Plant Molecular Biology 36: 323–328, 1997. 323c 1997 Kluwer Academic Publishers. Printed in Belgium.

Short communication

Analysis of the isopentenyl diphosphate isomerase gene family fromArabidopsis thaliana

Michael Campbell1;�, Frederick M. Hahn2, C. Dale Poulter2 and Thomas Leustek3

1Division of Science, Pennsylvania State University-Erie, The Behrend College, Erie, PA 16563, USA (�author forcorrespondence); 2Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA; 3Center forAgricultural Molecular Biology, Rutgers University, New Brunswick, NJ 08903, USA

Received 23 May 1997; accepted in revised form 18 September 1997

Key words: isoprenoid biosynthetic pathway, functional complementation, isopentenyl diphosphate isomerase,higher plants, yeast

Abstract

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase)were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAsencode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of theIPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurspredominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.

Compounds derived from the isoprenoid pathway inplants are highly diverse and include such products ashormones, photosynthetic pigments, electron carriers,structural membrane components, and a large array ofsecondary metabolites [16]. These secondary metabol-ites in particular are of keen interest due to their role ininterorganismic interactions such as allelopathy, insectattraction, antiherbivory, and because of the commer-cial value of plant compounds such as essential oils,waxes, rubber and human medicinals [16, 2, 13].

In most eukaryotes the isoprenoids are derived fromacetate via the mevalonic acid pathway leading to thesynthesis of isopentenyl diphosphate (IPP). In plantsa separate non-mevalonate pathway, which utilizespyruvate and dihydroxyacetone phosphate as startingpoints, produces IPP in the chloroplast (reviewed in[2]). Despite the metabolic approach for IPP produc-tion, the conversion to more complex isoprenoid com-pounds usually requires the activation of IPP by IPP

The nucleotide sequence data reported will appear in the EMBL,GenBank and DDBJ Nucleotide Sequence Databases under theaccession numbers U47324 and U49259.

isomerase (EC 5.3.3.2) creating dimethylallyldiphos-phate (DMAPP). DMAPP then loses inorganic pyro-phosphate to form isoprene, which can condensewith IPP to form polyisoprenoid chains, or canbecome incorporated into a variety of other metabol-ites. Recently, there has been an increasing interestin the molecular aspects of the isoprenoid pathway inhigher plants. Most efforts have focused on HMG-CoA reductase because in animals this enzyme cata-lyzes the key regulatory step in the synthesis of ster-ols [10]. In plants this enzyme is encoded by a largefamily of differentially regulated genes that respondto development, pathogen attack and various otherstresses [16]. IPP isomerase catalyzes a key activa-tion step of the basic five-carbon isoprene unit in thesynthesis of isoprenoids, so it is of interest to determ-ine the nature of the gene or gene family encodingthis protein, and information that can be derived fromthese genes, such as cellular localization and regulationof steady state mRNA levels. IPP isomerase activityhas been identified in plants [21, 7, 15]. The enzymefrom yeasts and animals uses an unusual protonation-

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Figure 1. Comparison of IPP isomerase from a variety of organisms.A selection of IPP isomerases in the GenBank database were ana-lyzed by pair-wise alignment using the Genetics Computer Groupprogram PileUp. A dendogram of the sequence relationships isshown with the length of horizontal lines being proportional tothe percentage identity between each sequence as indicated belowthe dendogram. The species names, and cDNA name, are shownon the right. The GenBank or PIR accession numbers for eachsequence from top to bottom are as follows: A. thaliana IPP1 andIPP2, U47324 and U49259; Clarkia breweri IPI1, S49588; Clarkiaxantiana, U48962; Schizosaccharomyces pombe, U21154; Sac-charomyces cerevisiae, U21154; Caenorhabiditis elegans, S44843,Rhodobacter capsulatus; Escherichia coli U28375.

deprotonation mechanism [17]. To date, genes for IPPisomerase have been isolated from Rhodobacter cap-sulatus [12], Saccharomyces cerevisiae [1], Schizosac-charomyces pombe [11], man [25], and the floweringplant Clarkia breweri [5]. Genome and EST sequen-cing projects have identified putative IPP isomerase-encoding genes from Escherichia coli, Caenorhabidit-is elegans, Oryza sativa and Arabidopsis thaliana. Theclone from S. pombe was isolated by a complement-ation method involving plasmid shuffling in a strainof S. cerevisiae in which the chromosomal copy ofthe IDI1 gene was disrupted, and the wild-type geneplaced on an episomal plasmid [11]. The general use-fulness of this technique provided the opportunity torapidly clone and functionally analyze IPP isomerasegenes from higher plants.

