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Assessing the Biosynthetic Capabilities of SecretoryGlands in Citrus Peel1[W][OA]

Siau Sie Voo, Howard D. Grimes, and B. Markus Lange*

Institute of Biological Chemistry (S.S.V., B.M.L.), M.J. Murdock Metabolomics Laboratory (B.M.L.), and Schoolof Molecular Biosciences (H.D.G.), Washington State University, Pullman, Washington 99164–6340

Epithelial cells (ECs) lining the secretory cavities of Citrus peel have been hypothesized to be responsible for the synthesis ofessential oil, but direct evidence for such a role is currently sparse. We used laser-capture microdissection and pressurecatapulting to isolate ECs and parenchyma cells (as controls not synthesizing oil) from the peel of young grapefruit (Citrus 3paradisi ‘Duncan’), isolated RNA, and evaluated transcript patterns based on oligonucleotide microarrays. A Gene Ontologyanalysis of these data sets indicated an enrichment of genes involved in the biosynthesis of volatile terpenoids and nonvolatilephenylpropanoids in ECs (when compared with parenchyma cells), thus indicating a significant metabolic specialization in thiscell type. The gene expression patterns in ECs were consistent with the accumulation of the major essential oil constituents(monoterpenes, prenylated coumarins, and polymethoxylated flavonoids). Morphometric analyses demonstrated that secretorycavities are formed early during fruit development, whereas the expansion of cavities, and thus oil accumulation, correlates withlater stages of fruit expansion. Our studies have laid the methodological and experimental groundwork for a vastly improvedknowledge of the as yet poorly understood processes controlling essential oil biosynthesis in Citrus peel.

Members of the genus Citrus (Rutaceae) producesome of the commercially most important tree fruitcrops, which are grown in over 100 countries world-wide, most prominently in Brazil, the Mediterraneanbasin, the United States, and China. The two majormarkets in the Citrus sector are fresh fruit for directconsumption and fruit juice. Roughly 50% of the fruitweight consists of pulp, seeds, and peel, which arefurther processed into value-added by-products suchas molasses, pectins, fiber, seed oils, fermentationproducts, and essential oils (Laufenberg et al., 2003).Citrus essential oils are obtained on an industrial scaleby cold extraction and generally contain two classes ofconstituents: a volatile fraction consisting of mono-terpenoids and small amounts of sesquiterpenoids(totaling 94%–98% of the oil) and a nonvolatile residuecontaining fatty acids, sterols, carotenoids, waxes,coumarins, and polymethoxylated flavonoids (2%–6%of the oil; Mondello et al., 2002). These oils are pro-cessed into various formulations for industrial

cleaning applications and as sustainable alternatives totraditional solvents. The volatile fraction, which isgathered by steam or vacuum distillation, has importantuses in the flavor, fragrance, aromatherapy, and agro-chemical industries (Carson and Hammer, 2011).

Collaborative projects to generate genomic resourcesfor Citrus, including EST libraries from various organsand cDNA microarrays (Forment et al., 2005; Reis et al.,2007), have yielded data sets that have been mined byseveral groups to discover genes with potential relevanceto essential oil biosynthesis in the peel. For example,transcripts related to terpenoid synthases, which catalyzethe first committed step in the biosynthesis of terpenoidessential oil constituents, were highly expressed in Citrusfruit peel when compared with other tissues and organs(Berger et al., 2007; Dornelas andMazzafera, 2007; Takitaet al., 2007). More recently, genome-wide oligonucleotidemicroarray analyses have been conducted with Citruspeel tissue (Maul et al., 2008; González-Candelas et al.,2010; Matas et al., 2010; Ballester et al., 2011; Hershkovitzet al., 2012). However, these studies were aimed atevaluating specific developmental processes or stress/defense responses of Citrus and did not provide directinsights into peel essential oil biosynthesis. The fewfunctionally characterized genes involved in Citrus es-sential oil biosynthesis encode monoterpene and ses-quiterpene synthases with activities for the synthesis of(+)-limonene, (2)-b-pinene, g-terpinene, (E)-b-ocimene,1,8-cineole, and (E)-b-farnesene (Maruyama et al., 2001;Lücker et al., 2002; Shimada et al., 2004, 2005).

In Citrus fruit, the pigmented region of the pericarpis called the flavedo and contains numerous oil glandsconsisting of secretory cavities that are lined by several

1 This work was supported by the Division of Chemical Sciences,Geosciences, and Biosciences, Office of Basic Energy Sciences, U.S.Department of Energy (grant no. DE–FG02–09ER16054 to B.M.L.).

* 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: B.Markus Lange ([email protected]).

[W] The online version of this article contains Web-only data.[OA] OpenAccess articles can be viewed onlinewithout a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.112.194233

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layers of specialized epithelial cells (ECs; Fahn, 1979).Various authors have hypothesized that the ECs areresponsible for essential oil biosynthesis (Esau, 1965;Schnepf, 1974; Turner et al., 1998; Lücker et al., 2002).It was also shown that plastid preparations obtainedfrom the outer peel of Citrus could convert iso-pentenyl diphosphate into monoterpenes (Gleizeset al., 1983; Pauly et al., 1986). Because of the en-richment of this preparation in leucoplasts likelyoriginating from ECs, the authors hypothesized thatthe ECs were the main location for essential oil bio-synthesis. However, the only direct evidence for the siteof essential oil biosynthesis comes from the in situ lo-calization of transcripts for putative monoterpene syn-thases to ECs of rough lemon (Citrus jambhiri; Yamasakiand Akimitsu, 2007).

This study was initiated as a first step to compre-hensively characterize essential oil biosynthesis and itsregulation in Citrus, using peel from grapefruit (Citrus3 paradisi ‘Duncan’) as an experimental model system.We employed an integrative approach to evaluate,throughout fruit development, the numbers, volume,and volume distribution of secretory cavities (micro-scopy and morphometrics), essential oil contents (gaschromatography [GC]-mass spectrometry [MS] andHPLC-MS), and global transcript profiles in isolatedECs (laser-capture microdissection and pressure cata-pulting followed by oligonucleotide microarray anal-ysis). These data sets are invaluable resources forcorrelating relevant processes at the microscopic level(metabolic dynamics in ECs) with quantifiable out-comes at the macroscopic level (essential oil quantityand composition).

