Cuticle Biosynthesis in Tomato Leaves Is DevelopmentallyRegulated by Abscisic Acid1[OPEN]

Laetitia B. B. Martin ,a,2 Paco Romero,a Eric A. Fich,a David S. Domozych,b and Jocelyn K. C. Rosea,3

aPlant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York 14853bDepartment of Biology, Skidmore College, Saratoga Springs, New York 12866

ORCID IDs: 0000-0002-6921-7246 (L.B.B.M.); 0000-0002-7811-0897 (P.R.); 0000-0002-8948-7112 (E.A.F.); 0000-0001-8800-0061 (D.S.D.);0000-0003-1881-9631 (J.K.C.R.).

The expansion of aerial organs in plants is coupled with the synthesis and deposition of a hydrophobic cuticle, composed ofcutin and waxes, which is critically important in limiting water loss. While the abiotic stress-related hormone abscisic acid (ABA)is known to up-regulate wax accumulation in response to drought, the hormonal regulation of cuticle biosynthesis during organontogeny is poorly understood. To address the hypothesis that ABA also mediates cuticle formation during organ development,we assessed the effect of ABA deficiency on cuticle formation in three ABA biosynthesis-impaired tomato mutants. The mutantleaf cuticles were thinner, had structural abnormalities, and had a substantial reduction in levels of cutin. ABA deficiency alsoconsistently resulted in differences in the composition of leaf cutin and cuticular waxes. Exogenous application of ABA partiallyrescued these phenotypes, confirming that they were a consequence of reduced ABA levels. The ABA mutants also showedreduced expression of genes involved in cutin or wax formation. This difference was again countered by exogenous ABA, furtherindicating regulation of cuticle biosynthesis by ABA. The fruit cuticles were affected differently by the ABA-associatedmutations, but in general were thicker. However, no structural abnormalities were observed, and the cutin and waxcompositions were less affected than in leaf cuticles, suggesting that ABA action influences cuticle formation in an organ-dependent manner. These results suggest dual roles for ABA in regulating leaf cuticle formation: one that is fundamentallyassociated with leaf expansion, independent of abiotic stress, and another that is drought induced.

The plant cuticle, a hydrophobic extracellular layerthat coats the epidermis of aerial organs, is composed ofa cutin matrix of ester-linked v-hydroxylated fattyacids that is covered by epicuticular waxes and infil-trated with intracuticular waxes. Polymeric cutin hasalso been reported to contain relatively low levels ofphenolic compounds, such as caffeic, ferulic, and cou-maric acid esters (Baker et al., 1982; Rautengarten et al.,

2012). The cuticle is a key barrier to desiccation andpathogen entry and so is required from the onset oforgan formation throughout ontogeny (Yeats and Rose,2013). In addition to being an intrinsic part of organdevelopmental programs, cuticle formation is dynamicas its composition and coverage change in response toenvironmental conditions, such as light/dark, ultra-violet radiation, and drought (Hooker et al., 2002;Cameron et al., 2006; Shepherd and Wynne Griffiths,2006; Kosma et al., 2009; Go et al., 2014; Martin andRose, 2014). For example, water deficit in Arabidopsis(Arabidopsis thaliana) triggers an increase in the accu-mulation of both cutin monomers and waxes, resultingin a thicker, less permeable cuticle (Kosma et al., 2009).

A key factor in the mediation of responses to waterdeficit is the phytohormone abscisic acid (ABA; Liuet al., 2005), and the consequences of ABA treatment oncuticle composition and associated gene expressionhave been investigated (Kosma et al., 2009). While thelevels of waxes, and in particular alkanes, were repor-ted to increase following ABA application, no suchchange in the abundance of cutin monomers was ob-served, suggesting that other factors trigger changes inthe cuticle following water stress (Kosma et al., 2009).Furthermore, the expression levels of numerous waxbiosynthesis-related genes are known to respond toABA treatment (Kosma et al., 2009; Seo et al., 2009,2011; Wang et al., 2012). For example, the expression ofECERIFERUM1 (CER1), which encodes an enzymethat generates alkanes from very-long-chain acyl-CoA

1 This work was supported by the Agriculture and Food ResearchInitiative Competitive Grants Program (2015-06803) of the USDANational Institute of Food and Agriculture and by a grant fromthe US National Science Foundation (Plant Genome Research Pro-gram; IOS-1339287). P. Romero acknowledges the funding from the3F:FutureFreshFruit Project in the framework of the MarieSkłodowska-Curie Actions and the European Horizon 2020 pro-gram (H2020-MSCA-IF-656127).

2 Current address: Université de Strasbourg, Centre National de laRecherche Scientifique, Institut de Biologie Moléculaire des PlantesUnité Propre de Recherche 2357, F-67000 Strasbourg, France.

3 Address correspondence to jr286@cornell.edu.The 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:Jocelyn K.C. Rose (jr286@cornell.edu).

L.B.B.M. designed and performed the research, analyzed data, andwrote the article; P.R. performed the research and analyzed data; E.A.F.analyzed data; D.S.D. performed the research; and J.K.C.R. designedthe research and wrote the article.

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(Bernard et al., 2012), is induced by ABA treatments(Kosma et al., 2009). In addition, the presence of cis-ABA-responsive elements in the promoters of somewax-related genes, such as CER6 (Hooker et al., 2002),suggests that ABA directly regulates wax synthesis.Moreover, some Arabidopsis transcriptions factors thatare associated with cuticle biosynthesis, such asMYB96, are known to be regulated by ABA and tobe part of the ABA-mediated signaling pathway(Cominelli et al., 2008; Seo et al., 2009; Lee et al., 2015).Lastly, Cui et al. (2016) showed that the expression ofsome cuticle related genes was affected in ArabidopsisABA mutants, further suggesting a role for ABA incuticle development.Several recent reports indicate that the relationship

between ABA and cutin is complex, and ABA biosyn-thesis and signaling are impaired in mutants withdisrupted cutin biosynthesis, suggesting that cutincomponents may be involved in mediating osmoticstress signaling (Wang et al., 2011). In addition, thetranscription factor NF-X LIKE2, which binds to thepromoters of a cutin-related gene and a transcriptionfactor regulating cutin formation (Lisso et al., 2012),suppresses ABA accumulation and ABA responses,possibly to avoid osmotic stress response under normalconditions (Lisso et al., 2011). Thus, NF-X LIKE2 sup-presses both ABA signaling and cutin biosynthesis.However, ABA has not been shown to have a directeffect on cutin formation.Collectively, these studies demonstrate that ABA

plays a role in the regulation of cuticular wax forma-tion, but it is not known whether this is only in thecontext of responses to environmental stress or whetherABA influences the developmental regulation of cuticlebiosynthesis. Indeed, in addition to its role in plant re-sponses to water stress, ABA is also known to regulatemany aspects of development, including the mainte-nance of shoot growth in well-watered plants (Sharpet al., 2000), the promotion of seed dormancy andthe synthesis of seed storage proteins and lipids(Finkelstein et al., 2002). Consequently, a goal of thiscurrent study was to test the hypothesis that ABA isrequired for cuticle formation independent of waterstress.To this end, we studied the cuticle structure, com-

position, and properties of three tomato (Solanum lyco-persicum) ABA-deficient nonallelic mutants: notabilis,flacca, and sitiens. The notabilis (not) mutant has a mu-tation in a 9-cis-epoxycarotenoid dioxygenase gene,which is believed to encode a key regulatory enzyme ofthe ABA biosynthesis pathway (Burbidge et al., 1999;Thompson et al., 2000). The mutation in sitiens (sit) af-fects an aldehyde oxidase that catalyzes the last step ofthe ABA synthesis pathway (Harrison et al., 2011),while flacca (flc) has a mutation in a molybdenum co-factor sulfurase that is required for the activation bysulfuration of the molybdenum cofactor of aldehydeoxidase (Sagi et al., 2002). The severity of the pheno-types of these mutants correlates with the ABA con-centrations in shoots of the 5-week-old plants: not has

the mildest phenotype and the highest level of ABA,while sit has the most severe phenotype and the lowestABA levels (Tal and Nevo, 1973; Taylor and Tarr, 1984).

