Deﬁciency in a Very-Long-Chain Fatty Acid b-Ketoacyl ...Deﬁciency in a Very-Long-Chain Fatty...

Deficiency in a Very-Long-Chain Fatty Acidb-Ketoacyl-Coenzyme A Synthase of Tomato ImpairsMicrogametogenesis and Causes Floral Organ Fusion1[W]

Anna Smirnova*, Jana Leide, and Markus Riederer

Universität Würzburg, Julius-von-Sachs-Institut für Biowissenschaften, D–97082 Wurzburg, Germany

Previously, it was shown that b-ketoacyl-coenzyme A synthase ECERIFERUM6 (CER6) is necessary for the biosynthesis of very-long-chain fatty acids with chain lengths beyond C28 in tomato (Solanum lycopersicum) fruits and C26 in Arabidopsis (Arabidopsisthaliana) leaves and the pollen coat. CER6 loss of function in Arabidopsis resulted in conditional male sterility, since pollen coatlipids are responsible for contact-mediated pollen hydration. In tomato, on the contrary, pollen hydration does not rely on pollencoat lipids. Nevertheless, mutation in SlCER6 impairs fertility and floral morphology. Here, the contribution of SlCER6 to thesexual reproduction and flower development of tomato was addressed. Cytological analysis and cross-pollination experimentsrevealed that the slcer6 mutant has male sterility caused by (1) hampered pollen dispersal and (2) abnormal tapetumdevelopment. SlCER6 loss of function provokes a decrease of n- and iso-alkanes with chain lengths of C27 or greater and ofanteiso-alkanes with chain lengths of C28 or greater in flower cuticular waxes, but it has no impact on flower cuticle ultrastructureand cutin content. Expression analysis confirmed high transcription levels of SlCER6 in the anther and the petal, preferentially insites subject to epidermal fusion. Hence, wax deficiency was proposed to be the primary reason for the flower fusionphenomenon in tomato. The SlCER6 substrate specificity was revisited. It might be involved in elongation of not only linearbut also branched very-long-chain fatty acids, leading to production of the corresponding alkanes. SlCER6 implements a functionin the sexual reproduction of tomato that is different from the one in Arabidopsis: SlCER6 is essential for the regulation of timelytapetum degradation and, consequently, microgametogenesis.

Terrestrial plants have developed the cuticle, a barrierthat restrains water movements between the outer cellwall of the epidermis and the surrounding atmosphere,thus preventing uncontrolled water loss (Riederer, 2006;Samuels et al., 2008; Domínguez et al., 2011). Besides this,the cuticle protects the plant from biotic stresses andprevents ectopic interorgan fusion during flower devel-opment (Lolle et al., 1992; Lolle and Pruitt, 1999; Tanakaand Machida, 2006; Pollard et al., 2008; Javelle et al.,2011). The cuticle is a complex matrix of cutin, waxes, andpolysaccharides. Cutin is a biopolymer consisting of C16and C18 fatty acids with a varying degree of oxygenation(Pollard et al., 2008; Domínguez et al., 2011). Cuticularwax is a general term for a complex mixture of hydro-phobic compounds, including homologous series of very-long-chain aliphatic lipids. The wax composition variesnot only among different species but also from one organto another and throughout the course of plant develop-ment. Environmental conditions also have an impact onthe wax composition (Samuels et al., 2008). Therefore, the

mechanisms by which plants control wax biosynthesisare of considerable interest.

Long-chain acyl-coenzyme A synthetases (LACS) ac-tivate the precursors of wax biosynthesis, C16 and C18fatty acids, on the outer membrane of the plastid. Theresulting long-chain acyl-CoAs undergo elongation tovery-long-chain fatty acids (VLCFAs) in the endoplasmicreticulum. VLCFA biosynthesis might be a major meta-bolic route in the epidermis, since VLCFAs are the pri-mary compounds for all other wax components (Kunstet al., 2006; Samuels et al., 2008). The initial condensationreaction catalyzed by b-ketoacyl-CoA synthases wasshown to be rate limiting in the elongation process.b-Ketoacyl-CoA synthases form a family of proteins thatare thought to have different substrate specificities (Millarand Kunst, 1997; Joubès et al., 2008). To date, three of thecondensing enzymes were shown to be involved in cu-ticle formation in Arabidopsis (Arabidopsis thaliana) shoots:KETOACYL-COENZYME A SYNTHASE1 (KCS1; Toddet al., 1999), FIDDLEHEAD (FDH; Lolle et al., 1992; Pruittet al., 2000; Voisin et al., 2009), and ECERIFERUM6(CER6 [also known as CUT1 or KCS6]; Jenks et al., 1995;Millar et al., 1999; Fiebig et al., 2000; Hooker et al., 2002).Furthermore, KCS20 and KCS2/DAISY were recentlydescribed as redundant condensing enzymes implicatedin VLCFA biosynthesis in shoots (Lee et al., 2009).

The importance of the contribution of cutin and cu-ticular waxes in cell-cell interactions remains unclear(Javelle et al., 2011). A number of cuticular mutants wereisolated, but only some of them showed postgenital

1 This work was supported by the Deutsche Forschungsgemein-schaft (Sonderforschungsbereich 567).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Markus Riederer ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.112.206656

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organ fusion, joining organ primordia after their incep-tion at the shoot apical meristem (Lolle et al., 1998).Among Arabidopsis mutants deficient in waxes, cer10,cer13, wax1, wax2/cer3/yre, and deadhead exhibited theorgan fusion phenotype (Jenks et al., 1995, 1996; Lolleet al., 1998; Yephremov et al., 1999; Zheng et al., 2005).Another contact-mediated developmental process is thepollen-stigma interaction. CER1, CER3, CER6, andCER10, which contribute to the biosynthesis and/ordeposition of cuticular waxes in Arabidopsis, wereshown to be required for early pollen recognition on thestigma (Preuss et al., 1993; Aarts et al., 1995; Hülskampet al., 1995; Fiebig et al., 2000). Mutations in any of thesegenes, besides the waxless shoot, resulted in conditionalsterility. The mutants produced viable pollen, but itfailed to hydrate on the dry stigma because of alteredpollen coat lipids. Under conditions of high humidity,fertility was restored. The same phenotype was ob-served in the lacs1 lacs4 double mutant (Jessen et al.,2011). Formation of the sporopolleninic pollen exine isanother aspect of the involvement of some enzymescontributing to the biosynthesis of cuticle components insexual plant reproduction (Ariizumi et al., 2003; Junget al., 2006; Zhang et al., 2008; Shi et al., 2011).A substantial part of the data presented so far were

