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 phosphatidylcholine (PC) before the rate of de novo 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 (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 , 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 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. The substantial rates of β-oxidation during seed de- oil accumulation within developing seeds (SI Appendix, Fig. S2). velopment did not reduce total oil accumulation in these seeds, The net rate of TAG accumulation over seed development of

implying that a concomitant increase in FA synthesis compen- CL37 was approximately half the rate of accumulation in the PLANT BIOLOGY sated for a futile cycle of 12:0 synthesis/degradation (21). To- fae1 background line (SI Appendix,Fig.S2A and B). In con- gether these results demonstrate that even though seed tissue trast, the PDAT and DGAT lines accumulated TAG at ∼0.7 can accumulate unusual FAs within glycerolipids, the proportion and 0.9 the net rate of fae1, respectively (SI Appendix, Fig. S2 C and content of unusual FAs within TAG may still be limited by and D). The correlation between reduced CL37 seed oil levels selective β-oxidation of the unusual FAs. (Fig. 1) and net rates of TAG accumulation over seed de- The apparent increased FA synthesis that is proposed to offset velopment (SI Appendix,Fig.S2) suggests that the reduced seed unusual FA degradation makes it unclear how production of oil content of CL37 versus fae1 is mostly due to less TAG accu- HFA or epoxy–FA has led to substantially reduced levels of seed mulation during seed development, rather than increased TAG oil in multiple transgenic plants (8–11). To better understand the breakdown during late seed maturation. metabolic processes that control TAG accumulation in seeds we investigated the basis of reduced oil accumulation in Arabidopsis FA Synthesis Rate Is Reduced When HFA Are Inefficiently Incorporated plants expressing the castor FA hydroxylase (12). Castor seeds into TAG. The oil content and net rate of TAG accumulation accumulate oil containing ∼90% HFA. However, heterologous of CL37 was approximately half of that in the fae1 control. expression of the FA hydroxylase in different plants has led to However, this does not necessarily imply that half of the FAs a relatively low proportion of HFA in the oil of transgenic seeds, synthesized in CL37 are broken down through β-oxidation. with the best lines producing ∼17% HFA in TAG (9, 18, 20, 23, Previously, β-oxidation induced by unusual FA production or FA 24). The accumulation of HFA within transgenic Arabidopsis synthesis mutants has led to increased levels of FA synthesis to seeds is also accompanied by a 30–50% reduction in total seed replenish cellular FA content (21, 27). Therefore, we hypothe- oil (8–10). Interestingly, endeavors to circumvent the mecha- sized that the rate of FA synthesis within CL37 may also be nisms limiting the proportion of HFA within TAG through coexpression of the castor FA hydroxylase with HFA-selective TAG-synthesis enzymes increased not only the proportion of HFA in the TAG (to over 25%) but also mostly recovered the AB seed oil content to that of nontransgenic lines (9, 25). These previous results suggest that inefficient utilization of unusual FAs leads to the reduced seed oil levels. However, it is unknown if the reduced oil content is due to increased catabolism of the unusual FAs through β-oxidation, impairment of FA synthesis, or both. Here we report that production of HFA within the ER of developing Arabidopsis seeds by castor FA hydroxylase expres- sion alone induced a large reduction in FA synthesis and seed oil content, apparently by posttranslational inhibition of plastid lo- calized acetyl–CoA carboxylase (ACCase) activity. Our results indicate that the reduction in FA synthesis is a primary mecha- nism for the reduced seed oil. Additionally, more efficient in- corporation of HFA into TAG by HFA-selective TAG-synthesis Fig. 1. Oil content of wild-type and transgenic Arabidopsis lines. (A)Micro- enzymes alleviates the inhibition of ACCase activity and increases grams of total FA per seed. (B) Oil content calculated as FA percentage of seed oil content. Thus, we demonstrate in vivo that developing seed weight (seed weights are given in SI Appendix, Fig. S1). CL37 and CL7 oilseeds can detect inefficient glycerolipid within the express the castor fatty acid hydroxylase (RcFAH12) in the fae1 background . ER and respond by posttranslational down-regulation of de novo PDAT contains RcPDAT1a in CL37, and DGAT contains RcDGAT2 in CL7 . Data FA synthesis within the plastid. are mean ± SEM of 15–18 replicates for each plant line.

