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Fatty Acid Synthesis Is Inhibited by Inefficient Utilization of Unusual Fatty Acids for Glycerolipid Assembly

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Fatty Acid Synthesis Is Inhibited by Inefficient Utilization of Unusual Fatty Acids for Glycerolipid Assembly Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly Philip D. Batesa,b,1, Sean R. Johnsonb, Xia Caoc, Jia Lid, Jeong-Won Namd, Jan G. Jaworskid, John B. Ohlroggec, and John Browseb aDepartment of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, MS 39402; bInstitute of Biological Chemistry, Washington State University, Clark Hall, Pullman, WA 99164; cDepartment of Plant Biology, Michigan State University, MI 48824; and dDonald Danforth Plant Science Center, St. Louis, MO 63132 Edited by Chris R. Somerville, University of California, Berkeley, Berkeley, CA, and approved December 13, 2013 (received for review October 1, 2013) Degradation of unusual fatty acids through β-oxidation within understanding of mechanisms that control seed FA synthesis transgenic plants has long been hypothesized as a major factor and accumulation. limiting the production of industrially useful unusual fatty acids The net accumulation of a metabolic product is controlled by in seed oils. Arabidopsis seeds expressing the castor fatty acid the combined action of anabolic and catabolic pathways. The hydroxylase accumulate hydroxylated fatty acids up to 17% of FAs that accumulate within TAG are initially synthesized up to total fatty acids in seed triacylglycerols; however, total seed oil 18C and 0–1 double bonds within the plastid. Upon exiting the is also reduced up to 50%. Investigations into the cause of the plastid, newly synthesized FAs may be further modified (desa- 14 3 turated, hydroxylated, etc.) while esterifed to endoplasmic re- reduced oil phenotype through in vivo [ C]acetate and [ H]2O metabolic labeling of developing seeds surprisingly revealed that ticulum (ER) membrane lipid phosphatidylcholine (PC) before the rate of de novo fatty acid synthesis within the transgenic incorporation into TAG (12, 13). FAs esterifed to glycerolipids seeds was approximately half that of control seeds. RNAseq anal- have long half-lives (14), with minimal turnover in most tissues ysis indicated no changes in expression of fatty acid synthesis (15). A prominent exception takes place in germinating seedlings where TAG is broken down through β-oxidation to produce genes in hydroxylase-expressing plants. However, differential acetyl–CoA for energy production and gluconeogenesis (16). In [14C]acetate and [14C]malonate metabolic labeling of hydroxylase- – preparation for germination, enzymes for TAG degradation ac- expressing seeds indicated the in vivo acetyl CoA carboxylase ac- cumulate during seed development and lead to a loss of ∼10% of tivity was reduced to approximately half that of control seeds. seed oil reserves during late seed maturation (17). Thus, oil levels Therefore, the reduction of oil content in the transgenic seeds is of mature seeds result from a combination of both FA synthesis consistent with reduced de novo fatty acid synthesis in the plastid and FA catabolism, and an alteration of either process could lead rather than fatty acid degradation. Intriguingly, the coexpression to the reduced oil phenotypes of some transgenic oilseeds. of triacylglycerol synthesis isozymes from castor along with the The selective breakdown of unusual FAs within transgenic plants fatty acid hydroxylase alleviated the reduced acetyl–CoA carbox- has long been suggested as a major factor limiting production of ylase activity, restored the rate of fatty acid synthesis, and the oilseed crops containing industrial oils (12, 18). Multiple lines of accumulation of seed oil was substantially recovered. Together evidence support this hypothesis. The castor (Ricinus communis) these results suggest a previously unidentified mechanism that detects inefficient utilization of unusual fatty acids within the Significance endoplasmic reticulum and activates an endogenous pathway for posttranslational reduction of fatty acid synthesis within Many plants produce valuable fatty acids in seed oils that the plastid. provide renewable alternatives to petrochemicals for pro- duction of lubricants, coatings, or polymers. However, most β-oxidation | feedback inhibition | metabolic engineering plants producing these unusual fatty acids are unsuitable as crops. Metabolic engineering of oilseed crops, or model spe- atty acids (FAs) that accumulate as triacylglycerols (TAGs) in cies, to produce the high-value unusual fatty acids has pro- Fseeds of plants represent a major source of renewable re- duced only low yields of the desired products, and previous duced carbon that can be used as food, fuel, or industrial feed- research has indicated fatty acid degradation as a potential stocks. Within the plant kingdom there are greater than 300 major factor hindering oilseed engineering. By contrast, we different types of “unusual FAs” that contain functional groups here present evidence that inefficient utilization of unusual (e.g., hydroxy, epoxy, and cyclopropane) or have physical prop- fatty acids within the endoplasmic reticulum can induce post- erties useful for replacing petroleum in the chemical industry (1, translational inhibition of acetyl–CoA carboxylase activity in 2). Unfortunately, most plants which naturally produce these the plastid, thus inhibiting fatty acid synthesis and total unusual FAs have agronomic features which make them un- oil accumulation. suitable as major crops. Over the past 2 decades, most attempts to genetically engineer unusual FAs into oilseed crops or model Author contributions: P.D.B. and J.B. designed research; P.D.B., S.R.J., J.L., and J.