obtained from studies on Arabidopsis. This species,however, has a very thin cuticle, and its cutin compo-sition is rather different from other plants (Bonaventureet al., 2004). Our knowledge of wax biosynthesis in othermodel species commonly used in cuticle research islimited. Tomato (Solanum lycopersicum) is an importanthorticultural crop and a valuable tool for plant research.Previously, the use of a slcer6 tomato mutant (formerlylecer6) allowed the demonstration of a direct relationshipbetween the cuticular transpiration barrier propertiesand modification of the cuticular wax compositionduring fruit development (Leide et al., 2007). Interest-ingly, besides alterations in cuticular waxes, the muta-tion in SlCER6 impacts flower organ morphology andfertility. Reasons for the reduced fertility in the slcer6mutant could not be the same as for atcer6 plants be-cause tomato has a wet stigma and, consequently, adifferent system of pistil-pollen interaction (Heslop-Harrison, 1977). In contrast to atcer6, the slcer6 mutantdemonstrates flower organ fusion and thus appears tobe a perfect model for investigating cuticle integrityregulation. The study presented in this paper showsthe contribution of SlCER6, previously only known tobe involved in cuticle formation in vegetative organs,to the sexual reproduction and flower development intomato.

RESULTS

Flower Phenotype of the slcer6 Mutant

The MicroTom wild-type flower contains five sepalsalternating with five petals and a pistil surrounded by fiveanthers coupled by epidermal hairs into an anther cone. Atthe time of anthesis, the wild-type flowers opened whilethe slcer6 flowers stayed closed (Fig. 1A). In the wild type,

anthers were opened by longitudinal slits that servefor anther desiccation and pollen release, whereas theslcer6 anthers remained closed (Fig. 1B). Stomium cellsdegenerated in slcer6 without visible disorders. How-ever, due to fusion between the external anther walls,the mutant pollen could only move to the neighborlocule but failed to escape from the anther cone (Fig.1C). The petals were fused with each other and partlyattached to the anther epidermis (Fig. 1, D–F). Thus,the slcer6 flowers demonstrated reproducible petal-to-petal, petal-to-anther, and anther-to-anther epidermalfusions. The calyx, the stigma, and the flower budswere not subject to this process.

To further characterize the slcer6 mutant, the fusedorgans at different flower developmental stages wereexamined by scanning electron microscopy. These stageswere correlated to the bud length (Table I). Epidermalcells of the wild-type anther underwent elongation at thetime of microspore maturation (Fig. 2, A and B). Typicalnanoridges emerged later during pollen development(Fig. 2C). In slcer6, misshapen epidermal cells were foundin immature anthers (Fig. 2, D and E). The mutant epi-dermis at anthesis also displayed nanoridges, but itssurface was covered with petal imprints (Fig. 2F). In bothlines, epidermal cells of the adaxial petal surface werehardly distinguishable at the stage of microspore becauseof numerous anther imprints (Fig. 2, G and J). At thestage of binucleate pollen, the outlines of petal epidermalcells became apparent in the wild type, while in the slcer6mutant, the imprints persisted (Fig. 2, H and K). At an-thesis, petal epidermal cells were easily discernible inboth lines. However, the petal epidermis of the mutantshowed remnants of anther cells that stayed attached aftermechanical disconnection of the fused organs (Fig. 2, Iand L). Thus, the anther abaxial and petal adaxial surfacesdynamically interact in the course of flower development.At the time of anthesis, this connection is abolished inthe MicroTom wild type but persists in the slcer6 mutant,resulting in coalescence of the anther cone and the corolla.

Sterility of the slcer6 Mutant

Seeds were present in all MicroTom wild-type fruits,while in the slcer6 mutant, seeds were found only in27% of fruits. The quantity of seeds per fruit in themutant was reduced to 5% 6 1% (mean 6 SE) of thewild type (Fig. 3A). In order to verify whether femalefertility and the pollen-stigma interaction are affectedby the mutation, the slcer6 plants were pollinated withthe wild-type pollen. The slcer6 stigma fully supportedgermination of the wild-type pollen (Fig. 3B). The numberof seeds in fruits obtained by the cross pollination was thesame as in the wild type (Fig. 3A). To verify whetherpollen release could be impaired in the mutant, slcer6plants were hand pollinated with the slcer6 pollen. Thepollen germinated on the stigma, but pollen tubes werenot as abundant as in the cross-pollination experiments(Fig. 3B). The number of seeds per fruit increased almost10 times, but it was still two times lower compared withthe MicroTom wild type (Fig. 3A).

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Pollen Development and Exine Formation in theslcer6 Mutant

Microscopic analysis showed that a large number ofthe slcer6 pollen grains are misshapen and highly vari-able in size (Fig. 4, A and B). Some mutant pollen grainswere stained neither with 49,6-diamidino-2-phenylindole(DAPI) nor with fluorescein diacetate. Such cells wererepresented by pollen walls without nuclei or cytoplasm(Fig. 4, C and D). On average, the degraded pollen madeup 68% 6 1% (mean 6 SE). Microsporogenesis, begin-ning with meiosis of the microsporocyte and completedwith microspore formation, occurred without visibledisorders. Microgametogenesis, which includes micro-spore maturation and mitosis with subsequent binuclearpollen grain formation, was heavily impaired in themutant. Already at the stage of the early microspore,nuclei were degraded in some cells (Fig. 4, E and F). Latemicrospores had a highly vacuolated cytoplasm and nonuclei (Fig. 4, G and H).

In order to verify whether the malfunction in slcer6pollen development is caused by impairments in sporo-polleninic exine formation, scanning and transmissionelectron microscopy of the pollen wall were performed.Neither the slcer6 pollen exine nor that of the microsporewere different from the MicroTom wild type. Pollen re-sistance to acetolysis was also unchanged (SupplementalFig. S1).