Bates et al. PNAS | January 21, 2014 | vol. 111 | no. 3 | 1205 Downloaded by guest on September 30, 2021 increased, and the total amount of CL37 FAs degraded by not indicate a change in PC catabolism or acyl editing (28) as β-oxidation would be the difference between total FA synthesis might be expected if there was a large flux of HFA from PC and total FA accumulation. To estimate the amount of FA into β-oxidation. Therefore, the reduced rate of [14C]acetate 3 synthesis in fae1 and CL37 developing seeds we measured the in and [ H]2O incorporation into of CL37 compared with vivo rate of FA synthesis during the phase of TAG accumulation fae1 is consistent with a reduced rate of de novo FA synthesis through metabolic labeling. Newly synthesized FAs can be la- in CL37. Thus, the reduced TAG accumulation in CL37 seeds beled with [14C]acetate in vivo through incorporation of [14C] is more likely due to less FA synthesis rather than increased FA acetate into the acetyl–CoA substrate for FA synthesis (28). Fig. β-oxidation. 2A demonstrates that the rate of [14C]acetate incorporation into both fae1 and CL37 FAs was linear from 0 to 60 min, indicating Expression of FA Synthesis-Related Genes is Not Changed Between very little degradation of the newly synthesized FAs over the the fae1, CL37, and PDAT Lines. TAG deposition within oil-bearing labeling time period. However, surprisingly, the rate of CL37 FA plant tissues is correlated to large increases in genes involved in labeling was approximately half that of fae1. This result is more FA synthesis including acetyl–CoA carboxylase (ACCase) which consistent with reduced FA synthesis in CL37 rather than in- is the first committed step in FA synthesis (35–37). We used creased FA β-oxidation. whole-transcriptome RNAseq to determine if transcriptional Acetyl–CoA is not only the substrate for FA synthesis but also regulation of FA synthesis is involved in the altered rates of FA the product of FA β-oxidation. Therefore, to control against synthesis between fae1, CL37, and PDAT developing seeds. If possible differences in acetate metabolism between fae1 and a particular change in gene expression between fae1 and CL37 is CL37 (such as dilution of [14C]acetate due to high rates of associated with the reduced FA synthesis rates, we would expect β-oxidation) we also measured the rate of FA synthesis using to see the opposite expression change between CL37 and PDAT 3 3 3 [ H]2O. Nascent FAs can be labeled with H from [ H]2O during where the rates of FA synthesis and oil levels are recovered. the reduction steps of FA synthesis (29). Fig. 2B demonstrates Because FA synthesis gene expression changes temporally in the accumulation of 3H-labeled FAs over 30 min in fae1 and developing seeds we analyzed gene expression at three de- CL37 at three different developmental stages during the period velopmental stages. The fold change in gene expression between of TAG accumulation in developing seeds. The rate of CL37 FA fae1 and CL37 or CL37 and PDAT for the four nuclear encoded synthesis was consistently less than half that of fae1 at all de- genes of ACCase is given in Table 1. Surprisingly, there was velopmental stages. Additionally, the rates of FA synthesis in the very little difference in ACCase gene expression between each of PDAT and DGAT lines were significantly increased from CL37, the three lines at each developmental stage. Additionally, there but not statistically different from fae1 (Fig. 2B). Together, the was no consistent opposite regulation of FA synthesis genes metabolic labelings of FA synthesis are consistent with a reduced between CL37/fae1 and PDAT/CL37 as hypothesized. Very rate of de novo FA synthesis in CL37 that is responsible for the similar results were found for all other known genes involved reduction in seed oil accumulation. Additionally, the more effi- in FA synthesis (Dataset S1). Additionally, there was little cient incorporation of HFA into TAG of the PDAT and DGAT change in expression of β-oxidation genes (Dataset S2) between lines and the recovered seed oil content (Fig. 1) are consistent fae1, CL37, and PDAT. Therefore, the large changes in lipid with recovered rates of FA synthesis within these lines (Fig. 2B). metabolism between fae1 and CL37 as indicated by TAG accu- It is possible that increased β-oxidation activity due to HFA mulation (Fig. 1) and metabolic labeling (Fig. 2) are most likely production channels nascent FAs exported from the plastid di- due to posttranscriptional regulation of FA synthesis rather than rectly into β-oxidation [similar to the effect of MCTE expression transcriptional regulation. in leaves (19)] and thus produces an apparent reduction in the FA synthesis labeling. However, this scenario is unlikely for two Acyl–CoA and acyl–ACP Compositions. Recent fatty acid-feeding reasons. First, MCTE-expressing leaves produce an incomplete studies of B. napus embryo-derived microspore cultures indicate product of FA