Cloning and Characterization of Mevalonate Pathway Genes

in a Natural Rubber Producing Plant, Hevea brasiliensis

Tomoki SANDO,1;* Chika TAKAOKA,1;** Yukio MUKAI,2 Atsushi YAMASHITA,3

Masahira HATTORI,4 Naotake OGASAWARA,5 Eiichiro FUKUSAKI,1;y and Akio KOBAYASHI1

1Department of Biotechnology, Graduate School of Engineering, Osaka University,

2-1 Yamadaoka, Suita, Osaka 565-0871, Japan2Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology,

1266 Tamuracho, Nagahama, Shiga 526-0829, Japan3Kitasato Institute for Life Sciences, Kitasato University,

1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan4Graduate School of Frontier Sciences, The University of Tokyo,

5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561, Japan5Department of Bioinformatics and Genomics, Graduate School of Information Science,

Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

Received March 17, 2008; Accepted May 13, 2008; Online Publication, August 7, 2008

[doi:10.1271/bbb.80165]

Hevea brasiliensis Mull. Arg. is a tree that produces

natural rubber, an industrially vital isoprenoid poly-

mer. Biosynthesis of natural rubber is known to take

place biochemically by a mevalonate (MVA) pathway,

but molecular biological characterization of related

genes has been insufficient. From H. brasiliensis, we

obtained full-length cDNA of genes encoding all of the

enzymes that catalyze the six steps of the MVA pathway.

Alignment analysis and phylogenetic analysis revealed

that in H. brasiliensis there are three acetyl-CoA

acetyltransferase genes, two HMG-CoA synthase

(HMGS) genes, and four HMG-CoA reductase (HMGR)

genes. Gene expression analysis by type of tissue

indicated that MVA pathway genes were highly ex-

pressed in latex, as compared to other types of tissue

and that HMGS and HMGR, which exist in multiple

copies, have different expression patterns. Moreover,

these MVA pathway genes in H. brasiliensis were found

to complement MVA pathway deletion mutations in

yeast.

Key words: Hevea brasiliensis; natural rubber; cis-poly-

isoprene; mevalonate (MVA) pathway;

yeast complementation

Natural rubber is present as rubber particles in latex,the fluid cytoplasm of laticifers in H. brasiliensis.Natural rubber produced by H. brasiliensis has superiorphysical properties in comparison to synthetic rubberand is an important raw material for rubber products,particularly truck, bus, and airplane tires and medicalgloves. Nevertheless, numerous uncertainties regardingthe pathway of natural rubber biosynthesis remain, andnumerous studies have sought to examine this pathway.Prior to the 1960s, tracer experiments using radio-

isotope tracers and biochemical experiments with latexfound that IPP, a crucial component of natural rubberbiosynthesis in H. brasiliensis, is synthesized by amevalonate (MVA) pathway.1–3) Molecular biologystudies have recently reported the sequences of theHMGS genes4,5) and HMGR genes6,7) and the se-quences of the genes of the entire MVA pathway8) inH. brasiliensis. In addition, large-scale transcriptomeanalyses of the latex of H. brasiliensis have beenperformed,9,10) but molecular biological characterizationof the MVA pathway as a whole has been inadequate. Inorder to obtain greater knowledge of the molecularevents involved in rubber biosynthesis, the mainauthor’s research group performed comparative ESTanalysis as between latex, a rubber-producing site, and

* Present address: Bridgestone Corporation, 3-1-1 Ogawahigashi, Kodaira, Tokyo 187-8531, Japan** Present address: Tamanoi Vinegar Corporation, 100 Nishimachi, Yamatokooriyama, Nara 639-1038, Japan

y To whom correspondence should be addressed. Tel/Fax: +81-6-6879-7424; E-mail: fukusaki@bio.eng.osaka-u.ac.jp

Abbreviations: CTAB, cetyl trimethyl ammonium bromide; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag; MVA,

mevalonate; AACT, acetyl-CoA acetyltransferase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MVK, mevalonate kinase; PMK,

phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; IDI,

IPP isomerase

Biosci. Biotechnol. Biochem., 72 (8), 2049–2060, 2008

xylem, a non-rubber-producing site. This EST analysisrevealed partial sequence information for all of thegenes related to the MVA pathway, which is crucial tothe biosynthesis of natural rubber.

The current study obtained full-length sequences forgenes related to the MVA pathway and compared themto those in previous reports. In addition, the expressionpatterns of MVA pathway genes in various tissues werealso investigated in order to characterize individualMVA pathway genes of H. brasiliensis in detail. More-over, complementation assays using yeast lacking MVApathway genes confirmed the individual functions ofthe genes.

Materials and Methods

Plant materials. Various tissue samples were obtainedfrom 10-year-old trees of Hevea brasiliensis (cloneRRIM600) growing on commercial plantation PTPNVIII, Cikumpay, Purwakarta, West Java, Indonesia. Thelatex exuding from the tapped trees was collected andmixed with equal volumes of 2 � RNA extractionbuffer (0.1 M Tris–HCl, 0.3 M LiCl, 0.01 M EDTA, and10% SDS, pH 9.5), as described previously.11) Xylemtissues were obtained from young branches at an earlystage of leaf development by peeling the phloem,including laticifers, and scraping both exposed surfaceswith a scalpel. The collected samples were immediatelyfrozen in liquid nitrogen and shipped back to thelaboratory on dry ice.