Water Content, Water Loss, and Cuticle PermeabilityChanges during slcer6 Flower Bud Development

Since an alteration of the flower cuticle may provokeenhanced transpiration and subsequent untimely pollendehydration, the water status of the slcer6 flower budswas addressed. The water content did not differ in thewild-type and slcer6 young flower buds at the stage ofmicrosporogenesis (Fig. 5A). Further on, the water con-tent in slcer6 decreased to 87% of the MicroTomwild type(bud length, 6–8 mm). Water loss in the buds with youngpollen (bud length, 6.5 mm) was 1.34-fold higher in themutant compared with the wild type (Fig. 5B). The cuticleof the slcer6 young buds at the stage of microsporogenesis(bud length, 1–4 mm) was more permeable for chloro-phylls and carotenoids as compared with the MicroTomwild type: the amount of diffused pigments was 1.3-foldhigher after 150 min of extraction (Fig. 5C). This differ-ence increased 3-fold in flower buds with young pollen(bud length, 6–7 mm). The total pigment content was notaltered (Supplemental Fig. S2).

Anther Tapetum Development in the slcer6 Mutant

Since male sterility is usually associated with ab-normal tapetum development, transmission electronmicroscopy was performed to characterize the slcer6tapetum ultrastructure. In the MicroTom wild type atthe early microspore stage, the cytoplasm of tapetalcells contained mitochondria, plastids, extensive en-doplasmic reticulum, and vacuoles with inclusions.

Figure 1. Flower morphology of the MicroTom wild type (WT) and theslcer6 mutant. A, Opened wild-type flower and slcer6 flower withfused organs at anthesis. The arrow indicates the suture between fusedpetals in the mutant. B, Wild-type anthers with longitudinal stomiumslits, which serve for pollen release (arrows), and slcer6 anthers closedlengthwise. C, Cross sections of anthers at anthesis. In the wild type,the anther walls bend inward. Epidermal fusion in slcer6 (arrowheads)impedes curling of the anther walls, and the pollen stays inside jointlocules of adjacent anthers. Most of the pollen has been lost duringspecimen preparation. Ec, Epidermal cells; L, locule; S, splitted sto-mium. D, Cross section of a slcer6 flower bud at the stage of micro-spore formation. The left box marks the site of petal fusion depicted inE. The right box marks the site of petal-to-anther epidermal fusiondepicted in F. AnEp, Anther epidermis; P, petal.

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The cells had an irregular shape, and their walls werelined with electron-dense orbicules (Fig. 6A), tapetum-produced globular structures consisting of sporopol-lenin. In the slcer6mutant, tapetum cells almost devoidof cytoplasm were detected already at the stage ofearly microspore (Fig. 6B). The outer tapetum facing

the epidermis was generally represented by such emptycells. At the stage of late microspore, degenerating tapetalcells contained multiple spheroid lipid bodies in the wildtype (Fig. 6C). In the slcer6 mutant, the cytoplasm of theinner tapetum cells contained irregularly shaped prom-inent bodies, while the outer tapetum cells were mostlyempty (Fig. 6, D–F). At the time of anthesis, tapetal cellswere devoid of cytoplasm in both lines (Fig. 6, G andH). Thus, a substantial part of the outer tapetum un-derwent untimely degradation in slcer6, while the in-ner tapetum demonstrated altered morphology.

Composition of Cuticular Waxes, Cutin, and CuticleUltrastructure in the slcer6 Flower

Analysis of anther cuticular waxes revealed six com-pound classes in both lines: (1) n-alkanes, (2) iso-alkanes,(3) anteiso-alkanes, (4) alkanoic acids, (5) sterols, and (6)

Table I. Stages of microsporogenesis and microgametogenesis inMicroTom flowers categorized by bud length (n = 10)

Stage Bud Length

mm

Microsporocyte meiosis 2.0–2.5Tetrad 2.5–3.0Early microspore 3.0–3.5Vacuolated microspore 3.5–4.0Late microspore 4.0–5.0Binucleate pollen 5.0–6.0Mature pollen $7.0

Figure 2. Scanning electron micros-copy of the abaxial anther and theadaxial petal epidermis at differentstages of flower development in theMicroTom wild type (WT) and theslcer6 mutant. A and D, Anther epi-dermal cells in the flower bud at thestage of late microspore. In slcer6, cellsare misshapen (arrows). B and E, Inboth lines, anther epidermal cells be-come wider at the stage of young pol-len. Misshaping of cells in the mutantis indicated (arrows). C and F, At thetime of anthesis, epidermal cells havelobed margins and show cuticularridges (arrowheads). slcer6 anther epi-dermal cells display petal imprints leftafter mechanical separation of thefused organs (arrows). G and J, Thepetal surface displays anther epidermalcell imprints in the flower buds at thestage of late microspore (dotted lines).Contours of petal epidermal cells areindistinguishable. H and K, The petalpattern becomes visible in the wild typeat the time of young pollen formation. Inslcer6, the petal surface still showsconspicuous anther imprints (dottedline). I and L, At the time of anthesis,petal epidermal cells are apparent inboth lines. In slcer6, the remnants ofanther cells left after mechanical dis-connection are found between petalepidermal cells (arrows).





triterpenoids. In the MicroTom wild type, the mostprominent compounds were n- and iso-alkanes, mainlywith chain lengths of C29 and C31, while in the mutant,alkanoic acids represented the major compound class(Fig. 7A). The strongest effect of the slcer6 mutation onwax constituents was found for the n-alkanes. Their totalcontent was diminished to 19% of the wild type (TableII). The mutation affected n-alkanes with chain lengthsof C25 and C27 or greater. Branched alkanes in slcer6reached only around one-third of their amount in thewild type. The content of iso-alkanes with chain lengthsof C25, C27, and C29 or greater and anteiso-alkanes withchain lengths of C28 and C30 or greater was lowered in

comparison with the wild type. Deposition of n-C32 andC33 in all groups of alkanes was exclusively seen in thewild type. The content of other compound classes wascomparable in both lines.