synthesis (12:0). The 12:0 exported from the that accumulation of oleate (18:1) within the acyl–CoA pool plastid is not used for membrane lipid synthesis in the ER (30) could lead to reduced export of oleate from plastid, accumula- and therefore is rapidly degraded. In contrast, the castor FA tion of oleoyl–ACP within the plastid, and product inhibition of hydroxylase can only produce HFA after newly synthesized 18:1 FA synthesis by interaction of oleoyl–ACP with ACCase (38). is exported from the plastid and incorporated into PC within the We hypothesized that inefficient utilization of HFA–CoA for ER. The major product of FA synthesis is 18:1 and thus it is un- TAG synthesis may also cause a backup of oleate export from likely to be selectively degraded. Second, the major flux of nascent the plastid and reduced FA synthesis within CL37. Therefore, we FAs exported from the plastid is into PC through acyl editing in measured the acyl–CoA and acyl–ACP compositions of de- Arabidopsis (31, 32) and in other plants (33, 34). Therefore, the veloping fae1, CL37, PDAT, and DGAT lines (Fig. 3). The removal of HFA from PC and their subsequent β-oxidation would proportion of oleoyl–CoA was similar in all lines (Fig. 3A), and require an increased flux of newly synthesized FAs into PC to HFA–CoA accumulated to less than 10% in CL37, similar to maintain membrane integrity. Analysis of nascent [14C]FA levels of HFA in developing castor seeds (39). Additionally, the incorporation into glycerolipids of fae1 and CL37 (SI Appendix, acyl–ACP compositions of all four lines were very similar (Fig. 3B), Fig. S3 and Fig. S3 Discussion), as well as previous analysis of and there was no statistically significant change in the acyl– [14C]glycerol labeling of fae1 and CL37 (10), together does ACP pool sizes (SI Appendix, Fig. S4). Therefore, it is unlikely

ABC

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 3 14 [ H]2O at three developmental stages. (C) Relative [ C] acetate and [14C]malonate incorporation into FAs of developing seeds (n = 5). Total incorporation of each radioisotope is in SI Appendix,Fig.S5.Dataaremean of replicates ± SEM.

1206 | www.pnas.org/cgi/doi/10.1073/pnas.1318511111 Bates et al. Downloaded by guest on September 30, 2021 Table 1. Absence of significant changes in ACCase gene reduced rate of FA synthesis (Fig. 2B), and substantially alle- expression in developing seeds viates the reduced seed oil accumulation (Fig. 1). Similar oil CL37 vs. fae1 PDAT vs. CL37 phenotypes have also been reported in soybeans expressing a FA epoxygenase alone and with coexpression of an epoxy–FA se- ACCase subunit Fold change q value Fold change q value lective DGAT (11). Together, these results suggest that plants can detect inefficient glycerolipid synthesis within the ER and 7–8 Days after flowering can respond by posttranslational down-regulation of ACCase BC −0.10 0.99 −0.37 0.70 activity, and thus de novo FA synthesis, within the plastid. BCCP1 0.04 1.00 −0.19 0.86 BCCP2 0.01 1.00 −0.25 0.82 FA Synthesis Inhibition and β-Oxidation Represent Differential α-CT 0.05 1.00 −0.24 0.83 Responses to Unusual FA Production in Transgenic Plants. Pre- 9–10 Days after flowering viously, futile cycles of unusual FA synthesis and β-oxidation BC 0.09 0.98 0.04 0.99 have been indicated as factors limiting the accumulation of BCCP1 0.07 0.98 0.04 0.99 HFA, epoxy–FA, or medium-chain FAs in the seeds or leaves BCCP2 0.14 0.95 0.04 0.99 of transgenic plants (19, 21, 22, 44). However, in the previous α-CT 0.04 0.99 0.27 0.71 studies with transgenic plants producing HFA or epoxy–FA, very ∼ 11–12 Days after flowering low levels of the unusual FAs were produced in the seeds ( 6% ∼ – BC −0.36 0.63 0.12 0.97 HFA and 3% epoxy FA), and no report was made on the seed ∼ BCCP1 −0.26 0.74 0.21 0.86 oil levels (22). CL37 seeds accumulate 17% HFA and have BCCP2 −0.75 0.06 0.23 0.86 reduced seed oil (Fig. 1), suggesting that higher levels of unusual α-CT −0.67 0.24 0.16 0.95 FAs may induce different changes in seed metabolism than do low levels. At low levels, the HFA or epoxy–FA that are in- Three biological replicates were analyzed at three different stages of efficiently used for ER glycerolipid synthesis may be shunted into development. Fold change (Log2) in gene expression calculated between β-oxidation with a corresponding small increase in de novo FA indicated plant lines is presented. The q value is calculated considering false synthesis required to achieve the WT level of TAG accumulation discovery rates. A q value > 0.05 indicates the fold change in gene expression (21). However, high levels of HFA production and inefficient is not statistically significant. BC, Biotin carboxylase (AT5G35360); BCCP, incorporation into TAG interferes with ER glycerolipid synthesis Biotin carboxyl carrier protein, BCCP1 (AT5G16390), BCCP2 (AT5G15530); (10) and may induce the ER-to-plastid feedback inhibition of FA α-CT, alpha-carboxyltransferase, (AT2G38040).

