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AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum Sangwoo Kim a , Yasuyo Yamaoka b , Hirofumi Ono b , Hanul Kim a , Donghwan Shim a , Masayoshi Maeshima c , Enrico Martinoia a,d , Edgar B. Cahoon e , Ikuo Nishida b,1,2 , and Youngsook Lee a,f,1,2 a Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea; b Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan; c Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; d Institute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland; e Center for Plant Science Innovation, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588; and f Pohang University of Science and TechnologyUniversity of Zurich Global Research Laboratory, Division of Integrative Biology and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Korea Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved November 28, 2012 (received for review August 17, 2012) Fatty acids, the building blocks of biological lipids, are synthesized in plastids and then transported to the endoplasmic reticulum (ER) for assimilation into specic lipid classes. The mechanism of fatty acid transport from plastids to the ER has not been identied. Here we report that AtABCA9, an ABC transporter in Arabidopsis thali- ana, mediates this transport. AtABCA9 was localized to the ER, and atabca9 null mutations reduced seed triacylglycerol (TAG) content by 35% compared with WT. Developing atabca9 seeds incorpo- rated 35% less 14 C-oleoyl-CoA into TAG compared with WT seeds. Furthermore, overexpression of AtABCA9 enhanced TAG deposi- tion by up to 40%. These data strongly support a role for AtABCA9 as a supplier of fatty acid substrates for TAG biosynthesis at the ER during the seed-lling stage. AtABCA9 may be a powerful tool for increasing lipid production in oilseeds. ABCA transporter | ABCA9 | acyl-CoA | fatty acid transporter F atty acids are essential for all forms of life, serving as com- ponents of membrane and storage lipids and as precursors of signaling molecules. Photosynthetic organisms, including plants and algae, are major suppliers of biological fatty acids for other organisms. Fatty acids are synthesized in plastids, and a subset is incorporated into plastidial glycerolipids. However, the majority of de novo synthesized fatty acids is assembled into phospholipids and neutral lipids at the endoplasmic reticulum (ER) (1), ne- cessitating the transport of fatty acids from the plastid to the ER. Although each step of fatty acid and lipid synthesis has been studied extensively for many decades, the process of fatty acid transport into the ER remains obscure. The identication of transporters of fatty acids or other lipophilic compounds is in- trinsically difcult owing to the lack of an appropriate assay system for the transport of such compounds across membranes. Thus, the identication of such transporters often depends on biochemical analyses of the lipid levels in selected candidate mutants. Trans- porters identied for lipophilic compounds belong mostly to ATP- binding cassette (ABC) proteins in ABCA and ABCG subfamilies in animals (2) and in the ABCG subfamily in plants (3). ABC proteins are ubiquitous in all living organisms, and their structures and functions are highly conserved (4). In animals, many important genetic diseases are associated with defects in lipid- transporting ABC proteins (2, 5). Changing the activity of some animal ABCA proteins causes overaccumulation of lipids, such as triacylglycerol (TAG) and cholesterol esters, in specic tissues (6). In plants, many ABCG proteins secrete lipidic molecules that form cutin and wax layers (3). Defects in the expression of such proteins result in reduced surface lipids and/or developmental defects, such as organ fusion (7, 8). To identify the transporters that deliver fatty acid substrates to the ER for glycerolipid synthesis, we focused on ABCA and ABCG proteins. Our database searches revealed that no ABCG proteins and only one animal ABCA protein, ABCA17, localize at the ER membrane (9). In the model plant Arabidopsis thaliana, 12 genes encode ABCA transporters, none of which has been characterized previously (3, 10). We hypothesized that ABCA proteins in plants mediate the transport of fatty acids into the ER, and that the activity of such proteins is a limiting step in TAG biosynthesis in oilseed plants during the seed-lling stage, when TAG biosynthesis is greatest, and thus the ux of acyl-CoAs from the plastid to the ER is greatest as well. This hypothesis predicts that a knockout of such a transporter would decrease TAG synthesis, resulting in re- duced seed storage lipid content. Results Screening of ABCA Subfamily Genes for Altered Seed Phenotypes. To identify the ABC transporters involved in fatty acid transport to the ER, we grew seeds of KO mutants of eight ABCA family members on half-strength Murashige and Skoog (1/2 MS) medium with or without sucrose, and compared early seedling growth with WT. The rationale behind this test is that early seedling growth of oilseed plants depends on storage lipids in the absence of sucrose, but not in its presence (11, 12). Thus, plants defective in expression of fatty acid-transporting ABC transporter would be expected to exhibit reduced growth in the absence of sucrose, but normal growth in its presence. Among the mutants tested, the growth of abca9-1 seedlings was most retarded on medium lacking sucrose (Fig. 1A). In contrast, the growth of abca9-1 seedlings on medium containing 1% sucrose was comparable to that of WT. The mutant phenotypes were conrmed for two other abca9 alleles (abca9-2 and abca9-3; Fig. S1). Thus, abca9 seeds are defective in the accumulation of storage lipids or the conversion of lipids to sucrose. abca9 Seeds Exhibit Reduced Seed Size and/or Abnormal Morphology. Given that storage lipids account for 3540% of the dry seed weight of Arabidopsis thaliana (1), seeds defective in storage lipid accumulation are expected to be smaller or misshapen. Among the eight ABCA KO seeds tested, abca9-1 seeds displayed the greatest variability in size and shape (Fig. 1B). Using a dissecting micro- scope, we classied abca9 seeds into four groups: normal, at, small, and brown and shrunken. Only 20% of abca9 seeds were normal, compared with 95% of WT seeds (Fig. S2A). Image Author contributions: S.K., M.M., E.M., E.B.C., I.N., and Y.L. designed research; S.K., Y.Y., H.O., H.K., and D.S. performed research; and S.K., E.M., E.B.C., I.N., and Y.L. wrote the paper. Conict of interest statement: S.K., Y.Y., H.O., I.N., and Y.L. have led patent PCT/KR2011/ 006826 and the title of patent is Composition for increasing seed size or content of storage lipid in seed, comprising the ABC transporter protein-coding gene. This article is a PNAS Direct Submission. 1 I.N. and Y.L. contributed equally to this work. 2 To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1214159110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1214159110 PNAS | January 8, 2013 | vol. 110 | no. 2 | 773778 PLANT BIOLOGY Downloaded by guest on June 16, 2021
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AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum · Fatty acids, the building blocks of biological lipids, are synthesized in plastids and

