A Farnesene Bio Synthesis

Cloning of hmg1 and hmg2 cDNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase and their expressionand activity in relation to α-farnesene synthesis in apple

Handunkutti P.V. Rupasinghea§, Kurt C. Almquist b‡, Gopinadhan Paliyathb*, Dennis P. Murr a

a Department of Plant Agriculture (Horticultural Science Division), University of Guelph, Guelph, Ontario N1G 2W1, Canadab Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Received 9 April 2001; accepted 2 July 2001

Abstract – In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyses the synthesis of mevalonate from3-hydroxy-3-methylglutaryl coenzyme A. It has been reported to be the rate-limiting enzyme in sesquiterpene and triterpenebiosynthesis and is encoded by a small gene family. The accumulation ofα-farnesene in the skin tissue of apple fruit duringstorage appears to be predominantly through the classical mevalonate pathway and not through the novel glyceraldehyde-3-phosphate/pyruvate pathway independent of HMGR action. The content ofα-farnesene in the skin tissue increased during thefirst 8 weeks of storage at 0 °C in air, and started declining after 12 weeks. In contrast, HMGR activity in the total membraneand soluble fractions, was the highest at the time of harvest, but decreased during the first 8 weeks in storage and remained stablethereafter. The potent ethylene action inhibitor 1-methylcyclopropene inhibitedα-farnesene evolution and HMGR activity by 97and 30 %, respectively. As a first step in studying the molecular mechanism of apple HMGR regulation, we have isolated andcloned a full-length cDNA (hmg1) as well as a fragment (hmg2), using apple skin mRNA and RT-PCR in the presence ofdegenerate oligonucleotides designed against conserved regions of plant HMGR genes. Genomic Southern analysis using probesdesigned for the 3’-end of the two cDNA clones confirmed the presence of at least two HMGR genes in apple. The cDNA forhmg1 has an open reading frame of 1 767 nucleotides. Analysis of the nucleotide sequence revealed that the cDNA encodes apolypeptide of 589 residues with a relative molecular mass of 62.7 kDa. The hydropathy profile of the putative polypeptideindicated the presence of two highly hydrophobic domains near the amino terminus. Northern blot analysis confirmed that bothhmg1 andhmg2 transcripts possessed a size of 2.4 kb. The two genes are differentially expressed during low temperature storageand in response to C2H4, with hmg1 being expressed constitutively andhmg2 being relatively more sensitive to developmentalstimuli and ethylene. © 2001 Éditions scientifiques et médicales Elsevier SAS

α-farnesene / apple / ethylene / HMGR /Malus × domestica / 1-MCP / post-harvest storage

DIG, digoxigenin / GAP, glyceraldehyde-3-phosphate / HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase / IPP,isopentenyl pyrophosphate / 1-MCP, 1-methylcyclopropene / MVA, mevalonate / RACE, rapid amplification of cDNAends

1. INTRODUCTION

A highly conserved enzyme in eukaryotes,3-hydroxy-3-methylglutaryl coenzyme A reductase

(HMGR), catalyses the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to meva-lonate (MVA), the proposed rate-limiting step ofisopentenyl pyrophosphate (IPP) biosynthesis [6, 21,24]. However, in addition to the mevalonate pathway,IPP is derived through a mevalonate-independent path-way (Rohmer pathway or glyceraldehyde-3-phosphate(GAP)/pyruvate pathway) for the biosynthesis of cer-tain isoprenoids [13, 24] (figure 1). In general, theclassical cytoplasmic mevalonate pathway providesIPP for sesquiterpene and triterpene biosynthesis [24].The Rohmer pathway is believed to be responsible for

*Correspondence and reprints: fax +1 519 824 6631.E-mail address: [email protected] (G. Paliyath).§ Present address: Guelph Centre for Functional Foods,Laboratory Services, University of Guelph, 95 Stone RoadWest, Guelph, Ontario N1H 8J7, Canada.‡ Present address: Department of Environmental Biology,University of Guelph, Guelph, Ontario N1G 2W1, Canada.

Plant Physiol. Biochem. 39 (2001) 933−947© 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS098194280101316X/FLA

the formation of all plastid-derived isoprenoid com-pounds in plants, including carotenoids, plastoquino-nes, the prenyl side chain of chlorophyll, as well asmonoterpenes and diterpenes [19, 21, 24]. In higherplants, HMGR is encoded by a multigene family innuclear DNA [24, 39]. HMGR is encoded by at leasttwo distinct genes in Arabidopsis thaliana [4], cotton(Gossypium hirsutum L.) [26], and rice (Oryza sativa)[31]; by three genes in rubber (Hevea brasiliensis)[11], tomato (Lycopersicon esculentum) [30], andpotato (Solanum tuberosum) [9]; and an even largercomplex multiple gene family in maize (Zea mays)and pea (Pisum sativum) [39].

HMGR isoforms are expressed differentially inresponse to a variety of developmental and environ-mental stimuli, such as fruit development, phytohor-mones, endogenous protein factors, light and pathogeninfection [39]. In tomato, hmg1 is highly expressedduring early stages of fruit development, when sterolbiosynthesis is required for membrane biogenesisduring cell division and expansion [30]. The expres-sion of hmg2 is not detectable in young fruit, but isactivated during fruit maturation and ripening [18, 32].Hmg1 mRNA of A. thaliana accumulates in all parts of

the plant, while the presence of hmg2 mRNA isrestricted to young seedlings, roots and inflorescence[14]. Cotton hmg2 encodes the largest of all plantHMGR enzymes described to date and contains sev-eral functional specialization features that include aunique 42 amino acid sequence located in the regionseparating the amino-terminal domain and carboxy-terminal catalytic domain, that is absent in hmg1 [26].