RNA isolation and isolation of cDNA encoding MVApathway genes. Total RNA from latex was isolated by amodification of the Cetyl trimethyl ammonium bromide(CTAB) method,12) after the latex mixture was centri-fuged at 10;000� g for 20min to remove the rubbercomponent. Total RNA from other tissues was isolatedusing an RNeasy Plant Mini Kit (Qiagen, Tokyo Japan),and purified using an RNase-Free DNase Set (Qiagen)according to the manufacturer’s protocol. For 30-rapidamplification of cDNA ends (30-RACE), the 30-FullRACE Core set (Takara, Shiga, Japan) was used withlatex and xylem total RNA as the template and a set ofspecific primers, S1 for the first PCR, and S2 for thesecond PCR: HbAACT 30S1 (50-CACCTAAATACC-TGGCAGAAGC-30) and HbAACT 30S2 (50-AAGGG-ATCACGACTTGGACAT-30), HbHMGS 30S1 (50-AC-TTGGACAGGATTGCAT-30) and HbHMGS 30S2 (50-TGCAAATTTTCGAGAAATTCG-30), HbHMGR1 30S1(50-CTACCTCATCGATGAAGATCACC-30) and HbH-MGR1 30S2 (50-CGGAGGAAGACGAAATG-30), HbH-MGR 30S1 (50-TTCCTTCATTTACCTTCTTGGC-30) andHbHMGR 30S2 (50-AACAAATTCTGCTCAAGGA-30),HbMVK 30S1 (50-AAGCCTGCCACCGTAG-30) andHbMVK 30S2 (50-TCTGATCTTCCACTGGGTTCA-30),HbPMK 30S1 (50-CAGTGACTGCTATTCATG-30) andHbPMK 30S2 (50-TTGACACCAGAATCATTGGCT-30),and HbMVD 30S1 (50-CAGCTGCTGGTTTTGCT-30)and HbMVD 30S2 (50-GCTGATGAATGCTAAAGAA-GATCA-30). All PCR products were cloned into pT7BlueT-vector (Takara) and sequenced. The sequence datawere combined with partial EST sequences to obtainthe full length sequence of each H. brasiliensis MVApathway gene.

Gene expression analysis by real-time PCR. Forquantification of Hevea MVA pathway genes transcriptsin the various tissues, quantitative real-time PCR wasperformed with primers designed using Primer Express(Applied Biosystems, CA, USA), as follows: HbAACT1-qFw (50-GCATTGGCTATACCTAAAACGATTG-30) andHbAACT1-qRv (50-GCGAAGGCTTCATTTATTTCA-

O O O-

O

O- O-

O

IPP

acetoacetyl-CoA

HMG-CoA

mevalonate

mevalonate phosphate

mevalonate diphosphate

AACT

HMGS

HMGR

MVK

PMK

MVD

acetyl-CoA

CoA

ATP

ADP

NADPH

NADP

SCoA

O

SCoA

O

SCoA

HO

CO2H

O

OH

HO

CO2H

O

HO

CO2H

P -

O

O-

O

HO

CO2H

P

O

O-

P -

O

O-

acetyl-CoA

CoA

acetyl-CoA

ADP

ATP

ADP

ATP

O O O-

O

O- O-

O

DMAPP

P

IDI

O

O

O O

P P P

Fig. 1. The MVA Pathway of Biosynthesis of the Isoprenoid

Precursors IPP and DMAPP.

Gene products implicated in the pathway are boxed.

2050 T. SANDO et al.

TAGT-30), HbAACT2-qFw (50-GCTCCGACCAAGTTT-CAAAGAG-30) and HbAACT2-qRv (50-ATTACAGC-AGCAGCACCATCAC-30), HbAACT3-qFw (50-GCAT-CTGCCCTTGTTCTTGAG-30) and HbAACT3-qRv (50-GCTGATGTTAGAGTGACCAATTTGA-30), HbHM-GS1-qFw (50-CGGGTGACACTGTTCTCTTATGG-30)and HbHMGS1-qRv (50-GGGATGTTGGCCTTCATG-TAGT-30), HbHMGS2-qFw (50-CTTGGCGCCTGGAA-CATACTA-30) and HbHMGS2-qRv (50-TGTATCGCC-AACAGCCTTCTG-30), HbHMGR1-qFw (50-GTCTT-GTTGAAGGATGGCATGAC-30) and HbHMGR1-qRv(50-TCAACTCCGCGGCTCTAGTC-30), HbHMGR3-qFw(50-AGTGTCCATGCCTTCCATTGA-30) and HbHM-GR3-qRv (50-CAGACAAGCTGATTGAGATGCAA-30),HbHMGR4-qFw (50-TGGAGCCACAAGTGTCCTGTT-30) and HbHMGR4-qRv (50-TTGCTGTCCCAAACCT-AACGA-30), HbHMGR5-qFw (50-CCCTGGCTGGAG-CTCTAGGT-30) and HbHMGR5-qRv (50-TCTTGGCC-TGTCGCTATGTAGAT-30), HbMVK-qFw (50-GTCT-GGTAATCTGACTCGCATCAA-30) and HbMVK-qRv(50-GTTCCTCCCAACTCTTGTGTTAGTG-30), HbPMK-qFw (50-GAAATGCCATGCTTCAGATCAG-30) andHbPMK-qRv (50-TGATTCAGGCTCTATCGGAACA-30), HbMVD-qFw (50-AGCTTTCTGCTATTGCAAGA-CAAG-30) and HbMVD-qRv (50-CCTTTCCCATTTT-CCACTTCAC-30), and HbGAPDH2-qFw (50-CTTGGC-GCCTGGAACATACTA-30) and HbGAPDH2-qRv (50-TGTATCGCCAACAGCCTTCTG-30). TheWT-OvationRNA Amplification System (Nugen Technologies, CA,USA) was used with 50 ng of each Total RNA tosynthesize single-strand cDNA. The reaction was per-formed on a GeneAmp 5700 Sequence DetectionSystem (Applied Biosystems) using SYBR Greendetection with a reaction mixture (20 ml) containing1 � SYBR Green PCR Master mix, 0.2 mM each offorward and reverse primers, and 1 ng/ml of templatecDNA. PCR amplification was performed under the

following conditions: 50 �C for 2min, and 95 �C for10min, followed by 50 cycles of 95 �C for 15 sec and60 �C for 1min. The relative level of amplified mRNAwas normalized to mRNA expression of the house-keeping gene, GAPDH. Gene expression was evaluatedby a modification of the 2���Ct method, as describedpreviously.13)