synthesis measured in CL37. It appears that production of me- PLANT BIOLOGY dium-chain FAs within transgenic plants can produce different metabolic responses. Up to 60% of the seed oil can be accu- that the reduced rates of FAS in CL37 are due to posttransla- mulated by 12:0 within transgenic B. napus plants and induce tional regulation of FA synthesis through oleoyl–ACP feedback futile cycles of increased rates of FA synthesis and β-oxidation, inhibition of ACCase. yet without an effect on oil levels (21). The differences in met- abolic responses to medium-chain FAs and HFA may be due to ACCase Activity Is Down-Regulated in CL37. Several studies have their chemical structure or their sites of synthesis. Medium-chain indicated ACCase as a major posttranslationally regulated en- FAs are produced within the plastid as intermediates of FA zyme of FA synthesis (38, 40–43). Therefore, to estimate in vivo synthesis. The shuttling of incomplete products of FA synthesis ACCase activity under the same conditions that indicated changes 14 exported from the plastid into β-oxidation (or sequestering them in the FA synthesis rate we used differential [ C]acetate and 14 in TAG) may represent an endogenous pathway to limit their [ C]malonate labeling (38) of fae1, CL37, PDAT, and DGAT developing seeds (Fig. 2C). Acetyl–CoA and malonyl–CoA are the substrates and products of ACCase, respectively. A change in ACCase activity will affect the incorporation of [14C]acetate into newly synthesized FAs, but it will not affect the incorporation A of [14C]malonate into FAs. Relative to fae1 labeling, the in- corporation of [14C]acetate into CL37 FAs was approximately half that of [14C]malonate incorporation into FAs. However, there were no differences between the radiolabeled substrates for the incorporation into PDAT and DGAT FAs, indicating that ACCase activity is reduced in CL37 but not in the PDAT or DGAT lines. Discussion Inefficient Glycerolipid Synthesis Induces Down-Regulation of de Novo FA Synthesis. Previously, β-oxidation of unusual FAs in transgenic plants has been hypothesized as a major factor lim- B iting efficient oilseed engineering. We cannot completely rule out β-oxidation of HFA to some extent during CL37 seed de- velopment. However, CL37 plants expressing the castor FA hy- droxylase, compared with the fae1 control, have approximately half as much seed oil accumulation (Fig.1), half the rate of FA synthesis (Fig. 2A), and half the ACCase activity (Fig. 2C), yet transcript levels for ACCase subunits are not changed (Table 1). Together these results suggest the major mechanism for reduced oil in transgenic plants accumulating HFA (8–10) is reduced FA synthesis through posttranslational inhibition of ACCase, rather than primarily through degradation of HFA via β-oxidation. Intriguingly, more efficient utilization of HFA for TAG synthesis by coexpression of castor DGAT or PDAT with the FA hy- Fig. 3. Acyl–CoA and acyl–ACP compositions in developing seeds. (A) acyl– droxylase alleviates the reduced ACCase activity (Fig. 2C), the CoA. (B) acyl–ACP. A and B,mean(n = 5) ± SEM.

