Increase in lysophosphatidate acyltransferase activity in oilseed rape (Brassica napus) increases seed triacylglycerol content despite its low intrinsic flux control coefficient Helen K. Woodfield 1 * , Stepan Fenyk 2 * , Emma Wallington 3 , Ruth E. Bates 3 , Alexander Brown 2 , Irina A. Guschina 1 , Elizabeth-France Marillia 4 , David C. Taylor 4 , David Fell 5 , John L. Harwood 1 and Tony Fawcett 2 1 School of Biosciences, Cardiff University, Cardiff, CF10 3AX, UK; 2 Department of Biosciences, Durham University, Durham, DH1 3LE, UK; 3 The John Bingham Laboratory, NIAB, Huntingdon Road, Cambridge, CB3 0LE, UK; 4 National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S79 0W9, Canada; 5 Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, OX3 0BP, UK Authors for correspondence: John L. Harwood Tel: +44 29 2087 4116 Email: Harwood@cardiff.ac.uk Tony Fawcett Tel: +44 191 3341328 Email: tony.fawcett@durham.ac.uk Received: 1 May 2019 Accepted: 23 July 2019 New Phytologist (2019) 224: 700–711 doi: 10.1111/nph.16100 Key words: Brassica napus, crop improve- ment, flux control coefficient, lysophosphati- date acyltransferase, metabolic control analysis, storage oil, TAG, triacylglycerol. Summary  Lysophosphatidate acyltransferase (LPAAT) catalyses the second step of the Kennedy path- way for triacylglycerol (TAG) synthesis. In this study we expressed Trapaeolum majus LPAAT in Brassica napus (B. napus) cv 12075 to evaluate the effects on lipid synthesis and estimate the flux control coefficient for LPAAT.  We estimated the flux control coefficient of LPAAT in a whole plant context by deriving a relationship between it and overall lipid accumulation, given that this process is a exponential.  Increasing LPAAT activity resulted in greater TAG accumulation in seeds of between 25% and 29%; altered fatty acid distributions in seed lipids (particularly those of the Kennedy path- way); and a redistribution of label from 14 C-glycerol between phosphoglycerides.  Greater LPAAT activity in seeds led to an increase in TAG content despite its low intrinsic flux control coefficient on account of the exponential nature of lipid accumulation that ampli- fies the effect of the small flux increment achieved by increasing its activity. We have also developed a novel application of metabolic control analysis likely to have broad application as it determines the in planta flux control that a single component has upon accumulation of storage products. Introduction Oilseed rape (Brassica napus L.) is one of the major oil crops, accounting currently for c. 15% of total global vegetable oil pro- duction (Maheshwari & Kovalchuk, 2016; Weselake et al., 2017). Because of the inexorable rise in demand for vegetable oils (Gunstone et al., 2007) coupled to limited agricultural land, efforts are continually being made to improve production. While basic characteristics of the metabolic pathway involved in oil accumulation are known (Weselake et al., 2009; Lu et al., 2011; Bates et al., 2013), new enzymes are still being discovered. More- over, subtle differences in compartmentation (Chapman & Ohlrogge, 2012) and in the contribution of competing biosyn- thetic pathways add complexity to the overall process (Bates & Browse, 2012; Chen et al., 2015). Metabolic engineering strategies can be designed using knowl- edge of the distribution of control within a pathway, and both theoretical and experimental insights into this can be obtained within the framework of Metabolic Control Analysis (MCA). This approach assigns a numerical value, the flux control coeffi- cient, to the influence that a single enzyme, or a block of enzymes, can have on the metabolic flux. The value of the flux control coefficient can be used to estimate the response of a metabolic flux to overexpression of a particular enzyme (Small & Kacser, 1993; Fell, 2005). There are two major approaches in MCA: top-down and bottom-up. In the former, a high-level, modular view of the metabolic pathway is obtained, and this method has another advantage in that specific ways of manipulat- ing the activity of individual enzymes are not needed. Bottom-up control analysis builds up the picture from the response of the metabolic system to changes in the activity of individual enzymes, using specific inhibitors or selective changes in expression. *These authors contributed equally to this work. 700 New Phytologist (2019) 224: 700–711 Ó 2019 The Authors New Phytologist Ó 2019 New Phytologist Trust www.newphytologist.com This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Research