In this study, the role of ABA in cuticle formationwasinvestigated by assessing the structure and compositionof leaf and fruit cuticles from the three ABA-deficientmutants. Additionally, the expression of genes in-volved in cuticle biosynthesis, transport, and regulationwas evaluated to determine the consequence of ABAdeficiency on the cuticle biosynthesis pathway. Finally,to determine whether the functions of the ABA-deficient fruit cuticles were impaired, we measuredwater loss, from which we inferred changes in cuticlepermeability. Based on the results, we propose a dual,but organ-specific, role for ABA in the control of cuticleformation.

RESULTS

ABA Deficiency Leads to a Reduction in Plant andOrgan Size

For the purposes of this study, we compared not andflc in the Ailsa Craig (AC) genetic background [not(AC)and flc(AC), respectively] as well as sit and flc inthe Rheinlands Ruhm (RR) background [sit(RR) andflc(RR), respectively]. We also included the RR and ACwild-type genotypes in the comparison. Importantly,this allowed an assessment of conserved phenotypes indifferent genetic backgrounds. Most of the publishedanalyses of ABA levels in the ABA-deficient not, sit, andflc tomato mutants have focused on their leaves (Tal,1966; Tal et al., 1979; Nagel et al., 1994). We observedthat this reduction in ABA levels was consistentthroughout leaf development and in ripening fruits(Fig. 1), indicating a constitutive perturbation of ABAbiosynthesis. Proportionally, the reduction in ABAlevels was greater in Pink (Pk) stage fruit, ranging froma 89% decrease in flc(AC) to a 99% decrease in sit(RR),than in leaves. However, the wild-type levels of ABAwere substantially lower in Pk fruit than they were inleaves, regardless of the developmental stage. Fur-thermore, leaf ABA levels were lower in sit(RR) than inflc(RR), and lower in flc(AC) than in not(AC), as waspreviously reported (Tal and Nevo, 1973; Taylor andTarr, 1984). Plant growth was generally reduced in theABA-deficient plants (Supplemental Fig. S1), whichhad smaller curled leaves (Fig. 2A) and smaller fruits(Fig. 2B; Supplemental Fig. S2) than those of the re-spective wild types. A reduction in organ size, includ-ing roots, of these mutants has previously beenreported (Ran�ci�c et al., 2010; Sharp et al., 2000; Nitschet al., 2012).

Effect of ABA Deficiency on Leaf and Fruit CuticleUltrastructure and Distribution

The ultrastructure of the leaf and fruit cuticles offlc(AC) and sit(RR) and their respective wild-type

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genotypes were assessed using transmission electronmicroscopy (TEM). The cuticles of small expandingmutant fruits of similar diameters (;13.5 mm) werethicker than those of the wild-type fruit (SupplementalFig. S3); specifically, an increase of 364% and 220% forflc(AC) and sit(RR), respectively (Supplemental TableS1). In contrast, the cuticles of the adaxial and abaxialfully expanded leaf surfaces were thinner in themutants than in the wild-type genotypes (Fig. 3;Supplemental Fig. S4). We observed a reduction inthe leaf adaxial cuticular thickness of 31% and 48%(significant for P , 0.05) for flc(AC) and sit(RR), re-spectively, and of 21% and 42% for the abaxial surfaceof flc(AC) and sit(RR), respectively (SupplementalTable S1).

The ultrastructure of the wild-type and the mutantfruit cuticles were indistinguishable (Supplemental Fig.S3), but various abnormalities were observed in themutant leaf cuticles. Most strikingly, while the cuticlesof the wild-type leaves had a uniformly reticulate ap-pearance between the polysaccharide cell wall andthe cuticle proper layer, the cuticles of the mutantsshowed an accumulation of electron-translucentglobules (Fig. 3, C, G, and H; Supplemental Fig.S4G). In addition, the structure of the leaf cuticles wasoccasionally disrupted (Fig. 3H). Irregularities in theappearance of the cuticle and the polysaccharide cellwall have previously been reported for sit in theMoneymaker background (Curvers et al., 2010), but inthat analysis, themutant leaf cuticle was reported to be

thicker than the wild-type cuticle, which is contrary tothe results of this study. This apparent contradictionmay be explained by insufficient contrast in the TEMimage shown in Figure 2 of Curvers et al. (2010) be-tween the cuticle and the polysaccharide cell wall ofthe wild type, making it difficult to distinguish thecuticle. Indeed, it appears that the thin region thatthe authors defined as the cuticle may have been in-advertently mislabeled, and a re-examination of theimages published in the article suggests to us thatthe wild-type cuticle was not thinner than that of themutants.

To further assess possible cuticular abnormalities inthe ABA mutants, we examined the cuticles of fullyexpanded fruit at the Mature Green (MG) stage usinglight microscopy (Supplemental Fig. S5). The mutantcuticles did not show a major difference in thickness,based on the region overlying the center point of theouter epidermal cells, compared with their respectivewild types: flc(RR) and sit(RR) cuticles were 41% (P ,0.0001) and 40% (P , 0.0001) thicker, respectively,while those of not(AC) and flc(AC) were not signifi-cantly thicker than the wild type (Supplemental TableS2). However, the cuticles of the mutants showedmore substantial penetration through the anticlinaland periclinal walls of additional subepidermal layers(Supplemental Fig. S5). Image analysis indicated anincreased penetration of 35% (P , 0.0001), 40% (P ,0.0001), and 22% (P , 0.001) for not(AC), flc(RR), andsit(RR), respectively, but the average increase (13%)

Figure 1. ABA levels in fruit and four leaf developmental stages of ABA-deficient tomato lines. The label “emerging” correspondsto developing leaves of fully mature plants (leaf size ,2 cm). The labels “small,” “medium,” and “large” represent three suc-cessive developmental stages of leaf development of 5-week-old plants, as described in the “Materials andMethods” section. Thelabel “Fruit” corresponds to Pk stage fruit pericarp. Differences between themutants and their correspondingwild-type genotypeswere assessed using Dunnett test if the equal variances hypothesis of the Levene test was not rejected at a = 0.05, and by a Steeltest otherwise, with *P, 0.05, **P, 0.01, and ***P, 0.001, respectively. The error bars represent SEs of the mean for n = 4. ng/gFW, ng of ABA per g of fresh weight; AC, Ailsa Craig; not(AC), notabilis in AC background (AC); flc(AC), flacca in (AC); RR,Rheinlands Ruhm; flc(RR), flacca in (RR); sit(RR), sitiens in (RR).