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  • AtABCA9 transporter supplies fatty acids for lipidsynthesis to the endoplasmic reticulumSangwoo Kima, Yasuyo Yamaokab, Hirofumi Onob, Hanul Kima, Donghwan Shima, Masayoshi Maeshimac,Enrico Martinoiaa,d, Edgar B. Cahoone, Ikuo Nishidab,1,2, and Youngsook Leea,f,1,2

    aDivision of Molecular Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea; bDivision of Life Science, Graduate School ofScience and Engineering, Saitama University, Saitama 338-8570, Japan; cGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601,Japan; dInstitute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland; eCenter for Plant Science Innovation, Department of Biochemistry, Universityof Nebraska-Lincoln, Lincoln, NE 68588; and fPohang University of Science and Technology–University of Zurich Global Research Laboratory, Division ofIntegrative Biology and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Korea

    Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved November 28, 2012 (received for review August 17, 2012)

    Fatty acids, the building blocks of biological lipids, are synthesizedin plastids and then transported to the endoplasmic reticulum (ER)for assimilation into specific lipid classes. The mechanism of fattyacid transport from plastids to the ER has not been identified. Herewe report that AtABCA9, an ABC transporter in Arabidopsis thali-ana, mediates this transport. AtABCA9 was localized to the ER, andatabca9 null mutations reduced seed triacylglycerol (TAG) contentby 35% compared with WT. Developing atabca9 seeds incorpo-rated 35% less 14C-oleoyl-CoA into TAG compared with WT seeds.Furthermore, overexpression of AtABCA9 enhanced TAG deposi-tion by up to 40%. These data strongly support a role for AtABCA9as a supplier of fatty acid substrates for TAG biosynthesis at the ERduring the seed-filling stage. AtABCA9 may be a powerful tool forincreasing lipid production in oilseeds.

    ABCA transporter | ABCA9 | acyl-CoA | fatty acid transporter

    Fatty acids are essential for all forms of life, serving as com-ponents of membrane and storage lipids and as precursors ofsignaling molecules. Photosynthetic organisms, including plantsand algae, are major suppliers of biological fatty acids for otherorganisms. Fatty acids are synthesized in plastids, and a subset isincorporated into plastidial glycerolipids. However, the majorityof de novo synthesized fatty acids is assembled into phospholipidsand neutral lipids at the endoplasmic reticulum (ER) (1), ne-cessitating the transport of fatty acids from the plastid to the ER.Although each step of fatty acid and lipid synthesis has been

    studied extensively for many decades, the process of fatty acidtransport into the ER remains obscure. The identification oftransporters of fatty acids or other lipophilic compounds is in-trinsically difficult owing to the lack of an appropriate assay systemfor the transport of such compounds across membranes. Thus, theidentification of such transporters often depends on biochemicalanalyses of the lipid levels in selected candidate mutants. Trans-porters identified for lipophilic compounds belong mostly to ATP-binding cassette (ABC) proteins in ABCA and ABCG subfamiliesin animals (2) and in the ABCG subfamily in plants (3).ABC proteins are ubiquitous in all living organisms, and their

    structures and functions are highly conserved (4). In animals, manyimportant genetic diseases are associated with defects in lipid-transporting ABC proteins (2, 5). Changing the activity of someanimal ABCA proteins causes overaccumulation of lipids, such astriacylglycerol (TAG) and cholesterol esters, in specific tissues (6).In plants, many ABCGproteins secrete lipidic molecules that formcutin and wax layers (3). Defects in the expression of such proteinsresult in reduced surface lipids and/or developmental defects, suchas organ fusion (7, 8).To identify the transporters that deliver fatty acid substrates to

    the ER for glycerolipid synthesis, we focused on ABCA andABCG proteins. Our database searches revealed that no ABCGproteins and only one animal ABCA protein, ABCA17, localizeat the ER membrane (9). In the model plant Arabidopsis thaliana,

    12 genes encode ABCA transporters, none of which has beencharacterized previously (3, 10).We hypothesized that ABCA proteins in plants mediate the

    transport of fatty acids into the ER, and that the activity of suchproteins is a limiting step in TAG biosynthesis in oilseed plantsduring the seed-filling stage, when TAG biosynthesis is greatest,and thus the flux of acyl-CoAs from the plastid to the ER isgreatest as well. This hypothesis predicts that a knockout of sucha transporter would decrease TAG synthesis, resulting in re-duced seed storage lipid content.