RESULTS

Correlation of the Distribution of Secretory Cavitiesand Essential Oil Quantities in the Peel ofDeveloping Grapefruit

The grapefruit tree for this investigation was main-tained under controlled greenhouse conditions. The fruitgrowth curve, monitored for two growth seasons (2009and 2010) as equatorial diameter (ED), resembled theshape of a logistic function, with fairly rapid growthfor the first 100 d post anthesis and a subsequentlyslower growth rate toward mature size (Fig. 1A). Theaverage final size of fruit was about 15% larger in 2010compared with 2009, but the growth trends were thesame (Fig. 1A). Fruit from smaller clusters (one to threefruits per cluster) were generally of bigger size comparedwith those from larger clusters (four or more fruits). Thetransition from green to yellow peel color began between80 and 110 d post anthesis and was completed within90 d (170–200 d post anthesis). Secretory cavities werecounted in analogy to Knight et al. (2001) from 6 to 133mm ED (for details, see “Materials and Methods”). Thecavity numbers increased rapidly from 6,840 (ED 6 mm)to 25,000 (ED 43 mm) and then much slower toward

fruit maturity (Fig. 1B). A natural logarithmic functionreflected these trends (r2 = 0.66). The variation in thenumber of cavities in mature fruit (29,040 6 4,159 cavi-ties per fruit [14.3%]) was larger than the variation infinal fruit size (113 6 10.1 mm ED [8.9%]).

In addition to counting the total number of cavitiesper fruit, we also determined the volumes of cavities.To the best of our knowledge, such a survey has notbeen performed previously; thus, we will briefly out-line our experimental strategy. Specimens were handsectioned, rapidly placed under immersion oil to en-sure optimal preservation of cavity shapes, and imageswere taken immediately thereafter. All immature cavi-ties had prolate spheroid shapes, whereas mature cav-ities approached the shape of a regular sphere or wereof an oblate spheroid shape (Fig. 1, C and D). Volumeswere calculated for all cavities in a defined area of thefruit flavedo. The total cavity volume in this area wasthen used to extrapolate the total amount of oil perfruit (Fig. 1E). To evaluate the accuracy of these esti-mations experimentally, we determined the quantitiesof volatile essential oil components by hydrodistillationof finely ground peel tissue, followed by GC coupledwith flame-ionization detection (FID; SupplementalTable S1). We also determined the contribution ofnonvolatile constituents to the oil (which ranged be-tween 2.5% and 3.4% [w/v] of the total oil), as describedin the next paragraph. The oil quantities estimated basedon secretory cavity volumes were very similar to themeasured oil yields throughout fruit development,with final oil volumes of 1,738 6 319 mL per fruit(estimated) and 1,586 6 356 mL per fruit (measured)at maturity (Fig. 1E). The developmental changes ofestimated oil quantities were fitted to a logistic func-tion, with an initial lag phase (less than 70 mL per fruitat 24 mm ED), a subsequent more rapid increase infruit of 40 to 100 mm ED, and then slower growthtoward maturity (127–135 mm ED; Fig. 1E).

To evaluate why the rapid initial increase in thenumber of cavities (Fig. 1B) did not result in an equallyfast increase in the accumulation of essential oil (Fig. 1E),we tested if the developmental distribution of cavitiesof different volumes might play a role in determiningoil quantities (Fig. 1, F and G). Cavities were dividedinto different volume categories, and the volume dis-tribution was determined throughout fruit develop-ment (Supplemental Protocol S1). In very young fruit(6–8 mm ED), cavity volumes ranged from 0.15 to2.4 nL per fruit (distribution apex at 1.3 nL per fruit);in medium sized fruit (51–53 mm ED), the range wasfrom 0.15 to 111 nL per fruit (distribution apex at12.3 nL per fruit); and in mature fruit (99–136 mm),volumes from 0.15 to 1,000 nL per fruit (distributionapex at 64.2 nL per fruit) were obtained (Fig. 1H). Thesedata indicate that the volume distribution of secretorycavities does indeed change, generally from smallerto larger cavities throughout fruit development. Oilyields estimated based on both cavity numbers andvolume distribution are very similar to those measuredexperimentally (Fig. 1E).

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Chemical Analysis of Grapefruit Peel Essential OilCollected Directly with Microcapillaries

Studies on the nonvolatile constituents of Citrus es-sential oil began in the 1960s (for review, see Dugo et al.,2009), but reliable quantifications for grapefruit were notreported until much later (McHale and Sheridan, 1989;Frérot and Decorzant, 2004; Dugo et al., 2010). All ofthese studies used cold-pressed grapefruit oil for

phytochemical and chemometric analyses. It is con-ceivable, however, that some components are not pre-sent in the essential oil itself but are instead dissolvedfrom other peel tissues during the cold-pressing pro-cess. In the context of this work, which was aimed atcharacterizing the biosynthetic capabilities of ECs, itwas critical to directly determine the oil composition insubepidermal cavities, which was achieved usingmicrocapillaries (Fig. 2). We used this approach

Figure 1. Developmental patterns of essential oil accumulation in grapefruit peel. A, Fruit growth in 2009 (white circles) and2010 (black circles). B, Oil gland formation in 2009 (white circles) and 2010 (black circles). C, Prolate spheroid shape ofsecretory cavities in peel of young fruit (28 mm ED). D, Spheroid shape of secretory cavities in peel of mature fruit (100 mm ED).E, Oil yield as a function of fruit development (estimated based on cavity volume measurements as white diamonds andmeasured by chemoanalytical means as black diamonds). F, Peel surface in young fruit (28 mm ED). Mostly small and medium-sized cavities are visible. G, Peel surface in mature fruit (100 mm ED). Almost all cavities are fairly large. H, Volume distributionof cavities as a function of fruit development.

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Citrus Essential Oil Biosynthesis



successfully before to analyze the contents of essentialoil-accumulating glandular trichomes (Rios-Estepaet al., 2008). The main volatile terpenoids in themicrocapillary-collected oil were (+)-limonene (93%–94%), myrcene (3%), and sabinene (0.7%–2.1%; Table

I), which is in agreement with published data on themost abundant volatile constituents in cold-pressed oils(Kirbaslar et al., 2006; Espina et al., 2010). Sesquiterpenesoccurred as minor components (Table I), which is also inaccordance with previous reports (Flamini and Cioni,2010). Oxygen-containing heterocyclic metabolites,consisting of coumarins, furanocoumarins, and poly-methoxylated flavones, were present in the oil at 4.9%(w/v) before the peel started yellowing (70 mm ED)and then decreased slightly to 4.1% (w/v) in oilobtained from mature fruit (100 mm ED; Table I). Thisis consistent with prior studies (McHale and Sheridan,1989; Frérot and Decorzant, 2004; Dugo et al., 2010).Carotenoids had been described as minor compo-nents of cold-pressed Citrus oils (Dugo et al., 2006)but were undetectable in our microcapillary-collectedgrapefruit oil samples (Table I). Sterols and fattyacids, which had been quantified in Citrus seed oils(El-Adawy et al., 1999), were present at very lowlevels in the grapefruit peel oil we collected withmicrocapillaries (0.38% or less and 0.003% or less,respectively; Table I).