Strains. Yeast knockout strains, as listed Table 1,were obtained from the Open Biosystems Yeast KnockOut Strain Collection (Open Biosystems, AL, USA).Then the hmg1 hmg2 double knockout mutant wascreated by mating hmg1 and hmg2 mutants and dissect-ing tetrads.

Construction of expression plasmids for yeast com-plementation. The fragment containing coding region ofeach gene was amplified by PCR using a pair of specificprimers with 40 bp of vector sequences: HbAACT1 cS(50-TAGCAGCTGTAATACGACTCACTATAGGGAA-TATTAAGCTTTTGCAGTAATCCATGGCCC-30) andHbAACT1 cA (50-GTAAGCGTGACATAACTAATT-ACATGATGCGGCCCTCTAGTTTATCGTCCACAA-GAGTTC-30), HbAACT2 cS (50-TAGCAGCTGTAA-TACGACTCACTATAGGGAATATTAAGCTGCTCTT-CTTCCCAGTAATCC-30) and HbAACT2 cA (50-GT-AAGCGTGACATAACTAATTACATGATGCGGCCC-TCTAGTTGAACCAAATAGCAAAATG-30), HbHM-GS1 cS (50-TAGCAGCTGTAATACGACTCACTATA-GGGAATATTAAGCTGGAATTGGAGAGGCGTAG-AG-30) and HbHMGS1 cA (50-GTAAGCGTGACAT-AACTAATTACATGATGCGGCCCTCTAGTTGAAGG-AAACACAATTGTC-30), HbHMGS2 cS (50-TAGCAGC-TGTAATACGACTCACTATAGGGAATATTAAGCT-GAGGAGGAATTCGACAGGCG-30) and HbHMGS2 cA(50-GTAAGCGTGACATAACTAATTACATGATGC-GGCCCTCTAGTGTTACATTACTTCACTTTG-30),

Table 1. Yeast Deletion Mutant Strain Information

DisruptedMutant

gene Standard Systematic Genotype

name name

AACT ERG10 YPL028W BY4743; MAT a/�; his3D1/his3D1; leu2D0/leu2D0; lys2D0/LYS2;MET15/met15D0; ura3D0/ura3D0; ypl028w::kanMX4/YPL028w

HMGS ERG13 YML126C BY4743; MAT a/�; his3D1/his3D1; leu2D0/leu2D0; lys2D0/LYS2;MET15/met15D0; ura3D0/ura3D0; yml126c::kanMX4/YML126c

HMGR HMG1 YML075C BY4741; MAT a; his3D1; leu2D0; met15D0; ura3D0; YML075c::kanMX4

HMG2 YLR450W BY4742; MAT �; his3D1; leu2D0; lys2D0; ura3D0; YLR450w::kanMX4

MVK ERG12 YMR208W BY4743; MAT a/�; his3D1/his3D1; leu2D0/leu2D0; lys2D0/LYS2;

MET15/met15D0; ura3D0/ura3D0; ymr208w::kanMX4/YMR208w

PMK ERG8 YMR220W BY4743; MAT a/�; his3D1/his3D1; leu2D0/leu2D0; lys2D0/LYS2;

MET15/met15D0; ura3D0/ura3D0; ymr220w::kanMX4/YMR220w

MVD MVD1 YNR043W BY4743; MAT a/�; his3D1/his3D1; leu2D0/leu2D0; lys2D0/LYS2;

MET15/met15D0; ura3D0/ura3D0; ynr043w::kanMX4/YNR043w

MVA Pathway Genes in Hevea brasiliensis 2051

HbHMGR1 cS (50-TAGCAGCTGTAATACGACTC-ACTATAGGGAATATTAAGCTTTACATGGACACC-ACCGGCC-30) and HbHMGR1 cA (50-GTAAGCGT-GACATAACTAATTACATGATGCGGCCCTCTAG-TTTTACATTGCTGGGACCAG-30), HbHMGR4 cS(50-TAGCAGCTGTAATACGACTCACTATAGGG-AATATTAAGCTGTCGCTCCAAAATGGATGTG-30)and HbHMGR4 cA (50-GTAAGCGTGACATAACTAA-TTACATGATGCGGCCCTCTAGAAAAGAGTATAC-AGAAGTTC-30), HbHMGR5 cS (50-TAGCAGCTGT-AATACGACTCACTATAGGGAATATTAAGCTCCG-CTAGCTGGTCTATAATG-30) and HbHMGR5 cA (50-GTAAGCGTGACATAACTAATTACATGATGCGG-CCCTCTAGAATCAATTTACCTCAATAGA-30), HbM-VK cS (50-TAGCAGCTGTAATACGACTCACTATA-GGGAATATTAAGCTGAGGAAATTGGGATTTTC-AG-30) and HbMVK cA (50-GTAAGCGTGACATAA-CTAATTACATGATGCGGCCCTCTAGGATCTTAT-AATTTATTGGTG-30), HbPMK cS (50-TAGCAGCT-GTAATACGACTCACTATAGGGAATATTAAGCT-GAGGCGGAGAGAGAGAAAAG-30) and HbPMK cA(50-GTAAGCGTGACATAACTAATTACATGATGC-GGCCCTCTAGATGCCTTTTGCTCAAATATG-30),and HbMVD cS (50-TAGCAGCTGTAATACGACTC-ACTATAGGGAATATTAAGCTGTTGGTGGGTTGC-TTGCTGT-30) and HbMVD cA (50-GTAAGCGTGAC-ATAACTAATTACATGATGCGGCCCTCTAGAACA-GAAGTGAAAGAAAAGG-30). Each amplified frag-ment and pYES2 vector (Invitrogen, Tokyo, Japan),digested with HindIII and XbaI, was transformed into aheterozygous yeast mutants directly using a Frozen-EZYeast Transformation II Kit (ZymoResearch) accordingto the manufacturer’s protocol.