    ResultsScreening of ABCA Subfamily Genes for Altered Seed Phenotypes. Toidentify the ABC transporters involved in fatty acid transport tothe ER, we grew seeds of KO mutants of eight ABCA familymembers on half-strengthMurashige and Skoog (1/2MS)mediumwith or without sucrose, and compared early seedling growth withWT. The rationale behind this test is that early seedling growth ofoilseed plants depends on storage lipids in the absence of sucrose,but not in its presence (11, 12). Thus, plants defective in expressionof fatty acid-transporting ABC transporter would be expected toexhibit reduced growth in the absence of sucrose, but normalgrowth in its presence.Among the mutants tested, the growth of abca9-1 seedlings was

    most retarded on medium lacking sucrose (Fig. 1A). In contrast,the growth of abca9-1 seedlings onmedium containing 1% sucrosewas comparable to that of WT. The mutant phenotypes wereconfirmed for two other abca9 alleles (abca9-2 and abca9-3; Fig.S1). Thus, abca9 seeds are defective in the accumulation of storagelipids or the conversion of lipids to sucrose.

    abca9 Seeds Exhibit Reduced Seed Size and/or Abnormal Morphology.Given that storage lipids account for ∼35–40% of the dry seedweight of Arabidopsis thaliana (1), seeds defective in storage lipidaccumulation are expected to be smaller ormisshapen. Among theeight ABCA KO seeds tested, abca9-1 seeds displayed the greatestvariability in size and shape (Fig. 1B). Using a dissecting micro-scope, we classified abca9 seeds into four groups: normal, flat,small, and brown and shrunken. Only 20% of abca9 seeds werenormal, compared with 95% of WT seeds (Fig. S2A). Image

    Author contributions: S.K., M.M., E.M., E.B.C., I.N., and Y.L. designed research; S.K., Y.Y.,H.O., H.K., and D.S. performed research; and S.K., E.M., E.B.C., I.N., and Y.L. wrotethe paper.

    Conflict of interest statement: S.K., Y.Y., H.O., I.N., and Y.L. have filed patent PCT/KR2011/006826 and the title of patent is “Composition for increasing seed size or content ofstorage lipid in seed, comprising the ABC transporter protein-coding gene”.

    This article is a PNAS Direct Submission.1I.N. and Y.L. contributed equally to this work.2To whom correspondence may be addressed. E-mail: [email protected] [email protected].

    This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1214159110/-/DCSupplemental.

    www.pnas.org/cgi/doi/10.1073/pnas.1214159110 PNAS | January 8, 2013 | vol. 110 | no. 2 | 773–778

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  • analysis using ImageJ (http://rsbweb.nih.gov/ij) revealed thatabca9 seeds were smaller and more varied in size than WT seeds(Fig. S2B). Such variability in seed phenotype was observed insubsequent generations as well, irrespective of the size and shapeof the seeds from which the plants were generated (Fig. S2C).To demonstrate genetic complementation of abca9, we trans-

    formed the mutant plants with ABCA9 under the control of itsnative promoter.We generated two independent complementationlines for each of the three alleles of KO mutants. The resulting sixtransgenic lines produced normal seeds and grew normally onmedium lacking sucrose (Fig. 1B and Fig. S3), proving that ABCA9is required for proper seed morphology and early seedlingestablishment.

    ABCA9 Is Expressed Specifically in Maturing Seeds.We evaluated thespatial and temporal patterns ofABCA9 expression by quantitativeRT-PCR analysis. ABCA9 transcripts were amplified only slightlyfrom the whole seedlings, rosette leaves, stems, and flowers,whereas high transcript levels were found in siliques, especiallyduring the middle (S2) and late (S3) stages of seed development(Fig. 1C). Together with the defective seed morphology, this ex-pression pattern suggests that ABCA9 functions during seed de-velopment. To test this, we evaluated developing seeds at 4, 8, 12,16, and 20 d after flowering (DAF) (Fig. 1D). Approximately 50%of abca9 seeds were paler and/or smaller than WT seeds at 8, 12(early S2 stage), and 16 (S3 stage) DAF. The temporal manifes-tation of abnormal seed morphology in mutant siliques coincidedwith the timing of ABCA9 transcript accumulation in WT siliques,supporting our conclusion that the abnormal seed phenotypes arecaused by disruption of ABCA9.

    abca9 Seeds Have Reduced TAG Content. We then tested whetherthe abca9 seeds indeed had reduced lipid content. Dry seedweight was 20% lower and total lipid content per seed was 16%

    lower in mutant seeds compared with WT seeds, but proteincontent did not differ (Fig. 1E). TAG, the major component oftotal lipids in seeds, was ∼35% less abundant in abca9 seedscompared with WT (Fig. 1F); however, the fatty acid compositionof TAG was comparable in dry mutant and WT seeds (Fig. 1G),except for slightly lower linolenate (18:3) levels in the mutants.These findings indicate that abca9 does not affect the selectivity offatty acid incorporation into TAG or further desaturation steps.