Analysis of Global Transcript Expression Patterns in ECsSurrounding Secretory Cavities

To test the hypothesis that ECs of peel glands areresponsible for the biosynthesis of the oil accumulatedin secretory cavities, it was crucial to study this celltype in isolation, without interference from neighbor-ing cells. Previous transcriptome studies of Citrus fruitpeel and peel sections were designed to investigatestress and defense responses but did not provide directinsights into essential oil biosynthesis (Maul et al.,2008; González-Candelas et al., 2010; Matas et al., 2010;Ballester et al., 2011; Hershkovitz et al., 2012). Weoptimized a protocol for the collection of intact ECsfrom the flavedo layer of grapefruit peel by lasermicrodissection and pressure catapulting, subsequentisolation of RNA, and linear amplification of tran-scripts for analysis on Affymetrix Citrus genome oli-gonucleotide microarrays. As negative controls, wecollected parenchyma cells (PCs) of the albedo layer,which were processed in the same way (Fig. 3). Forthe microarray analysis, we selected fruit right beforeand during the highest oil accumulation (28 and 40mm ED, respectively; Fig. 1E). Microarray data wereuploaded into the Partek Genomics Suite softwarepackage for background correction and statistical pro-cessing. In 28 mm ED fruit, the expression levels of4,079 transcripts were significantly different betweenECs and PCs (Supplemental Table S2). The same com-parison for transcripts from 40 mm ED fruit revealed3,082 differentially expressed genes. This preliminaryanalysis provided evidence that there are substantialdifferences between gene expression patterns in ECsand PCs.

To categorize the differentially expressed genes,gene lists were processed with the Web-based AgriGO

Figure 2. Collection of essential oil directly from secretory cavities ofCitrus fruit peel using microcapillaries. A, Custom-made glass micro-capillary (i.d. 756 15 mm). B, Citrus peel section with secretory cavity.C, Citrus peel section after collection of secretory cavity contents witha microcapillary. Note that the cells surrounding the cavity are mostlyundamaged.


Voo et al.




tool (Du et al., 2010), which allows users to perform asingular enrichment analysis for Gene Ontology (GO)terms. The most positively enriched GO terms formetabolic genes in ECs (compared with PCs) for bothfruit sizes were “phenylpropanoid metabolic process”(GO:0009698) and “isoprenoid metabolic process”(GO:0006720; Supplemental Table S3). A morecomprehensive list of genes involved in these path-ways is given in Table II and provides further evidencefor the fairly high expression levels, in ECs, of genesinvolved in the biosynthesis of monoterpenes and

sesquiterpenes, coumarins, and polymethoxylatedflavones, the main components of Citrus peel essentialoil. The list of metabolic genes with lower expres-sion levels in ECs (when compared with PCs) wasenriched in the GO categories “response to stimulus”(GO:0050896) and “polysaccharide metabolic pro-cess” (GO:0005976; Supplemental Table S3), whichboth contained genes related to cell wall biosynthesis.The expression levels of only 143 genes were differentbetween ECs of fruits with EDs of 28 and 41 mm(26 higher and 117 lower in ECs of 28-mm compared

Table I. Composition of grapefruit peel essential oil obtained directly from secretory cavities using microcapillaries

ConstituentsConcentration

ED 70 mm SD ED 80 mm SD ED 100 mm SD

mg mL21 oil

MonoterpenesLimonene 645.1 81.0 670.3 62.4 820.8 57.0Myrcene 23.2 3.1 23.7 2.2 29.3 2.4Sabinene 16.6 5.2 8.4 1.4 6.9 2.5g-Terpinene 2.7 0.7 2.0 0.6 7.1 1.8a-Terpinene 1.6 0.4 1.2 0.5 5.9 2.0a-Pinene 3.9 1.0 3.7 0.3 4.6 0.4b-Pinene 1.3 0.4 0.6 0.2 0.6 0.1Terpinolene 0.8 0.8 1.7 0.5 3.1 0.9Sum 695.3 85.1 711.5 65.7 878.2 60.6Percentage (total) 94.8 11.6 94.1 8.7 95.6 6.6

Sesquiterpenesb-Caryophyllene 0.23 0.05 0.21 0.04 0.21 0.03b-Farnesene 0.17 0.06 0.09 0.03 0.09 0.04Sum 0.4 0.11 0.3 0.07 0.3 0.07Percentage (total) 0.05 0.01 0.04 0.01 0.03 0.01

Coumarins and furanocoumarinsAurapten 9.0 1.1 16.0 2.6 14.0 6.5Epoxyaurapten 4.9 0.8 4.6 1.0 3.9 1.7Meranzin 8.4 2.1 9.0 3.4 9.4 2.1Isomeranzin 1.0 0.3 1.1 0.2 1.2 0.0Bergamottin 0.6 0.2 1.3 0.1 1.3 0.8Epoxybergamottin 9.9 3.2 8.5 0.2 7.0 2.9Osthol 0.2 0.1 0.2 0.0 0.2 0.0Sum 34.1 7.7 40.6 7.6 37.0 14.0Percentage (total) 4.6 1.0 5.4 1.0 4.0 1.5

Polymethoxylated flavonesNobiletin 1.1 0.3 0.6 0.1 0.5 0.2Tangeritin 0.6 0.1 0.3 0.0 0.3 0.1Heptamethoxyflavone 0.5 0.1 0.4 0.1 0.3 0.0Sum 2.2 0.5 1.3 0.1 1.0 0.3Percentage (total) 0.3 0.07 0.2 0.02 0.1 0.03

Sterolsb-Sitosterol 1.15 0.09 1.53 0.60 1.59 0.68Cycloartenol 0.88 0.40 1.18 0.41 0.80 0.21Campesterol 0.03 0.01 0.04 0.01 0.04 0.01Stigmasterol 0.02 0.01 0.03 0.01 0.03 0.01Sum 2.08 0.47 2.78 1.02 2.47 0.55Percentage (total) 0.3 0.06 0.4 0.13 0.3 0.06

Fatty acidsPalmitic acid (C16:0) 0.003 0.001 0.005 0.000 0.006 0.001Stearic acid (C18:0) 0.001 0.001 0.002 0.000 0.002 0.001Linoleic acid (C18:2) 0.007 0.003 0.011 0.003 0.013 0.003a-Linolenic acid (C18:3) 0.005 0.002 0.007 0.003 0.007 0.002Sum 0.016 0.007 0.024 0.006 0.028 0.004Percentage (total) 0.002 0.0009 0.003 0.0008 0.003 0.0004







with 41-mm fruit), and there was no significant en-richment in any GO categories. An analogous analy-sis of PCs revealed the differential expression of356 genes in fruit with EDs of 28 and 41 mm (103higher and 253 lower in PCs of 28-mm comparedwith 41-mm fruit), and there was also no enrichmentof GO terms. These results indicate that there were nomajor differences between gene expression patternsat the two selected developmental stages.