In order to exclude possible effects of the alteredmorphology of the slcer6 anthers on the surface area, thesame experiment was performed for anthers and corollasharvested from closed green flowers before anthesis. Thedata were comparable to those presented above, but theimpact of the slcer6 mutation was detected for n- andiso-alkanes with chain lengths of C27 or greater and foranteiso-alkanes with chain lengths of C26 or greater(Supplemental Fig. S3A). Analysis of the wild-type

Figure 3. Seed production and in vivo pollen germination in the MicroTom wild type (WT) and the slcer6 mutant. A, Seedproduction in the wild type (n = 47), the slcer6 mutant (n = 129), and slcer6 plants hand pollinated (HP) with wild-type pollen(n = 28) or slcer6 pollen (n = 51). Means6 SE are shown. Significant differences are marked with different letters (P, 0.05, one-way ANOVA followed by Tukey’s HSD test). B, In vivo pollen germination. The slcer6 flowers were hand pollinated with wild-type (n = 29) or slcer6 (n = 31) pollen. Pistils were divided in three classes: pistils with numerous pollen tubes (more than 10;black bars), few pollen tubes (gray bars), and no pollen tubes (white bars).

Figure 4. Microgametogenesis in the MicroTom wild type (WT) and the slcer6 mutant. A and B, Wild-type pollen with veg-etative and generative nuclei and misshapen slcer6 pollen grains stained with DAPI. C and D, Semisections of wild-type pollenwith electron-dense cytoplasm and the degraded slcer6 pollen. Pollen walls without cytoplasm are marked (arrows). E and F,Wild-type and slcer6 early microspores stained with DAPI. Microspores without nuclei are indicated in the mutant (arrows). Gand H, Semisections of wild-type and slcer6 late microspores. Wild-type cells have structured nuclei and cytoplasm withvacuoles and mitochondria. Mutant microspores are highly vacuolated and have no nuclei.


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corolla waxes revealed that the contribution of branchedalkanes in total wax coverage in petals is more prominentthan in the anther and makes up 56%, although the waxpattern was similar for both organs (Supplemental Fig.S3B).Analysis of the monomeric composition of cutin

was performed for anthers and corollas harvested atanthesis. In the first experimental setup, enzymaticallyisolated cuticles were analyzed in order to determinethe relative proportions of cutin monomers (SupplementalFig. S4). In the second, freeze-dried material was usedfor quantitative estimation of the cutin content (Fig. 7B).The major monomer in both lines was (9)10,16-dihy-droxyhexadecanoic acid; its portion made up 25% to30% of the total cutin content in freeze-dried materialand more than 90% in the isolated cuticles. The rel-ative proportions of different cutin monomers for theMicroTom wild type and the slcer6 mutant were es-sentially comparable in both types of samples.To verify whether the adhesion response in the slcer6

mutant is associated with defects of the cuticularmembrane, the cuticle ultrastructure was inspected(Supplemental Fig. S5). The cuticle was observed as a thincontinuous and regular layer with a thickness around

60 nm in the wild type anther with early microspores.At the time of anthesis, the cuticle membrane thickened to270 6 41 nm. However, the slcer6 anther cuticle mem-brane showed the same structure throughout develop-ment, even in the fusion suture zone. The cuticle thicknessin the mutant was not altered and made up 2706 26 nmat anthesis.

Expression of the SlCER6 Gene

Quantitative reverse transcription (qRT)-PCR anal-ysis was carried out to examine the expression profileof SlCER6 among flower organs, pollen, immaturegreen fruits, the peel of mature green and red fruits,young leaves, stems, and roots (Fig. 8). SlCER6 tran-scripts were detected in all MicroTom wild-type sam-ples except the pollen. The highest level of SlCER6transcripts was found in the anther. High SlCER6 ex-pression was evident in young leaves and mature andimmature green fruits, where it made up 76% and 57%to 59% of the expression in the anther, respectively. In themature red fruit, the transcript amount decreased to 32%.The SlCER6 expression pattern for the wild-type fruits

Figure 5. Analysis of the water content, transpirational water loss, and pigment leaching in MicroTom wild-type (WT) andslcer6 flower buds. A, Water content changes in the course of flower development (means 6 SE, n = 20–30). Significant dif-ferences in the water content are marked with asterisks (P , 0.05, one-way ANOVA followed by Tukey’s HSD test). B, Tran-spirational water loss of flower buds with binucleate pollen (bud length, 6.5 mm; means 6 SD, n = 5; P , 0.05, Mann-WhitneyU test). DW, Dry weight. C and D, Chlorophyll and carotenoid leaching for the flower buds at early stages of microsporogenesis(bud length, 1–4 mm) and for flower buds with young pollen (bud length, 6 mm). Means 6 SD are shown; n = 5. Each time point issignificantly different between the wild type and the slcer6mutant for both pigments (P, 0.05, Mann-WhitneyU test). FW, Fresh weight.










was similar to the one obtained earlier (Mintz-Oron et al.,2008). SlCER6 was expressed at intermediate levels insepals and petals of 39% to 42% compared with the anther.The root and the pistil expressed SlCER6 only weakly. Inthe slcer6 mutant, SlCER6 expression was negligible in allsamples and did not exceed 0.01%; only in the anther, itreached 6% of the expression level in the wild-type anther.

To characterize SlCER6 spatial expression in theMicroTom flower, transgenic plants containing a GUSreporter construct fused to the upstream genomic

promoter sequence of SlCER6 were analyzed. GUSexpression was found in anthers, sepals, and petals offlowers at anthesis (Fig. 9A). Positive blue GUS stain-ing bordered the anther stomial slit coinciding with thesites of anther-to-anther epidermal fusion in the slcer6mutant (Fig. 9B). The most intensive GUS staining wasobserved at the distal part of the anther where twoapical pores are located (Fig. 9C). Examination of crosssections revealed that GUS is expressed in the epi-dermal cells of anther, sepal, petal, and in the ovary

Figure 6. Transmission electron microscopy of anther tapetum cells during microgametogenesis in the MicroTom wild type andthe slcer6 mutant. A, Wild-type tapetum at the stage of early microspore. The tapetum cell is irregularly shaped, and electron-dense cytoplasm contains mitochondria (M), vacuoles (V), and other organelles. The tapetal wall (TW) facing the locule is linedwith electron-dense orbicules (Or). B, slcer6 tapetum at the stage of early microspore. The tapetal cell is almost devoid ofcytoplasm. C, Wild-type tapetum at the vacuolate microspore stage. Cytoplasm becomes more disintegrated and containsspheroid lipid drops (arrowheads). D, slcer6 inner tapetum cell with irregularly shaped prominent bodies (asterisks) at thevacuolate microspore stage. E, Outer tapetum at the vacuolate microspore stage in slcer6. The tapetum cell lined with orbiculesalong the wall has almost no cytoplasm. F, Cross section of a slcer6 anther with microspores (MS). The outer tapetum (OtT) islocated at the side of contacting anthers. The inner tapetum (InT) is located closer to the connective tissue. G, Wild-type ta-petum at anthesis and mature pollen grain (PG) with highly structured electron-dense cytoplasm. The tapetal wall facing thelocule is appressed to that of the middle layer. H, slcer6 tapetum and degenerated pollen grain (DP) at anthesis.


