Oil-Producing Metabolons Containing DGAT1 Use Separate Substrate Pools from those Containing DGAT2 or PDAT.

Anushobha Regmi,Jay Shockey,Hari Kiran Kotapati,Philip D Bates

doi:10.1104/pp.20.00461

Abstract

Seed triacylglycerol (TAG) biosynthesis involves a metabolic network containing multiple different diacylglycerol (DAG) and acyl donor substrate pools. This network of pathways overlaps with those for essential membrane lipid synthesis and utilizes multiple different classes of TAG biosynthetic enzymes. Acyl flux through this network ultimately dictates the final oil fatty acid composition. Most strategies to alter seed oil composition involve the overexpression of lipid biosynthetic enzymes, but how these enzymes are assembled into metabolons and which substrate pools are used by each is still not well understood. To understand the roles of different classes of TAG biosynthetic acyltransferases in seed oil biosynthesis, we utilized the Arabidopsis (Arabidopsis thaliana) diacylglycerol acyltransferase mutant dgat1-1 (in which phosphatidylcholine:diacylglycerol acyltransferase (AtPDAT1) is the major TAG biosynthetic enzyme), and enhanced TAG biosynthesis by expression of Arabidopsis acyltransferases AtDGAT1 and AtDGAT2, as well as the DGAT2 enzymes from soybean (Glycine max), and castor (Ricinus communis), followed by isotopic tracing of glycerol flux through the lipid metabolic network in developing seeds. The results indicate each acyltransferase has a unique effect on seed oil composition. AtDGAT1 produces TAG from a rapidly produced phosphatidylcholine-derived DAG pool. However, AtPDAT1 and plant DGAT2 enzymes utilize a different and larger bulk phosphatidylcholine-derived DAG pool that is more slowly turned over for TAG biosynthesis. Based on metabolic fluxes and protein:protein interactions, our model of TAG synthesis suggests that substrate channeling to select enzymes and spatial separation of different acyltransferases into separate metabolons affect efficient TAG production and oil fatty acid composition.

Highlights

Seed triacylglycerol (TAG) biosynthesis involves a metabolic network containing multiple different diacylglycerol (DAG) and acyl donor substrate pools
The dgat1-1 pdat1-1 double mutant is lethal (Zhang et al, 2009), and suppression of AtPDAT1 expression by RNAi interference in the dgat11 genetic background reduces oil accumulation by 70% to 80%, suggesting that PDAT rather than DGAT2 or DGAT3 supports TAG biosynthesis when DGAT1 is lacking (Zhang et al, 2009). Together these results indicate that utilization of PC-derived DAG by DGAT1 for TAG biosynthesis contributes to the accumulation of polyunsaturated fatty acids (PUFA) in TAG in wild-type Arabidopsis, and likely in camelina and soybean as well
To better understand the role of PDAT and plant DGAT2s in the utilization of the multiple possible DAG pools for seed oil biosynthesis in an environment without competing DGAT1 activity, we produced Arabidopsis lines that contained endogenous PDAT activity paired with transgenic DGAT1 or DGAT2 expression as the major TAG biosynthetic enzymes expressed during the oil biosynthetic stages of seed development

Summary

Introduction

Seed triacylglycerol (TAG) biosynthesis involves a metabolic network containing multiple different diacylglycerol (DAG) and acyl donor substrate pools This network of pathways overlaps with those for essential membrane lipid synthesis and utilizes multiple different classes of TAG biosynthetic enzymes. Oil biosynthesis involves a metabolic network that directly overlaps with essential membrane lipid biosynthesis involving multiple different DAG and acyl donor substrate pools, and various different TAG biosynthetic acyltransferases (Fig. 1; Li-Beisson et al, 2013; Bates, 2016). To effectively control the fatty acid composition of plant oils through bioengineering, we need to understand the path of substrate flux through the seed lipid metabolic network, and the utilization of various substrate pools by each type of TAG biosynthetic acyltransferase (Fig. 1). PDAT activity efficiently transfers fatty acids from highly desaturated species of PC to TAG and contributes to the high levels of PUFA in seed oils of camelina (Marmon et al, 2017) and flax (Linum usitatissimum; Pan et al, 2013; Wickramarathna et al, 2015)

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Conclusion