Bates et al. PNAS | January 21, 2014 | vol. 111 | no. 3 | 1207 Downloaded by guest on September 30, 2021 incorporation into ER membrane lipids, and the corresponding increased rates of FA synthesis within these plants may restore the flux of FAs into ER glycerolipid synthesis. However, HFA are produced on PC which is a key intermediate of cellular membrane and storage lipid synthesis (13). Many PC-derived unusual FAs are thought to affect membrane structure and function and are typically maintained at very low levels within A membrane lipids, even in plant species that naturally accumulate PC-derived unusual FAs within TAG (30). Thus, the inefficient flux of HFA from PC into TAG (9, 10) may be identified by the plant as a problem with membrane lipid synthesis or lipid oxi- dation and elicit a differential metabolic response than that of incomplete FA synthesis. B Possible Mechanisms of ACCase Inhibition from Inefficient ER C Glycerolipid Synthesis. Excess FA accumulation (from oleate feeding) has been shown to induce posttranscriptional down- regulation of ACCase activity and reduce de novo FA synthesis in tobacco and B. napus cell cultures (38, 40). However, the cells used the supplied FAs for growth, and thus overall lipid accu- mulation was not affected by the reduction in de novo FA syn- thesis. Additionally, the reduction in FA synthesis was recently attributed to a build-up of oleate in the acyl–ACP pool and Fig. 4. Proposed model of CL37 . (A) HFA enters the acyl– CoA pool by acyl editing after HFA production on PC. (B) Red arrows, futile may lead to direct ACCase inhibition through interaction with – – oleoyl–ACP (38). CL37 did not have an increased proportion of cycle of de novo HFA DAG synthesis and turnover; red X, bottleneck in HFA oleoyl–ACP relative to fae1, PDAT, or DGAT lines (Fig. 3B), 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- and thus this mechanism is unlikely to be responsible for the phosphatidylcholine; LPA, lyso-phosphatidic acid; PA, phosphatidic acid. CL37 phenotype. Additionally, it is unlikely that HFA–CoA is directly related to the reduction in FA synthesis rate in CL37 because both the PDAT and the DGAT lines have recovered metabolic analysis of transgenic plants producing HFA pre- FA synthesis rates, but only DGAT has a reduced proportion of – – sented here enhances our knowledge of the mechanisms limiting HFA CoA (Fig. 3A). The reduction in HFA CoA by the castor the accumulation of TAG containing unusual FAs. In addition, DGAT is consistent with its enzymatic activity that selectively – the differential responses to various transgenically produced uses HFA CoA to produce TAG (25). unusual FAs measured here and by others may be even more Fig. 4 displays a possible model for CL37 lipid metabolism useful for understanding the basic biochemistry of how plants based on the results reported here and previous research (10). deal with changes to cellular FA content. The diacylglycerol (DAG) substrate used for TAG production can be synthesized de novo from glycerol-3-phosphate (G3P) Methods – and acyl CoAs or it can be derived from PC (28). In wild-type Plants, Oil Content, and Metabolic Labeling. All six plant lines were grown Arabidopsis, TAG is primarily produced from PC-derived DAG randomized across a growth chamber under constant ∼100–170 μE light, ∼22 °C, (10), indicating a de novo DAG → PC → PC-derived DAG → ∼ – and 60% humidity. Seed oil was determined by whole-seed transmethylation TAG pathway (Fig. 4). We recently demonstrated that HFA and analysis by gas chromatography with flame ionization detection (46). For CoA [produced from PC acyl editing (Fig. 4A)] is efficiently used labeling, developing seeds were collected from each plant line grown to- for de novo DAG synthesis; however, de novo HFA–DAG is not gether as in ref. 10. The [14C]acetate time course labeling was conducted as in efficiently used for membrane lipid or TAG synthesis within ref. 10 using 1 mM [14C]acetate within the labeling media; data represent 3 CL37 seeds and is rapidly turned over, limiting the flux of glyc- mean ± SEM (n = 4) from two separate experiments (each n = 2). The [ H]2O erol into PC and thus TAG (Fig. 4, red X) (10). Because we and [14C]acetate vs. [14C]malonate labelings each consisted of five individual conclude here that reduced FA synthesis (Fig. 2) is the major 30-min labelings for each plant line/growth stage/radioisotope. Each in- 3 μ 14 mechanism limiting oil accumulation in CL37, the FAs released dividual labeling in 0.2 mL of