To further test the robustness of microarray data setsfor transcripts highly expressed in ECs, real-timequantitative PCR (qPCR) analyses were performed forselected genes with relevance to Citrus essential oilbiosynthesis. Because the laser microdissection andpressure catapulting of thousands of ECs had alreadybeen an enormously time-consuming task, we decidedto compare gene expression patterns in thin sections(roughly 600–700 mm) of flavedo (exocarp layer con-taining ECs and secretory cavities) and upper albedo(mesocarp layer devoid of oil glands), which shouldprovide similar insights into the apparent specializa-tion of ECs. We selected three different developmentalstages of grapefruit (34, 43, and 120 mm ED), whichencompassed both early time points used in ourmicroarray study and an additional time point close to

maturity. Primers were designed to amplify fragmentsof genes representing the most highly enriched tran-scripts of ECs as indicated by our microarray data(Supplemental Table S4). Three monoterpene synthasegenes [encoding (+)-limonene synthase, myrcene syn-thase, and b-pinene synthase], one characterized ses-quiterpene synthase [(E)-b-farnesene synthase], andtwo putative sesquiterpene synthases (Citrus Chipidentifiers Cit.29748.1.S1_s_at [orange1.1g015245m] andCit.17284.1.S1_at [orange1.1g039366m]) were highlyenriched in 34- and 43-mm fruit peel flavedo (Fig. 4),which is in full accordance with the microarray data.Two of these genes were also enriched in 120-mm fruit[(+)-limonene synthase and a putative sesquiterpenesynthase of as yet unknown function (Cit.29748.1.S1_s_at)]. As a negative control, we evaluated the tran-script levels of a putative terpene synthase (Cit.38319.1.S1_s_at) that, based on microarray results, was notfound to be preferentially expressed in ECs, and weobserved that its expression levels, as determined byqPCR, also did not differ significantly in the albedo andflavedo layers. We also selected six genes involved inphenylpropanoid/flavonoid biosynthesis (4-coumarate:CoA ligase [two genes], hydroxycinnamoyl-CoA:quinate transferase, chalcone synthase, and flavone

Figure 3. Collection of grapefruit peel ECs andPCs using LMPC.


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Table II. Enrichment of genes involved in essential oil biosynthesis in ECs compared with PCs of grapefruit peel at two differentstages of development (28 mm ED and 41 mm ED), expressed as fold change

Annotation Citrus Affy Chip IdentifierEC versus PC Enrichment

28 mm 41 mm

Terpenoid biosynthesisMVA pathwayAcetyl-CoA thiolase Cit.1287.1.S1_s_at 6.941 4.1173-Hydroxy-3-methylglutaryl-CoA synthase Cit.29397.1.S1_s_at 12.272 11.972

Cit.1280.1.S1_s_at 6.551 5.119Cit.29249.1.S1_at 4.446 5.080

3-Hydroxy-3-methylglutaryl-CoA reductase Cit.17889.1.S1_s_at 14.403 8.488Cit.23097.1.S1_s_at 10.603 6.856

MVA kinase Cit.2723.1.S1_s_at 5.617 3.106Phosphomevalonate kinase Cit.37962.1.S1_at 2.891 4.609Diphosphomevalonate decarboxylase Cit.20947.1.S1_s_at 3.849 2.764

MEP pathway1-Deoxy-D-xylulose 5-phosphate synthase Cit.10053.1.S1_at 49.532 12.021

Cit.10054.1.S1_s_at 12.292 4.887Cit.10056.1.S1_at 11.740 8.216

1-Deoxy-D-xylulose-5-phosphate reductoisomerase Cit.23507.1.S1_at 12.309 11.740Cit.4968.1.S1_s_at 5.384 3.434Cit.26985.1.S1_at 3.538 2.404

2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase Cit.14478.1.S1_at 5.452 4.3754-(Cytidine 59-diphospho)-2-C-methyl-D-erythritol kinase Cit.29567.1.S1_s_at 6.659 10.413

Cit.3446.1.S1_s_at 7.803 5.0972-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase Cit.1430.1.S1_s_at 5.645 4.0464-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase Cit.985.1.S1_at 8.931 4.8784-Hydroxy-3-methylbut-2-en-1-yl diphosphate reductase Cit.17714.1.S1_at 29.980 8.382

Cit.9279.1.S1_at 4.626 3.123Cit.9280.1.S1_s_at 4.483 2.612

Prenyltransferases and terpene synthasesIsopentenyl diphosphate isomerase (putative) Cit.9842.1.S1_s_at 3.430 2.800

Cit.27203.1.S1_s_at 2.875 2.350Cit.9841.1.S1_s_at 2.679 2.232

Farnesyl diphosphate synthase (putative) Cit.1688.1.S1_s_at 2.806 1.963Cit.35197.1.S1_at 2.333 2.917

Geranylgeranyl pyrophosphate synthase (putative) Cit.20724.1.S1_at 25.436 7.288Cit.14050.1.S1_at 9.553 3.298Cit.16303.1.S1_at 3.544 3.159

(+)-Limonene synthase (monoterpene synthase) Cit.9964.1.S1_s_at 9.540 3.194Cit.23560.1.S1_at 9.116 4.338

b-Pinene synthase (monoterpene synthase) Cit.2694.1.S1_at 29.767 17.103(E)-b-Ocimene synthase (monoterpene synthase) Cit.31295.1.S1_at 3.119 1.821Monoterpene synthase (putative) Cit.31559.1.S1_at 2.158 1.416b-Farnesene synthase Cit.2936.1.S1_at 53.196 15.623Sesquiterpene synthase (putative) Cit.17284.1.S1_at 49.647 14.095

Cit.30774.1.S1_at 41.631 21.153Cit.29748.1.S1_s_at 27.134 17.274

Coumarin and flavonoid biosynthesisMalonyl-CoA biosynthesisATP citrate lyase subunit B Cit.11635.1.S1_at 3.384 2.668

Cit.30906.1.S1_s_at 2.948 3.718Cit.14414.1.S1_at 2.796 3.078

Acetyl-CoA carboxylase (biotin carboxylase subunit) Cit.26354.1.S1_s_at 2.771 2.853Cit.26354.1.S1_s_at 2.771 2.853

General phenylpropanoid pathwayPhe ammonia lyase Cit.9590.1.S1_s_at 4.452 5.469

Cit.2241.1.S1_s_at 2.810 2.6544-Coumarate CoA ligase Cit.25648.1.S1_s_at 3.473 4.061

Coumarin and furanocoumarin pathwayCYP82C (hydroxylates psoralens) Cit.5945.1.S1_at 56.028 14.715

Cit.29478.1.S1_s_at 26.793 7.596Cit.2373.1.S1_s_at 17.277 4.347

(Table continues on following page.)





synthase [two genes]) that were highly expressed inECs (as determined by microarray analysis). All ofthese genes were found to be expressed at elevatedlevels in flavedo (compared with albedo) in qPCRassays (Fig. 4). Interestingly, transcript levels stayedat high levels throughout fruit development for fiveof the six selected phenylpropanoid/flavonoid path-way genes.