The presence of multiple HMGR genes in plants isconsistent with the hypothesis that different isoformsof HMGR could be involved in separate subcellularpathways to produce specific isoprenoid end-productsthrough metabolic channels, or ‘metabolons’ [6, 39]. Anumber of investigators have reported a correlationbetween the induction of isoprenoid biosynthesis,particularly that of sesquiterpenes, and HMGR enzymeactivity [6]. In potato, the expression of specificHMGR genes has been correlated with the accumula-tion of steroids or sesquiterpenes [9]. Chye et al. [11]observed that only hmg1 was inducible by C2H4

among HMGR genes, and also speculated that distinctisoprenoid pathways do occur for rubber biosynthesisin H. brasiliensis. In cotton, hmg2 has been associatedwith the synthesis of specific sesquiterpenes in devel-oping embryos [26]. These results support the conceptof metabolic channels, or arrays of isoenzymes that areindependently-regulated and dedicated to the produc-tion of specific isoprenoids [6].

The acyclic sesquiterpene α-farnesene (C15H24;[3E,6E]-3,7,11-trimethyl-1,3,6,10-dodecatetraene) ac-cumulates in the skin of apple fruit exclusively afterharvest and during prolonged low temperature (0 to1 °C) storage [37]. The extent of oxidation ofα-farnesene to conjugated trienes has been shown tobe proportional to the development and severity of thepost-harvest physiological disorder superficial scald[8, 16, 28]. Biosynthesis of α-farnesene in apple skinis highly regulated by temperature [37] and C2H4 [34,35]. However, biochemical and molecular mechanismsof modulation of HMGR activity in relation toα-farnesene biosynthesis in apple has not been reported.Identification of regulatory factors of HMGR andcloning of HMGR gene(s) that may potentially bedirected to α-farnesene biosynthesis, will provideimportant insight into the understanding and the roleof α-farnesene in superficial scald development inapple fruit. Therefore, as a first step towards under-standing the regulation of α-farnesene accumulationby HMGR, total in vitro HMGR enzyme activity andexpression of two novel cDNAclones encoding HMGRin apple, hmg1 and hmg2, were studied in relation tolow temperature storage and C2H4 action.

Figure 1. A simplified biosynthetic pathway for plant isoprenoids.PP, pyrophosphate; HMGR, 3-hydroxy-3-methylglutaryl coenzyme Areductase.

934 H.P.V. Rupasinghe et al. / Plant Physiol. Biochem. 39 (2001) 933–947

2. RESULTS

2.1. HMGR activity in apple skin during storage

To decipher the relation between HMGR activityand α-farnesene synthesis, the levels of α-farneseneand HMGR activity were analysed. Previous studieshave shown that α-farnesene is predominantly local-ized in the skin tissue of apples [36]. The content ofα-farnesene in the skin tissue of ‘Delicious’ applesincreased during the first 8–10 weeks of storage at0 °C in air, and decreased after 12 weeks (figure 2). Incontrast, HMGR activity, as determined by the con-version of [4-3H]-HMG CoA to MVA in the totalmembrane and soluble fraction, was the highest at thetime of harvest and gradually decreased during the first8 weeks, and then remained constant during the rest ofthe storage period (20 weeks).

2.2. Incorporation ofradiolabelled-IPP precursors

The substrates that are channelled through theisoprenoid pathway may originate from differentsources, the first being acetyl CoA and the secondbeing glyceraldehyde-3-phosphate and pyruvate. Thelatter step bypasses the HMGR and leads directly tothe formation of isopentenyl pyrophosphate (figure 1).Since there was an inverse correlation betweenα-farnesene levels and HMGR activity, it was neces-sary to determine whether the increase in α-farnesenewas due to metabolite channelling through theGAP/pyruvate pathway. To determine whether theGAP/pyruvate pathway was present in the apple skintissue, and the relative contributions of the HMGRpathway and GAP/pyruvate pathway to α-farnesenebiosynthesis, R[5-3H]-mevalonic acid or a mixture of[2-14C]-pyruvic acid and GAP, was supplied to iso-lated apple skin tissue under in vitro conditions.Incorporation of radiolabel into α-farnesene was 105-fold higher with mevalonic acid compared with themixture of pyruvate and GAP (table I). In addition, in

a separate study when apple skin discs were incubatedwith unlabelled MVA, or a mixture of GAP andpyruvate, α-farnesene levels (HPLC analysis) werehigher in MVA-treated skin tissue than that of GAPand pyruvate-treated skin tissue (data not presented).

2.3. Inhibition of HMGR activity by Lovastatin

To further confirm the role of HMGR in α-farnesenebiosynthesis, apple fruits were treated with Lovastatin,a specific inhibitor of HMGR [1], and the levels ofα-farnesene in the skin tissue determined after storagefor 8, 12 or 16 weeks at 0–1 °C in air. When apple fruitwere treated with Lovastatin (200 mg·L–1), α-farneseneaccumulation in the skin was suppressed by 25 to 54 %during storage (table II). Lack of a complete inhibitionof α-farnesene synthesis by Lovastatin could be due toincomplete uptake and incorporation of the inhibitorinto the cells of apple skin where HMGR is located.

Table I. Incorporation of radiolabel from precursors of isopentenyl pyrophosphate into α-farnesene in isolated skin tissue of Lovastatin-treated(200 mg·L–1) apples. Six discs (1 cm in diameter and 2–3 mm in thickness) isolated from three apples were incubated in the presence of radiolabel.Data represent the mean of three such replicates. The α-farnesene formed was extracted with hexane, concentrated by evaporation in a stream ofnitrogen and subjected to separation using thin layer chromatography. Region corresponding to authentic α-farnesene was scraped and mixed withscintillation fluid to quantify the radiolabel.

Precursor Radioactivity incorporated into α-farnesene (pmol)

R[5-3H]-mevalonic acid 741A mixture of [2-14C]-pyruvic acid and 25 mM glyceraldehyde-3-phosphate 0.01

Figure 2. α-Farnesene content (A) and in vitro HMGR activity in themembrane fraction and the soluble fraction (B), in ‘Delicious’ applesduring storage at 0 °C in air. All parameters are expressed on a freshweight basis and each data point is the mean of three replicates.

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These results further imply that, in apple fruit, thebiosynthesis of α-farnesene occurs predominantlythrough the classical MVA pathway and not throughthe GAP/pyruvate pathway.