media contained either 1 mCi [ H]2O, 10 Ci [ C] from HFA–DAG turnover likely reenter glycerolipid metabo- acetate, or 10 μCi [14C]malonate. The incorporation of radiolabel into FAs was lism. The synthesis of HFA-containing de novo DAG and its determined by transmethylation of the total lipid extract as above, and pu- subsequent turnover produces a futile cycle of inefficient glyc- rification of the fatty acid methyl esters by TLC on silica gel 60 20 × 20 cm glass erolipid synthesis within the ER (Fig. 4B, red lines) which leads plates (EMD, www.emdmillipore.com/) developed in hexane/diethyl ether/ to posttranslational regulation of ACCase activity in the plastid acetic acid, 70/30/1 (vol/vol/vol). Radiolabel was quantified by liquid scin- (Fig. 4C) via phosphorylation, , binding , enzyme tillation counting on a Tri–CARB liquid scintillation analyzer (Packard turnover, or other regulators of ACCase activity (41–43). Further Instrument Company). identification of the metabolic signals which control ACCase – – – regulation, and thus FA synthesis, in response to unusual FA RNA Expression Analysis. Frozen developing seeds of 7 8, 9 10, and 11 12 production may generate alternative bioengineering strategies days after flowering (DAF) were collected from liquid N2 frozen siliques (47). for increasing total oil accumulation in plants (45). Approximately 100 mg of frozen seed samples (three biological replicates for each plant line/developmental stage) were ground to a fine power, RNA extracted (48), and DNA removed (DNA-Free RNA Kit, Zymo Research). Li- Closing Remarks. β It has long been proposed that -oxidation is braries were prepared from 2 to 4 μg total RNA (Illumina TruSeq RNA kits) a major factor limiting the engineering of unusual FAs into crop and sequenced with Illumina HiSeq2000. Reads (50 nt) were trimmed, fil- plants. Here we demonstrate that posttranslational down-regulation tered, and mapped against TAIR10 genome (www.arabidopsis.org/) with of FA synthesis by unusual FAs can also significantly inhibit Bowtie (v. 2.0.0-beta7). Only alignments consistent with annotated transcripts the accumulation of industrial oils within transgenic plants. The were considered valid. Each sample and biological replicate was aligned sep- induced down-regulation of plastidial ACCase, due to in vivo arately. All alignments were then used as input to Cuffdiff (v. 2.0.1) (49) to metabolic changes within the ER of CL37 seeds, suggests that calculate per sample, per gene fragments per kilobase of exon per million this transgenic system may be responding to an endogenous fragments mapped (FPKM), and statistical significance (q value) of differential intercompartmental posttranslational regulatory pathway that expression between transgenic lines. Cufflinks averages the FPKM across bi- would otherwise be difficult to study in wild-type plants. The ological replicates within each sample.

1208 | www.pnas.org/cgi/doi/10.1073/pnas.1318511111 Bates et al. Downloaded by guest on September 30, 2021 Acyl–CoA and Acyl–ACP Analysis. Acyl–CoA and acyl–ACP were extracted from to a final concentration of 10%, frozen (−80 °C, 10 min), thawed on ice, and

developing seeds of 9–12 DAF that were collected from liquid N2 frozen centrifuged again. The pellet was washed with 1% TCA, centrifuged again, siliques (47). Acyl–CoA were exacted as in ref. 50. Extracted acyl–CoA and dissolved in Mops buffer again. The protein samples were digested with – dried under N2 were suspended in 100 μL of 50% (vol/vol) MeOH and Asp N endoproteinase and analyzed on a 4000 QTRAP LC/MS/MS system analyzed as in ref. 51, except HPLC solvents A and B contained an addi- (Applied Biosystems). tional 7.5 mM ammonium hydroxide. An acyl–ACP-enriched protein fraction was collected by vortexing finely ground frozen seed tissue in 5% ACKNOWLEDGMENTS. We are grateful to the Research Technology Support Facility at Michigan State University for sequencing and to Kevin Carr for (vol/vol) trichloroacetic acid (TCA) and centrifuged 21,000 g (10 min, 4 °C). bioinformatics analysis. This work was supported by the National Science The pellet was dissolved in 1% TCA and centrifuged again. The pellet was Foundation (Grant DBI-0701919), the Agricultural Research Center at Wash- dissolved in 50 mM Mops, pH 7.6, checked for pH above 6.5, incubated on ington State University, and Department of Energy–Great Lakes Bioenergy ice (30 min), and centrifuged again. 100% TCA was added to the supernatant Research Center Cooperative Agreement DE-FC02-07ER6449.