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measured in flc(AC) was not statistically significantfrom wild type (Supplemental Table S2).

Effect of ABA Deficiency on Cutin and Wax ChemicalComposition of Fruit and Leaf Cuticles

To confirm the observation, made by light micros-copy, that the distribution of cuticles of the mutantMG fruit was more extensive than in the respectivewild-type fruit, we performed a compositional analysis

of isolated cuticles. The amount of total cutin per squarecm of the mutant MG fruit was significantly higher fornot(AC) and flc(RR) with an increase of 38% and 21%,respectively, and slightly higher for flc(AC), with an in-crease of 17%, but not for sit(RR) (Supplemental Fig. S6).Similarly, the increases of levels of coumaric acids (125%)and of 10(9),16-dihydroxyhexadecanoic acid (33%) werestatistically significant for not(AC), and no differenceswere observed in the levels of hexadecanoic acid (C16)and 1,16-hexadecanedioic acid (C16 di-ac). The onlycutin-associated compound that was consistently dif-ferent in the fourmutant genotypes comparedwith theirrespective wild types was v-hydroxyhexadecanoic acid

Figure 2. ABA-deficient mutant leaf and fruit phenotypes. A, Photo-graphs of the youngest mature (large) leaf of 5-week-old seedlings arerepresented. Scale bar = 5 cm. B, Pictures of representative Red Ripestage fruits of the ABA-deficient genotypes and their respective wildtypes. Scale bar, 1 cm. AC, Ailsa Craig; not(AC), notabilis in AC back-ground (AC); flc(AC), flacca in (AC); RR, Rheinlands Ruhm; flc(RR),flacca in (RR); sit(RR), sitiens in (RR).

Figure 3. TEM micrographs of the cuticle of the adaxial epidermis offully expanded leaflets. A, C, E, andG,Outer epidermal cell junction. B,D, F, and H Outer periclinal wall of an epidermal cell. Scale bars =500 nm. Cl, Chloroplast; Cp, cuticle proper; cut, cuticular membrane;cyt, cytoplasm; dis, disrupted cuticle; gl, cuticular globules; pcw,polysaccharide cell wall; V, vacuole. AC, Ailsa Craig; flc(AC), flacca in(AC); RR, Rheinlands Ruhm; sit(RR), sitiens in (RR).

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(v-OHC16): not(AC), flc(AC), and flc(RR) had increases of16%, 160%, and 99%, respectively, but sit(RR) showed adecrease of 18% (Supplemental Fig. S6). These bio-chemical data were consistent with the light microscopyanalysis, indicating that the mutant fruit cuticles weresomewhat thicker than those of the respective wildtypes.

The total amounts of fruit cuticular waxes per squarecm did not significantly differ between the mutants andtheir respective wild-type genotypes (SupplementalFig. S7). However, some compositional differenceswere observed, as the mutant cuticles tended to havelower amounts of alkanes, with a significant decrease inthe levels of the C30 and C29 alkanes in either three[flc(AC), flc(RR), and sit(RR)]or one [flc(AC)] of themutant genotypes, respectively. In contrast, levels of

amyrins were generally higher in the mutants, with asignificant increase, compared to thewild-type fruits, inthe amounts of a-amyrin and d-amyrin in two [not(AC)and flc(RR)] and one [flc(RR)] of the mutants, respec-tively. These results suggest that wax components ofMG fruit cuticles are only moderately affected by ABAdeficiency.

We then measured cutin levels in leaves at threedistinct developmental stages (the youngest leaf of a5-week-old plant, the youngest fully expanded leaf, andthe leaf located in between them, referred as small,large, and medium in Fig. 4, respectively), whichrevealed four distinct patterns of compound accumu-lation (Fig. 4). The levels of coumaric acids, caffeic acid,v-OH C16, 10(9),16-diOH C16, 2-OH C24 and C32 wereconsistently lower in the mutant genotypes at all

Figure 4. Cutin analysis of ABA-deficientlines for three leaf developmental stages.Amount of cutin monomers per cm2 ofsmall (A), medium (B), and large (C) leavesof 5-week-old plants (n = 6). Significantdifferences between the mutants and theirwild types were given by a Dunnett testif the equal variances hypothesis of theLevene test was not rejected at a = 0.05and by a Steel test otherwise (*P , 0.05,**P , 0.01, and ***P , 0.001). The errorbars represent SE of the mean. “Total iden-tified” refers to the sum of the compoundsshown in the graphs, and “total” refers tothe sum of the peaks detected by GC. AC,Ailsa Craig; not(AC), notabilis in ACbackground (AC); flc(AC), flacca in (AC);RR, Rheinlands Ruhm; flc(RR), flacca in(RR); sit(RR), sitiens in (RR) and ac, acid.

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developmental stages than those in the wild types.These decreases resulted in a decrease in total cutinlevels for the three stages of development in the mu-tants. In contrast, the levels of C16, dimethylester C16,C18 diene, and C18 triene were consistently higher. Thelevels of 2-OH C25 and 2-OH C26 were unaffected inthe mutants and those of ferulic acid, C18, C16, andC30 showed no consistent difference through leafdevelopment.Unlike the effect on cutin, the effect of ABA defi-

ciency on wax levels became more marked during leafdevelopment. Amyrins and alkanes of chain lengthfrom C31 to C33 were the only constituents showing aconsistent change between the small leaf mutants andtheir respective wild types (Supplemental Fig. S8).Medium and large mutant leaves also generally had

lower levels of these compounds (Supplemental Fig. S9;Fig. 5). Additionally, in medium and large leaves, al-kanes from C25 to C30, isoalkanes from C29 to C33,OH-C22 and the fatty acid C22 tended to be moreabundant in the mutants than in their wild types(Supplemental Fig. S9; Fig. 5). In contrast, the increaseof the levels of aiC28, aiC30, and aiC31 anteisoalkaneswas the most pronounced for the mutant medium leafand the decrease of the longer anteisoalkanes levels(aiC32, aiC33, and aiC34) was the most consistent in largeleaves. These results indicate that the composition ofthe leaf cuticular waxes was altered by ABA deficiency.In summary, ABA deficiency had a moderate effect onthe cuticles of MG fruits and resulted in a reduction inthe levels of cutin and changes in wax and cutin com-position in leaf cuticles. Notably, the observed cuticle

Figure 5. Analysis of large leaf cuticu-lar waxes from ABA deficient mutants.Amount of waxes per cm2 of the youngestfully expanded leaf of 5-week-old plants(n = 5). Significant differences between themutants and their wild types were given bya Dunnett test if the equal variances hy-pothesis of the Levene test was not rejectedat a = 0.05, and by a Steel test otherwise(*P, 0.05, **P, 0.01, and ***P, 0.001).The error bars represent SE of the mean.Outlier values were removed. “Totalidentified” refers to the sum of the com-pounds shown in the graph, and “total”refers to the sum of the peaks detected byGC. AC, Ailsa Craig; not(AC), notabilis inAC background (AC); flc(AC), flacca in(AC); RR, Rheinlands Ruhm; flc(RR), flaccain (RR); sit(RR), sitiens in (RR). ‘i’ refers tothe iso form of the alkane and ‘ai’ to theanteiso form.