2.4. Relationship between C2H4and HMGR activity

HMGR activity is influenced by various environ-mental and hormonal signals [29, 30, 39]. Ethylenebiosynthesis increases during ripening and post-harveststorage, which enhances metabolic activity, and thisincreased metabolic activity may involve the partici-pation of HMGR. To understand the relationshipsbetween ethylene, HMGR activity and α-farnesenelevels, apples were exposed to the gaseous ethyleneaction inhibitor 1-MCP, and HMGR activity andα-farnesene levels were determined. Apples treatedwith 1-MCP (600 nL·L–1) after harvest, exhibitedcomplete inhibition (100 %) of C2H4 production uponremoval from storage (table III). α-Farnesene contentin the skin and in the head-space volatiles of 1-MCP-treated apples was inhibited by 72 and 100 %, respec-tively. In vitro activity of HMGR in apple skin tissue

was inhibited by 30 % when C2H4 action was blockedby 1-MCP. In addition, 1-MCP inhibited respiratoryCO2 evolution by nearly 60 % (table III) whichsuggests that inhibition of α-farnesene synthesis inapple by 1-MCP, could also be mediated throughrespiratory regulation, which provides C2 skeletons(acetyl CoA) for isoprenoid biosynthesis.

2.5. Isolation and analysis of apple hmg1and hmg2 cDNAs

To understand the molecular regulation of HMGR,experiments were designed to isolate the cDNA forHMGR from apple tissue. Degenerate primers weredesigned based on the CODEHOP algorithm [33]using the sequence information available on higherplant HMGR genes. Using mRNA isolated from appleskin tissue and RT-PCR in the presence of the primers,a 549-bp PCR product was obtained from the cDNAlibrary. The fragment was sequenced and its identityconfirmed as an HMGR cDNA fragment. HMGR-specific primers for 5’ - and 3’ -RACE were designedbased on the sequences of this HMGR cDNA frag-ment. Forward and reverse primers were designed tothe 5’ - and 3’ -RACE products. Using these cDNAfragments and PCR amplification, a full-length cDNA(hmg1) with an open reading frame of 1 767 bp (figure3) was isolated. Apple hmg1 cDNA encodes a putativepolypeptide of 589 amino acids with an estimatedmolecular mass of 62 727 Da and an estimated iso-electric point (pI) of 5.79. As a comparison, theapparent molecular mass of HMGR protein ofH. brasiliensis estimated by western blot analysis is59 000 Da [10].

The deduced amino acid sequence of apple hmg1cDNA was aligned with that of other plant HMGRgenes and showed very close similarity (figure 4). Thepredicted amino acid sequence of apple hmg1 shares79 % homology with the hmg1 gene of Camptothecaacuminata [3], 75 % homology with hmg1 of H. bra-siliensis [10], 80 % homology with hmg1 of G. hirsu

Table II. α-Farnesene content in the skin of apples treated with orwithout Lovastatin (200 mg·L–1), a specific HMGR inhibitor. Appleswere treated soon after harvest and stored at 0 °C in air for 8, 12 or16 weeks. Data represent mean of three independent estimations ± SD.For each estimation, a total of six discs (1 cm in diameter and 2–3 mmin thickness) were excised from three apples and extracted with 3 mLhexane overnight. After clarification by filtration through a 0.22-micron filters, an aliquot was subjected to HPLC separation using aNova-Pak C18 column and acetonitrile as the mobile phase.α-Farnesene was quantified using a standard curve.

Treatment α-Farnesene content in the skin (µg·g–1 FW)Storage period (weeks)

Total

8 12 16

Control 396 ± 35 567 ± 48 446 ± 39 1 409Lovastatin 182 ± 21 277 ± 31 338 ± 26 797

Table III. Metabolic responses of apples treated with the C2H4 action inhibitor, 1-methylcyclopropene (1-MCP, 600 nL·L–1). Each variaterepresents the mean value of response measured in triplicate after 5 d at 20 °C following removal from storage for 60 d at 0 °C in air. One unit ofvolatile α-farnesene = peak area × 10–6. *, **, *** Significant at P < 0.05, 0.01 or 0.001, respectively.

Metabolic response Untreated MCP Inhibition Significance

C2H4 production (µL·h–1·kg–1) 114 0.49 ∼ 100 % ***HMGR activity (pkat·mg–1 protein) 10.3 7.25 30 % *α-Farnesene content in the skin (µg·g–1) 621 126 72 % *α-Farnesene evolution (unit·kg–1) 7.23 0.01 ∼ 100 % ***CO2 production (mL·h–1·kg–1) 8.6 3.5 59 % ***


Figure 3. Combined nucleotide sequence and the predicted amino acid sequence of the encoded product of the cDNA corresponding to hmg1 fromM. × domestica cv. Delicious. The amino acid sequence is shown in one-letter code below the corresponding codons. Lower case letters indicateuntranslated regions and the asterisk (*) indicates the stop codon. Regions used to design the oligonucleotide primers to synthesize hmg1 specificRNA probe are underlined. GenBank accession number is AF315713.


tum [25] and 72 % homology with hmg1 of A. thaliana[4]. Among all the identified plant HMGR genes, theN-terminal region differs greatly both in length andamino acid sequence (figure 4).

The hydropathy profile of the protein, deduced bythe algorithm of Kyte and Doolittle [20], shows closesimilarity to the typical structural features of otherplant HMGR genes; e.g. hmg1 of A. thaliana [22], H.

Figure 4. Alignment and comparison of deduced amino acid sequence of M. × domestica hmg1 with hmg1 sequences of A. thaliana (GI 123340;[4]), C. acuminata (GI 289881; [27]), G. hirsutum (GI 2935298; [26]) and H. brasiliensis (GI 18835; [11]). Dots denote spaces introduced tomaximize alignment and black shaded amino acid stretches indicate regions identical among the five comparisons.


brasiliensis [10], and hmg3 of C. acuminata [27](figure 5A). The two hydrophobic regions locatedbetween residues 22 to 46 and 67 to 86 (figure 5B),each of which is long enough to span a membranebilayer, are conserved in other HMGRs characterizedin plants. HMGR is a membrane-bound enzyme [1,15]. It has previously been postulated that theseN-terminal hydrophobic regions could correspond totrans-membrane domains [4, 11, 26, 30]. Furthermore,the carboxyl-termini of all plant HMGRs are highlyconserved and the catalytic site of the enzyme islocated within this region [11].