1. Badami RC, Patil KB (1980) Structure and occurrence of unusual fatty acids in minor 26. Kunst L, Taylor DC, Underhill EW (1992) Fatty-acid elongation in developing seeds of seed oils. Prog Lipid Res 19(3–4):119–153. Arabidopsis-thaliana. Plant Physiol Biochem 30(4):425–434. 2. Vanhercke T, Wood CC, Stymne S, Singh SP, Green AG (2013) Metabolic engineering 27. Bonaventure G, Bao X, Ohlrogge J, Pollard M (2004) Metabolic responses to the re- of plant oils and waxes for use as industrial feedstocks. Plant Biotechnol J 11(2): duction in palmitate caused by disruption of the FATB gene in Arabidopsis. Plant – 197 210. Physiol 135(3):1269–1279. 3. Cahoon EB, et al. (2007) Engineering oilseeds for sustainable production of industrial 28. Bates PD, Browse J (2012) The significance of different diacylgycerol synthesis path- and nutritional feedstocks: Solving bottlenecks in fatty acid flux. Curr Opin Plant Biol ways on plant oil composition and bioengineering. Front Plant Sci 3:147. – 10(3):236 244. 29. Jungas RL (1968) Fatty acid synthesis in adipose tissue incubated in tritiated water. 4. Napier JA (2007) The production of unusual fatty acids in transgenic plants. Annu Rev Biochemistry 7(10):3708–3717. Plant Biol 58:295–319. 30. Millar AA, Smith MA, Kunst L (2000) All fatty acids are not equal: Discrimination in 5. Haslam RP, et al. (2013) The modification of plant oil composition via metabolic plant membrane lipids. Trends Plant Sci 5(3):95–101. engineering—better nutrition by design. Plant Biotechnol J 11(2):157–168. 31. Tjellström H, Yang Z, Allen DK, Ohlrogge JB (2012) Rapid kinetic labeling of Arabi- 6. Knutzon DS, Hayes TR, Wyrick A, Xiong H, Maelor Davies H, Voelker TA (1999) Ly- dopsis cell suspension cultures: implications for models of lipid export from plastids. sophosphatidic acid acyltransferase from coconut endosperm mediates the insertion – of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase Plant Physiol 158(2):601 611. total laurate levels. Plant Physiol 120(3):739–746. 32. Bates PD, et al. (2012) Acyl editing and headgroup exchange are the major mecha- 7. Larson TR, Edgell T, Byrne J, Dehesh K, Graham IA (2002) Acyl CoA profiles of trans- nisms that direct polyunsaturated fatty acid flux into triacylglycerols. Plant Physiol genic plants that accumulate medium-chain fatty acids indicate inefficient storage 160(3):1530–1539. lipid synthesis in developing oilseeds. Plant J 32(4):519–527. 33. Bates PD, Durrett TP, Ohlrogge JB, Pollard M (2009) Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean embryos. Plant 8. Dauk M, Lam P, Kunst L, Smith MA (2007) A FAD2 homologue from Lesquerella PLANT BIOLOGY lindheimeri has predominantly fatty acid hydroxylase activity. Plant Sci 173(1):43–49. Physiol 150(1):55–72. 9. van Erp H, Bates PD, Burgal J, Shockey J, Browse J (2011) Castor phospholipid:diac- 34. Bates PD, Ohlrogge JB, Pollard M (2007) Incorporation of newly synthesized fatty ylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in acids into cytosolic glycerolipids in pea leaves occurs via acyl editing. J Biol Chem transgenic Arabidopsis. Plant Physiol 155(2):683–693. 282(43):31206–31216. 10. Bates PD, Browse J (2011) The pathway of triacylglycerol synthesis through phos- 35. Bourgis F, et al. (2011) Comparative transcriptome and metabolite analysis of oil palm phatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual and date palm mesocarp that differ dramatically in carbon partitioning. Proc Natl – fatty acids in transgenic seeds. Plant J 68(3):387 399. Acad Sci USA 108(30):12527–12532. 11. Li R, et al. (2012) Vernonia DGATs can complement the disrupted oil and protein 36. Troncoso-Ponce MA, et al. (2011) Comparative deep transcriptional profiling of four – metabolism in epoxygenase-expressing soybean seeds. Metab Eng 14(1):29 38. developing oilseeds. Plant J 68(6):1014–1027. 12. van de Loo FJ, Broun P, Turner S, Somerville C (1995) An oleate 12-hydroxylase from 37. Ruuska SA, Girke T, Benning C, Ohlrogge JB (2002) Contrapuntal networks of gene Ricinus communis L. is a fatty acyl desaturase homolog. Proc Natl Acad Sci USA 92(15): expression during Arabidopsis seed filling. Plant Cell 14(6):1191–1206. 6743–6747. 