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compositional changes showed opposite patterns infruits and leaves: The C29 and C30 alkanes were lessabundant in the mutant fruit cuticles but more abun-dant in mutant leaf cuticles, while a- and d-amyrins,coumaric acids, 10(9),16-diOH C16, and v-OH C16 weremore abundant in the mutant fruit cuticles but lessabundant in leaf cuticles.

Effect of ABA Application on ABA-Deficient Leaves

Exogenous application of ABA to the ABA-deficientmutants resulted in a slight, but consistent increase inleaf total cutin levels, with an increase of 20% and 11%for flc(AC) and sit(RR), respectively (Fig. 6). More spe-cifically, ABA application resulted in an increase in theabundance of coumaric acids (93% and 50%), caffeicacids (83% and 18%), and 10(9),16-diOH C16 (58% and30%), for flc(AC) and sit(RR), respectively, suggesting apartial rescue of the cuticle deficiency (Fig. 6). Appli-cation of ABA treatment to wild-type leaves resulted ina minor increase in total cutin for AC (16%), but not forRR (25%), but had little effect on the wild-type cutic-ular waxes (Fig. 7). This result was unexpected, as itwas reported that applying ABA to Arabidopsis leavesresulted in an increase in the levels of alkanes (Kosmaet al., 2009). We considered the possibility that theamount of ABA penetrating the tomato leaves in ourstudy was insufficient to induce the expected increasein alkane levels. However, the same ABA treatmentwas able to partially restore the normal amounts ofwaxes in the leaves of the mutant lines (Fig. 7). Indeed,

ABA application resulted in an increase in the abun-dance of most of the compounds that were reducedby ABA deficiency (C31–C33 alkanes, C32–C34 ante-isoalkanes, amyrins and taraxasterol) and in a decreasein the abundance of most of the compounds that wereincreased by ABA deficiency (C26 and C27 alkanes andC29–C31 isoalkanes). These results confirmed that ABAdeficiency alters the cuticular waxes composition oftomato leaves (Fig. 7).

The Cuticle Biosynthesis Pathway Is Down-Regulated inABA-Deficient Lines

To better understand the complex changes in leafcutin and cuticular wax composition resulting fromABA deficiency, we investigated whether therewere corresponding variations in the expressionof cuticle biosynthesis genes. Some of these geneshave previously been characterized in tomato (CUTINSYNTHASE1 [CUS1]; Yeats et al., 2012; CUTINDEFICIENT3 [CD3]; Isaacson et al., 2009; Shi et al.,2013; and CER6; Vogg et al., 2004), but other genes thatwe targeted were identified using the amino acid se-quences of cuticle-related proteins from Arabidopsisto search the Sol Genomics Network database (http://solgenomics.net/) with the BLAST tool, thereby obtain-ing the closest tomato homolog. Most of the surveyedgenes involved in cutin and/or wax monomer bio-synthesis, transport, deposition, and regulation wereexpressed at lower levels in the emerging leaves of themutants than in those of the wild types (Supplemental

Figure 6. Leaf cutin phenotype is partially rescued by ABA application. Cutin analysis was performed on the youngest mature(large) leaf of 5-week-old plants. Black and red stars represent statistical differenceswith wild type-ctr andwith the correspondingcontrol genotype, respectively. Control plants (-ctr) were sprayed with water + 0.1% ethanol; -ABA plants were sprayed with100 mmABA + 0.1% ethanol. Significant differences between the treated plants and their controls were given by a Dunnett test ifthe equal variances hypothesis of the Levene test was not rejected at a = 0.05 and by a Steel test otherwise (*P, 0.05, **P, 0.01,and ***P, 0.001; n= 6). The error bars represent SE of themean. ctr, Control. “Total identified” refers to the sumof the compoundsshown in the graph, and “total” refers to the sum of the peaks detected by GC. AC, Ailsa Craig; flc(AC), flacca in (AC); RR,Rheinlands Ruhm; sit(RR), sitiens in (RR).

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Figure 7. Leaf wax phenotype is partially rescued by ABA application. Wax analysis was performed on the youngest mature(large) leaf of 5-week-old plants. Black and red stars represent statistical differenceswith wild type-ctr andwith the correspondingcontrol genotype, respectively. Control plants (-ctr) were sprayed with water + 0.1% ethanol; -ABA plants were sprayed with

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Fig. S10A). Of particular note, the expression levels of CD3and GLYCEROL-3-PHOSPHATE ACYLTRANSFEREASE6(GPAT6), which catalyze cutin monomer formation(Shi et al., 2013; Yang et al., 2012), were significantlylower (2- to 4.5-fold) in the ABA-deficient mutants thanin their respective wild-type genotypes (SupplementalFig. S10A). Additionally, the expression levels of waxbiosynthesis genes, such as CER3 or CER6, and ofthe CUTICLE MONOMER ATP-BINDING CASSETTETRANSPORTER11 (ABCG11), were also substantiallylower in the mutants than in the wild-type leaves(Supplemental Fig. S10A; Samuels et al., 2008). In ad-dition, the mean expression levels of CUS1, CYP77A6,ABCG12,GPAT4, LONG-CHAINACYL-COA1 (LACS1),and CER1 were consistently lower in the emergingleaves of the mutants, although in each case this wasstatistically significant for only one, or none, of themutant genotypes. The expression levels ofHOTHEAD,FIDDLEHEAD,WSD1, LACS2, GPAT4, and of the geneexpression regulators SHINE1, CD2, and CER7 didnot differ between the mutants and their wild types.However, the overall decrease in the expression of wax-and cutin-related genes in the ABA-deficient mutantscorrelated with a decrease in the expression of threetranscription factors that are known to regulate cuticleformation: MYB41, MYB30/96, and MYB16/106 (thesame tomato gene is the closest homolog of bothAtMYB30 and AtMYB96, and the same is the case forAtMYB16 and AtMYB106; Supplemental Fig. S10A).

As a consequence of the ABA deficiency, the mutantgenotypes are more likely to suffer from water stressthan the wild-type plants (Tal, 1966), which might beexpected to induce the expression of genes involved incuticle biosynthesis (Kosma et al., 2009). However, thefact that most of the tested cuticle-related genes showedeither no change in expression or decreased expressionin the mutants suggested that the ABA deficiency, andnot the secondary effects of water stress, was respon-sible for the observed differences in expression. Tofurther test this possibility, we evaluated the expressionof a set of genes that are known to be responsiveto drought (Supplemental Fig. S11A). We did notdetect the expression of the drought-responsive geneSolyc03g116390 (Gong et al., 2010) in emerging leavesof the mutant or wild-type genotypes, suggesting thatnone of the plants were water stressed. Moreover, theexpression levels of two other drought-responsivegenes Solyc02g063520 and Solyc02g076690 (Gonget al., 2010) were not affected or were lower in the ABAmutants than in their respective wild-type genotypes.This further indicates that the plants were not drought

stressed and/or that these two genes are likely regulatedby ABA. In addition, the expression of Solyc06g050520,which is known to be drought induced, and the ex-pression of Solyc06g065970, which is suppressed bydrought (Liu et al., 1998; Gong et al., 2010), were notconsistently affected in the mutants. The only expres-sion pattern that was consistent with water stress wasobserved in not in the case of Solyc06g050520. Takentogether, these data indicate that the leaves of thewild-type and mutant genotypes that were subjectedto the various cuticle analyses described above werenot experiencing water stress.