HMGR belongs to a multigene family. As a strategyto isolate other HMGR genes from apple fruit, addi-tional degenerate oligonucleotide primers (forward)were designed for the 3’ -end region, and 3’ -RACEperformed. A 565-bp fragment of cDNA (hmg2) wasisolated (figure 6), and the coding region (303 bp) ofhmg2 fragment showed 79 % nucleotide sequencehomology and 87 % amino acid homology to the applehmg1.

2.6. Southern blot analysisof apple genomic DNA

To verify that the cDNAs of hmg1 and hmg2 arederived from two different genes, genomic Southernanalyses were performed using gene-specific DIG-UTP RNA probes as described in Methods. Thehybridizations, performed under high stringency con-ditions where probes show high specificity for eachgene, indicated that each probe identified a differentset of genomic fragments (figure 7). Under low-stringency hybridization conditions, a mutually comple-mentary pattern of bands was observed, indicating thatboth genomic sequences are closely related (data notshown). The results confirm that apple hmg1 and hmg2

Figure 5. Sequence and domain characteristics of HMGRs. A,Hydropathy index plot of the predicted amino acid residues. Applehmg1 (I), A. thaliana hmg1 (II), H. brasiliensis hmg1 (III), and C.acuminata hmg3 (IV). The average hydrophobicity of each amino acidwas calculated using the algorithm of Kyte and Doolittle [20] over awindow size of nine residues, and was plotted as a function of aminoacid position. Positive values indicate that free energy is required fortransfer to water, characteristic of hydrophobic regions. H1 and H2indicate two probable trans-membrane domain (membrane-spanning)regions. B, Schematic representation of the domain structure of appleHMGR isoform encoded by hmg1. Abbreviations: H1 and H2, highlyconserved membrane-spanning sequences; LS, highly conservedlumenal sequence. Numbers inside the domains indicate the respectivenumber of amino acid residues.

Figure 6. Nucleotide sequence ofthe fragment of hmg2 fromM. × domestica cv. Delicious, andthe predicted amino acid sequenceof the encoded product. The aminoacid sequence is shown in one-letter code below the correspond-ing codons. Lower case lettersindicate untranslated regions andthe asterisk (*) indicates the stopcodon. Regions used to design theoligonucleotide primers to synthe-size hmg2 specific RNA probe areunderlined. GenBank accessionnumber is AF316112.


cDNAs are from two independent genes, and there areat least two genes in the HMGR gene family of apple.Several well-documented studies confirm the presenceof multiple copies of HMGR genes in plants [4, 9, 11,24, 26, 31, 39, 40]. The detection of HMGR activity incytosolic and membrane fractions (figure 2) also sug-gests that these enzymes might have derived fromdifferent HMGR genes in apple fruit.

2.7. Northern blot analysis on apple total RNAThe transcript size of hmg1 and hmg2 was estimated

by northern blot analysis. RNA was isolated from theapple skin tissue and subjected to agarose gel electro-phoresis. After blotting to nylon membranes, the RNAwas allowed to hybridize with DIG-labelled hmg1 andhmg2 RNA probes. Under high stringency conditionswhere the probes behave as specific as possible foreach gene, both hmg1 and hmg2 genes showed asimilar size of 2.4 kb (figure 8). This is similar to theresults obtained from H. brasiliensis [10, 11] and A.thaliana [4] where the transcript size was found to beof the order of 2.4 kb. The relative abundance of hmg1transcript in the skin of apple fruit was considerablyhigher than that of hmg2.

2.8. Expression of hmg1 and hmg2in relation to storage and C2H4 production

To study the expression of hmg1 and hmg2 genesduring post-harvest storage, RNA was isolated from

apple skin tissue at harvest and at 4-week intervalsduring the 16-week storage period and subjected tonorthern blot analysis (figure 9). Abundance of thehmg1 transcript was nearly constant during the first8 weeks of storage, and declined during further storageup to 16-weeks. In contrast, hmg2 showed a relativelylesser abundance of transcript during early periods ofstorage, with increasing abundance noticed at 4 weeksand a peak of accumulation at 8-week after harvest.Thus, it appears that hmg1 is expressed constitutivelyduring storage of apples, whereas the accumulation ofhmg2 mRNA, appears to peak at the time when fruitα-farnesene content (figure 2) and C2H4 production(data not shown) are also high during storage. In ourmodel system, the C2H4 action inhibitor 1-MCP sup-pressed the expression of hmg2 completely, but onlypartially that of hmg1 (figure 10).

3. DISCUSSION

The development of superficial scald in apples hasbeen extensively studied, however, the explanationsfor the biochemical mechanisms of scald developmentare still hypothetical. The biosynthesis and degrada-tion of the sesquiterpene α-farnesene has drawn con-siderable attention from researchers. Recent studiesfrom our laboratory have revealed several interestingaspects on the biosynthesis of α-farnesene [34–37].α-Farnesene is synthesized by the enzyme α-farnesene

Figure 7. Genomic Southern blot hybridization analysis to verify thenumber of HMGR genes in apple genome. Apple genomic DNA(10 µg per lane) was digested with BamHI (lanes 1), EcoRI (lanes 2)and PstI (lanes 3). Fragments were electrophoretically separated,transferred to a positively charged membrane (BrightStar™, Ambion)and hybridized with DIG-labelled RNA probe representing either thehmg1 or hmg2 cDNA. DNA molecular size markers are indicated onthe right.

Figure 8. Northern blot-hybridization of apple total RNA (10 µg perlane) probed with hmg1 (A) and hmg2 (B) specific DIG-labelled RNAprobes to show the size(s) of transcripts. Mobility of molecular sizemarkers (bp) is indicated on the right.


synthase, which catalyses the direct conversion offarnesyl pyrophosphate to α-farnesene through anallylic carbocation intermediate which possesses apositive charge at its C2 position. An elimination of aproton from the C1 position and rearrangement wouldcreate double bonds at C1 and C3 positions leading tothe formation of farnesene (3,7,11-trimethyl, 1,3,6,10-dodecatetraene) [36]. α-Farnesene synthase activityincreased during storage of apples, almost in parallelwith the accumulation of α-farnesene in the fruit skin[37]. However, the existence of additional regulatorysteps became apparent when studies revealed thatinhibition of ethylene biosynthesis in apples by 1-MCPtreatment reduced α-farnesene accumulation but didnot alter α-farnesene synthase activity [34]. A potentialregulatory site that could affect α-farnesene biosynthe-sis is at the level of HMGR, through regulating the

flow of substrates. Thus, these studies were performedin apple fruit to understand the potential regulation ofisoprenoid pathway and α-farnesene biosynthesis atthe level of HMGR.