    ABCA9 Is Localized at the ER. If ABCA9 is involved in fatty acidtransport to theER, then it should be localized at this organelle.Weexamined the subcellular localization of ABCA9 using transgenicplants expressing ABCA9-sGFP under the control of the CaMV35S promoter (Pro35S::ABCA9gDNA-sGFP). Green fluorescencewas observed in the petioles (Fig. 2A) and isolated mesophyll pro-toplasts (Fig. 2B) of the transgenic plants, in a distribution re-sembling that of the ER.To further test the possibility that ABCA9 is localized to the

    ER, we fractionated crude membrane samples from Pro35S::ABCA9gDNA-sGFP transgenic plants by sucrose density gradientcentrifugation and evaluated ABCA9-sGFP using anti-sGFP an-tibody. The distribution of ABCA9-sGFP matched that of the ERmembrane marker BiP (Fig. 2C). To confirm the ER localizationof ABCA9, we performed an ER membrane-shifting assay byadding EDTA, which causes ribosomes to dissociate from the ER,thereby shifting the distribution of ER markers to lower-densityfractions. Under these conditions, both the BiP andABCA9 bandsshifted to lower sucrose density fractions than in the presence of4 mM MgCl2, further supporting the ER localization of ABCA9(Fig. 2D).

    abca9 Is Defective in TAG Synthesis. Our findings of reduced TAGcontent in abca9 seeds and localization of ABCA9 to the ERsupport the idea that ABCA9 is involved in TAG biosynthesis at

    **

    **

    A

    abca9-1WTabca9-1WT

    1% sucroseno sucroseB

    1-C1 2-C1

    abca9-1WT

    DC

    **

    **

    *

    E GF

    Fig. 1. Characterization of ABCA9 KO mutants. (A)abca9-1 is delayed in seedling growth on 1/2 MSmedium without sucrose (Left), but not on mediumcontaining 1% sucrose (Right). (B) Seed phenotypeof the WT, abca9-1, and two complementation lines,1-C1 and 2-C1. (Scale bar: 500 μm.) (C) Transcriptlevels of ABCA9 in various tissues of WT. WS, wholeseedlings; L, rosette leaves; S, stems; F, flowers; S1,siliques from 4∼6 DAF; S2, siliques from 10∼12 DAF;S3, siliques from 16∼18 DAF. (D) WT and abca9 seedsduring the seed-filling stages. Red arrows indicatedefective seeds. (E) Seed, lipid, and protein weightper seed. (F) TAG content per seed. (G) Fatty acidcomposition of TAG. *P < 0.05; **P < 0.01. Error barsindicate SEs from three replicates.

    774 | www.pnas.org/cgi/doi/10.1073/pnas.1214159110 Kim et al.

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  • the ER membrane, the site of TAG biosynthesis (1). To furthertest this possibility, we fed 14C-acetate to actively growing seeds(at 10 DAF) isolated from WT and abca9 siliques, and thenmeasured the radioactivity incorporated into the TAG fraction.We used 14C-acetate because it is readily converted into acetyl-CoA and malonyl-CoA for fatty acid biosynthesis (13). Theamount of radioactivity incorporated into TAG in developingabca9 seeds was approximately half that in WT seeds (Fig. 3A).Reduced TAG synthesis in abca9 seeds was confirmed in time-chasing experiments (Fig. 3B).

    To test whether ABCA9 can supply fatty acids for TAG syn-thesis, we examined whether ABCA9 facilitates assimilation ofexogenously supplied acyl-CoAs and free fatty acids into TAG. At10 DAF, developing seeds of WT and abca9 were fed 14C-oleoyl-CoA and 14C-oleic acid. The amount of radioactivity incorporatedinto TAG from 14C-oleoyl-CoA and oleic acid was significantlylower in abca9 seeds than in WT seeds (Fig. 3A). These results arein agreement with the reduced TAG content in dry abca9 seeds,indicating that ABCA9 supplies fatty acid substrates for seed TAGbiosynthesis at the ER membrane.

    A

    C

    ABCA9-sGFP

    BiP

    PIP2;1

    γ-TIPER

    PMVM

    17.8

    19.0

    20.6

    21.4

    22.4

    23.6

    24.6

    26.0

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    29.6

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    39.4

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    42.8

    43.8

    44.8

    16.0Sucrose

    w/w (%)

    (4 mM MgCl2)

    ER

    PM

    VM

    ABCA9-sGFP

    BiP

    PIP2;1

    γ-TIP

    Sucrosew/w (%)17

    .6

    19.0

    20.0

    21.0

    22.4

    23.4

    24.6

    25.6

    27.0

    28.6

    29.4

    30.8

    32.4

    34.0

    35.2

    36.6

    38.0

    39.6

    41.0

    42.0

    42.6

    44.0

    45.0

    16.0

    (4 mM EDTA)D

    ABCA-GFP BiP-RFP MergedB

    Fig. 2. Localization of ABCA9 at the endoplasmicreticulum. (A) sGFP signal from the petiole of thePro35S::ABCA9gDNA-sGFP T3 single homozygousline. (Scale bar: 20 μm.) (B) Overlap of GFP andRFP fluorescence in protoplasts isolated from thePro35S::ABCA9gDNA-sGFP T3 single homozygousline and transformed with the ER marker Pro35S::BiP-RFP. (Scale bar: 5 μm.) (C and D) Intracelluar lo-calization of ABCA9-sGFP detected by fractionationof microsomes on a sucrose density gradient in thepresence of 4 mM MgCl2 (C) or 4 mM EDTA (D). The24 fractions were collected and immunoblotted withantibodies to sGFP and ER (BiP) and with vacuolarmembrane (VM; γ-TIP) and plasma membrane (PM;PIP2;1) markers.