To examine the effects of ABA deficiency on cuticle-related genes in leaf development in more detail, weselected a subset of the genes that were tested inemerging leaves of mature plants and evaluated theirexpression in the three stages of leaf development usedin this study (Supplemental Fig. S12). The expression ofthe selected genes was lower in the mutants than in thewild-type genotypes at the small stage of development,as was previously observed (Supplemental Fig. S10). Inaddition, the decrease of expression of CUS1 andLACS1, which was suggested but was not statisticallysignificant in Supplemental Figure S10, was confirmedin this subsequent experiment (Supplemental Fig. S12).Two distinct expression patterns were observed: CUS1and CER3 had consistently lower expressions in themutants in the three stages of leaf development, whilethe relative expression of CD3, GPAT6, ABCG11,LACS1, CER6, and MYB30/96 steadily increased as theleaf developed (Supplemental Fig. S12).

The expression of cuticle associated genes in 15 dpost-anthesis fruits generally showed a similar patternto that in leaves (Supplemental Fig. S10), although thereduction in their expression levels was less significant,and no difference was seen for the transcription factorgenes (Supplemental Fig. S10B). Indeed, several cuticle-associated genes (CUS1, CD3, CYP77A6, GPAT6,ABCG12, and CER3) were expressed at lower levels, inthe mutants compared with their respective wild types,as was the case in leaves. Quantitative PCR (qPCR)analysis of the drought-responsive genes describedabove showed no differences in expression between themutants and their wild types, suggesting that thefruits did not experience water stress (SupplementalFig. S11B).

Taken together, these gene expression analysessuggest that ABA deficiency resulted in down-regulation of genes in the cuticle biosynthesis path-way, particularly in leaves, which is consistent withthe original hypothesis that ABA regulates cuticle

Figure 7. (Continued.)100 mmABA + 0.1% ethanol. Significant differences between the treated plants and their controls were given by a Dunnett test ifthe equal variances hypothesis of the Levene test was not rejected at a = 0.05 and by a Steel test otherwise (*P, 0.05, **P, 0.01,and ***P , 0.001; n = 6). The error bars represent SEs of the mean. ctr, Control. “Total identified” refers to the sum of the com-pounds shown in the graph, and “total” refers to the sum of the peaks detected by GC. AC, Ailsa Craig; flc(AC), flacca in (AC); RR,Rheinlands Ruhm; sit(RR), sitiens in (RR).

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formation during organ development independentlyof water stress.

Effect of ABA Application on the Expression of Cuticle-Associated Genes

The cutin and cuticular wax phenotypes were par-tially rescued following an application of ABA (Figs. 6and 7). To determine the effect of such treatment oncuticle-associated gene expression, we examined theexpression patterns of seven genes in the four mutantgenotypes (Fig. 8) and of 16 genes in the mutants of RRgenetic background (Supplemental Fig. S13) through-out leaf development. To increase the likelihood thatABA penetrated the plant surface, we dipped the leavesinto an ABA solution, rather than spraying them. The

expression of CER3was strongly increased by the ABAtreatment, in all genotypes at all stages of leaf devel-opment. The expression of CD3, CYP77A6, GPAT6,ABCG11, CER1, and CER6 was increased by the appli-cation of ABA to a lesser extent, while the expression ofLACS1 and CER4 was not affected by the ABA treat-ment. The transcription factors MYB41 and MYB30/90were strongly induced to the ABA treatment in themutants and wild-type genotypes, whereas the ABAtreatment only slightly affected MYB16/106 expressionin the mutant genotypes and not at all in the wild-typegenotypes. We concluded that ABA treatment inducedthe expression of genes contributing to several stepsof the synthesis of cutin precursors (specifically, thehydroxylation of fatty acids and the production ofacylglycerols), the elongation of C16 and C18 fatty acids

Figure 8. Effect of ABA treatment on cuti-cle-related gene expression of ABA defi-cient lines through leaf ontogeny. In small(A), medium (B), and large (C) leaves of5-week-old plants. The REST-mcs softwarewas used to normalize gene expression onRPL2 and ACTIN and to determine theirrelative expression using the correspondingwild types. The log2 values of the ratios areplotted in this figure. Significant differencesbetween the mutants and their wild typeswere given by the pairwise fixed realloca-tion randomization test incorporated inREST-mcs (*P , 0.05, **P , 0.01, and***P, 0.001; n= 3). AC,AilsaCraig;not(AC),notabilis in AC background (AC); flc(AC),flacca in (AC); RR, Rheinlands Ruhm; flc(RR),flacca in (RR); sit(RR), sitiens in (RR).

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into very long-chain fatty acids for wax biosynthesis,the formation of alkanes, and to the transport of cutinprecursors and waxes into the apoplast.

ABA Deficiency Affects the Water and Barrier Functions ofthe Cuticle

The wilting phenotype of the ABA-deficient mutantshas been attributed to their inability to prevent waterloss by stomatal closure (Tal, 1966). Nevertheless, waterloss through the leaf cuticle was reported to be slightlyhigher in the three tomato mutants (Tal, 1966), and thesit leaf cuticle has been reported to be more permeableto water (Curvers et al., 2010). We compared the cuticlepermeability of detached Red stage fruits, which lackstomata, from the wild-type and ABA-deficient lines,by measuring water loss under controlled temperatureand humidity conditions. The stem scar was sealed toensure that any transpirational water loss occurredthrough the cuticle. The mutant fruits showed greaterresistance to water loss than the respective wild-typefruits, with not(AC) having an intermediate phenotype,and flc(AC), flc(RR), and sit(RR) fruits losing the leastwater (Supplemental Fig. S14).

DISCUSSION

ABA Regulates Cuticle Biosynthesis duringLeaf Development

The key question underlying this studywaswhetherABA regulates cuticle formation during the course oforgan development, in addition to its established rolein modifying cuticle formation as a consequence ofabiotic stress. We addressed this question using arange of ABA-deficient tomato mutants. The bio-chemical analyses indicated that ABA regulates cutinbiosynthesis during leaf development and exoge-nous ABA application resulted in a slight but con-sistent increase in the levels of the predominanttomato cutin monomer, 10(9)-16,dihydroxyhexadecanoicacid, and of coumaric acids, in the two tested mutants,flc(AC) and sit(RR). We conclude that ABA is requiredfor the synthesis of wild-type levels of cutin duringleaf development. The reduced expression in theABA mutants, compared to their respective wild-typegenotypes, of a number of genes involved in cutinformation revealed that the pathway as a whole isinfluenced by ABA, from the formation of cutin pre-cursors (LACS1, CD3, CYP77A6, and GPAT6) tomonomer transportation (ABCG11) and polymeriza-tion (CUS1). The regulatory role of ABA on these geneswas further demonstrated for CD3, CYP77A6, GPAT6,and ABCG11, whose expression was induced by theABA treatment (Fig. 8; Supplemental Fig. S13). Wenote that not all cutin-associated genes showed alteredexpression in the ABA mutants or responsiveness toABA. This was not unexpected, given the likely com-plexity in the regulatory pathways involved in cuticle

formation, and future studies may uncover additionalregulatory mechanisms.