38. Andre C, Haslam RP, Shanklin J (2012) Feedback regulation of plastidic acetyl-CoA 13. Bates PD, Stymne S, Ohlrogge J (2013) Biochemical pathways in seed oil synthesis. Curr carboxylase by 18:1-acyl carrier protein in Brassica napus. Proc Natl Acad Sci USA Opin Plant Biol 16(3):358–364. 109(25):10107–10112. 14. Roughan PG (1970) Turnover of the glycerolipids of pumpkin leaves. The importence 39. Brown AP, et al. (2012) Tissue-specific whole transcriptome sequencing in castor, di- of phosphatidylcholine. Biochem J 117(1):1–8. 15. Gerhardt B (1992) Fatty acid degradation in plants. Prog Lipid Res 31(4):417–446. rected at understanding triacylglycerol lipid biosynthetic pathways. PLoS ONE 7(2): 16. Theodoulou FL, Eastmond PJ (2012) Seed storage oil catabolism: A story of give and e30100. take. Curr Opin Plant Biol 15(3):322–328. 40. Shintani DK, Ohlrogge JB (1995) Feedback inhibition of fatty-acid synthesis in tobacco 17. Baud S, Boutin JP, Miquel M, Lepiniec L, Rochat C (2002) An integrated overview of suspension cells. Plant J 7(4):577–587. seed development in Arabidopsis thaliana ecotype WS. Plant Physiol Biochem 40(2): 41. Feria Bourrellier AB, et al. (2010) Chloroplast acetyl-CoA carboxylase activity is 2- 151–160. oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit. 18. Broun P, Somerville C (1997) Accumulation of ricinoleic, lesquerolic, and densipolic Proc Natl Acad Sci USA 107(1):502–507. acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase 42. Sasaki Y, Nagano Y (2004) Plant acetyl-CoA carboxylase: Structure, biosynthesis, cDNA from castor bean. Plant Physiol 113(3):933–942. regulation, and gene manipulation for plant breeding. Biosci Biotechnol Biochem 19. Eccleston VS, Cranmer AM, Voelker TA, Ohlrogge JB (1996) Medium-chain fatty acid 68(6):1175–1184. biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl carrier 43. Nikolau BJ, Ohlrogge JB, Wurtele ES (2003) Plant biotin-containing carboxylases. Arch protein thioesterase. Planta 198(1):46–53. Biochem Biophys 414(2):211–222. 20. Lu CF, Fulda M, Wallis JG, Browse J (2006) A high-throughput screen for genes from 44. Poirier Y, Ventre G, Caldelari D (1999) Increased flow of fatty acids toward beta- castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabi- oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransfer- – dopsis. Plant J 45(5):847 856. ase activity or synthesizing medium-chain-length fatty acids. Plant Physiol 121(4): 21. Eccleston VS, Ohlrogge JB (1998) Expression of lauroyl-acyl carrier protein - 1359–1366. ase in brassica napus seeds induces pathways for both fatty acid oxidation and bio- 45. Weselake RJ, et al. (2009) Increasing the flow of carbon into seed oil. Biotechnol Adv synthesis and implies a set point for triacylglycerol accumulation. Plant Cell 10(4): 27(6):866–878. 613–622. 46. Li-Beisson Y, et al. (2013) Acyl-lipid metabolism. Arabidopsis Book 11:e0161. 22. Moire L, Rezzonico E, Goepfert S, Poirier Y (2004) Impact of unusual fatty acid syn- 47. Bates PD, Jewell JB, Browse J (2013) Rapid separation of developing Arabidopsis seeds thesis on futile cycling through beta-oxidation and on gene expression in transgenic from siliques for RNA or metabolite analysis. Plant Methods 9(1):9. plants. Plant Physiol 134(1):432–442. 48. Suzuki Y, Kawazu T, Koyama H (2004) RNA isolation from siliques, dry seeds, and 23. Smith MA, Moon H, Chowrira G, Kunst L (2003) Heterologous expression of a fatty – acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta 217(3): other tissues of Arabidopsis thaliana. Biotechniques 37(4):542 544, 544. 507–516. 49. Trapnell C, et al. (2013) Differential analysis of gene regulation at transcript resolu- – 24. Lu CF, Kang JL (2008) Generation of transgenic plants of a potential oilseed crop tion with RNA-seq. Nat Biotechnol 31(1):46 53. Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep 27(2): 50. Larson TR, Graham IA (2001) Technical advance: A novel technique for the sensitive 273–278. quantification of acyl CoA esters from plant tissues. Plant J 25(1):115–125. 25. Burgal J, et al. (2008) Metabolic engineering of hydroxy fatty acid production in 51. Han J, et al. (2010) The cytochrome P450 CYP86A22 is a fatty acyl-CoA ω-hydroxylase plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. Plant Bio- essential for Estolide synthesis in the stigma of Petunia hybrida. J Biol Chem 285(6): technol J 6(8):819–831. 3986–3996.

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