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(Fig. 9, D–H). Positive blue staining was also detectedin the endothecium, a subepidermal cell layer specifi-cally formed in the upper one-third of the tomato an-ther, but neither in the developing pollen nor in tapetalcells. For further analysis, GUS expression was exam-ined in flower buds at early developmental stages.GUS staining became visible in sepals and anthers

already in 1-mm flower buds with newly differenti-ated organs (Fig. 9I). Blue positive staining highlightedthe whole sepal surface before meiosis of microspo-rocytes (Fig. 9J). In contrast to the sepals, GUS stainingbecame initially apparent at petal edges (Fig. 9, K–M),which are subject to the organ fusion in the slcer6mutant.

Figure 7. Cuticular wax and cutin composition of MicroTom wild-type (WT) and slcer6 flowers at anthesis. A, Composition ofthe anther cuticular waxes. B, Cutin composition of the anther and the corolla (freeze-dried material). Means 6 SD are shown;n = 4 to 5. Significant differences are marked with asterisks (P , 0.05, Mann-Whitney U test). DW, Dry weight.





DISCUSSION

Contribution of Cuticular Waxes to the Prevention ofFlower Organ Fusion in Tomato

The slcer6 tomato exhibits prominent epidermal fu-sion of flower organs. Intimate interaction betweenepidermal cells is well reflected in changes of the petaland anther surface architectures in the course of flowerdevelopment. SlCER6 transcripts are detected in thepetal and the anther fusion sites in tomato, similar tothe CER6 transcripts in Arabidopsis (Hooker et al.,2002). Additionally, SlCER6 is expressed in organs notsubject to the fusion process (e.g. sepals). This might beexplained by early and asynchronous formation of thesepal primordia in flower development. As a result,each sepal stays apart from the other organs (Sekharand Sawhney, 1984). SlCER6 transcripts were alsodetected in the walls of the multilocular ovary, whichis formed as a result of a routine carpel fusion process.Similarly, expression of a FDH ortholog was found inthe Antirrhinum majus ovary, which has the same on-togeny as tomato (Efremova et al., 2004). The data onthe SlCER6 expression pattern in vegetative organs oftomato are similar to the results obtained for Arabi-dopsis by qRT-PCR and the analysis of GUS expres-sion under the control of the CER6 promoter (Hookeret al., 2002; Joubès et al., 2008).

The hypothesis of the involvement of cuticular waxes inpostgenital organ fusion was proposed some time ago(Lolle and Pruitt, 1999; Tanaka and Machida, 2006).However, it was found that the abnormal adhesion re-sponse in Arabidopsis is not necessarily accompaniedby defective wax accumulation but rather is char-acterized by impairments in cutin formation and/orthe inability to form the continuous cuticle proper.

Irregular depositions in the cuticle were observed inwax2 (Chen et al., 2003), hothead/ace (Kurdyukov et al.,2006b), bodyguard (Kurdyukov et al., 2006a), lacerate(Voisin et al., 2009), cutinase-expressing plants (Sieberet al., 2000), and atwbc11 defective in the wax trans-porter (Luo et al., 2007). The wax-deficient fdh mutant

Table II. Total cuticular wax quantities and relative wax composition of MicroTom wild-type and slcer6anthers at anthesis (n = 5)

Values shown are means 6 SD.

Compound Class MicroTom Wild Type MicroTom slcer6 Ratio

mg g21 dry wt

n-AlkanesEven chained 142 6 11 71 6 5 0.50a

Odd chained 565 6 39 61 6 1 0.11a

Total 707 6 46 (33%) 132 6 5 (13%) 0.19a

iso-AlkanesEven chained 90 6 5 42 6 1 0.47a

Odd chained 628 6 30 209 6 6 0.33a

Total 718 6 34 (33%) 251 6 6 (25%) 0.35a

anteiso-AlkanesEven chained 108 6 5 29 6 2 0.27a

Odd chained 17 6 1 12 6 2 0.71a

Total 125 6 5 (6%) 41 6 3 (4%) 0.33a

Alkanoic acidsTotal 445 6 55 (21%) 445 6 23 (44%) 1.00

Triterpenoids and sterolsTotal 160 6 11 (7%) 148 6 15 (15%) 0.93a

Wax coverage 2,155 6 92 (100%) 1,017 6 49 (100%) 0.47a

aP , 0.05, Mann-Whitney U test.

Figure 8. qRT-PCR analysis of SlCER6 transcriptional level in differentorgans of the MicroTom wild type (WT) and its slcer6 mutant. Flowerorgans (sepal, petal, pistil, anther, and pollen), fruits (whole immaturegreen, peel of mature green, and mature red fruits), young leaf, stem,and root were sampled in independent biological replicates (n = 3 forthe wild type, n = 2 for slcer6). Petal and pollen are shown only for thewild type. Means 6 SD are shown.


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showed an unaltered cuticle structure in the fusion zonesbut, in contrast to slcer6, accumulated abnormally highlevels of cutin and wax (Voisin et al., 2009). It is a pecu-liarity of slcer6 that it has a reduced wax content butunchanged structure and quantity of cutin. Thus, neitherperturbation in the cuticle structure nor altered cutin,but a malfunction of wax biosynthesis, is primarily re-sponsible for the flower fusion phenomenon in slcer6.

Putative Role of the SlCER6 Enzyme in Cuticular WaxProduction in Tomato

Research on wax biosynthetic pathways focuses mainlyon the biosynthesis of linear aliphatics, since they arecharacteristic components of plant cuticular waxes(Samuels et al., 2008). However, large amounts of iso-and anteiso-alkanes were found in waxes of Solanaceae

members, specifically in leaves of tobacco (Nicotianatabacum) and eggplant (Solanum melongena; Grice et al.,2008; Hali�nski et al., 2009). The content of branched al-kanes is low in leaves andmature fruits of tomato, as in themajority of model plants used in cuticle research so far(Vogg et al., 2004; Leide et al., 2007). It is a new finding thatthese compounds are abundant in tomato flower waxes.