ABA application did not result in consistent differ-ences in wax composition in the two wild-type geno-types following ABA application. This result wassomewhat unexpected since an ABA treatment ofArabidopsis was reported to cause an increase in cu-ticular alkane levels (Kosma et al., 2009). We do notbelieve the lack of such a response in our studywas dueto insufficient diffusion of ABA into the leaf since weobserved a partial rescue of the wax composition phe-notypes of the ABA-deficient lines, indicating that ABAis needed for cuticular wax deposition during leaf on-togeny. The ABA mutants showed an overall increasein the levels of C25 to C30 alkanes and of the C29 to C33isoalkanes in medium and large leaves, compared totheir respective wild types. In contrast, the longer-chainalkanes (C31–C33), which are the main wax constituents,were less abundant in the three stages of leaves of theABA mutants, as were the amyrins. Our data suggestthat during leaf development, ABA promotes very-long-chain fatty acid elongation and the accumulationof triterpenoids, while repressing isoalkane production.In contrast, Kosma et al. (2009) suggested that ABAtreatment mainly triggers an increase of alkane accu-mulation. This discrepancymay indicate distinct effectsof ABA action in leaves as a response to water stressversus during normal leaf development or differencesin ABA effects between tomato and Arabidopsis.

The consequences of ABA deficiency on the profilesof the leaf cuticular waxes were complex but can bepartially explained by the gene expression analysis.Notably, the expression levels of CER1, CER3, andCER6 were reduced in the ABA mutants and increasedin response to the ABA application. AtCER1 andAtCER3 are known to interact to synthesize alkanes(Bourdenx et al., 2011; Bernard et al., 2012), and over-expression of AtCER1 was reported to substantiallyincrease the levels of isoalkanes (Bourdenx et al., 2011).The decreased expression of CER1 and CER3 in themutant leaves may therefore partly explain their ab-normal levels of alkanes and isoalkanes. The reducedexpression levels of CER6 in the ABA mutant leavesmay underlie the decrease in the abundance of longer-chain alkanes and the increased levels of shorter-chainalkanes, as this shift in chain length suggests a reduc-tion in very-long-chain fatty acid elongation. Indeed,CER6 is a component of the fatty acid elongase com-plex, which has previously been shown to be involvedin the production of alkanes longer than C30 in tomatoleaves (Vogg et al., 2004). In summary, the data par-tially explain some of the differences in cutin and waxcomposition observed in the mutant leaves, although abroader survey of gene expression may more fully ex-plain the compositional variations.

Our analysis also identified three transcription fac-tors (MYB41, MYB30/96, and MYB16/106) whoseexpression was lower in the ABA-deficient mutants,suggesting an indirect regulation of the cuticle-associated genes by ABA. Moreover, the expression of

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two of these transcription factors, MYB30/96 andMYB41, was responsive to ABA treatment in all testedgenotypes. However, the expression of other regulatoryfactors, such as CER7, CD2, and SHINE1, was unaf-fected, suggesting that ABA regulates cuticle formationby influencing the expression of a subset of the tran-scription factors associated with cuticle formation, butnot the entire regulatory network. In Arabidopsis,AtMYB41, AtMYB30, AtMYB16, AtMYB106, andAtMYB96 have been shown to regulate cuticular waxformation and, with the exception of AtMYB96, to alsoregulate cutin synthesis (Cominelli et al., 2008; Raffaeleet al., 2008; Seo et al., 2011; Oshima et al., 2013). Sincethe tomato gene Solyc03g116100 was identified as theclosest homolog of both AtMYB30 and AtMYB96, itmay regulate both leaf cutin and wax formation; how-ever, this remains to be tested.

ABA Deficiency Has Distinctly Different Effects onTomato Fruit and Leaf Cuticles

Amajor conclusion from our study was that the fruitcuticle was not as affected by ABA deficiency as the leafcuticle: No ultrastructure abnormalities were detected,and cuticle-related gene expression did not differ sub-stantially from wild-type levels. TEM analysis indi-cated that the cuticles of expanding ABA-deficientmutant fruits were thicker than those of the wild-typegenotypes of the same size. However, since the mutantfruits did not expand to the same extent as those ofwild-type fruit, our comparison of similarly sized fruitswas likely of younger wild-type and older mutantfruits. The latter therefore had more time to deposit acuticle, hence the thicker appearance. However, lightmicroscopy imaging and cutin biochemical analysisshowed that the mutant mature fruits tended to accu-mulate more cuticle than the wild-type fruits. In par-ticular, the mutant fruit cuticles tended to penetratedeeper into the subepidermal cell layers (SupplementalFig. S5). This phenotype may be related to the obser-vation that the mutant fruits generally lost less waterthan those of their respective wild types (SupplementalFig. S14). These results are contrary to what was seenfor the leaf cuticle of the mutants, which had thinnercuticles (Fig. 4) and increased permeability (Tal, 1966).Chemical analysis of fruit cuticles also revealed oppo-site trends for a range of cutin and cuticular wax com-ponents, including examples with higher or lowerlevels in the mutant fruits than in the respective wildtypes. Notably, these trends were reversed for the leafcuticles, highlighting the organ-specific regulation ofcuticle formation by ABA.The lack of a substantially altered fruit cuticle com-

position in the mutants may be considered counterin-tuitive, given that the reduction in ABA levels wasgreater in fruit than in leaves (Fig. 1) and that the se-verity of the shoot phenotypes of the ABA mutants hasbeen correlated with the magnitude of ABA reduction(Tal and Nevo, 1973; Taylor and Tarr, 1984). This

apparent contradiction suggests that cuticle formationin fruits is less correlated with ABA levels than it is inleaves, and indeed, the levels of ABA in Pk stage fruitswere substantially lower than those of leaves. It may bethat fruit cuticle does not need to be as dynamicallyregulated by water stress as the leaf cuticle, since thesurface/volume ratio of leaves is far higher than that offruits and the leaves have stomata, both of which arefactors that make leaves more prone to desiccation.Moreover, fruit cuticles of various species are, onaverage, thicker than leaf cuticles, but are also morepermeable (Becker et al., 1986; Araus et al., 1991;Schreiber and Riederer, 1996), suggesting that limitingwater loss is more important in leaves than in fruits.Indeed, it has been suggested that transpirational waterloss may be necessary for fruit growth (Lee, 1989;Schreiber and Riederer, 1996). Given these factors, itmight be expected that water stress would have lessof an effect on fruit cuticles than on leaf cuticles. Thishypothesis has yet to be tested in fleshy fruit, althoughwater stress was reported to result in an increase in totalcuticular wax levels of cotton (Gossypium hirsutum)leaves and bracts, but not bolls (Bondada et al., 1996),illustrating the potential for differences in cuticle re-sponses to water stress. We propose that the leaf cuticlebiosynthesis pathway is more sensitive to changes inABA levels than the analogous pathway in fruits.