-W.N. species have produced only low proportions of the desired FA performed research; P.D.B., S.R.J., X.C., J.L., J.-W.N., J.G.J., J.B.O., and J.B. analyzed data; and P.D.B., J.B.O., and J.B. wrote the paper. within TAG (2–5). Additionally, in many cases, accumulation of unusual FAs in transgenic plants is accompanied by a reduction The authors declare no conflict of interest. of total seed oil (6–11); in some instances reductions of up to This article is a PNAS Direct Submission. 50% of total seed oil have been reported (7, 10). The endoge- Data deposition: The RNAseq data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo. nous mechanisms that recognize and respond to unusual FAs 1To whom correspondence should be addressed at the present address: Department of and result in reduced seed oil accumulation in transgenic plants Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, MS are unknown. These limited successes and adverse outcomes 39406. E-mail: [email protected]. of oilseed engineering highlight our lack of knowledge on This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. how plants accumulate TAG and indicate a need for better 1073/pnas.1318511111/-/DCSupplemental. 1204–1209 | PNAS | January 21, 2014 | vol. 111 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1318511111 Downloaded by guest on September 30, 2021 FA hydroxylase which produces hydroxylated FAs (HFA) and Results – the California bay medium-chain acyl acyl carrier protein thio- Reduced Oil Accumulation in Plants Producing HFA. We initially set esterase (MCTE) which produces 12:0 (FA nomenclature, # out to quantify the effect of HFA β-oxidation on mature seed oil carbons: # double bonds) have been constitutively expressed in levels and the accumulation of TAG during seed development. tobacco and Brassica napus, respectively (12, 19). In each case, Fig. 1 displays the oil content of wild-type and transgenic Ara- small amounts of the unusual FAs were found in the seeds but bidopsis seeds producing HFA. There is no difference in oil con- not in the leaves. Biochemical analysis of MCTE leaves indicated tent between wild-type Col-0 and the fatty acid elongation 1 (fae1) high MCTE activity and 12:0 production, but no accumulation mutant (26) which is the background for the transgenic lines. Two (19). Thus, the lack of unusual FA accumulation in leaves sug- independent transformations of fae1 with the castor FA hydroxy- gests that unusual FAs are rapidly degraded after synthesis. The lase using a seed-specific promoter (CL37 and CL7) each contain accumulation of HFA and 12:0 to significant levels within seed ∼17% HFA within seed oil (20). Both hydroxylase-expressing lines TAG of transgenic plants has been achieved by the use of strong have an ∼50% reduction in total FAs per seed (Fig. 1A)aswellas seed-specific promoters (18, 20, 21). Sequestration of unusual a reduction in oil content as a percent of total seed weight (Fig. FAs in TAG of transgenic plants may limit their adverse effects 1B). In an attempt to increase the proportion of HFA in TAG on membranes and thus allow accumulation in seeds. However, HFA-selective TAG-synthesis enzymes from castor [phospholipid: some evidence suggests that unusual FAs are also broken down diacylglycerol acyltransferase (PDAT) and acyl-CoA:diacylglycerol by β-oxidation in developing seeds. Degradation of HFA through acyltransferase (DGAT)] were coexpressed with the FA hy- β-oxidation was indicated in the seeds of Arabidopsis plants droxylase within the CL37 and CL7 backgrounds, respectively (9, coexpressing the castor FA hydroxylase along with a bacterial 25). The total micrograms of HFA per seed in the PDAT and polyhydroxyalkanoate (PHA) synthase, which produces PHA DGAT lines was doubled (Fig. 1A), indicating a more efficient from intermediates of β-oxidation (22). However, it is unclear if incorporation of HFA into TAG. However, HFA content as the PHA accumulation in the transgenic seeds was due to a percent of total FA only increased up to ∼25% due to an even β-oxidation during the TAG accumulation phase of seed de- larger increase in the micrograms of normal FA (9). In the velopment or during the TAG breakdown phase of late seed PDAT and DGAT lines the oil content of the seeds is recovered maturation. B. napus embryos accumulating 12:0 up to 60% of to 75 and 85% of the control, respectively (Fig. 1A). We also seed FA had increased levels of a 12:0 specific β-oxidation ac- measured the net accumulation of TAG during the stage of rapid tivity.
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