3.1. Characterization of HMGR activityin apples

HMGR of apple tissue appears to be highly con-served and similar in properties to several otherHMGRs reported in plants. Plant HMGR is membrane-bound [1, 15], and the presence of two trans-membranedomains in HMGR protein suggests that the enzyme isassociated with the endoplasmic reticulum (ER) [10,14]. Recently, Campos and Boronat [5] demonstratedthat in A. thaliana, these sequences function as internalsignal sequences by specifically interacting with thesignal recognition particle (SRP), and mediating thetargeting of the protein to ER-derived membranes.Although it is evident that HMGR is associated withmicrosomal membranes, the enzyme activity has alsobeen detected in mitochondrial and plastid membranes[2, 6]. The presence of HMGR activity in the solublefraction of apple skin tissue extracts suggests that thesoluble fraction prepared after 105 000 × g centrifuga-tion, may potentially contain non-sedimentable frag-ments of cellular membranes formed during the homog-enization process of extraction. Alternatively, HMGRprotein may proteolytically release a cytosolic frag-ment containing the active site that is fully functionalas the native enzyme. The enzyme responsible for thebiosynthesis of the C15 isoprenoid intermediate, farne-syl pyrophosphate (FPP), FPP synthase, is observed tobe in the cytoplasmic compartment and its productFPP serves as a precursor for sesquiterpene and sterolbiosynthesis in cytoplasm/ER [23]. Similarly, the high-est specific and total activity of trans,trans-α-farnesenesynthase, which catalyses the conversion of FPP intoα-farnesene, was located in the cytosolic fraction inapples [37]. Therefore, it could be speculated thatenzymes involved in α-farnesene biosynthesis arelocalized in the cytosol/ER boundary and could oper-ate as a highly organized supramolecular complex [6,39].

Accumulation of α-farnesene in the skin of apples,is triggered by low temperature storage and reaches apeak during 8 to 12 weeks in storage. By contrast, invitro HMGR activity was the highest at the time ofharvest, gradually decreased during the first 8 weeksof storage, and then remained constant during theremainder of the storage period. Very similar changesin HMGR activity and accumulation of abscisic acidhave been observed during the development of maize

Figure 9. Expression of apple hmg1 and hmg2 during 0, 4, 8, 12, and16 weeks of storage at 0 °C in air. Total RNA was extracted from theskin tissue of apple fruits and stored at –70 °C until used for thenorthern blot hybridization. RNA samples were hybridized with theDIG RNA-labelled probe specific to hmg1 and hmg2 cDNA. The 18SrRNA stained with ethidium bromide and photographed before blot-ting is shown below the blot.

Figure 10. Effect of the C2H4 action inhibitor 1-methylcyclopropene(1-MCP) on the expression of apple hmg1 and hmg2 (lane 1,untreated; lane 2, 1-MCP-treated). Apples were exposed to 0 or600 nL·L–1 1-methylcyclopropene after harvest for a period of 18 hand placed in storage at 0 °C in air with 90 to 95 % relative humidityfor 60 d.


seeds, wherein, the HMGR activity was the highest10–12 d after pollination, when the mitotic activitywas the highest. The endosperm-localized HMGRactivity declined by over 80 % during maturation.However, during the same period the level of abscisicacid, an isoprenoid product, increased [29]. The lackof a positive correlation between HMGR activity andα-farnesene accumulation also raises the questionregarding the origin of isopentenyl pyrophosphatesupply for α-farnesene biosynthesis, whether it occursthrough the HMGR-dependent pathway or through theHMGR-independent Rohmer pathway during the periodwhen the decline in HMGR activity was observed. Theexistence of this novel pathway for IPP formation fromGAP and pyruvate, which bypasses the HMGR reac-tion, is well established in higher plants (figure 1) [13,19, 21, 24, 32]. However, the present results indicatethat incorporation of radiolabelled or unlabelled meva-lonic acid into α-farnesene is relatively favoured overa mixture of GAP and pyruvic acid in isolated appleskin tissues, even considering the differences in spe-cific activity and potential internal dilution. Therefore,it is evident that the biosynthesis of α-farnesene occurspredominantly through the classical MVA pathway inapple fruit. This conclusion is also supported by theobservation that Lovastatin, a competitive inhibitor ofHMGR [1], inhibits α-farnesene accumulation signifi-cantly (by 25 to 54 %) in apple skin during storage.Recently, Ju and Curry [18] found that when Lova-statin was applied to apple fruit tissue at high concen-trations (1 g·L–1), α-farnesene biosynthesis was sup-pressed to undetectable levels in ‘Delicious’ and‘Granny Smith’ apples. The GAP/pyruvate pathway isresponsible mainly for the formation of plastid-derivedisoprenoid compounds in plants, including carotenoids,plastoquinones, the prenyl side chains of chlorophyll,[19, 24, 32], monoterpenes and diterpenes [13]. How-ever, the classical cytoplasmic mevalonate pathway isspecialized in providing IPP for sesquiterpenes andtriterpenes [24]. Together, these results imply that inapple fruit, the biosynthesis of α-farnesene occurspredominantly through the classical MVA pathway.

3.2. Isolation of hmg1 and hmg2 cDNAsfrom apple and their expression during storage

To further study the regulation of HMGR activity inrelation to the accumulation of α-farnesene in applefruit, cDNAs of two HMGR genes, the first having afull length (hmg1) and the second being a fragment(hmg2), were cloned. All plant HMGR genes identifiedto date share some common structural features [27].