Previous studies have shown that environmentalstresses, such as drought, increase ABA levels, resultingin the increased production of cuticular waxes (Kosmaet al., 2009; Seo et al., 2009, 2011; Wang et al., 2012). Inthis study, we conclude that ABA regulation of cuticleformation is an intrinsic aspect of leaf development.Specifically, our data suggest that ABA regulates thestructure of the cuticle, the amount of cutin, and thecomposition of cutin and waxes during tomato leafdevelopment, while abiotic stress exerts a secondarylevel of ABA-mediated control.

MATERIALS AND METHODS

Plant Materials

The tomato wild-type genotypes AC and RR and their mutants, not, flc, andsit, were obtained from the University of California Davis/C.M. Rick TomatoGenetics Resource Center (http://tgrc.ucdavis.edu/). Plants were grown in agreenhouse under 16 h of light at 22°C during the day and 19°C during the nightwith 150 ppm of 15-5-15 (N-P-K), 4Ca 2Mg fertilizer (Jack’s Professional LX; J. R.Peters) being applied twice a day. The leaves of 5-week-old plants provided thesmall, medium, and large developmental stages, with “large” being theyoungest fully expanded leaf, “medium” being leaves halfway through ex-pansion, and “small” being leaves starting their expansion. Emerging leaveswere very young leaves (length,2 cm) harvested frommature plants. Ripeningstages were determined as follows: MG stage, the fruit has reached its full sizeand the surface and interior tissues are entirely green; Pk stage, 30% to 60% ofthe fruit surface is pink or red in color; Red stage,more than 90% of the surface isred; and Red Ripe, the fruit surface is entirely red.

ABA Measurements

Plantmaterialwasflash frozen and stored at280°C before being ground intoa fine powder in liquid nitrogen. Around 200 to 300 mg of this powder was

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mixed with 500 mL of 1-propanol:water:concentrated HCl (2:1:0.002). Aliquotsof 80 ng of d6-ABA (Toronto Research Chemicals) were added to each sample asan internal standard. The samples were shaken at 4°C for 30 min, centrifuged5 min at 13,000 rpm, and 800 mL of dichloromethane was added to the super-natant. The shaking and centrifugation steps were repeated as above and thelower organic phase was then collected and dried at 35°C under a gentle streamof nitrogen gas. The extracts were resuspended in 100 mL of methanol andanalyzed using a triple-quadrupole liquid chromatography-tandem massspectrometry system (Quantum Access, Thermo Scientific) as described byThaler et al. (2010) at the Chemical Ecology Group Core Facility, with the se-lected reaction monitoring of compound-specific parent/product ion transi-tions ABA 263 → 153; d6-ABA 269 → 159.

Exogenous ABA Treatments

For the biochemical analyses, 3-week-old tomato seedlings were sprayedwith 100 mM (6)-ABA (Sigma-Aldrich) in 0.1% ethanol every 5 d for 2 weeks, tothe extent that entire leaf adaxial surfaces were covered, as described by Achuoet al. (2006) and Curvers et al. (2010). Control plants were similarly sprayedwith 0.1% ethanol. Cutin and wax samples were extracted as described in the“cutin monomer and wax analysis” section from the youngest fully expandedleaf of 5-week-old plants, using the two leaflets closest to the distal leaflet. Thesix biological replicates per genotype of cutin analysis were constituted ofleaflets from independent plants. Each biological replicate of the wax analysiswere constituted of the leaflets of at least three independent plants. For thestatistical analyses of these experiments, the Levene test was first used to test forequal variances. If the null hypothesis was not rejected at a = 0.05, comparisonof means for all pairs using Tukey-Kramer’s HSD test was performed. If thevariances were unequal for a = 0.05, nonparametric comparisons for each pairusing Wilcoxon method were done.

For the gene expression experiments, 5-week-old tomato plants were sub-merged for few seconds into 100 mM (6)-ABA (Sigma-Aldrich) in 0.1% ethanolor into a 0.1% ethanol solution for controls. The treatment was repeated 3 hlater. An hour after the second treatment, the leaves were harvested and flash-frozen in liquid nitrogen. The samples were processed according to the protocoldescribed in “Gene Expression Analysis.”

Gene Expression Analysis

Total RNA sampleswere extracted fromemerging, small,medium, and largeleaves (as described in “Plant Materials”) and from the fruit pericarp usingTRIzol reagent (Life Technologies) and following themanufacturer’s directions.cDNAs were generated using SuperScript II reverse transcriptase (Life Tech-nologies) according to the manufacturer’s instructions. A Life Technologies/ABI Viia7 instrument at Cornell’s Institute of Biotechnology Genomics Facilitywas used to generate the data. A list of the primers used in this study is given inSupplemental Table S3, together with gene identification numbers (SGN;http://solgenomics.net/). The REST-MC software was used to determine geneexpression levels and to apply its pairwise fixed reallocation randomizationstatistic test on the results (Pfaffl et al., 2002).

Water Loss Measurements

Red fruitswereharvested, theirpedicel scarswere coveredwithhighvacuumgrease (silicone lubricant, Dow Corning) and they were placed in an incubationchamber at 35% humidity and 22°C in the dark. Fruit surface area was deter-mined by measuring fruit diameter. The fruits were weighed every day, anddecreases in weight were considered to be equivalent to the amount of waterloss.

Light Microscopy

Tissue fixation and embedding were performed as by Buda et al. (2009). Asaturated solution of Oil RedO (Alfa Aesar) in isopropyl alcohol was diluted 3:2with distilled water, mixed well, left for 30 min at room temperature, and thenfiltered with a syringe filter of 0.8/0.2 mm pore size (Acrodisc syringe filters;Pall Corporation) to remove precipitates. The solution was then applied on6 mm sections, generated as described in Yeats et al. (2012), for 30 min at 100%humidity. The slides were rinsedwith a series of 50, 30, 22, 15, and 8% isopropylalcohol and mounted in the last dilution. The stained slides were viewed on anAxioImager A1microscope (Zeiss) using Zeiss EC-PlanNeoFluar 403/0.75 dry

objective, a Zeiss AxioCamMRc color video camera, andZeiss Axio Vs40 4.6.3.0software. Photoshop CS5 software (Adobe) was used to adjust the image levelsand color balance.

