Using flux control analysis to improve oilseed rape

Oil crops are one of the most important agricultural commodities. In the U.K. (and Northern Europe and Canada) oilseed rape is the dominant oil crop and worldwide it accounts for about 12% of the total oil and fat production. There is an increasing demand for plant oils not only for human food and animal feed but also as renewable sources of chemicals and biofuels. This increased demand has shown a doubling every 8 years over the last four decades and is likely to continue at, at least, this rate in the future. With a limitation on agricultural land, the main way to increase production is to increase yields. This can be achieved by conventional breeding but, in the future, significant enhancements will need genetic manipulation. The latter technique will also allow specific modification of the oil product to be achieved.
In order for informed genetic manipulation to take place, a thorough knowledge of the biosynthesis of plant oils is needed. Crucially, this would include how regulation of oil quality and quantity is controlled. The synthesis of storage oil in plant seeds is analogous to a factory production line, where the supply of raw materials, manufacture of components and final assembly can all potentially limit the rate of production. Recently, we made a first experimental study of overall regulation of storage oil accumulation in oilseed rape, which we analysed by a mathematical method called flux control analysis. This showed that it is the final assembly that is the most important limitation on the biosynthetic process.
The assembly process requires several enzyme steps and we have already highlighted one of these, diacylglycerol acyltransferase (DGAT), as being a significant controlling factor. We now wish to examine enzymes, other than DGAT, involved in storage lipid assembly and in supply of component parts. This will enable us to quantify the limitations imposed by different enzymes of the pathway and, furthermore, will provide information to underpin logical steps in genetic manipulation leading to plants with increased oil synthesis and storage capabilities.
We will use rape plants where the activity of individual enzymes in the biosynthetic pathway have been changed and quantify the effects on overall oil accumulation.
To begin with we will use existing transgenic oilseed rape, with increased enzyme levels, where increases in oil yields have been noted; these are available from our collaborators (Canada, Germany). For enzymes where there are no current transgenic plants available, we will make these and carry out similar analyses.
Although our primary focus is on enzymes that increase oil yields, we will also examine the contribution the enzyme phospholipid: diacylglycerol acyltransferase (PDAT) makes to lipid production because this enzyme controls the accumulation of unsaturated oil, which has important dietary implications. In the analogous model plant Arabidopsis, PDAT and DGAT are both important during oil production.
Once we have assembled data from these transgenic plants we will have a much better idea of the control of lipid production in oilseed rape. Our quantitative measurements will provide specific targets for future crop improvements. In addition, because we will be monitoring oil yields as well as flux control we will be able to correlate these two measures. Moreover, plants manipulated with multiple genes (gene stacking) will reveal if there are synergistic effects of such strategies.
Because no one has yet defined quantitatively the oil synthesis pathway in crops, data produced in the project will have a fundamental impact in basic science. By combining the expertise of three important U.K. labs. with our world-leading international collaborators, this cross-disciplinary project will ensure a significant advance in knowledge of direct application to agriculture.

We have pioneered the use of flux control analysis in the understanding of lipid biosynthesis, which has led to the quantitative measure that 70% of the regulation is in the enzymes catalysing triacylglycerol assembly, with the remainder lying in substrate supply. Further we have shown that terminal enzyme in the assembly (Kennedy) pathway exerts significant flux control and that increasing the level of DGAT results in improvements in oil yield in rapeseed.
We will systematically quantify the control that enzymes exert over storage lipid synthesis by over-expressing them individually, or in combination in transgenic plants and measuring pathway flux. Target enzymes will be in the Kennedy pathway (GPAT, LPAT, PDAT), in glycerol-3-phosphate supply (G-3-P dehydrogenase), or which have been shown to divert carbon from central metabolism into the Kennedy pathway (antisense mitochondrial pyruvate dehydrogenase kinase; mtPDCK). The potential synergy of multigene cassettes will be probed, using plants that contain both LPAT and DGAT, either alone or in combination with antisense mtPDCK. We will also quantify the importance of the supply of glycerol 3-phosphate in storage lipid synthesis; over-expression of glycerol-3-phosphate dehydrogenase has previously been seen to increase seed fatty acid content of oil-seed rape, indicating that enzymes outside of the Kennedy pathway can influence oil yield.
Throughout, we will compare gene expression-induced changes in flux, with independent measures of regulation and analyse transcripts, enzyme activities and seed characteristics for ancillary effects.The data produced will give a comprehensive picture of the regulation of oil accumulation and indicate which combinations of over-expressed genes result is the optimal increase in pathway flux and overall yield without a growth penalty.
This research will allow informed advice to be given to maximise agricultural production of rapeseed through breeding and biotechnology approaches.

Beneficiaries from this project will be industry, the academic community and the general public. Oil crops are one of the most important features of agriculture in the UK, Northern Europe and North America and are of primary importance for food security. In addition, the possibilities of using rapeseed oil as a sustainable source of petrochemical substitutes are becoming better known. With the tight limits on agricultural land, most people realise that understanding how to maintain (or enhance) crop yields is vital; in this regard, there is both academic and commercial interest in understanding and alleviating the constraints in metabolic pathways to produce commercially valuable crop products.
Our previous work about flux control analysis has been noticed by industry and, indeed, partly funded therefrom (see supporting letters attached). Clearly, increases in crop yields are an important target for the agrochemical industry and the demonstrated elevated yields for several new transgenic lines in oilseed rape are already promising. In this project, defining the impact of increasing the activity of individual enzymes and two- and three-gene stacks will allow us to move forward immediately in a progressive way to inform future crop modifications. As an example, field trials of our first transgenic rapeseed line, with increased diacylglycerol acyltransferase, gave an 8% increase in oil yield. At current market prices (Dec. 2012) this is worth about £980M for rape oil.
By the end of the project we will have identified new target enzymes and will have transgenic plants available for field trials. We will discuss these results and their potential commercialization, initially, with our existing industrial contacts, utilising the expertise within the Technology Transfer teams of both Cardiff and Durham Universities. It is likely that commercialization of this research would be on a medium (5-10 years) timescale.
The combination of biochemistry, molecular biology and systems control analysis is a strong one which will give fundamental understanding about the regulation of a primary pathway of metabolism. The results of the work will be reported at both national and international conferences and the significance of the work is likely to continue to attract invitations to important international meetings. This research will have an impact outside of the lipid field, as it will illustrate a systematic way of increasing flux through a metabolic pathway.
The project would involve researchers in state-of-the-art techniques, leading to highly skilled and trained individuals. Since the project integrates biochemistry/molecular biology with systems modelling approaches, the biologists will benefit from practical knowledge of how mathematics can assist in biological research. These types of integrated skill sets acquired by the researchers are essential for a highly trained and flexible workforce that will be required to deliver the KBBE and contribute to future economic development and associated social benefits. Such individuals also enhance the skills and knowledge base available and, therefore, further contribute to the UK's attractiveness for international collaboration and for outside investment in R&D.
We are strong advocates of discussing the results of research with the general public. For example, Harwood has already written invited articles for popular science, given interviews for radio and TV and presented talks about the work for local science societies and in outreach activities for schools. These avenues will be followed also for this project and, in addition, we will utilise the Innovation Farm at NIAB in the third year of the project to disseminate the results. This will enable us to engage in a discussion with members of the public about the potential benefits of GM crops.