    A

    B C

    **

    *

    *

    abca9

    Acyl-CoAs Fatty acids

    WT

    Normal acyl-CoA Pool

    TAG

    Reduced acyl-CoA Pool

    TAG

    ER

    ER

    Cytosol

    Cytosol

    ADPATP

    ADPATP

    ABCA9

    Fig. 3. Reduced TAG biosynthesis in developingabca9 seeds. (A) Incorporation into TAG from 14C-acetate, 14C-oloeyl-CoA, and 14C-oleic acid was testedusing 50 developing seeds at 10 DAF. Total proteincontent did not differ between the WT and abca9seeds (WT, 637.8 ± 19.61 μg; abca9, 618.2 ± 15.73 μg).Six replicates were averaged, and the SE is shown.*P < 0.05; **P < 0.01, Student t test. (B) Time de-pendence of 14C-acetate incorporation into TAG. (C)Working hypothesis. The ABCA9 transporter (in red)transports both acyl-CoAs and fatty acids to the ER,thereby facilitating TAG synthesis.

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  • Overexpression of ABCA9 Increases Seed Oil Content. To testwhether ABCA9 mediates a limiting step in lipid accumulationduring seed filling, we generated Arabidopsis thaliana plantsexpressing full-length genomic DNA of ABCA9 under the CaMV35S promoter (Pro35S::ABCA9gDNA) (Fig. S4A). Interestingly,the ABCA9-overexpressing plants produced enlarged seeds (Fig.4A), with dry weight up to 126% of that of WT (Fig. 4D). Cot-yledon cells of ABCA9-overexpressing embryos were larger thanthose of WT (Fig. 4B), and ABCA9-overexpressing cells weremore densely packed with oil bodies compared with WT (Fig.4C). In contrast, protein body frequency and size did not sig-nificantly differ between ABCA9-overexpressing and WT cells(Fig. 4C). The TAG content per seed in ABCA9-overexpressinglines was up to 140% of that of WT, with no significant differ-ences in fatty acid composition (Fig. 4E and Fig. S4B).We performed biochemical analyses of protein and carbohy-

    drates to investigate whether increased lipid levels affected otherseed reserves. Comparison of ABCA9-overexpressing andWT linesrevealed significantly increased lipids in ABCA9-overexpressinglines with no reduction in other seed reserves (Table S1). Given that

    silique number per plant (average±SE, 130.7±10.5 forWT, 125.4±9.1 for overexpression line 2, 112.4 ± 11.2 for overexpression line4, and 116.2± 10.2 for overexpression line 6) and seed number perhalf-silique (average ± SE, 26.7 ± 0.8 for WT, 26.9 ± 0.4 foroverexpression line 2, and 26.6 ± 0.9 for overexpression line 6)were not significantly different between ABCA9-overexpressinglines and WT (Fig. S4 C and D), we conclude that overexpressionof ABCA9 can increase the total seed oil yield per plant.

    DiscussionA transporter that mediates the transport of acyl-CoAs and/orfree fatty acids from plastids to the ER has not yet been iden-tified in any living system. A recent transcriptome analysis ofoilseed-specific gene expression revealed increased levels ofmRNAs involved in fatty acid synthesis and acyl-CoA efflux fromplastids during seed filling (14); however, cytosolic acyl-CoAsinhibit plastidial fatty acid biosynthesis by inhibiting plastidialmetabolite transporters (15, 16). In addition, cytosolic acyl-CoAsand/or free fatty acids are toxic to the cell, and thus must be

    ** ** **

    ** **** **

    D

    WT OX2OX2 OX5OX2 WTWTA B

    Line Area of cotyledon, mm2 Area per cell, μm2

    WT 0.09 (100) 150.23 4.31 (100)OX2 0.11 (122) 179.15 4.49** (119)

    COX4WT

    PB

    PBOB

    * *** ***

    E*

    Fig. 4. ABCA9-overexpressing plants produce enlarged seedswith elevated averageweight and lipid content. (A) Developing seeds at 12DAF (Left) and dry seeds(Right) fromWTandABCA9-overexpressing plants. (B)Mature embryos isolated frommature dry seeds, imbibed for 1 h, of theWTandABCA9–overexpressing line(Upper) and cotyledon cells from these embryos (Lower). (Scale bars:Upper, 100 μm; Lower, 10 μm.) Surface areas of cotyledons andof individual cells of cotyledonsmeasured from images of the embryos are listed in the table. (C) Increased density of oil bodies in anABCA9-overexpressing line. Note that OX4 cells containmanyoil bodies that exclude toluidine blue dye (Right), which stains the cytosol ofWT cells purple (Left). OB, oil body; PB, protein body. (Scale bars: 5 μm.) (D and E) Dryseed weight (D) and TAG level (E) in WT and ABCA9-overexpressing plants. *P < 0.05; **P < 0.01, Student t test. Error bars indicate SE from four replicates.