DISCUSSION

Citrus Fruit Peel Oil Glands Develop Early, Butthe Filling of Secretory Cavities Correlates with LaterStages of Development

Our experiments showed that the increase in cavitynumbers (Fig. 1B) in young grapefruit was muchfaster than the growth rate of the entire fruit (Fig. 1A),indicating that most cavities are formed early duringfruit development. This is in agreement with previousdata obtained with orange (Citrus sinensis) fruit(Knight et al., 2001). We also attempted to learn moreabout the process of cavity filling, which could not bediscerned based on previously published data. In theyoungest fruit (6 mm ED), we counted roughly 6,000cavities, but their volumes were not uniform. Thecalculated volumes ranged from 0.15 to 2.4 nL, cor-responding to a 16-fold difference from the lowest tothe highest volume (Fig. 1, C and H). Interestingly,we found a distribution of vastly different cavityvolumes at different stages of fruit development,most notably during the intermediate growth phase(50–90 mm ED), when volumes of 0.15 to 192 nL perfruit were observed (1,280-fold difference from lowestto highest volume). As expected, the distribution wasshifted toward larger cavities at maturity, but therewas still a considerable distribution of different vol-umes (1.4–1,000 nL per fruit; 714-fold difference; Fig.1, D and H). These data indicate that, although the

total number of cavities began to level off at ap-proximately 60 mm ED (average of 29,040 cavities perfruit), the filling continued as long as the fruit wasstill growing.

While determining cavity diameters for the calcu-lation of oil accumulated in all cavities of a fruit, wenoticed that younger fruit, when compared withmature fruit, had a substantially higher proportion ofcavities with fairly narrow, prolate spheroid shapes(Fig. 1C). As the fruit expanded, the cavities contin-ued to fill until reaching the shape of a regular sphereor an oblate spheroid (Fig. 1H). It is generally ac-cepted that the secretory cavities in Citrus are formedschizogenously (Thomson et al., 1976; Turner et al.,1998), that is, by a separation of gland cells at an earlystage, which results in the formation of a storagespace lined by ECs. It is thus tempting to speculateabout the mechanism that leads to the observedcavity volume increases. We noted that the expansionof the peel during fruit growth was marked by theformation of air spaces between the exocarp PCs(Supplemental Fig. S1). Air spaces were most prom-inent in the albedo, which consists of an extensiveaerenchyma. Flavedo air spaces were not apparent insmall fruit (up to about 50 mm ED) but constitutedmore than half of the volume of the flavedo in maturefruit (Supplemental Fig. S1). No air spaces formedbetween the thick-walled sheath cells of the secretorycavities. Their expansion was apparently directedagainst each other, tangentially to the cavity surface,thereby causing the sheath cells to bow outward,whereas the secretory cavity assumed a more spher-ical shape. Whether the final cavity shape is deter-mined by the expansion of sheath cells, theaccumulation of secretion within the cavity, or bothremains an open question that we are currently in-vestigating.

Our estimations of the oil volume per fruit, based onthe experimentally determined volume distribution of

Table II. (Continued from previous page.)

Annotation Citrus Affy Chip IdentifierEC versus PC Enrichment

28 mm 41 mm

Flavonoid pathwayChalcone synthase Cit.1966.1.S1_s_at 14.402 1.299

Cit.10216.1.S1_at 3.008 2.146Chalcone isomerase Cit.5577.1.S1_at 2.145 1.632

Cit.29773.1.S1_s_at 2.045 2.161Flavanone 3-hydroxylase Cit.2890.1.S1_s_at 15.901 7.209

Cit.2889.1.S1_at 14.602 6.341Cit.2890.1.S1_at 8.796 5.203

Flavone synthase (CYP93D) Cit.29489.1.S1_at 26.434 13.133Cit.2475.1.S1_at 21.786 13.355Cit.29489.1.S1_s_at 13.944 10.741

Flavonoid 39-monooxygenase Cit.11243.1.S1_at 12.247 3.884Cit.30703.1.S1_at 31.822 26.116Cit.4610.1.S1_s_at 49.621 12.614Cit.4610.1.S1_at 46.816 10.041

TT1 (glabra transcription factor) Cit.2963.1.S1_s_at 2.375 1.433


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secretory cavities at different developmental stages,were in very good agreement with the experimentallydetermined oil yields (Fig. 1D). Similar observationswere made in previous work with peppermint(Mentha 3 piperita [Lamiaceae]; Turner et al., 2000;Rios-Estepa et al., 2010), which demonstrated that theaccumulation of leaf essential oil correlated with thedevelopmental distribution of glandular trichomes.These anatomical structures harbor secretory cellsthat are hypothesized to be functionally related toECs in Citrus glands (Fahn, 1988). Our study hereprovides, to our knowledge, the first direct evidencethat the control of essential oil accumulation mightfollow similar patterns in the Lamiaceae and Ruta-ceae. It is also interesting that secretory cells in theLamiaceae (Lange et al., 2000; Gang et al., 2001; Laneet al., 2010) and Citrus ECs (this work) are bothhighly enriched in transcripts related to essential oilbiosynthesis. We are now expanding our analyses tofurther assess the evidence for generalizable patternsof metabolic specialization in cells involved in terpe-noid oil and resin biosynthesis across different plantphyla.

Gene Expression Patterns in Citrus Fruit Peel ECsGenerally Correlate with Essential Oil Composition

Although ECs surrounding secretory cavities havebeen hypothesized to be responsible for oil biosyn-thesis in Citrus peel (Turner et al., 1998; Lücker et al.,2002; Evert, 2006; Yamasaki and Akimitsu, 2007), ourstudy has provided, to our knowledge, the first directevidence for the biosynthetic capabilities of thesehighly specialized cells. It is important to note thatthe cavity filling in Citrus peel appears to be a rela-tively slow process when compared with the accu-mulation of essential oil in peppermint glandulartrichomes (mean filling time of 20 h; Turner et al.,2000). The genes encoding all previously character-ized terpene synthases of Citrus peel were expressedat high levels in ECs (when compared with PCs,which do not produce essential oil). Interestingly,although most terpene synthases are not expressedat appreciable levels in maturing fruit, the transcriptfor (+)-limonene synthase, which catalyzes the reac-tion to the major constituent of the essential oil ofmature fruit [(+)-limonene; Supplemental Table S1],

Figure 4. Gene expression patterns of genes in-volved in grapefruit peel essential oil biosynthe-sis, as determined by real-time qPCR (n = 3),using the b-actin transcript (corresponding Chipidentifier Cit.16435.1.S1_at) as an endogenouscontrol. The log2 (relative quantification [RQ])values are indicators of the fold change betweenthe expression levels of a gene in the essential oil-producing flavedo and nonproducing albedolayers of the peel. Transcript abundance wasquantified in peel of fruit at three developmentalstages: 34 mm ED (white bars), 43 mm ED (graybars), and 120 mm ED (black bars). Asterisks in-dicate statistical significance: * P , 0.05, ** P ,0.01, *** P , 0.001. n.d., Not determined.






stays at high levels throughout fruit development(Fig. 4).