Complementation assays. Yeast transformants ofknockout mutants with plasmid harboring the MVApathway genes were spotted on complete medium YPD(1% yeast extract, 2% Bacto-Peptone, and 2% glucose)at 30 �C to analyze functional complementation of theH. brasiliensis genes. Transformed heterozygous diploidcells were forced to sporulate to obtain viable trans-formed haploid cells by dissecting tetrads on YPG (1%yeast extract, 2% Bacto-Peptone, and 2% galactose).Haploid transformant cells bearing both the disruptedallele and the plasmid-borne mevalonate pathway geneswere selected on minimal medium SC (-Ura) (6.7%yeast nitrogen base w/o amino acid, 2% galactose) andYPG + Geneticin (1% yeast extract, 2% Bacto-Peptone,2% galactose, and 200mg/l geneticin). Plasmid losswas analyzed on SC (-Ura) medium containing 0.1%5-fluoro-orotic acid (5-FOA).14)

Results and Discussion

Isolation of MVA pathway genes in H. brasiliensisMVA pathway candidate genes in H. brasiliensis

were identified by searching an in-house EST Databaseconstructed with cDNA libraries of latex and xylem of

H. brasiliensis RRIM600. Partial sequences of AACT,HMGS, HMGR, MVK, PMK, and MVD, the enzymesthat catalyze the six reactions in the MVA pathway,were determined, and are shown in Table 2.To obtain the full-length DNA sequences encoding

AACT, HMGS, HMGR, MVK, PMK, and MVD inH. brasiliensis, 30-RACE was performed using se-quence-specific primers. Based on these sequences,full-length cDNA was amplified by PCR, and the PCRproducts were cloned into pT7Blue vector and se-quenced. Ultimately, full-length sequences of MVApathway genes in H. brasiliensis were obtained. Theseconsisted of two AACT genes, two HMGS genes, threeHMGR genes, and one MVK, one PMK, and one MVDgene. The ORFs of these genes were designatedHbAACT1, HbAACT2, HbHMGS1, HbHMGS2,HbHMGR1, HbHMGR4, HbHMGR5, HbMVK,HbPMK, and HbMVD. The cDNA lengths, ORFlengths, numbers of amino acid residues, theoreticalisoelectric points (pIs), MW, and accession numbers forthese genes are shown in Table 3.The isolated sequences of these MVA pathway genes

were aligned first with those of H. brasiliensis, aspreviously reported, and secondly with those of yeast,Arabidopsis thaliana, and Oryza sativa based on aminoacid sequence similarities (Fig. 2).With regard to AACT in H. brasiliensis, one AACT

(accession no. AF429383) has been reported.8) Thissequence was designated HbAACT3 for convenience.HbAACT1 and HbAACT2 in the current study showed82–83% identity to HbAACT3 in a previous report.HbAACT1 showed 96% identity to HbAACT2. Inaddition, the AACTs in H. brasiliensis showed 76%–86% identity to those in A. thaliana and O. sativa, and

Table 2. H. brasiliensis EST Clones Annotated as MVA Pathway

Genes

Gene name Tree no.

Number of

EST clones

library distribution

(Latex:Xylem)

Acetyl-CoA acetyltransferase

(AACT)01617 9 (7:2)

02858 9 (9:0)

HMG-CoA synthase (HMGS) 00889 5 (5:0)

12176 1 (1:0)

HMG-CoA reductase (HMGR) 09997 3 (3:0)

08135 1 (1:0)

07803 2 (0:2)

Mevalonate kinase (MVK) 06169 1 (0:1)

Phosphomevalonate kinase

(PMK)00526 1 (0:1)

Mevalonate diphosphate

decarboxylase (MVD)08776 2 (2:0)

Data are from our two cDNA libraries, latex and xylem. Tree no. represents

arbitrary allocated numbers, when clustering analysis were performed based

on sequence homology using VISUAL-BIO Clustering software (NTT

SOFT). Numbers in brackets refer to the number of ESTs sequenced in each

library.

2052 T. SANDO et al.

Table 3. MVA Pathway Genes in H. brasiliensis

Tree no. Enzyme Full length (ORF)Amino acid

residuepI

Mr

(kDa)Accession no.