The slcer6mutation mostly affects n-alkanes in flowers,leaves, and fruits. However, the slcer6 mutation has ahigh impact on the content of branched alkanes in flowersand immature green fruits (Leide et al., 2007). Since waxesof leaves andmature fruits contain branched alkanes onlyin trace amounts (Vogg et al., 2004; Leide et al., 2007),the effect of the slcer6 mutation is masked in theseorgans. One can suggest that SlCER6 is involved inthe production of both linear and branched alkanes withchain lengths of C27 or greater. Importantly, most of these

Figure 9. Temporal and spatial expression pattern of SlCER6 detected by aGUS reporter gene assay. A, Inflorescence at anthesiswith reporter gene expression in sepals, petals, and the upper part of the anther (blue). B, Adaxial side of the anther at anthesis.The staining borders longitudinal stomium slits, sites of anther desiccation and pollen distribution (arrows). C, Androecium andgynoecium at the stage of young microspore. GUS is expressed in the region of the future apical pores, longitudinally betweenanthers (arrow), and in the ovary. D, Fragments of an anther cross section showing GUS staining in the epidermal and endo-thecial cells with characteristic ribs. En, Endothecium; Ep, epidermis; L, locule; T, tapetum. E, Cross section of the anther apexabove the pores showing GUS expression in the epidermis. F and G, Cross sections with staining of the petal (F) and the sepal(G) epidermis in flower buds with microspores. H, Cross section of an ovary with GUS staining in the ovary wall in a flower atanthesis. EW, External ovary wall; IW, internal ovary wall. I, Cross section of a flower bud with newly formed flower organs.GUS is expressed in the sepal epidermis and the anther. An, Anther; S, sepal. J, Flower bud with microsporocytes. Sepals arestained. K to M, Development of GUS expression in petals. The expression emerges initially in petal margins.





alkanes are still present in the mutant, although in re-duced quantities, indicating that redundant enzymes orother b-ketoacyl-CoA synthases, whose substrate speci-ficities overlap with SlCER6, might contribute to thebiosynthesis of these compounds.

The current vision of the biosynthesis of branchedaliphatics states that iso- and anteiso-long-chain fattyacids are synthesized by the successive elongation ofCoA thioesters of short branched-chain acids. Thesefatty acids are derived from branched-chain aminoacids (Val, Leu, and Ile). Thus, iso-, anteiso-, and n-alkaneshave different CoA precursors, while the subsequentelongation reactions are similar (Kolattukudy, 1970;Grice et al., 2008). Recent research on Arabidopsisshowed that overexpression of CER1, involved in thereduction reaction of VLCFAs to alkanes (Bernardet al., 2012), sharply increases the production of n- andiso-alkanes in leaves, indicating that CER1 is able toproduce both linear and branched hydrocarbons(Bourdenx et al., 2011). This presumed ability to workwith different substrates might be shared between theSlCER6 and CER1 enzymes.

Thus, the SlCER6 enzyme specificity seems to bebroader than was suggested before. The data testify thatSlCER6 elongates fatty acids leading to n- and iso-alkaneswith total chain lengths of C27 or greater and to anteiso-alkanes with total chain lengths of C28 or greater. Prob-ably, the resulting product of the condensation reactiondepends not only on the specificity of the enzyme butalso on the accessibility of linear or branched-chain acids,which in turn depends on the CoA precursors.

The Impact of the SlCER6 Gene on Sexual Reproductionin Tomato

Hand-pollination experiments revealed that reducedseed production in the slcer6mutant is caused exclusivelyby male sterility. One could assume that the SlCER6 geneproduct is involved in the pollen exine formation becausemost male-sterile mutants are impaired in sporopolleninbiosynthesis or transport (Piffanelli et al., 1998; Ariizumiand Toriyama, 2011). Enzymes participating in both waxbiosynthesis and pollen exine formation are known forArabidopsis and rice (Oryza sativa), such as FACELESSPOLLEN1 (Ariizumi et al., 2003), WAX DEFICIENTANTHER1 (Jung et al., 2006), and DEFECTIVE POLLENWALL (Shi et al., 2011). However, this suggestion is notconfirmed, since the slcer6 exine does not differ from thewild type. The absence of SlCER6 transcripts in the de-veloping pollen indicates that SlCER6-derived aliphaticsare not produced by the male gametophyte itself, as inArabidopsis (Hooker et al., 2002).

Successful production of viable pollen depends onthe programmed cell death of the tapetum. Both pre-mature and delayed death of tapetal cells result inmalfunction of the nutrient supply followed by deathof the microgametophyte (Parish and Li, 2010; Wilsonet al., 2011). We found that impairment in the tapetalcell degeneration process contributes to male sterility

in the slcer6 tomato. In contrast to slcer6, the atcer6mutant has viable pollen and its tapetum does notdisplay any abnormalities (Preuss et al., 1993). How-ever, the content of long aliphatics is reduced in theatcer6 pollen coat, causing conditional sterility (Preusset al., 1993; Fiebig et al., 2000). The finding of CER6transcripts in the tapetum by in situ hybridization ledto the conclusion that CER6 is necessary for the bio-synthesis of pollen coat lipids by tapetal cells (Hookeret al., 2002). Surprisingly, SlCER6 expression is notdetected in the tapetum of the tomato GUS reporterline. Probably, this reflects different contributions ofthe condensing enzymes to the formation of the lipidicpollen coat in these two species. Indeed, the pollen coatis feebly developed in tomato pollen, while the Arabi-dopsis pollen grain has a thick pollen coat with lipidicdroplets (Preuss et al., 1993). Zheng et al. (2005) foundthat the cer10 mutant, deficient in an enoyl-CoA reduc-tase required for VLCFA biosynthesis, has unviable pol-len similar to slcer6. One can speculate that aliphaticsproduced by the fatty acid elongase complex are involvedin the regulation of tapetum disintegration. A few yearsago, evidence for the involvement of VLCFAs in the reg-ulation of cell death appeared. It was shown that epider-mal misexpression of the FATTY ACID ELONGATION1gene in Arabidopsis leads to the death of trichomes viaa lipidic pathway. An assumption was made that thedeath program is triggered by the presence of a criticalthreshold of certain fatty acids or other lipids (Reina-Pinto et al., 2009).