To our surprise, although grapefruit essential oilobtained from fruit of a greenhouse-grown tree con-tained less than 0.05% sesquiterpenes (Table I), twoputative sesquiterpene synthases were enriched in theEC data set (Supplemental Table S2). In this context, itis important to note that the absolute expression valuesfor the corresponding probe sets on the Affymetrixmicroarray were fairly low (15.2 for Cit.17284.1.S1_atand 14.7 for Cit.2936.1.S1_at) or very low (2.0 forCit.29748.1.S1_s_at) in PCs, which were used as ref-erence cells. A 10-fold increase of sesquiterpene syn-thase expression levels in ECs (compared with PCs), asdetermined by qPCR (Fig. 4), still means that the ab-solute expression levels were much lower than thosefor the most abundant genes, such as (+)-limonenesynthase (expression level of 2,192.9 for Cit.9964.1.S1_s_at in ECs). Be that as it may, the enrichment oftranscripts putatively related to sesquiterpene biosyn-thesis is interesting, and a possible explanation mightbe the contribution of ECs to terpenoid volatile for-mation. The complex terpenoid mixture emitted bygrapefruit prior to maturity is significantly differentfrom that of the oil accumulated in secretory cavities,with a fairly high proportion of sesquiterpenes (24% ofemitted terpenoids as opposed to less than 2% in theoil; Flamini and Cioni, 2010).

A significant enrichment in ECs was also found forgenes involved in terpenoid precursor biosynthesisvia the mevalonate (MVA) and methylerythritol 4-phosphate (MEP) pathways. Both pathways areexpressed in all cells, as they are involved in the

biosynthesis of essential terpenoid end products(Phillips at al., 2008). In most plants, monoterpenes(the major constituents of Citrus oil) are derived pri-marily from the MEP pathway, with only little con-tribution of the MVA pathway (Phillips at al., 2008).Although the relative contributions of the MVA andMEP pathways for Citrus essential oil biosynthesisremain to be determined, it is possible that the MVApathway, which is relevant for sesquiterpene bio-synthesis in many plant species (Phillips at al., 2008),may contribute more to the sesquiterpene-rich terpe-noid emissions.

Citrus oil also contains prenylated coumarins andpolymethoxylated flavones (Fig. 5). The core skeletonsof both metabolite classes are derived from the phen-ylpropanoid pathway, and the corresponding geneswere also expressed at high levels in ECs (Table II).Very few coumarin-specific genes have been charac-terized thus far (Bourgaud et al., 2006). A gene of theCYP82C subfamily, members of which have beendemonstrated to be involved in the biosynthesis of5-hydroxylated coumarins (Kruse et al., 2008), wasfound to be highly enriched in ECs (Table II). Poly-methoxylated flavonoids are formed by successivehydroxylations and O-methylations (Willits et al.,2004; Schmidt et al., 2011), and candidate genes forenzymes involved in these steps were also found tobe highly enriched in ECs (Supplemental Table S5).These observations indicate that our gene expressiondata from Citrus glandular ECs reflect their specializationfor essential oil biosynthesis and present an excellentresource for gene discovery related to the biosynthesisof oil constituents.

Figure 5. Contribution of the terpenoid and phenylpropanoid biosynthetic pathways to the accumulation of the majorcomponents of grapefruit peel essential oil. A color code is used to indicate the origin of structural moieties in oil con-stituents: blue, terpenoid pathway; red, phenylpropanoid pathway; orange, derived from a polyketide synthase. Thethickness of the reaction arrows and the percentages next to them indicate the relative distribution of carbon flux through thedifferent pathway branches. Ac, Acetyl; DMAPP, dimethyl allyl diphosphate; DOXP, 1-deoxy-D-xylulose-5-phosphate;D-GAP, D-glyceraldehyde 3-phosphate; HMF, heptamethoxyflavone; IPP, isopentenyl diphosphate.


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MATERIALS AND METHODS

Plant Growth Conditions

Grapefruit (Citrus 3 paradisi ‘Duncan’) were harvested from a 30-year-old tree (height, 2.8 m; stem circumference, 12.5 cm) grown in thegreenhouse of the Institute of Biological Chemistry on the Pullman,Washington, campus. The mean temperature was 26°C, and the humiditywas set to 60%. Natural lighting was supplemented with sodium vaporlights that turned on at light intensities of 200 mmol m22 s21 or less, witha 14-h-day/10-h-night cycle. The tree was watered daily and fertilizedtwice a week with Peters 20/20/20. Fruits were labeled, and growthpatterns (polar diameter [PD] and ED) were measured weekly starting inearly June to late April of the following year for two growth seasons(2009/2010 and 2010/2011). The fruit surface area (S) was calculatedbased on the approximation for a specific shape, which depended on thedevelopmental stage as follows.

For the surface area of prolate spheroid:

S ¼ 2pb2 þ 2pabesin2 1 e; a ¼ PD

2; b ¼ ED

2;

where

e ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffia2 2 b2

p

a:

For the surface area of oblate spheroid:

S ¼ 2pa2 þ pb2

eln�1þ e12 e

�; a ¼ ED

2; b ¼ PD

2:

Morphometric Measurements

To determine the number of secretory cavities in grapefruit peel, a definedarea (seven to 10 patches of 300 mm2 each) of the flavedo layer (s) was handsectioned horizontally, and images were taken with a Jenoptik ProgResC12Plus camera mounted on a dissecting microscope. All cavities werecounted for the smallest fruit. In this context, it is important to note that thedensity of cavities for a fruit of a defined developmental stage did not varysignificantly in different positions of the fruit peel (assessed by countingcavities of fruit of five different developmental stages). The electronic imagefiles were uploaded into GIMP 2 (an open-source image-processing program),and the total number of secretory cavities (n) per defined area was counted.The total number of cavities per fruit (N) was then calculated based on thesurface area of the entire fruit (S):

N ¼ n 3 Ss

To measure the volumes of individual secretory cavities, images of trans-versely hand-sectioned grapefruit peel were taken with a Leica DFC425Ccamera, and the polar diameter (PD) and ED of each cavity were determinedusing the Leica Application Suite version 3 software. Assuming that the shapeof the cavities resembles that of a spheroid (of any kind), the cavity volume (V)can be estimated as follows:

V ¼ 16pED2 PD

The total volume of oil per fruit was then extrapolated by a series of cal-culations outlined in Supplemental Protocol S1.

For an evaluation of the volume distribution patterns, secretory cavities weredivided into 19 classes by volume (ranging from 0.15 to 1,000 nL) and the numberof cavities in each class was determined for fruit at various developmental stages(stage differentiation by size; 12 different sizes ranging from 6 to 136 mm ED).

Metabolite Analysis

Hydrodistillation of the Volatile Essential Oil Fraction

Three randomly taken sections of flavedo layer tissue (300 mm2 each) wereobtained from freshly harvested grapefruit using a razor blade, placed in a

mortar filled with liquid nitrogen, and ground to a fine powder. Samplematerial was then transferred to a glass flask for subsequent hydrodistillationusing a modified Likens-Nickerson apparatus (Ringer et al., 2003). An aliquotof the n-hexane fraction, which contained the volatile oil constituents andcamphor as an internal standard (final concentration at 5 ng mL21), wastransferred to a 2-mL glass vial for GC-FID analysis.