01617 AACT1 1518 (100-1314) 404 6.95 41.4 AB294686

02858 AACT2 1472 ( 41-1267) 408 6.25 41.6 AB294687

00889 HMGS1 1827 (241-1638) 465 6.33 51.3 AB294688

12176 HMGS2 1881 (302-1696) 464 6.20 51.3 AB294689

09997 HMGR1 2214 ( 63-1790) 575 6.44 61.6 AB294692

08135 HMGR4 2023 ( 54-1874) 606 6.38 64.7 AB294691

07803 HMGR5 1736 ( 51-1631) 526 5.92 56.7 AB294690

06169 MVK 1486 ( 52-1212) 386 5.63 40.8 AB294693

00526 PMK 1861 (134-1645) 503 5.92 53.9 AB294694

08776 MVD 1692 ( 95-1342) 415 6.80 45.8 AB294695

A AACT

B HMGS

Fig. 2. Continued.

MVA Pathway Genes in Hevea brasiliensis 2053

53–55% identity to that in yeast. Moreover, the AACTsequences in H. brasiliensis also had amino acidresidues crucial to the thiolase activity of acetoacetyl-CoA thiolase15) that were well conserved (Fig. 2A).

With regard to HMGS in H. brasiliensis, HMGS(accession no. AF429389) and HMGS2 (accessionno. AY534617) have been reported.4,5) In addition,HMGS showed 94% identity to HMGS2. HbHMGS1

C HMGR

Fig. 2. Continued.

2054 T. SANDO et al.

and HbHMGS2 in the current study were identical toHMGS and HMGS2 respectively in the previous reports.HbHMGS1 showed 95% identity to HbHMGS2 in asimilar fashion. In addition, HMGSs in H. brasiliensisshowed 78–82% identity to those in A. thaliana andO. sativa, and 47% identity to that in yeast. HMGSsin H. brasiliensis had amino acid residues crucial to theactivity of HMG-CoA synthase16) that were well con-served (Fig. 2B).

With regard to HMGR in H. brasiliensis, HMGR(accession no. X54659), HMGR2 (accession no.

X54658), and HMGR3 (accession no. M74800) havebeen reported,6–8) but the sequence information forHMGR2 was incomplete, so it was not dealth with in thecurrent study. HMGR showed 77% identity to HMGR3.Three HMGR genes were obtained, but only HbHMGR1was identical to HMGR (accession no. X54659). Hencetwo other HMGR genes in the current study weredesignated HbHMGR4 and HbHMGR5. HbHMGR1showed 73% identity to HbHMGR4 and 71% identity toHbHMGR5. HbHMGR3 showed 71% identity to bothHbHMGR4 and HbHMGR5. HbHMGR4 showed 90%

D MVK

E PMK

F MVD

Fig. 2. Amino Acid Alignment of MVA Pathway Genes of Plant Origin.

S. cerevisiae MVA pathway genes are shown for reference. Black stars indicate essential residues of catalytic activity.

MVA Pathway Genes in Hevea brasiliensis 2055

identity to HbHMGR5. In addition, the HMGRs inH. brasiliensis showed 67–80% identity to those inA. thaliana and O. sativa, and 47–52% identity to thosein yeast. Moreover, the HMGRs in H. brasiliensis alsohad motifs crucial to the activity of the C-terminalregion that were highly conserved, with the exceptionof HbHMGR5. But as in other plants and trees, theN-terminal region, which is needed to anchor enzymesto the ER membrane in yeast,17,18) was largely lacking(Fig. 2C). Even though HbHMGR5 lacks the C-terminalregion, the presence of a poly-A tail in the cDNA ofHbHMGR5 implies that this peptide is a genuineproduct of gene expression in H. brasiliensis.

With regard to other members of H. brasiliensis,MVK (accession no. AF429384), PMK (accessionno. AF429385), MVD (accession no. AF429386) havebeen reported. HbMVK, HbPMK, and HbMVD showedover 98% identity to those in a previous report.8) MVDin H. brasiliensis showed 76–79% identity to that inA. thaliana and O. sativa, and 55% identity to that inyeast. Nevertheless, MVK and PMK in H. brasiliensishad 61–73% identity to those in A. thaliana andO. sativa, but only 29–33% identity to those in yeast.The crystal structures and active sites of MVK, PMK,and MVD have been studied only to a small extent inanimals, even though the MVA pathway is an importantmetabolic pathway in many organisms. Moreover, theseenzymes share little identity in animals and plants, andmay have different crystal structures and active sites, sofurther study is needed.

Phylogenetic analysisTo elucidate the evolutionary relationships, between

yeast, as a typical eukaryote, and plants, a dendrogramwas created based on MVA pathway gene sequencesobtained from GenBank (Fig. 3). With regard to theseMVA pathway genes, the genes in H. brasiliensis werelocated in plant gene clusters and were clearly distinctfrom those of yeast. In addition, HbAACT1 andHbAACT2, HbHMGS1 and HbHMGS2, andHbHMGR4 and HbHMGR5 were located in the sameclades, but little sequence information was available forMVK, PMK, and MVD, and adequate examination ofmolecular evolution proved difficult. For all of thegenes, the sequences in H. brasiliensis were located nearthose for Vitis vinifera.

