements, and that they can be produced on sufficient scale to meet current and growing industrial demands. Replacement of 40% of the fossil oil used in the chemical industry with renewable plant oils, whilst ensuring that growing demand for food oils is also met, will require a trebling of global plant oil production from current levels of around 139 MT to over 400 MT annually. Realisation of this potential will rely on application of plant biotechnology to (i) tailor plant oils to have high purity (preferably >90%) of single desirable fatty acids, (ii) introduce unusual fatty acids that have specialty end-use functionalities and (iii) increase plant oil production capacity by increased oil content in current oil crops, and conversion of other high biomass crops into oil accumulating crops. This review outlines recent progress and future challenges in each of these areas. Practical applications: The research reviewed in this paper aims to develop metabolic engineering technologies to radically increase the yield and alter the fatty acid composition of plant oils and enable the development of new and more productive oil crops that can serve as renewable sources of industrial feedstocks currently provided by non-renewable and polluting fossil-based resources. As a result of recent and anticipated research developments we can expect to see significant enhancements in quality and productivity of oil crops over the coming decades. This should generate the technologies needed to support increasing plant oil production into the future, hopefully of sufficient magnitude to provide a major supply of renewable plant oils for the industrial economy without encroaching on the higher priority demand for food oils. Achievement of this goal will make a significant contribution to moving to a sustainable carbon-neutral industrial society with lower emissions of carbon dioxide to the atmosphere and reduced environmental impact as a result.

Acyl editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid flux into triacylglycerols.

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified “acyl-editing” process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [14C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis.

Metabolic Interactions between the Lands Cycle and the Kennedy Pathway of Glycerolipid Synthesis in Arabidopsis Developing Seeds

The Kennedy pathway and the Lands cycle are two principal metabolic modules of glycerolipid metabolism. This work examines the crosstalk of these two pathways and shows that loss of Lands cycle activity leads to an enhanced de novo phosphatidylcholine PC synthesis through the Kennedy pathway and PC turnover in Arabidopsis developing seeds. It has been widely accepted that the primary function of the Lands cycle is to provide a route for acyl remodeling to modify fatty acid (FA) composition of phospholipids derived from the Kennedy pathway. Lysophosphatidylcholine acyltransferase (LPCAT) is an evolutionarily conserved key enzyme in the Lands cycle. In this study, we provide direct evidence that the Arabidopsis thaliana LPCATs, LPCAT1 and LPCAT2, participate in the Lands cycle in developing seeds. In spite of a substantially reduced initial rate of nascent FA incorporation into phosphatidylcholine (PC), the PC level in the double mutant lpcat1 lpcat2-2 remained unchanged. LPCAT deficiency triggered a compensatory response of de novo PC synthesis and a concomitant acceleration of PC turnover that were attributable at least in part to PC deacylation. Acyl-CoA profile analysis revealed complicated metabolic alterations rather than merely reduced acyl group shuffling from PC in the mutant. Shifts in FA stereo-specific distribution in triacylglycerol of the mutant seed suggested a preferential retention of saturated acyl chains at the stereospecific numbering (sn)-1 position from PC and likely a channeling of lysophosphatidic acid, derived from PC, into the Kennedy pathway. Our study thus illustrates an intricate relationship between the Lands cycle and the Kennedy pathway.

Distribution of fatty acids in polar and neutral lipids during seed development in Arabidopsis thaliana genetically engineered to produce acetylenic, epoxy and hydroxy fatty acids

Genetically engineered plants of Arabidopsis thaliana containing either the Crepis alpina linoleate delta 12-acetylenase gene, the Crepis palaestina linoleate delta 12-epoxygenase gene under transcriptional control of a napin promoter or a Lesquerella fendleri oleate hydroxylase under the transcriptional control of the endogenous promoter were analysed for the distribution of fatty acids. Seed samples were analysed from 9 to 23 days after pollination (DAP) as well as mature seeds. The relative amount of oleate was higher and the amount of the polyunsaturated fatty acids was lower in the seeds of the transgenic plants expressing the fatty acid modifying genes than in seeds from the control plants. The degree of the changes was more pronounced in phosphatidylcholine (PC) than in the neutral fraction and varied considerably between the transgenic plants carrying the different genes. The unusual fatty acids (acetylenic, epoxy and hydroxy fatty acids) produced in the transgenic A. thaliana plants accumulated in significant amounts in seed phospholipids during seed development. Despite that the percentage of the unusual fatty acids accumulated in neutral lipids in the transgenic plants were 5–35 times less than in the wild plant species accumulating these acids, the relative levels of these acids in PC were approximately the same (epoxy and hydroxy fatty acids) or much higher (acetylenic fatty acids) than in that lipid in the wild plants. Since ricinoleic, vernolic and crepenynic acid are formed from precursor fatty acids esterified in PC catalysed by delta 12-desaturase like enzymes, it can be concluded that A. thaliana seeds have less efficient mechanisms for the specific channelling of these fatty acids from PC to triacylglycerol (TAG).

