Reducing Isozyme Competition Increases Target Fatty Acid Accumulation in Seed Triacylglycerols of Transgenic Arabidopsis

Harrie Van Erp,Neil D Adhikari,John Browse,Jay Shockey,Meng Zhang

doi:10.1104/pp.114.254110

Harrie Van Erp, Neil D Adhikari + Show 3 more

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https://doi.org/10.1104/pp.114.254110

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Abstract

One goal of green chemistry is the production of industrially useful fatty acids (FAs) in crop plants. We focus on hydroxy fatty acids (HFAs) and conjugated polyenoic FAs (α-eleostearic acids [ESAs]) using Arabidopsis (Arabidopsis thaliana) as a model. These FAs are found naturally in seed oils of castor (Ricinus communis) and tung tree (Vernicia fordii), respectively, and used for the production of lubricants, nylon, and paints. Transgenic oils typically contain less target FA than that produced in the source species. We hypothesized that competition between endogenous and transgenic isozymes for substrates limits accumulation of unique FAs in Arabidopsis seeds. This hypothesis was tested by introducing a mutation in Arabidopsis diacylglycerol acyltransferase1 (AtDGAT1) in a line expressing castor FA hydroxylase and acyl-Coenzyme A:RcDGAT2 in its seeds. This led to a 17% increase in the proportion of HFA in seed oil. Expression of castor phospholipid:diacylglycerol acyltransferase 1A in this line increased the proportion of HFA by an additional 12%. To determine if our observations are more widely applicable, we investigated if isozyme competition influenced production of ESA. Expression of tung tree FA conjugase/desaturase in Arabidopsis produced approximately 7.5% ESA in seed lipids. Coexpression of VfDGAT2 increased ESA levels to approximately 11%. Overexpression of VfDGAT2 combined with suppression of AtDGAT1 increased ESA accumulation to 14% to 15%. Our results indicate that isozyme competition is a limiting factor in the engineering of unusual FAs in heterologous plant systems and that reduction of competition through mutation and RNA suppression may be a useful component of seed metabolic engineering strategies.

Highlights

Further Increases hydroxy fatty acids (HFAs) LevelsExpression of RcPDAT1A in the dgat1 Chaofu Lu7 (CL7) RcDGAT2 background could shift the balance in substrate competition between castor and Arabidopsis enzymes further in favor of incorporation of HFA into TAG
AtDGAT1 and AtPDAT1 catalyze the final step in the synthesis of TAG in Arabidopsis seeds (Zhang et al, 2009)
We hypothesize that substrate competition for common FAs versus unusual FAs occurs between AtPDAT1/AtDGAT1 and the transgenic acyltransferases and that this competition limits the accumulation of unusual FAs in our FAH12 RcDGAT2 transgenic lines

Summary

Introduction

Expression of RcPDAT1A in the dgat CL7 RcDGAT2 background could shift the balance in substrate competition between castor and Arabidopsis enzymes further in favor of incorporation of HFA into TAG To test this hypothesis, RcPDAT1A was transformed in the dgat CL7 RcDGAT2 background using Discosoma spp. red fluorescent protein (DsRed) as a selection marker (Stuitje et al, 2003). There were no significant changes in the amount of total FA per seed (Fig. 3A), but there was a 23% increase in the amount of HFA per seed (Fig. 3B) in the dgat CL7 RcDGAT2 RcPDAT1A plants compared with the dgat CL7 RcDGAT2 segregants These increases are somewhat larger than observed previously (van Erp et al, 2011) when RcPDAT1A was expressed in the CL7 RcDGAT2 background (26.7% HFA). These improvements likely arise from the introduction of the dgat mutation, which shifts the balance in substrate competition further in favor of incorporation of HFA

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