Gene expression pattern of MVA pathway genesTo determine the expression patterns of MVA path-

way genes among different tissue types in H. brasilien-sis, total RNA was extracted from reddish young leaves,pale-green young leaves, mature leaves, latex, phloem,and xylem, and quantitative real-time PCR was per-formed for all of the MVA pathway genes. The PCRresults are shown in Fig. 4. InH. brasiliensis, HbAACT1was highly expressed in latex, HbAACT2 in latex andmature leaves, and HbAACT3 in mature leaves. HMGS1has been found to be highly expressed in latex rather than

in leaves, and HMGS2 has been found to be expressedin latex and petioles, but levels lower than that ofHMGS1 were reported.4,5) The current results are similarto previous results. Moreover, they revealed thatHbHMGS2 was more highly expressed in young leavesand phloem than in latex. Previously reported findingswere that HMGR1 is highly expressed in latex ratherthan leaves, that HMGR3 is constantly expressed inleaves and latex, and that expression of HMGR1 isinduced by ethylene treatment while that of HMGR3does not change.7) The results of the current studyindicate similar trends. In addition, the current studyrevealed that the HbHMGR4 and HbHMGR5 obtainedwere specifically expressed at high levels in matureleaves and xylem respectively, and that HbMVK,HbPMK, and HbMVD are all highly expressed in latex.This suggests that the genes for HbAACT1,

HbHMGS1, HbHMGR1, HbMVK, HbPMK, andHbMVD, which are highly expressed in latex, play animportant role in natural rubber biosynthesis in the latexof H. brasiliensis. In addition, we found that those genesthat were highly expressed in latex were expressed atrelatively high levels even in the xylem, which is notinvolved in rubber synthesis. HbMVK and HbPMK inparticular were expressed at the same levels as in latex,but xylem contains no rubber, and the two may insteadbe involved in the biosynthesis of other isoprenoids.

Functional complementation of MVA pathway genesin H. brasiliensisIn yeast, the MVA pathway is a pathway for biosyn-

thesis that is essential to survival; disrupting thesepathway genes in a strain proves lethal. Hence, acomplementation assay was performed using a hetero-zygous diploid strain lacking an allele of the target gene,and the functions of the H. brasiliensis MVA pathwaygenes obtained were confirmed.None of disrupted strains with empty pYES2 vector

were able to grow on either YPG expression medium orYPD non-expression medium (data not shown).A �erg10-disrupted strain with HbAACT1 and with

HbAACT2 grew on YPG expression medium, but not onYPD non-expression medium (Fig. 5A). This indicatesthat HbAACT1 and HbAACT2 have AACT activity.In addition, with HbAACT1, functional recovery of�erg10 took 1 d. With HbAACT2, in contrast, recoverytook 3 d, so HbAACT1 had a higher functional affinityfor AACT in yeast than HbAACT2.A �erg13-disrupted strain with HbHMGS1 and with

HbHMGS2 grew on YPG expression medium (Fig. 5B),but �erg13 with HbHMGS2 also grew on YPD non-expression medium. To confirm more completely thephenotypic differences resulting from the GAL1 promo-ter’s regulation of expression, growth on YPD mediumwith 5% glucose was verified; it was similarly possible.This can be explained by the fact that the low expressionof HbHMGS2 due to leakage of GAL1 promoter inglucose. This was sufficient for recovery of the HMGS

2056 T. SANDO et al.

ScHMG1

AtHMGR2

OsHMGR1

RsHMGRAtHMGR1

PsHMGR

ScHMG2

HbHMGR5

HbHMGR1

HbHMGR4

HbHMGR3

OsHMGR2

VvHMGR1CaHMGR1

CaHMGR2

GlHMGR

CrHMGRVvHMGR2

NtHMGR1NtHMGR2

LeHMGRCpaHMGR

VvHMGR3

EuHMGR

CmHMGR

ScPMK

OsPMK

AtPMK

HbPMK

VvPMK

ScMVD

HbMVD

LeMVD

AeMVD

VvMVD

GbMVD

AtMVD

OsMVD

VvHMG

AtHMGS

ScHMGS

OsHMGS1

HbHMGS2

LeHMGSBjHMGS

HbHMGS1

OsHMGS2

ScAACT

HbAACT1

NtAACT1

VvAACT PkAACT

AtAACT1RsAACT

OsAACT

AeAACT

AtAACT2

NtAACT2

HbAACT2

HbAACT3

HbMVK

OsMVK

MtMVK

VvMVK

AtMVK

ScMVKB

A

C

D

E

F

Fig. 3. Phylogenetic Relationships of the MVA Pathway Genes.

The dendrogram was created using the ClustalW program. The genes in H. brasiliensis are boxed. A black background represents genes

obtained successfully in the current study, and a gray background represents only genes obtained in the previous study. Key and GenBank

accession numbers are as follows: A, AACT, HbAACT1 and HbAACT2 (this study); AeAACT (Arnebia euchroma, ABD57973); AtAACT1 and

AtAACT2 (Arabidopsis thaliana, NP 851154 and NP 851150); HbAACT3 (H. brasiliensis, AAL18924); NtAACT1 and NtAACT2 (Nicotiana

tabacum, AAU95618 and AAU95619); OsAACT (Oryza sativa, NP 001041797); PkAACT (Picrorhiza kurrooa, ABC74567); RsAACT

(Raphanus sativus, CAA55006); ScAACT (Saccharomyces cerevisiae, P41338); VvAACT (Vitis vinifera, CAO41142). B, HMGS, HbHMGS1

and HbHMGS2 (this study); AtHMGS (A. thaliana, NM 117251); BjHMGS (Brassica juncea, AAG32924); LeHMGS (Lycopersicon

esculentum, ABX55778); OsHMGS1 and OsHMGS2 (O. sativa, EAZ25374 and ABG73437); ScHMGS (S. cerevisiae, P54839); VvHMGS

(V. vinifera, CAO63650). C, HMGR, HbHMGR1, HbHMGR4 and HbHMGR5 (this study); AtHMGR1 and AtHMGR2 (A. thaliana,

NM 106299 and NM 127292); CaHMGR1 and CaHMGR2 (Camptotheca acuminate, P48021 and AAB69726); CmHMGR (Cucumis melo,