They are highly conserved at the carboxyl-terminalregion, highly divergent at the amino-terminal region,and possess sequences for two putative trans-membranedomains, which is a common feature of all the HMGRgenes cloned to date from plants, but differ from theanimal HMGR genes which possess sequences forseven trans-membrane domains. HMGR is a membrane-bound enzyme [1, 15], and it was previously postu-lated that these amino-terminal hydrophobic regionscould correspond to trans-membrane domains [4, 11,26, 30]. The catalytic site of the enzyme is locatedwithin the highly conserved carboxyl-terminal region[11].

From our study, it appears that the induction ofspecific HMGR isozyme(s) could be involved in theaccumulation of α-farnesene in apple skin duringstorage. Northern blot analysis revealed that hmg1 andhmg2 were differentially expressed in apples duringcold storage. It is interesting to note that hmg1 isexpressed constitutively while hmg2 showed the high-est levels of transcript when the accumulation ofα-farnesene in the skin also attained a high levelduring storage [37]. In addition, the abundance ofhmg2 transcript increased in parallel with endogenousC2H4 production. However, the abundance of hmg2mRNA was relatively low compared with that ofhmg1. The differential regulation of hmg1 and hmg2expression in apple is consistent with the theory thatlevels of the different HMGR isozymes in plants, aremodulated in response to specific developmental andstress signals [39]. These may involve post-transcriptional events such as mRNA processing, tran-script stability, nucleo-cytoplasmic transport, transla-tional efficiency, and/or protein modification andproteolysis. HMGR is also regulated by a proteinkinase in which phosphorylation inactivates the enzyme.As an example, the catalytic domain of the HMGRenzyme from the hmg1 gene of A. thaliana expressedin E. coli, was reversibly inactivated by a Brassicaoleracea HMGR kinase in a cell-free system [12].Calcium, calmodulin and proteolytic degradation mayalso have a role in the regulation of plant HMGRs[39]. In tomato, both HMGR activity and mRNAlevels are high at early stages of fruit development,when rapid cell division occurs, as well as during theearly stages of cellular expansion [30]. Narita andGruissem [30] postulated that the final period of fruitexpansion and ripening is not dependent upon HMGRactivity, but instead utilizes a pre-existing pool ofpathway intermediates such as isoprene units. Thus,several internal and external factors regulate the expres-sion and activity of HMGR.


3.3. Ethylene stimulates HMGR activityand regulates hmg2 expression

Our results indicate that C2H4-mediated stimulationof α-farnesene biosynthesis is partly due to the induc-tion of HMGR activity by C2H4. In apples, the C2H4

action inhibitor 1-MCP suppressed the expression ofhmg2 completely and hmg1 partially. 1-MCP alsoinhibited respiratory CO2 evolution by 59 %, whichsuggest that MCP-mediated inhibition of α-farnesenesynthesis in apple, could also be regulated through theavailable acetyl CoA pool that is utilized in isoprenoidsynthesis. However, it is clear from the literature thatC2H4 or other stimuli which induce C2H4 production,can influence differential expression of the isogenes ofHMGR. In rubber, hmg1 is induced by C2H4 whilehmg3 expression remains stable, indicative of thehouse-keeping role of the gene [11]. Furthermore,hmg1 is expressed predominantly in laticifers, the cellsspecific to rubber biosynthesis [11]. Thus, it is postu-lated that hmg1 of rubber plants encodes the HMGRenzyme involved in rubber biosynthesis. In tomato,hmg1 expression is very high at early stages of fruitdevelopment but declines during ripening [17, 30], buthmg2 is highly expressed during ripening. In C.acuminata, hmg1 mRNA increased in response towounding, but hmg2 and hmg3 transcript levelsremained unaffected [27]. In stem apices, hmg1 isexpressed at high levels, hmg3 is moderately expressedand hmg2 transcripts are altogether absent [27]. Simi-larly, in potato, hmg2 mRNA levels are elevated inresponse to wounding or fungal elicitors, suggestingthat hmg2 is a defence-related or the major elicitor-induced isogene [40]. Thus, it appears that the expres-sion of specific HMGR genes in specific plant organsand tissues could be used as a mechanism for regulat-ing the supply of mevalonate to metabolic pathwayslocalized in those places. We anticipate identificationof regulatory factors of HMGR and cloning of HMGRgene(s) specifically directed to α-farnesene biosynthe-sis, which will provide important insight into under-standing the role of α-farnesene in apple fruit devel-opment and the susceptibility of apples to superficialscald.

4. METHODS

4.1. Plant material and storage

‘Delicious’ apples (Malus × domestica Borkh.) wereharvested on 5 October 1999, from the HorticulturalResearch Station of the University of Guelph, Simcoe,

Ontario (harvest maturity indices: firmness, 73.3 N;soluble solids, 10.3 %; starch-iodine index (Cornellchart), 3.2). Apples were transported to Guelph andwere cooled to 0 °C within 8 h of harvest and stored at0 to 1 °C in air with 90 to 95 % relative humidity.These apples were used for monitoring HMGR activ-ity and expression during storage.

4.1.1. Post-harvest treatments

For HMGR inhibition study, one sample of apples(120 fruits) was dipped for 20 min in deionized watercontaining 200 mg·L–1 Lovastatin (a generous giftfrom Merck Frosst) with 0.5 mL·L–1 ABG 7011™(Abbott Laboratories) as surfactant. A second sampleof 120 fruit was dipped only in D.I. water containingthe surfactant (control). To study the effect of C2H4

inhibition, apples were allowed to equilibrate to 20 °Covernight after harvest and exposed to 0 or 600 nL·L–1

1-MCP (EthylBloc™, generously supplied by Dr Har-low Warner, Rohm and Haas Inc., Philadelphia) for18 h. Following treatment, the apples were placed incold storage at 0 °C and 90 to 95 % relative humidityfor 60 d.