Collection of Citrus Essential Oil Using Microcapillaries

Microcapillaries of a defined size (internal diameter of 70–100 mm) were madeusing a custom-built capillary puller. Oil was collected by penetrating through thethin epidermal/subepidermal cell layers above each secretory cavity and allow-ing the liquid contents to enter the microcapillary. Roughly 4,000 cavities per fruitwere accessed from fruit representing three developmental stages (EDs of 70, 80,and 100 mm; three biological replicates each). The microcapillary contents wereemptied into a glass vial and spun at 4,000g for 2 min. A small volume of aqueousmaterial was visible at the bottom of the vial, which was most likely attributableto small amounts of cellular content from ruptured cells. It is important to note,however, that most of the cells surrounding secretory cavities remained unaf-fected by the oil collection procedure (Supplemental Fig. S1).

Terpenoids

The essential oil was diluted 100-fold with n-hexane and transferred to2-mL glass vials for GC-FID analysis. The separation of terpenoids was ac-hieved by GC-FID as described by Ringer et al. (2003). Individual componentswere quantified based on calibration curves with known amounts of authenticstandards and normalization to the peak area of camphor as an internalstandard. The sum of all components was used to determine the total oilamount injected onto the GC device, and by taking into account all dilutionsand losses, the total amount of oil recovered from each fruit was calculated.

Oxygen Heterocyclic Metabolites

Sample analysis was carried out using an ultra-high-pressure liquid chro-matograph with diode array detector (1200 series; Agilent Technologies) coupledto a quadrupole time-of-flight mass spectrometer (6520 series; Agilent Tech-nologies). The gradient separationwas performed on anEclipse Plus C-18 column(2.1 3 50 mm, 1.8 mm; Agilent Technologies) with a flow rate of 0.6 mL min21.Solvent A was 0.2% (v/v) acetic acid in water and solvent B was 0.2% (v/v)acetic acid in acetonitrile. Analytes were separated isocratically at 70% B and30% A (held for 16 min after injection). This was followed by a wash step at 98%B and 2% A (6 min) and a reequilibration at the initial conditions (5 min) be-tween runs. Authentic standards were purchased from Enzo Life Sciences(aurapten, greater than 95% purity), Selleckchem (nobiletin, greater than 99%purity; osthole, greater than 99% purity; tangeretin, greater than 99% purity),and Sigma-Aldrich (bergamottin, greater than 98% purity). Calibration curvesgenerated with authentic standards were used for the absolute quantification ofnonvolatile essential oil constituents by diode array detection at 310 nm. Massspectrometric data were used to confirm peak annotations. Additional minornonvolatile oil components were meranzin (mass-to-charge ratio [m/z] 261.113),isomeranzin (m/z 261.119), epoxyaurapten (m/z 315.160), epoxybergamottin (m/z355.155), and heptamethoxylated flavones (m/z 433.151), the concentrations ofwhich were estimated based on the published literature (McHale and Sheridan,1989; Frérot and Decorzant, 2004; Dugo et al., 2010).

Sterols

After addition of the internal standard (epi-cholesterol), sterol constituents of theoil collectedwith microcapillaries were saponified at 90°C for 60min in 2mL of 6%(w/v) KOH in methanol. Upon cooling to room temperature, 2 mL of n-hexanesand 2 mL of water were added, and the mixture was shaken vigorously for 20 s.After centrifugation (3,000g for 2 min) to separate the phases, the hexane phasewas transferred to a 2-mL glass vial. The hexane phase was then evaporated todryness using a SpeedVac concentrator, 50 mL of N-methyl-N-trimethylsilyltri-fluoroacetamide was added to the residue, the sample was shaken vigorously for20 s, and the mixture was transferred to a 2-mL autosampler glass vial with a 100-mL conical glass insert. After capping the vial, the reaction mixture was incubatedat 23°C for at least 5 min. GC-MS analyses were performed on an Agilent 6890Napparatus coupled to an Agilent 5973 inert mass selective detector (MSD) detector.Samples were loaded (injection volume of 1 mL) with a CombiPAL autosampler(LEAP Technologies) onto an HP-5MS fused silica column (30 m 3 250 mm, 0.25mm film thickness; J&W Scientific). The temperatures of the injector and MSDinterface were both set to 280°C. Analytes were separated at a flow rate of 1 mL







min21 using helium as the carrier gas and using a thermal gradient starting at 170°C (1.5 min), which was ramped first to 280°C at 37°C min21 and then to 300°C at1.5°C min21, where it was held for 5.0 min. Analytes were fragmented in electron-impact mode with an ionization voltage of 70 eV. Data were acquired using theMSD ChemStation (revision D.01.02.SP1) software. Background was subtractedand peaks were deconvoluted using the automated mass spectral deconvolutionand identification system (National Institute of Standards and Technology). An-alytes were identified based on their mass fragmentation patterns by comparisonwith those of authentic standards. Quantification was achieved based on cali-bration curves acquired with authentic standards.

Fatty Acids and Carotenoids

Fatty acids were separated and quantified as methyl ester derivatives basedon the protocol developed by Dyer et al. (2002). Carotenoid analysis wasperformed according to Fraser et al. (2000).

Gene Expression Analysis

Sample Processing for Laser Capture Microdissection andPressure Catapulting

Grapefruit peel flavedo was cut into 3- 3 3-mm sections and immediatelydipped into ethanol:acetic acid (5:1, v/v) fixative (Deeken et al., 2008). Samplespecimens were incubated in the fixative for 1 h under reduced pressure (23°C,0.78 bar) in a vacuum oven. This fixation step was repeated twice with freshfixative before incubation for 12 h at 4°C. The fixative was then replaced with100% ethanol, and specimens were incubated for 30 min at 23°C. This step wasalso repeated twice with fresh ethanol. Samples were then incubated in a se-ries of ethanol:xylene in different ratios (3:1, 1:1, and 1:3 [v/v]), each step for1 h at 23°C. For tissue embedding, samples were vacuum infiltrated in 100%xylene for 1 h, followed by a series of xylene and liquid paraffin (meltingtemperature of 45°C; Sigma) mixtures (3:1, 1:1, and 1:3 [v/v]), each step for 1 hat 23°C. Lastly, specimens were infiltrated in 100% liquid paraffin for 1 h at23°C. This step was repeated twice with fresh paraffin. Paraffin blocks werestored at 4°C in a sealed bag filled with silica gel desiccant.