Another parameter provoking untimely tapetumdegeneration in the slcer6 mutant could be water stressin flowers, similar to that in the fruits (Vogg et al., 2004;Leide et al., 2007). It was shown that anther water defi-ciency causes abnormal tapetal cell development in wheat(Triticum aestivum; Lalonde et al., 1997; Koonjul et al., 2005).Loss of turgor by anther epidermal cells, reduced watercontent, higher pigment leaching, and water loss might besigns of water stress in the slcer6 flower. It is hypothesizedthat this is mainly due to changes in cuticular wax chem-istry and amount. However, some contribution of stomataltranspiration cannot be excluded, since tomato sepals havestomata (Sekhar and Sawhney, 1984).

Morphological studies together with cross-pollinationexperiments showed that impediment in pollen releasefrom the anther is a further reason for male sterility in theslcer6 mutant. The absence of longitudinal stomium slitsin the mutant anther could also influence the pollendesiccation process, reducing pollen lifetime and viability.Tomato plants with a mutated POSITIONAL STERILE(PS) gene, responsible for blocking of the decarbon-ylation pathway leading to alkane, aldehyde, ketones,and secondary alcohol biosynthesis, have sterility andunopened flowers similar to slcer6 (Leide et al., 2011).However, the ps mutant produces ample seeds underartificial self-pollination. Natural pollination does nottake place in ps because the stomium cells stay intact,thereby preventing the anther split (Larson and Paur,1948). In contrast, breakage of the stomium cells in theslcer6 mutant occurs without abnormalities.


Smirnova et al.



Expression of the SlCER6 gene in the endotheciumposes a new question regarding the participation of al-iphatic compounds in anther dehiscence and opening.Endothecium development is coordinated with pollenmaturation and degeneration of the tapetum. At thestage of a young pollen grain, endothecium cells un-dergo secondary wall thickening by the incorporationof lignocellulose (Dawson et al., 1999). It imposes me-chanical stress on the anther, which leads to desiccationand opening (Bonner and Dickinson, 1989; Wilson et al.,2011). Interestingly, SlCER6 is expressed in endothecialcells already at the early microspore stage, before thebeginning of the secondary thickening. This may hint atthe presence of an aliphatic domain in these cells.However, SlCER6 loss of function does not prevent en-dothecium thickening. Similarly, specific expression ofthe SHINE2 transcription factor regulating wax biosyn-thesis was detected in the stomium region of the Arabi-dopsis anther (Aharoni et al., 2004). One can speculatethat the aliphatics produced by SlCER6 are necessaryto prevent water loss by endothecial cells before sec-ondary wall deposition.This work shows that SlCER6 is involved in the bio-

synthesis of cuticular waxes not only in vegetative or-gans, fruits, and leaves but also in flowers. Biosynthesis ofthe pollen coat lipids in Arabidopsis is not the onlyfunction of CER6 during pollen development: SlCER6appeared to be essential for timely tapetal cell degenera-tion and successful microgametogenesis in tomato. Here,we report that wax deficiency, but not disturbance incutin accumulation or cuticle formation, is the primaryreason for the adhesive response between epidermal cellsin the tomato flower. Based on chemical analysis, thesubstrate specificity of the SlCER6-condensing enzymemay be reevaluated. It might be involved in the elonga-tion of linear and branched VLCFAs with chain lengthsof C26 or greater, leading to the production of linear andbranched alkanes, respectively. These findings broadenour current knowledge of wax biosynthetic pathwaysand show new aspects of the involvement of wax-producing enzymes in sexual plant reproduction.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Plants of tomato (Solanum lycopersicum ‘MicroTom’) wild type and itsmutant slcer6 deficient in a b-ketoacyl-CoA synthase were investigated. Iso-lation of the mutant referred to as lecer6 and generation of the MicroTomplants with upstream CER6 fragments fused to a GUS reporter were describedpreviously (Vogg et al., 2004; Mintz-Oron et al., 2008). The plants were grownin a controlled-climate chamber at 75% to 80% relative humidity with a 14-hphotoperiod at 450 mmol m22 s21 and a day/night temperature regime of22°C/18°C. Plants were watered daily and fertilized with Hakaphos Blaunutrient solution once per week.

Scanning and Transmission Electron Microscopy

For scanning electron microscopy, plant material was fixed in 3% (v/v)glutaraldehyde, 0.1 M sodium phosphate buffer, pH 6.8, at 4°C for 4 h,washed in the same buffer, and postfixed in 2% (w/v) osmium tetroxide, 50mM cacodylate buffer, pH 6.8, at 4°C overnight. Samples were then dehydrated

in a graded acetone series and critical point dried with liquid CO2. Before ex-amination with the scanning electron microscope (Zeiss DSM 962), samples weresputtered with gold palladium at 25 mA for 300 s (Bal-Tec SCD 005).

For transmission electron microscopy, plant material was fixed in Kar-novsky solution at 4°C overnight and postfixed for 1.5 h as for scanningelectron microscopy. The samples were then dehydrated in a graded ethanolseries, treated with propylene oxide, and embedded in Spurr’s resin. Semi-sections were done for light microscopy. Ultrathin sections were cut, mountedon copper grids, and contrasted with 2% (w/v) uranyl acetate and lead citrate.Specimens were examined with a Leo 912 AB microscope (Carl Zeiss).

Fluorescence Microscopy

For the detection of pollen development stage and the pollen viability test,1 mg mL21 DAPI (Sigma) and 2 mg mL21

fluorescein diacetate (Sigma) wereused, respectively. The samples were analyzed with Leica DMR (LeicaMicrosystems) and Axio Observer Z1 microscopes supplied with AxioCamMrm or Mrc digital cameras and AxioVision 4.8 software (Zeiss).