BAA36291); CpaHMGR (Capsicum annuum, Q9XEL8); CrHMGR (Catharanthus roseus, AAT52222); EuHMGR (Eucommia ulmoides,

AAV54051); GlHMGR (Gentiana lutea, BAE92730); HbHMGR3 (H. brasiliensis, Q00583); LeHMGR (L. esculentum, AAB62581);

NtHMGR1 and NtHMGR2 (N. tabacum, AAB87727 and AAL54878); OsHMGR1 and OsHMGR2 (O. sativa, Q9XHL5 and A2X8W3);

PsHMGR (Pisum sativum, AAL37041); RsHMGR (R. sativus, CAA48611); ScHMG1 and ScHMG2 (S. cerevisiae, P12683 and P12684);

VvHMGR1, VvHMGR2 and VvHMGR3 (V. vinifera, CAO47551, CAO68514 and CAO66292). D, MVK, HbMVK (this study); AtMVK

(A. thaliana, X77793); MtMVK (Medicago truncatula, ABD32397); OsMVK (O. sativa, ABB47176); ScMVK (S. cerevisiae, P07277);

VvMVK (V. vinifera, CAO17636). E, PMK, HbPMK (this study); AtPMK (A. thaliana, NM 102927); OsPMK (O. sativa, ABF95008); ScPMK

(S. cerevisiae, P24521); VvPMK (V. vinifera, CAO63313). F, MVD, HbMVD (this study); AeMVD (A. euchroma, ABG24207); AtMVD

(A. thaliana, ATY14325); GbMVD (Ginkgo biloba, AAV32433); LeMVD (L. esculentum, ABW87316); OsMVD (O. sativa, BAD27942);

ScMVD (S. cerevisiae, P32377); VvMVD (V. vinifera, CAN82519).

MVA Pathway Genes in Hevea brasiliensis 2057

function in the �erg13-disrupted strain. In addition, atransformed haploid strain was inoculated on 5-FOAmedium, and the differences in growth when the plasmidwas lost were observed. On 5-FOA medium, in whichonly strains that had lost the plasmid grew, a haploidtransformant with HbHMGS2 did not grow. Thusgrowth recovery dependent on the plasmid harboringHbHMGS2 was confirmed. This indicates that bothHbHMGS1 and HbHMGS2 have HMGS activity.

A �erg8-disrupted strain with HbPMK grew on bothYPG expression medium and YPD non-expressionmedium with 5% glucose (Fig. 5C). This can beexplained by the fact that the low expression of HbPMKdue to leakage of GAL1 promoter in glucose is sufficient

to allow recovery of the PMK function in a �erg8-disrupted strain. In addition, a transformed haploid strainwas inoculated on 5-FOA medium, and the differencesin growth when the plasmid was lost were observed.A haploid transformant with HbPMK did not grow on5-FOA medium. Thus growth recovery dependent on theplasmid harboring HbPMK was confirmed. This indi-cates that HbPMK has PMK activity.A �mvd1-disrupted strain with HbMVD also grew

only on YPG expression medium (data not shown). Thisindicates that HbMVD has MVD activity.Yeast possesses two HMGR genes, HMG1 and

HMG2. An hmg1 hmg2 double mutant strain in whichthese two genes are disrupted dies, but it can survive if

Fig. 4. Gene Expression Patterns of MVA Pathway Genes in the Various Tissues of H. brasiliensis.

Data from RT-PCR were analyzed as expression ratios relative to the red young leaf, and are shown as the mean of three replicated assays.

Standard deviations of the means are shown as error bars. A, red young leaf; B, pale green young leaf; C, mature leaf; D, latex; E, phloem;

F, xylem.

2058 T. SANDO et al.

mevalonolactone is added. Thus differences in growthin the presence and the absence of mevalonolactoneconfirmed the functions of genes in H. brasiliensis. Adouble mutant strain without a plasmid and a doublemutant strain with only the pYES2 vector did not growwithout mevalonolactone; in contrast, a double mutantstrain with HbHMGR1 or HbHMGR4 grew even onYPG medium without mevalonolactone (Fig. 5D). Thisindicates that both HbHMGR1 and HbHMGR4 haveHMGR activity. A double mutant strain withHbHMGR5 did not provide any transformants, so itsfunction could not be confirmed. HbHMGR5 had 90%

identity to HbHMGR4 at the amino acid level, but theHbHMGR5 lacked the C-terminal region, which con-tains the catalytic domain of the enzyme.19) Thisindicates that HbHMGR5 is insufficient to complementyeast mutant.A �erg12-disrupted strain with HbMVK did not

provide any transformants, so its function could not beconfirmed. The existence of MVK activity in the latex ofH. brasiliensis has been confirmed.20) The HbMVKobtained in the current study had a low identity, of about30%, to MVK in yeast at the amino acid level, andfunctional confirmation by yeast complementation assay

Fig. 5. Functional Complementation.

The growth of haploid disrupted strains �erg10, �erg13, and �erg8, complemented with HbAACT1 or HbAACT2 (A), HbHMGS1 or

HbHMGS2 (B), or HbPMK (C) were assayed. The strains were grown on YPG and YPD at 30 �C for 1 d with the exception of �erg10 with

HbAACT2 3 d. D, The growth of double knockout mutant hmg1 hmg2 complemented with HbHMGR1 and HbHMGR4 was assayed. The strains

were grown on YPG with and without mevalonolactone at 30 �C for 4 d.

MVA Pathway Genes in Hevea brasiliensis 2059

was not possible, but it is believed to have MVKactivity. New knowledge of MVK in plants willprobably be obtained in the future, through work suchas detailed comparison of its active sites.