4.1.2. Incorporation of radiolabel into tissue

For the precursor channelling study, either 9.25 kBqR[5-3H]-mevalonic acid (740 GBq·mmol–1; ARC) or amixture of 9.25 kBq [2-14C]-pyruvic acid(18.5 MBq·mmol–1; ARC) and 25 mM GAP (Sigma)dissolved in 10 mM MES buffer (pH 5.6) were vacuuminfiltrated (2 min) into isolated apple skin tissues(6 discs, each 2 mm in thickness and 1 cm in diameter)and incubated at 20 °C for 90 min. α-Farnesene wasisolated by incubating the treated discs in hexaneovernight at 4 °C. Apples treated with Lovastatin(200 mg·L–1) at harvest and subjected to storage for10 weeks at 0 °C in air, were used for the experiment.To exclude the potential for non-enzymatic incorpora-tion of radiolabel into α-farnesene, control experi-ments with boiled apple skins were conducted.

4.2. HMGR enzyme assay

HMGR assays were conducted by a method essen-tially as described by Chappell et al. [7]. Approxi-mately 200 g outer cortical tissue (0.25 to 0.5 cmdepth) including the cuticle was removed from apples,cut into 0.25 to 0.5-cm3 pieces, and homogenizedusing a Polytron homogenizer (Brinkmann Instru-ments, model PT 10/35) for 1 min in 150 mL 100 mMMOPS buffer (pH 7.5) containing 0.25 M sucrose,1 mM EDTA, 5 mM DTT, 5 mM MgCl2, 1 mM PMSF,


3 % BSA, and 50 g·L–1 polyvinylpyrrolidone. Thehomogenate was filtered through four layers of cheese-cloth, and the filtrate was centrifuged at 3 000 × g for10 min to remove starch and debris. The resultingsupernatant was centrifuged at 105 000 × g for 45 minand the membrane pellet was resuspended in 0.1 Msodium phosphate buffer (pH 6.5) containing 2.5 mMDTT. The supernatant containing soluble proteins wascollected for further experiments. Aliquots of totalmembrane or supernatant, equivalent to 100 µg proteinwere incubated for 45 min at 30 °C in a final assayvolume of 250 µL containing 100 mM sodium phos-phate buffer (pH 7.0), 3 mM NADPH, 10 mM DTT,20 µM HMG-CoA, and 0.925 kBq DL-[3-14C]-HMG-CoA (215 MBq·mmol–1; Amersham). The assay wasterminated by the addition of 20 µL 5 mM mevalonatelactone and 50 µL 6 N HCl followed by vortex stir-ring. The mixture was incubated for an additional30 min at room temperature to allow the radiolabelledmevalonate formed to lactonize. After addition of100 µL saturated potassium phosphate (pH 6.0) and300 µL ethyl acetate, the samples were briefly vor-texed and centrifuged, and an aliquot of the upperorganic phase containing the mevalonate lactone, wasused to quantify the radiolabel by liquid scintillationcounting. The chemical nature of the product wasconfirmed by analysing the ethyl acetate fraction usingthin layer chromatography along with authentic meva-lonate lactone, in a solvent system containing chloro-form and acetone (2/1, v/v), and measuring the radio-activity in the mevalonate lactone-containing zone(Rf = 0.6). The assay was performed in triplicate.

4.3. RNA isolation and cDNA construction

Five grams of apple skin tissue (0.5 to 1 mmthickness) was ground to a fine powder with a mortarin a pestle cooled with liquid N2. The powder wasadded immediately to 15 mL preheated (65 °C) lysisbuffer (150 mM Tris HCl, pH 7.5, 50 mM EDTA, 4 %SDS, 2 % PVP, and 1 % (v/v) �-mercaptoethanol,adjusted to pH 7.5 with boric acid) and vortexed for30 s at room temperature. The suspension was homog-enized with a Polytron homogenizer at maximumspeed for 20 s. Cold absolute ethanol (0.25 vol) and5 M potassium acetate (0.1 vol) were added and mixedfor a further 30 s. One volume of chloroform/isoamylalcohol (24/1) (Sigma) was added to the homogenate,mixed by vortex stirring and centrifuged at 20 000 × gfor 10 min at 4 °C. The recovered aqueous phase wasextracted twice with an equal volume of 10 mM Tris(pH 8.0), equilibrated with phenol/chloroform/isoamylalcohol (25/24/1, v/v/v) (Sigma). The RNA was selec-

tively precipitated by adding LiCl2 to a final concen-tration of 3.2 M and incubated overnight at –20 °C.RNA was pelleted by centrifugation at 20 000 × g for20 min at 4 °C. The pellet was resuspended in 2 mLDEPC-treated water, and sodium acetate (final concen-tration of 0.3 M) and 6 mL cold absolute ethanol wereadded. After 1 h incubation at –20 °C, RNA was pel-leted by centrifugation for 10 min at 12 000 × g andthe pellet was resuspended in 200 µL RNase-freewater and stored at –80 °C. The messenger RNA wasisolated from the total RNA using an Oligotex™mRNA Spin-Column kit (Oligotex). The total RNAextracted from the skin tissue of ‘Delicious’ applesstored for 12 weeks in air at 0 °C, was used. Synthesisof cDNA by reverse transcription of mRNA wasperformed using a SMART™ RACE cDNA amplifi-cation kit (Clontech). A 20-µL aliquot of reactionmixture containing 1 µg mRNA, 1 µg oligo (dT) andsterile distilled water was heated to 70 °C for 2 minand chilled quickly on ice. After a brief centrifugationof the tube, 4 µL first strand buffer (5×), 2 µL 0.1 MDTT, and 1 µL 10 mM dNTP mix (10 mM each dATP,dGTP, dCTP, and dTTP at neutral pH) were added. Thecontents were mixed gently and RNase H reversetranscriptase was added and incubated for 90 min at42 °C. The reaction was inactivated by heating at72 °C for 7 min after adding 100 µL tricine-EDTAbuffer. The cDNA was precipitated with ethanol andstored at –20 °C.