TEM

Leafmaterialwasharvested fromtheyoungest fullyexpanded leaf of 6-week-old plants, using the two leaflets closest to the distal leaflet. Leaflets were cut inpieces of ;5 3 5 mm, and for the fruit pericarp samples, ;3 3 3-mm cubes oftomato pericarp were excised from pericarp sections using a razor blade. Theharvested tissue was placed in a fixative consisting of 1% glutaraldehyde in0.1 M sodium cacodylate buffer (pH 7.2) for 60 min on ice. The samples werewashed three times in wash buffer (0.1 M sodium cacodylate buffer, pH 7.2)for ;5 min each and subsequently postfixed in 0.5% OsO4 in 0.1 M sodiumcacodylate buffer (pH 7.2) for 60 min on ice. The washing step was then re-peated. Samples were dehydrated with a series of acetone dilutions (10, 30, 50,70, 90, and 100%) for 20 min each, with the 100% solution being replaced byfresh acetone after 10 min. Samples were then infiltrated by immersion in 75%acetone-25% Spurr’s Low Viscosity Resin (Electron Microscopy Sciences)overnight, followed by 2 h immersions in 50% resin/50% acetone and then 75%resin/25% acetone. Finally, the samples were infiltrated with 100% resinovernight. Polymerization of the resin was performed at 60°C for 10 h. Sixty-nanometer sections were cut with a diamond knife on a Reichert Jung Ultracut Eultramicrotome and collected on Formvar-coated nickel grids. The grids werestained in uranyl acetate/lead citrate, washed with deionized water, and dried.The sections were viewed with a Zeiss Libra 120 transmission electron micro-scope (Zeiss) at 120 kV.

The cuticle and the underlying cellwallwere visually distinguished based ondifferences in their optical contrast andby lookingatmultiple images of the samesample. Measurements of cuticular thickness were based on the TEM micro-graphs of leaflets from three independent plants per genotype and of three to sixfruits per genotype. At least three measurements per leaflet were taken. Wild-type and mutant fruits of ;14.4-, ;13.5-, and ;12.7-mm diameters werecompared using from 10 to 22 measurements per size and genotype. Student’st test was used to determine statistical significance of the results.

Cutin Monomer and Wax Analysis

Cutin and waxes were extracted from the youngest fully expanded leaf of6-week-old plants, using the two leaflets closest to the distal leaflet. For the leafsamples, cuticular waxes were extracted by dipping the leaflets in chloroformcontaining 50 or 100mg of tetracosane, for 30 s. Cutin samples were isolated asdescribed in Li-Beisson et al. (2013). For the fruit samples, the fruit cuticle wasisolated as described in Isaacson et al. (2009), with the peels being incubatedat 42°C instead of 35°C. Isolated cuticles were washed in distilled water, airdried, and rinsed three times with chloroform to remove cuticular waxes andcontaminating lipid residues. Fruit cuticular waxes were extracted by dip-ping intact fruits in two successive baths of chloroform for 1min each, the firstbath containing 50 or 100 mg of tetracosane as internal standard. The bathswere pooled, anhydrous sodium sulfate was added to remove any trace ofwater, and the solutions were passed through filter paper. Leaf and fruit cutinsamples were depolymerized as described in Li-Beisson et al. (2013). Cutinmonomers and cuticular waxes were derivatized as previously described(Li-Beisson et al., 2013)

The cutin and wax extracts were resuspended in 100 mL chloroform andinjected into a gas chromatography (GC) 6850 GC system (Agilent) with aHP-1 (30 m 3 320 mm 3 0.1 mm) column (Agilent). The wax samples wereinjected with an oven temperature of 50°C, and the temperature was heldconstant for 2 min. The temperature was then increased by 40°C per min up to200°C, and then by 4°C/min up to 235°C and held for 15 min, prior to anincrease of 10°C per min up to 315°C, which was then maintained for 15 min.The cutin monomer samples were injected with an oven temperature of 50°C,which was then held for 2 min. The temperature was then increased by 40°Cper min up to 120°C and held for 2 min prior to an increase of 10°C per min upto 320°C, which was then maintained for 15 min. Compounds were identifiedas described in Yeats et al. (2012).

Statistical Analyses

Unless stated otherwise in the text, the statistical tests were performed usingJMP software, by first performing an ANOVA on the mutants and their wild

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type. If the variances were equivalent for a = 0.05, a pairwise comparison testusingDunnett’smethod (using a control group) was performed. If the varianceswere unequal for a = 0.05, the nonparametric multiple comparison test “Steelwith control” was used.

Accession Numbers

Sequence data from this article can be found in the SGN data libraries underthe accession numbers Solyc11g006250 (CUS1), Solyc08g008610 (BDG),Solyc08g081220 (CD3), Solyc05g055400 (CYP77A6), Solyc01g094700 (GPAT4),Solyc09g014350 (GPAT6), Solyc01g079240 (LACS2), Solyc06g062600(HTH), Solyc03g005320 (FDH), Solyc03g019760 (ABCG11), Solyc11g065350(ABCG12), Solyc03g065250 (CER1), Solyc03g117800 (CER3), Solyc06g074390(CER4), Solyc02g085870 (CER6), Solyc01g079240 (LACS1), Solyc01g011430(WSD1), Solyc05g047420 (CER7), Solyc01g091630 (CD2), Solyc03g116610(SHN1), Solyc10g005460 (MYB41), Solyc03g116100 (MYB30/96), Solyc02g088190(MYB16/106), Solyc06g050520 (DREB2A), Solyc03g116390, Solyc02g076690,Solyc02g063520, Solyc06g065970, Solyc05g054480 (ACTIN), Solyc10g006580 (RPL2).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Whole-plant phenotype of the ABA-deficientmutants.

Supplemental Figure S2. ABA-deficient mutant fruit phenotype.

Supplemental Figure S3. TEM micrographs of the cuticle of small expand-ing fruit.

Supplemental Figure S4. TEM micrographs of the cuticle of the abaxialepidermis of fully expanded leaflets.

Supplemental Figure S5. Light microscopy micrographs of fruit cuticles.

Supplemental Figure S6. Analysis of ABA-deficient mutant fruit cutin.

Supplemental Figure S7. Analysis of fruit cuticular waxes in ABA-deficient mutants.

Supplemental Figure S8. Amounts of small leaf cuticular waxes fromABA-deficient mutants.

Supplemental Figure S9. Amounts of medium leaf cuticular waxes fromABA-deficient mutants.

Supplemental Figure S10. Cuticle related gene expression in developingleaves and fruits of ABA-deficient lines.

Supplemental Figure S11. Expression of drought-responsive genesdetected by qPCR.

Supplemental Figure S12. Expression of cuticle-related genes in ABA-deficient lines in three stages of leaf development.

Supplemental Figure S13. Response of cuticle-related genes to ABA treat-ment of ABA-deficient lines through leaf ontogeny.

Supplemental Figure S14. Analysis of transpirational water loss from theABA-deficient lines and their respective wild types.

Supplemental Table S1. Fruit and leaf cuticle thickness measured on TEMimages.

Supplemental Table S2. MG fruit cuticle penetration in betweensubepidermal cells and thickness, measured on light microscopyimages.

Supplemental Table S3. IDs and specific qPCR primer pairs of the tomatogenes analyzed in this study.

ACKNOWLEDGMENTS

We thank Daniel Evanich for help with the qPCR analyses, the C.M. RickTomato Genetics Resource Center (University of California, Davis) for provid-ing tomato seeds, and the Chemical Ecology Group Core Facility for ABAquantification.

Received March 20, 2017; accepted May 4, 2017; published May 8, 2017.

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