The current results lead to the conclusion that theHbAACT1, HbAACT2, HbHMGS1, HbHMGS2,HbHMGR1, HbHMGR4, HbPMK, and HbMVD genesinH. brasiliensis have corresponding enzymatic activity.

Acknowledgments

Part of this work was performed in the project‘‘Development of Fundamental Technologies for Con-trolling the Production of Industrial Materials by Plants’’supported by the New Energy and Industrial TechnologyDevelopment Organization of Japan (NEDO). We arevery grateful to Dr. T. Tajuddin (Badan Pengkajian danPenerapan Teknologi (BPPT), Indonesia) for provisionof plant materials, and to Mr. E. Hirosue (Department ofBiotechnology, Graduate School of Engineering, OsakaUniversity, Suita, Japan) for technical support.

References

1) Bandurski, R. S., and Teas, H. J., Rubber biosynthesis inlatex of Hevea brasiliensis. Plant Physiol., 32, 643–648(1957).

2) Lynen, F., Biochemical problems of rubber synthesis.J. Rub. Res. Inst. Malaya, 21, 389–406 (1969).

3) Archer, B. L., Polyisoprene. In ‘‘Encyclopedia of PlantPhysiology’’ Vol. 8, eds. Bell, E. A., and Charlwood,B. V., Springer-Verlag, Berlin, pp. 309–327 (1980).

4) Suwanmanee, P., Suvachittanont, W., and Fincher,G. B., Molecular cloning of 3-hydroxy-3-methylglutarylcoenzyme A synthase in Hevea brasiliensis Arg Mull.Sci. Asia, 28, 29–36 (2002).

5) Sirinupong, N., Suwanmanee, P., Doolittle, R. F., andSuvachitanont, W., Molecular cloning of a new cDNAand expression of 3-hydroxy-3-methylglutaryl-CoAsynthase gene from Hevea brasiliensis. Planta, 221,502–512 (2005).

6) Chye, M. L., Kush, A., Tan, C. T., and Chua, N. H.,Characterization of cDNA and genomic clones encoding3-hydroxy-3-methylglutaryl-coenzyme A reductase fromHevea brasiliensis. Plant Mol. Biol., 16, 567–577(1991).

7) Chye, M. L., Tan, C. T., and Chua, N. H., Three genesencode 3-hydroxy-3-methylglutaryl-coenzyme A reduc-tase in Hevea brasiliensis. hmg1 and hmg3 are differ-entially expressed. Plant Mol. Biol., 19, 473–484 (1992).

8) Hallahan, D. L., and Keiper-Hrynko, N. M., PCT. WO03/010294 A2 (Feb. 6, 2003).

9) Ko, J. H., Chow, K. S., and Han, K. H., Transcriptomeanalysis reveals novel features of the molecular eventsoccurring in the laticifers of Hevea brasiliensis (pararubber tree). Plant Mol. Biol., 53, 479–492 (2003).

10) Chow, K. S., Wan, K. L., Isa, M. N. M., Bahari, A., Tan,S. H., Harikrishna, K., and Yeang, H. Y., Insights intorubber biosynthesis from transcriptome analysis ofHevea brasiliensis latex. J. Exp. Bot., 58, 2429–2440(2007).

11) Kush, A., Goyvaerts, E., Chye, M. L., and Chua, N. H.,Laticifer-specific gene expression in Hevea brasiliensis(rubber tree). Proc. Natl. Acad. Sci. USA, 87, 1787–1790(1990).

12) Chang, S., Puryear, J., and Cairney, J., A simple andefficient method for isolating RNA from pine trees. PlantMol. Biol. Rep., 11, 113–116 (1993).

13) Livak, K. J., and Schmittgen, T. D., Analysis of relativegene expression data using real-time quantitative PCRand the 2���CT method. Methods, 25, 402–408 (2001).

14) Boeke, J. D., Trueheart, J., Natsoulis, G., and Fink,G. R., 5-Fluoroorotic acid as a selective agent in yeastmolecular genetics. Methods Enzymol., 154, 164–175(1987).

15) Haapalainen, A. M., Merilainen, G., Pirila, P. L., Kondo,N., Fukao, T., and Wierenga, R. K., Crystallographic andkinetic studies of human mitochondrial acetoacetyl-CoAthiolase: the importance of potassium and chloride ionsfor its structure and function. Biochemistry, 46, 4305–4321 (2007).

16) Campobasso, N., Patel, M., Wilding, I. E., Kallender, H.,Rosenberg, M., and Gwynn, M. N., Staphylococcusaureus 3-Hydroxy-3-methylglutaryl-CoA Synthase.J. Biol. Chem., 279, 44883–44888 (2004).

17) Istvan, E. S., Palnitkar, M., Buchanan, S. K., andDeisenhofer, J., Crystal structure of the catalytic portionof human HMG-CoA reductase: insights into regulationof activity and catalysis. EMBO J., 19, 819–830 (2000).

18) Wright, R., Basson, M., D’Ari, L., and Rine, J.,Increased amounts of HMG-CoA reductase induce‘karmellae’: a proliferation of stacked membrane pairssurrounding the yeast nucleus. J. Cell Biol., 107, 101–114 (1988).

19) Learned, R. M., and Fink, G. R., 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase from Arabidopsis thali-ana is structurally distinct from the yeast and animalenzymes. Proc. Natl. Acad. Sci. USA, 86, 2779–2783(1989).

20) Williamson, I. P., and Kekwick, R. G. O., The formationof 5-phosphomevalonate by mevalonate kinase in Heveabrasiliensis latex. Biochem. J., 96, 862–870 (1965).

2060 T. SANDO et al.