Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly

Abstract

Degradation of unusual fatty acids through β-oxidation within transgenic plants has long been hypothesized as a major factor limiting the production of industrially useful unusual fatty acids in seed oils. Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols; however, total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [(14)C]acetate and [(3)H]2O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants. However, differential [(14)C]acetate and [(14)C]malonate metabolic labeling of hydroxylase-expressing seeds indicated the in vivo acetyl-CoA carboxylase activity was reduced to approximately half that of control seeds. Therefore, the reduction of oil content in the transgenic seeds is consistent with reduced de novo fatty acid synthesis in the plastid rather than fatty acid degradation. Intriguingly, the coexpression of triacylglycerol synthesis isozymes from castor along with the fatty acid hydroxylase alleviated the reduced acetyl-CoA carboxylase activity, restored the rate of fatty acid synthesis, and the accumulation of seed oil was substantially recovered. Together these results suggest a previously unidentified mechanism that detects inefficient utilization of unusual fatty acids within the endoplasmic reticulum and activates an endogenous pathway for posttranslational reduction of fatty acid synthesis within the plastid.

Keywords: feedback inhibition; metabolic engineering; β-oxidation.

Fig. 1.

Oil content of wild-type and transgenic Arabidopsis lines. (A) Micrograms of total FA per seed. (B) Oil content calculated as FA percentage of seed weight (seed weights are given in SI Appendix, Fig. S1). CL37 and CL7 express the castor fatty acid hydroxylase (RcFAH12) in the fae1 background . PDAT contains RcPDAT1a in CL37, and DGAT contains RcDGAT2 in CL7 . Data are mean ± SEM of 15–18 replicates for each plant line.

Fig. 2.

Rates of FA synthesis in developing Arabidopsis seeds. (A) Time course of [¹⁴C]acetate incorporation into total FAs, n = 4. Seed age 9–10 d after flowering. (B) Thirty-minute labeling of FA synthesis (n = 5) with [³H]₂O at three developmental stages. (C) Relative [¹⁴C]acetate and [¹⁴C]malonate incorporation into FAs of developing seeds (n = 5). Total incorporation of each radioisotope is in SI Appendix, Fig. S5. Data are mean of replicates ± SEM.

Fig. 3.

Acyl–CoA and acyl–ACP compositions in developing seeds. (A) acyl–CoA. (B) acyl–ACP. A and B, mean (n = 5) ± SEM.

Fig. 4.

Proposed model of CL37 lipid metabolism. (A) HFA enters the acyl–CoA pool by acyl editing after HFA production on PC. (B) Red arrows, futile cycle of de novo HFA–DAG synthesis and turnover; red X, bottleneck in HFA–DAG flux into PC. (C) ER-to-plastid signaling that down-regulates ACCase activity and thus plastid FA synthesis. Abbreviations not in the text: LPC, lyso-phosphatidylcholine; LPA, lyso-phosphatidic acid; PA, phosphatidic acid.