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  • rapidly transported into the ER to maintain the high rates offatty acid and TAG biosynthesis during seed filling.We propose that ABCA9 acts as such a transporter.We base this

    conclusion on the following lines of evidence: (i) TAG levels arereduced in abca9 and increased in ABCA9-overexpressing seeds;(ii) ABCA9 is expressed specifically in seeds at the middle and latestages of maturation, when storage lipids are rapidly accumulatedand the rate of TAG synthesis is greatest (17); (iii) the temporalprofile ofABCA9 transcript accumulation is closely correlated withthe manifestation of abnormal seeds in developing abca9 siliques;(iv) TAG synthesis is reduced in abca9 seeds, as demonstrated byassimilation experiments with 14C-acetate, 14C-oleoyl-CoA, and14C-oleic acid; (v) ABCA9 is localized at the ER and belongs toa subfamily of lipid transporters, ABCA; and (vi) the relativeproportions of fatty acids in TAGs are similar in WT and abca9seeds, suggesting that the desaturation of esterified fatty acids onthe glycerol backbone is not affected in abca9 and in turn, that onlythefirst step at theER, but not the later steps of lipidmetabolism, isaltered in the mutant (18–21). If any steps further downstream inthe TAG biosynthesis pathway were defective, then a marked al-teration in the fatty acid composition of TAGs would be expected,because the ER is the site of extensive desaturation of fatty acids.Thus, we conclude that ABCA9 facilitates acyl-CoA uptake

    into the ER, thereby enhancing the assembly of acyl-CoAs intoTAGs. Cytosolic acyl-CoA binding proteins also may contributeto the transfer of acyl-CoAs from plastids to the ER (22). Theseproteins reportedly bind acyl-CoAs and maintain the acyl-CoApool size in the cytosol. Whether they are critical for TAG bio-synthesis in developing seeds remains to be determined, however.In ABCA9 overexpression lines, embryo cells are larger and

    contain more oil bodies than WT cells (Fig. 4C). As a result,ABCA9 overexpression increases seed size without changing thenumber of seeds per silique or the number of silique per plant(Fig. 4A and Fig. S4 C and D), resulting in an overall increase inseed oil yield per plant. Of note, there were no changes in proteinor carbohydrate levels in ABCA9-overexpressing seeds (Table S1).Thus, ABCA9 seems to increase sink capacity specific to storagelipids by facilitating the transport of activated fatty acids into theER. The highly expressed ABCA9most likely increases the size ofthe acyl-CoA pool in the ER, which may facilitate TAG synthesisby increasing substrate concentrations or extending the criticalperiod of TAG synthesis during seed filling (Fig. 3C). The in-creased oil production by ABCA9 overexpression suggests thatuptake of acyl-CoAs into the ER is a limiting step in metaboliteflux during seed filling.A previous genetic engineering strategy was to increase seed oil

    content by introducing enzymes and transcription factors involvedin lipid metabolism. Overexpression of acetyl-CoA carboxylase orfatty acid synthase had little effect on the level of lipid accumu-lation (23, 24), whereas overexpression of enzymes catalyzingTAG biosynthesis, such as glycerol-3-phosphate acyltransferase,lysophosphatidic acid acyltransferase, and diacylglycerol acyl-transferase, significantly increased seed oil yield, by 10–40% (25–29). Overexpression of the transcription factorWRINKLED1 alsoincreased seed oil yield by 20% (30). ABCA9 overexpressionprovides a valuable option for increasing the TAG content inseeds, demonstrating up to a 40% increase in seed oil yield with noreduction in protein and carbohydrate content. The use of a lipidtransporter is a unique approach to increasing the vegetable oilcontent of seeds that can be combined with other methodsenforcing seed metabolic functions. Given that the global con-sumption of vegetable oils is expected to double by 2030 (31),this strategy may be valuable in exploring ways to meet theurgent need for increased oil production.

    Materials and MethodsPlant Materials and Growth Conditions. Arabidopsis thaliana seeds were sur-face-sterilized, placed in the dark at 4 °C for 2 d, and then randomly sownon1/2MS-agar plates with 1% sucrose. Plates were incubated for 2–3 wk (22/18 °C;

    16/8 h day/night). For further analyses, plants were transferred to soil andgrown in a greenhouse (18/16 °C; 16/8 h day/night).

    Isolation of ABCA9 KO Mutants. Seeds of three alleles ofABCA9 KOArabidopsisthaliana mutants—SALK_058070, SALK_023744, and SALK_084342—wereobtained from the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/cgi-bin/tdnaexpress). Genomic DNA (gDNA) was extracted from plantsgrown for 4 wk on soil. Homozygous ABCA9 KO plants were isolated by PCRusing a T-DNA–specific primer (pROKLBb1: 5′-GCGTGGAACCGCTTGCTGCAACT-3′) and four ABCA9-specific primers (SALK_058070LP: 5′-CTACATATGGCTCGT-GGGAAC-3′; SALK_058070RP: 5′-AAAGAGGTGGAGGTGCTCTTC; SALK_084342LP:5′-ATGACTCTGCGAGAAGGCTT-3′; and SALK_084342RP: 5′-GAAAGAGACCAAA-CCACACC-3′).