GUS Histochemical Assay and Cryosectioning

For GUS staining, samples were placed in 90% (v/v) acetone and vacuuminfiltrated on ice, transferred to 50 mM sodium phosphate buffer, pH 7.2, con-taining 10 mM EDTA, 0.1% (w/v) Triton X-100, and 0.5 mM K3Fe(CN)6, andvacuum infiltrated. Samples were transferred to the buffer containing 0.5 mgmL21 5-bromo-4-chloro-3-indolyl b-D-glucuronide sodium salt (Sigma-Aldrich).After infiltration, the samples were incubated at 37°C overnight. Chlorophyllwas washed out in a graded series of ethanol. Afterward, samples were fixed informalin-acetic acid-alcohol solution at 4°C for 24 h. Prior to examination, sampleswere washed three times in sodium phosphate buffer. Flower buds were dis-sected with a Leica MZ16 stereomicroscope and a Leica DC500 digital camera.For cryosectioning, material was frozen in Tissue-Tek O.C.T. (Sakura FintekEurope) and mounted on stubs. Ten- to 30-mm sections were cut in a LeicaCM1900 cryostat (Leica Microsystems) at –20°C. Sections were transferred onglass slides, washed with sodium phosphate buffer, and analyzed.

Analysis of Pollen

To investigate pollen development, flower buds with different lengths wereharvested. At least 10 buds were analyzed for each developmental stage. Pollenwas collected from flowers at anthesis using a vacuum cleaner (Johnson-Brousseau and McCormick, 2004). For acetolysis, pollen grains were treatedwith a mixture of sulfuric acid and acetic anhydride at 100°C (Hesse et al.,2009). In vivo and in vitro pollen germination assays were performed (Moriet al., 2006; Smirnova et al., 2009).

Measurement of Water Content, Water Loss, and Leachingof Pigments

Immediately after harvesting, the flower bud length and fresh weight weredetermined. Afterward, samples were dried to a constant weight at 60°C.

Water loss was recorded for flower buds approximately 6.5 mm in length.For one sample, three buds from different plants were pooled. Pedicles weresealed with high-melting paraffin. The amount of water loss versus time (sevendata points per sample) was measured gravimetrically. Subsequently, the dryweight was determined. Between measurements, samples were stored at 25°Con silica gel.

The pigment-leaching protocol was adapted from Lolle et al. (1998). Threeflower buds per sample were weighed and immersed in 80% (v/v) ethanol.Aliquots of the supernatant were removed at different time points. To deter-mine the total pigment amount, plant material was frozen in liquid nitrogen,homogenized, dissolved in 100% ethanol, and pelleted. The content of chlo-rophylls and carotenoids in the supernatant was determined by measuringabsorption at 664, 649, and 470 nm (Lichtenthaler and Buschmann, 2001) on aUnicam UV/Vis Spectrophotometer UV4 (Thermo Scientific).

Analysis of Flower Cuticular Waxes and Cutin Monomers

About 10 to 30 mg of freeze-dried material was submersed in 10 mL ofchloroform containing 3 mg of heptatriacontane (Fluka) as an internal standard





for 1min andfiltered through apaperfilter. The solventwas evaporatedunder aflowof nitrogen. Hydroxyl-containing compounds were transformed into the corre-sponding trimethylsilyl derivatives using N,O-bis-trimethylsilyl-trifluoroacetamide(Macherey-Nagel) in pyridine (Merck). The wax composition was analyzed as de-scribed previously (Leide et al., 2007).

For cutin analysis, freeze-dried material or enzymatically isolated cuticleswere used. About 25 mg of freeze-dried anthers and corollas was washed withchloroform at room temperature and at 50°C for 30 min. Afterward, sampleswere incubated in chloroform, which was changed daily, for 1 week. Cuticlesfrom fresh anthers and corollas were isolated according to Leide et al. (2007).Dried samples were transesterified with 1 mL of 1.25 M methanol-HCl (Fluka)at 80°C overnight. Subsequent extraction of cutin components and derivati-zation were performed as described by Leide et al. (2007).

Gene Expression

Three biological samples for MicroTom wild type and two for the slcer6mutant were compared. Plant material was harvested from 2- to 4-month-oldplants. RNA isolation, complementary DNA synthesis, qRT-PCR, and dataanalysis were performed as described in detail by Leide et al. (2012). TIP41was chosen as a reference gene among RPS26C, EFa1, RPL2, CAC, EXP, TUB,and UBQ (Supplemental Table S1) according to the expression stability ana-lyzed by NormFinder version 20.

Statistical Analysis

Significant differences (P , 0.05) between multiple data sets were tested byone-way ANOVA followed by Tukey’s honestly significant difference (HSD)test for unequal n. Pairwise comparisons were tested by Mann-Whitney U test.Analysis was performed with STATISTICA 20 (StatSoft).

Sequence data from this article can be found in the GenBank databaseunder the following accession number: CER6, GQ214500.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Scanning and transmission electron microscopyof the pollen grains and microspores in the MicroTom wild type and theslcer6 mutant.

Supplemental Figure S2. Total content of chlorophylls and carotenoids inthe MicroTom wild type and the slcer6 flower buds at the stage of micro-gametogenesis (6.5 mm in length).

Supplemental Figure S3. Cuticular wax composition of the corollas andanthers harvested before flower opening in the MicroTom wild type andslcer6 and the wild-type corolla at anthesis.

Supplemental Figure S4. Cutin composition of the cuticles isolated fromanther and corolla of the MicroTom wild type and the slcer6 mutant.

Supplemental Figure S5. Transmission electron microscopy of the anthercuticle at different stages of flower development in the MicroTom wildtype and the slcer6 mutant.

Supplemental Table S1. Primer pairs used for gene expression analysis byqRT-PCR.

ACKNOWLEDGMENTS

We thank Asaph Aharoni for providing us with the GUS reporter plantsand Avraham A. Levy for supplying seeds for the slcer6 mutant (WeizmannInstitute of Science). We are indebted to Georg Krohne for the possibility toperform electron microscopy (Theodor-Boveri-Institut für Biowissenschaften).We thank Daniela Bunsen, Claudia Gehrig, and Olga Frank for technical assis-tance and Jutta Winkler-Steinbeck for plant care. We are grateful to Gerd Voggand Anton Hansjakob for criticism of the manuscript and to Ulrich Hildebrandt forcomments on the manuscript (Julius-von-Sachs-Institut für Biowissenschaften).

Received September 3, 2012; accepted November 8, 2012; published Novem-ber 9, 2012.

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Deﬁciency in a Very-Long-Chain Fatty Acid b-Ketoacyl ...Deﬁciency in a Very-Long-Chain Fatty...

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