Triacylglycerol synthesis by PDAT1 in the absence of DGAT1 activity is dependent on re-acylation of LPC by LPCAT2

BackgroundThe Arabidopsis thaliana dgat1 mutant, AS11, has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, AS11 still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of DGAT1, PDAT1 was essential for normal seed development, and is a dominant determinant in Arabidopsis TAG biosynthesis.MethodsBiochemical, metabolic and gene expression studies combined with genetic crossing of selected Arabidopsis mutants have been carried out to demonstrate the contribution of Arabidopsis PDAT1 and LPCAT2 in the absence of DGAT1 activity.ResultsThrough microarray and RT-PCR gene expression analyses of AS11 vs. WT mid-developing siliques, we observed consistent trends between the two methods. FAD2 and FAD3 were up-regulated and FAE1 down-regulated, consistent with the AS11 acyl phenotype. PDAT1 expression was up-regulated by ca 65% while PDAT2 expression was up-regulated only 15%, reinforcing the dominant role of PDAT1 in AS11 TAG biosynthesis. The expression of LPCAT2 was up-regulated by 50-75%, while LPCAT1 expression was not significantly affected. In vitro LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing AS11 seed vs WT. Co-incident homozygous knockout lines of dgat1/lpcat2 exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant dgat1/lpcat1 showed only marginally lower oil content than AS11.ConclusionsCollectively, the data strongly support that in AS11 it is LPCAT2 up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer sn-2 moieties to the sn-3 position of the enlarged AS11 DAG pool.

Transcriptional transitions in Nicotiana benthamiana leaves upon induction of oil synthesis by WRINKLED1 homologs from diverse species and tissues.

BackgroundCarbon accumulation and remobilization are essential mechanisms in plants to ensure energy transfer between plant tissues with different functions or metabolic needs and to support new generations. Knowledge about the regulation of carbon allocation into oil (triacylglycerol) in plant storage tissue can be of great economic and environmental importance for developing new high-yielding oil crops. Here, the effect on global gene expression as well as on physiological changes in leaves transiently expressing five homologs of the transcription factor WRINKLED1 (WRI1) originating from diverse species and tissues; Arabidopsis thaliana and potato (Solanum tuberosum) seed embryo, poplar (Populus trichocarpa) stem cambium, oat (Avena sativa) grain endosperm, and nutsedge (Cyperus esculentus) tuber parenchyma, were studied by agroinfiltration in Nicotiana benthamiana.ResultsAll WRI1 homologs induced oil accumulation when expressed in leaf tissue. Transcriptome sequencing revealed that all homologs induced the same general patterns with a drastic shift in gene expression profiles of leaves from that of a typical source tissue to a source-limited sink-like tissue: Transcripts encoding enzymes for plastid uptake and metabolism of phosphoenolpyruvate, fatty acid and oil biosynthesis were up-regulated, as were also transcripts encoding starch degradation. Transcripts encoding enzymes in photosynthesis and starch synthesis were instead down-regulated. Moreover, transcripts representing fatty acid degradation were up-regulated indicating that fatty acids might be degraded to feed the increased need to channel carbons into fatty acid synthesis creating a futile cycle. RT-qPCR analysis of leaves expressing Arabidopsis WRI1 showed the temporal trends of transcripts selected as ‘markers’ for key metabolic pathways one to five days after agroinfiltration. Chlorophyll fluorescence measurements of leaves expressing Arabidopsis WRI1 showed a significant decrease in photosynthesis, even though effect on starch content could not be observed.ConclusionsThis data gives for the first time a general view on the transcriptional transitions in leaf tissue upon induction of oil synthesis by WRI1. This yields important information about what effects WRI1 may exert on global gene expression during seed and embryo development. The results suggest why high oil content in leaf tissue cannot be achieved by solely transcriptional activation by WRI1, which can be essential knowledge in the development of new high-yielding oil crops.