    DNA Constructs. To generate complemented and tagged lines of abca9, sGFPwas amplified from the 326-sGFP (kindly providedby InhwanHwang, POSTECH,Korea) vector using primers containing SpeI and PmlI restriction sites (5′-ACTAGTATGGTGAGCAAGGGCGAGGA-3′ and 5′-CACGTGTTACTTGTACAGCTCGTCCATG-3′) and inserted into the SpeI and PmlI sites of pCAMBIA1302(Cambia, Canberra, Australia). For the complementation line, the ABCA9 pro-moter was amplified using primers containing KpnI and SpeI sites (5′-GGTACCGCACGGTGTGAACATTAATT-3′ and 5′-ACTAGTGATCACAGAGGAAGAAGAAG-3′)and inserted into the KpnI and SpeI sites of each construct. Finally, full-lengthgenomic DNAofABCA9was amplified usingprimers containing the SpeI site (5′-ACTAGTATGACTCTGCGAGAAGGCTT-3′ and 5′-ACTAGTTTCATTGTTAGATTCA-TAAT-3′) and ligated into the SpeI site of the construct.

    To generate the ABCA9 overexpression construct, genomic DNA of ABCA9was amplified using primers containing the SpeI site (5′-ACTAGTATGA-CTCTGCGAGAAGGCTT-3′ and 5′-ACTAGTTTCATTGTTAGATTCATAAT-3′) andinserted into the SpeI site of pCAMBIA1302. All constructs were verifiedby sequencing.

    Seed Lipid, Protein, and Carbohydrate Analysis. A total of 500 Arabidopsisthaliana seeds were immersed in 1 mL of boiling isopropanol and heated for 5min at 80 °C. After cooling, 2 mL of chloroform was added to the sample, andthe plant material was finely ground with a Polytron homogenizer (HitachiKoki). The extract was centrifuged at 1,600 × g for 10 min. The resultant su-pernatant was decanted to a new 10-mL screw-capped glass tube, and the pelletwas reextracted with 2 mL of chloroform and 1 mL of methanol by vortexing.After centrifugation at 1,600 × g for 10 min, the supernatant was recoveredby decantation, combined with the first supernatant, and then washed with 1.2mL of 0.9% KCl by vigorous shaking. Following centrifugation at 1,600 × g for15 min, the lower layer was recovered into a weighted 20-mL pear-shaped flask,and the solvent was evaporated on a rotary evaporator. The residual solventwas dried using a vacuum desiccator, and lipid dry weight was determined.Dried lipid residues were dissolved in chloroform at a concentration of 10 mg/mL and stored in a 1-mL screw-capped sample tube at −30 °C until use.

    Total lipids (1 mg) were separated by silica gel TLC using a solvent mixture[80:30:1 (by volume) hexane/diethylether/acetic acid] that facilitated theseparation of neutral lipids. Lipid spots were visualized by spraying with0.01% (wt/vol) primuline reagent (Sigma-Aldrich), and each lipid class wastransformed into fatty acid methyl esters at 80 °C for 3 h in 3 mL of 5% (wt/vol) HCl in methanol, with an additional 47.8 nmol pentadecanoic acid as aninternal standard and 50 nmol 2,6-di-t-butyl-4-methylphenol as an antioxi-dant. The resultant fatty acid methyl esters were extracted with 3 mL ofhexane and quantified by GC.

    For total protein assays, 50 seeds were homogenized in 200 μL of extractionbuffer [1% (wt/vol) SDS 6 M urea]. Protein content was measured in 200 μL ofthe crude homogenate using the Bio-Rad DC Protein Assay Kit with BSA asa standard. Carbohydrates were analyzed as described by Siloto et al. (32) withsome modifications. A total of 300 seeds were homogenized in 80% (vol/vol)ethanol and incubated at 70 °C for 90 min. After centrifugation at 16,000 × gfor 5 min, the supernatant was transferred to a new test tube. The pellet wasextracted three times with 500 μL of 80% (vol/vol) ethanol, and the solvent ofthe combined supernatants was evaporated at room temperature undera vacuum. This residue was dissolved in 0.1 mL of water and used for sucrosequantification. The pellet remaining after ethanol extraction was homoge-nized in 200 μL of 0.2 M KOH and then incubated at 95 °C for 1 h. After theaddition of 35 μL of 1 M acetic acid and centrifugation for 5 min at 16,000 × g,the supernatant was used for starch quantification. Sucrose content and starchcontent were measured using kits from Sigma-Aldrich.

    Tissue-Specific Expression of ABCA9. To investigate the level of ABCA9transcript in different tissues, real-time PCR was performed using totalRNA extracted from each organ. To eliminate the possibility of genomic

    Kim et al. PNAS | January 8, 2013 | vol. 110 | no. 2 | 777

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  • DNA contamination, real-time PCR was performed using ABCA9 cDNA-specific primers (5′-TGCTGTAAAGGGTTTGTGGA-3′ and 5′-TGCCAGTAG-TCGGTTCATCT-3′).