4.4. Cloning of apple hmg1 and hmg2

Conserved regions of the plant HMGR gene familywere identified by multiple sequence alignment of thepredicted amino acid sequences of five selected HMGRgenes (GI 167488, Catharanthus roseus; GI 169485,Solanum tuberosum; GI 1763234, Camptotheca acumi-nata; GI 19746, Nicotiana sylvestris; and GI 2072322,Oryza sativa) using the Block Maker program. Highlyconserved regions were assigned to the consensus-degenerate hybrid oligonucleotide primer (CODE-HOP) algorithm [33] and the following plant-specificHMGR degenerate oligonucleotide sequences weregenerated; the forward primer being, 5’ -GCTT-CTGTTATTTATCTGCTGGGATT(C/T)TT(C/T)GG-(A/G/C/T)(A/C/T)T-3’ ,corresponding to the amino acidsequence (ASVIYLLGFFGI) and the reverse primerbeing, 5’ -AGCAACCAGACATCCTTCAGTAGT-(A/G/C/T)GCCAT(A/G/C/T)GG-3’ corresponding tothe amino acid sequence (PMATTEGCLVA). Thepolymerase chain reaction (PCR) was carried outusing an Advantage™ cDNA PCR kit (Clontech) withapple cDNA as the template in the high fidelity buffer.


The PCR reaction conditions were: 1 min at 94 °C,plus 35 cycles at 94 °C (30 s), 68 °C (2.2 min) forannealing and a final extension step at 72 °C for 7 min.The resulting 549-bp PCR product (hmg1) was used todesign forward and reverse HMGR-specific primersfor 3’ - and 5’ -RACE, respectively (SMART™ RACEcDNA amplification kit, Clontech). The two fragmentsobtained after 3’ - and 5’ - RACE were then used todesign primers (forward 5’ -TGTCCTTTCCTCTTCT-CTCCTCCGCC-3’ and reverse 5’ -CTTCAAGCTCA-GAAGTTAGAGCCTTTCAAGTTC-3’ ) to obtain afull length cDNA clone (hmg1). To obtain the frag-ment of hmg2 cDNA clone, additional degenerateoligonucleotide primers (forward) was designed forthe 3’ region: 5’ -GCTCCACCGGTGACGCTATGGG-(A/G/C/T)TGAA-3’ corresponding to the amino acidsequence (CSTGDAMGMN), and 5’ -TCCCACTGC-ATCACCATGATGGA(A/G)GC-3’ corresponding tothe amino acid sequence (SHCITMMEA) to perform3’ -RACE by the procedure described above. DNAsequences were determined after subcloning the PCRproduct into E. coli using an AdvanTAge cloning kit™(Clontech), and by the chain termination method [38].The sequence of both strands was determined usingsynthetic oligonucleotide primers. DNA sequenceswere analysed using the MBS on-line tools.

4.5. Construction of DIG-UTP RNA probes

The hmg1 and hmg2 cDNA fragments were clonedinto a pCRII vector and gene segments correspondingto hmg1 (309 bp) and hmg2 (385 bp) were isolated.Gene-specific probes were prepared for hmg1 andhmg2, using the above gene segments and PCR witholigonucleotide primers designed to the carboxyl-terminal coding region (hmg1) and the 3’ -untranslatedregion (hmg2). The forward and the reverse primerpairs were: 5’ -CACGTGTCTGTCACCATGCCTTC-AATT-3’ and 5’ -CTTCAAGCTCAGAAGTTAGAG-CCTTTCAAGTTC-3’ for hmg1, and 5’ -CCTATCGA-CGGCAAGGACCTTCATGT-3’ and 5’ -CCCGAACC-GTCGAGCTTACTTATTTCTCT-3’ for hmg2. Anti-sense transcripts of each probe were made in vitro inthe presence of digoxigenin (DIG)-labelled-UTP usinga MAXIscript™ in vitro transcription Kit (Ambion)and DIG RNA labelling mix (Roche).

4.6. Southern blot analysisof apple genomic DNA

Genomic DNA was extracted using DNeasy PlantMaxi™ kit (Qiagen) from buds and young leaves of‘Delicious’ apple trees to improve the yield of DNA.

DNA was digested with three restriction endonu-cleases, EcoRI, BamHI, and PstI (Fermentas), and theresulting fragments were separated by electrophoresisin 0.8 % (w/v) agarose gels. The gels were blotted onto a positively-charged BrightStar-Plus™ membrane(Ambion) using SouthernMax™ Southern blotting kit(Ambion). The membranes were prehybridized inUltrahyb™ hybridization buffer (Ambion) for 1 h at50 °C, and then hybridized with digoxigenin-labelledapple hmg1 and hmg2 RNA probes at 50 °C for 16 h.The membrane was washed twice in 2× sodiumchloride/sodium citrate (SSC), 0.1 % SDS for 5 min atroom temperature (low stringency) and twice in0.1× SSC, 0.1 % SDS for 15 min at 50 °C (highstringency). Chemiluminescent detection was per-formed as described in the DIG Northern Starter kit(Roche).

4.7. Expression analyses using northern blots

For northern blot analysis, 10 µg total RNA fromapple skin was denatured at 65 °C for 10 min in RNAsample loading buffer (Sigma), separated by electro-phoresis in 1.0 % (w/v) formaldehyde agarose gels,and blotted onto BrightStar-Plus™ membrane(Ambion) using NorthernMax™ northern blotting kit(Ambion). The RNA bound to the membrane wascross-linked by baking at 80 °C for 15 min. Themembranes were prehybridized in Ultrahyb™ hybrid-ization buffer (Ambion) for 30 min at 68 °C, and thenhybridized with digoxigenin-labelled apple hmg1 andhmg2 RNA probes at 68 °C for 16 h. The membranewas washed twice in 2× SSC, 0.1 % SDS for 5 min atroom temperature (low stringency) and twice in0.1× SSC, 0.1 % SDS for 15 min at 68 °C (highstringency). Chemiluminescent detection was per-formed as described in the DIG Northern Starter kit(Roche).

Acknowledgments. This research was supported bygrants from the Ontario Ministry of Agriculture, Foodand Rural Affairs, and Agriculture and Agri-FoodCanada. We would like to thank Dr Geza Hrazdina,Cornell Agricultural Experimental Station, Geneva,New York, and Drs Mike McLean and Owen VanCauwenberge, Department of Plant Agriculture, fortheir expert advice and help. We are also grateful toDrs Judy Strommer and Barry Shelp, Department ofPlant Agriculture, for allowing us to use their labfacilities.


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A Farnesene Bio Synthesis