Laser Capture Microdissection and Pressure Catapulting ofEpithelial and PCs

Ribbons of paraffin sections (15 mm thickness) were cut with a rotarymicrotome, expanded by floatation on a bath of diethyl pyrocarbonate-treated water at 45°C, and placed onto polyethylene naphthalate (PEN)-membrane coated slides (Zeiss Microimaging). PEN slides were irradiatedwith UV light (254 nm) for 30 min and handled according to the manu-facturer’s instructions. Slides were air dried for 20 min at 23°C, followedby melting of the paraffin on a slide warmer at 45°C for 30 min. Thisresulted in a better adherence of sections to PEN slides while furtherdrying them. At this point, representative sections were scraped off fromthe slides for an assessment of RNA quality (RNA extraction using themethod as described below). The slides were then stored in the dark at 280°C in a plastic box filled with desiccant until further use. Prior to mi-crodissection, sample slides were deparaffinized in 100% xylene for 3 minat 23°C. This step was repeated three times with fresh xylene. Slides werethen air dried in a fume hood for 15 min at 23°C. Laser capture micro-dissection and pressure catapulting (LMPC) was carried out using thePALM MicroBeam system (Zeiss Microimaging). The laser energy was setto 74 (scale from 0 to 100) and the laser focus to 57 (scale from 0 to 100).The excised target cells were catapulted and collected into caps of 0.5-mLEppendorf vials containing lysis buffer (RLT buffer; Qiagen Micro RNeasykit). Approximately 100 target cells were catapulted into one vial cap. Thecell material was collected at the bottom of the tube by brief centrifugation(15,000g) and immediately stored in a 280°C freezer until enough cellshad been collected for RNA extraction (roughly 2,500 cells per sample).

RNA Extraction from Cells Isolated by LMPC, cDNASynthesis, Amplification, and Microarray Hybridization

RNA extractions were conducted using the RNeasy Micro kit (Qiagen).LMPC-derived cell material was incubated at 37°C for 5 min, followed byvigorous mixing for 1 min at 23°C. The suspension was centrifuged at 15,000g

for 1 min, the supernatant was transferred into a new vial, and RNA wasextracted according to the manufacturer’s protocol. The RNA yield andquality were assessed using a NanoDrop 2000c spectrophotometer (ThermoScientific). RNA samples were stored at 280°C. The Whole-TranscriptomeOvation Pico RNA Amplification system version 1.0 (NuGEN Technologies)was used for cDNA synthesis and simultaneous amplification. The protocolinvolved a three-step process: (1) generation of first-strand cDNA; (2) gener-ation of a DNA/RNA heteroduplex double-strand cDNA; and (3) linear iso-thermal DNA amplification. Prior to microarray hybridization, cDNAproducts were fragmented and biotin labeled using the FL-Ovation cDNABiotin Module V2 kit (NuGEN Technologies). Once biotin labeled, cDNAswere hybridized to GeneChip Citrus Genome Arrays (Affymetrix).

Microarray Data Analysis

Microarray data analysis was performed with the Genomics Suite software(Partek). Affymetrix .cel files were imported, probe intensities were adjusted,and a background correction was carried out using the robust multiarrayaverage algorithm, which included a correction on perfect match values,quantile normalization across all of the chips in the experiment, median polishsummarization, and log2 transformation (Irizarry et al., 2006). Probe sets la-beled as absent or marginal for any of the three replicate arrays for each timepoint and cell type were removed, leaving only those that were scored aspresent among all three biological replicates. A multiple testing correction wasperformed for assessing the false discovery rate (Benjamini and Hochberg,1995), and only probe sets below a false discovery rate threshold of 0.01 werekept. Probe sets were filtered for a greater than 2.5-fold expression level dif-ference in a two-way comparison between appropriate samples. Gene anno-tation was carried out based on similarity scores in BLASTX comparisonsagainst sequences contained in the HarvEST (Close et al., 2007), The Arabi-dopsis Information Resource (Rhee et al., 2003), and Citrus (http://www.citrusgenomedb.org/) genome databases. All raw data files (.cel and .chp)were submitted to the National Center for Biotechnology Information GeneExpression Omnibus repository (http://www.ncbi.nlm.nih.gov/geo/; accessionno. GSE33964).

RNA Extraction of Fruit Peel Flavedo and Albedo andSubsequent qPCR

Flavedo and albedo layers of grapefruit peel were excised with a scalpel andprocessed separately. Samples were placed in a mortar filled with liquidnitrogen and ground to a fine powder. RNA was extracted using the ConcertPlant RNAReagent (Invitrogen) and further purified using the RNeasyMini kit(Qiagen) according to the manufacturer’s instructions. Isolated RNA (500–700ng) was treated with RNase-free DNase (Fermentas Life Science), and first-strand cDNA was synthesized using SuperScript III reverse transcriptase(Invitrogen). RNA isolation and cDNA synthesis were carried out with threedifferent grapefruit sizes (ED 34, 43, and 120 mm). Three independent bio-logical replicates were processed for each fruit size. In a 10-mL qPCR, con-centrations were adjusted to 150 nM (primers), 13 SYBR Green PCR MasterMix, and 1003 diluted first-strand cDNA as template. Reactions were per-formed on a 96-well optical plate at 95°C for 10 min, followed by 40 cycles of95°C for 15 s and 60° for 10 min in a 7500 Real-Time PCR system (AppliedBiosystems). Fluorescence intensities of three independent measurements(technical replicates) were normalized against the ROX reference dye (RocheApplied Science). For each sample, the amounts of target and endogenouscontrol (b-actin gene selected from Citrus microarray: Cit.16435.1.S1_at) weredetermined using the comparative cycle threshold method according to themanufacturer’s instructions (Applied Biosystems).

Supplemental Data

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

Supplemental Figure S1. Microscopic evaluation of secretory cavity ex-pansion in grapefruit peel at different stages of fruit development.

Supplemental Table S1. Analysis of volatile essential oil components ofgrapefruit peel by hydrodistillation.

Supplemental Table S2. Microarray analysis of transcript patterns ingrapefruit peel ECs and PCs at different stages of fruit development(28 mm and 41 mm ED of fruit).


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http://www.citrusgenomedb.org/

http://www.citrusgenomedb.org/

http://www.ncbi.nlm.nih.gov/geo/





Supplemental Table S3. GO analysis of microarray data obtained withgrapefruit peel ECs and PCs at different stages of fruit development(28 mm and 41 mm ED of fruit).

Supplemental Table S4. Primers used for quantitative real-time PCR ana-lyses of gene expression patterns in grapefruit peel flavedo and albedotissues.

Supplemental Table S5. Cytochrome P450 and O-methyltransferase genesof unknown function enriched in grapefruit peel ECs compared with PCs.

Supplemental Protocol S1. Determining the volume distribution ofsecretory cavities in grapefruit peel.

ACKNOWLEDGMENTS

We thank Dr. Daniel Cuthbertson and Dr. Glenn Turner (Institute of BiologicalChemistry) for technical assistance and editorial comments regarding the man-uscript and Mr. Derek Pouchnik (School of Molecular Biosciences) for technicalassistance with microarray hybridization. We also thank Mr. Craig Whitney andMs. Amy Hetrick (Institute of Biological Chemistry) for maintaining the plantsused in this study. We are grateful for the technical support of the FranceschiMicroscopy and Imaging Center.

Received January 19, 2012; accepted March 19, 2012; published March 27,2012.

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