    Membrane Fractionation Using Sucrose Gradient Centrifugation Arabidopsisthaliana plants (Pro35S::ABCA9gDNA-sGFP) were cultured for 2 wk in liquid 1/2MS medium. Then 10 g of plant seedlings (50 seedlings) was homogenized in40mL of homogenized solution [250 mM sorbitol, 50 mM Tris-acetate (pH 7.5),1 mM EGTA-Tris (pH 7.5), 2 mM DTT, 1× protease inhibitor mixture (PIC, EDTA-free; Roche Applied Science), 2% (wt/vol) polyvinylpyrrolidone, and 4 mMEDTA or MgCl2] using a mortar and pestle. The sample was filtered throughnylon mesh and centrifuged at 2,000 × g for 10 min at 4 °C. The supernatantwas recentrifuged twice at 10,000 × g for 10 min at 4 °C. The supernatantwas ultracentrifuged at 100,000 × g for 1 h at 4 °C (SW28 rotor; BeckmanCoulter). The pellet was suspended in microsome resuspension buffer con-taining 5% (wt/vol) sucrose, 20 mM Tris-acetate (pH 7.5), 0.5 mM EGTA-Tris,1× PIC, and 4 mM EDTA or MgCl2. Then 2.5 mg of the crude membranesuspension was applied to the sucrose gradient solution [10–50% sucroselinear gradient, 20 mM Tris·HCl (pH 7.5), 0.5 mM EGTA-Tris, 1× PIC, and 4 mMEDTA or MgCl2], centrifuged (slow acceleration, no break) at 100,000 × g for16 h at 4 °C (SW41Ti rotor; Beckman Coulter) and fractionated into 28fractions of 410 μL each. The fractionated proteins were separated by SDS/PAGE and transferred to a PVDF membrane. The membrane was blockedwith 5% (wt/vol) skim milk and incubated with organelle-specific primaryantibodies (BiP, γ-TIP, and PIP2;1 for the ER, vacuole, and plasma membrane,respectively). HRP-conjugated goat anti-mouse or goat anti-rabbit IgG wasapplied as the secondary antibody.

    ABCA9:sGFP was detected using anti-sGFP (Clontech) and goat anti-mouseantibody. The transfer solution contained 48 mM Tris, 39 mM glycine, 0.02%SDS, and 20% (vol/vol) methanol. Blocking solution contained TBST buffer [0.5M Tris base (pH 7.6), 9% (wt/vol) NaCl, and 1% (vol/vol) Tween-20] and 5% (wt/vol) skim milk. The membrane was washed with TBST buffer. Antibody con-centrations were 1:2,000 for anti-sGFP and 1:3,000 for anti-BiP, anti-PIP2;1,anti–γ-TIP, anti-mouse, and anti-rabbit. Incubation with primary antibodieswas carried out overnight at 4 °C, and incubation with secondary antibodieswas done for 1 h at room temperature (∼25 °C) with gentle shaking.

    Assay Monitoring Incorporation of Precursors into TAG. To compare the ratesof incorporation of precursors into TAG, 50 Arabidopsis thaliana seeds were

    collected from WT and abca9 siliques at 10 DAF and transferred to 200 μL of20 mM MES buffer (pH 5.8). One of the following 14C-labeled compoundswas added at the specific activities indicated: 0.5 μCi of 14C-acetate (50 mCi/mmol), 0.5 μCi of 14C-oleoyl-CoA (40–60 mCi/mmol), or 0.5 μCi of 14C-oleicacid (40-60 mCi/mmol). The seeds were incubated for 18 h in the dark whilebeing rotated at 100 rpm.

    In the 14C-acetate time-course assay, the seeds were incubated for 9, 12,and 18 h under the same conditions. The seeds were then washed with 1 mLof ice-cold water and homogenized in 50 μL of chloroform:methanol:formicacid (10:10:1 by volume). The organic and aqueous phase were separated byadding 12.5 μL of solution consisting of 1 M KCl and 0.2 M H3PO4 andcentrifuging at 16,000 × g for 5 min. The lipids in the lower phase wereseparated on a silica TLC plate (1.05721.0001; Merck) with hexane:diethylether:acetic acid (80:30:1 by volume). Silica material containingthe TAG was scraped from the TLC plate and mixed with the scintillationmixture. Radioactivity was measured by scintillation counting.

    Oil Body Observation. To observe oil bodies in embryo cells, dry seeds of WTand ABCA9-overexpressing line were imbibed for 1 h and then cut in halfwith a razor blade. The samples were fixed overnight at 4 °C in 20 mMcacodylate buffer (pH 7.0) containing 3% (wt/vol) paraformaldehyde and2.5% (vol/vol) glutaraldehyde, and then rinsed in cacodylate buffer andfurther fixed in 1% (wt/vol) osmium tetraoxide for 1 h at 4 °C. The sampleswere dehydrated using a graded ethanol series and embedded in LR Whiteresin (Electron Microscopy Sciences). Serial sections of the samples werestained with 1% (wt/vol) toluidine blue and observed by light microscopy.

    ACKNOWLEDGMENTS. We thank the Salk Institute Genomic Analysis Labora-tory for the Arabidopsis thaliana mutant seeds and J. L. Harwood andW. Dewitte for their insightful comments on the manuscript. Research in theY.L. laboratory was supported by grants from the Global Research Laboratoryprogram of the Ministry of Science and Technology; the Next-Generation Bio-green 21 Program (Grant PJ008102), Rural Development Administration; andthe Global Frontier Program (Grant 2011-0031345) of the Republic of Korea.Research in the I.N. laboratory was funded by Grants-in-Aid for Scientific Re-search 21570034 and 24570040 from theMinistry of Education, Culture, Sports,Science and Technology of Japan. Research in the E.B.C. laboratory was sup-ported by the Center for Advanced Biofuel Systems, an Energy Frontier Re-search Center funded by the US Department of Energy, Office of Science,Office of Basic Energy Sciences (Award DE-SC0001295).

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