Isolation of cDNAs encoding IPP isomerase cDNAs

An Arabidopsis thaliana cDNA library, constructed inthe �YES yeast expression vector, was obtained fromDr Ron Davis (Stanford University, Stanford, CA) [8].

The 1YES vector was utilized because it containedthe transformation selection marker gene URA3 andresulted in expression of cDNA inserts controlled bythe GAL1 promoter. The library was used to transformyeast strain FH2-5b by electroporation [4]. Transform-ants were recovered on SD lacking uracil. The colon-ies were then replica plated onto SG lacking uracil toinduce cDNA expression. After a two day incubationat 30 �C the growing colonies were suspended in liquid�-AA medium and then the cells were pelleted at 3000rpm for 10 min. The cell pellets were resuspended inthe same medium and then plated onto agar solidified�-AA medium. Growth on �-AA medium, used herefor ‘plasmid shuffing’, resulted in yeast cells that lostpRS317:IDI1 containing the yeast IPP isomerase geneand gained a functional IPP isomerase gene from theA. thaliana cDNA. Growing colonies were obtainedfrom 18 out of 20 transformations (representing 22 000transformants per transformation) after 5 days incub-ation at 30 �C. To distinguish true positives a sampleof 5 colonies from each transformation was replicaplated onto the same medium or �-AA medium withglucose as a carbon source. Of the 18 transformationsshowing positives, 7 resulted in at least one colonythat grew on galactose-containing, but not on glucose-containing�-AA medium. Positive cDNA clones wererescued into E. coli [22] and the cDNA was subclonedinto the EcoRI site of pBluescriptSK(+) (Stratagene).These plasmids were mapped with restriction enzymesrevealing two classes of cDNAs. A representative clonefrom each class, named IPP1 and IPP2 were used forDNA sequencing.

The DNA sequence is 944 bp for IPP1 and 1053 bpfor IPP2. Both clones exhibit a continuous open read-ing frame (ORF) beginning with nucleotide 3, so iden-tification of the true initiation codon is problematic.Met codons exist, beginning at nucleotide 96 and 120of IPP1. However, the homologous Met codons arefound in IPP2 at nucleotides 171 and 195. IPP2 con-tains a Met codon further upstream beginning at nuc-leotide 18. This Met codon falls within a nucleotidecontext similar to that of the translational initiation siteof plant genes [14]. The first Met codon of IPP1 alsofalls within such a nucleotide context.

Structure of the IPP isomerase genes

A search of the GenBank database revealed that thetranslation products of the IPP clones are homologouswith genes encoding IPP isomerase in other organisms.Figure 1 shows a dendogram displaying the sequence

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relationship of the IPP1 and IPP2 amino acid sequenceswith IPP isomerases from other species. This analys-is indicates that the higher plant enzymes fall into aclass of high homology. The plant enzymes are moreclosely related to the enzymes from the yeasts thanto the prokaryotes, phototrophic or heterotrophic, orC. elegans. Interestingly, in addition to being diver-gent from plants, the prokaryotic sequences are quitedivergent from each other.

A comparison of the amino acid sequences is shownin Figure 2. A high level of homology is observedbetween amino acids 104 and 232 of IPP2. In particu-lar, Cys-139 and Glu-201 correspond to the active-siteresidues identified in yeast isomerase as being catalyt-ically important [23].

The amino acid alignment in Figure 2 shows that theamino terminal ends of IPP1 and IPP2 are not homo-logous with the other IPP isomerases but do resembletransit peptides for localization to chloroplasts [24].The features of plastid transit peptides include a highconcentration of hydroxyl amino acids, hydrophobicamino acids with small side chains, and acidic aminoacids such that there is an overall positive charge tothe transit peptide. Another feature of transit peptidesis that they are not necessary for catalytic function ofthe enzyme and are typically removed by proteolyt-ic cleavage by a specific plastid processing enzyme.The first 100 amino acids of both IPP clones containa number of potential processing sites so it is not pos-sible to guess exactly how long the transit peptide isbut chloroplast transit peptides usually fall within thisregion of a protein [9].

Southern analysis

Hybridization of IPP1 and IPP2 to Southern blotsof total genomic DNA resulted in different bandingpatterns for each clone (Figure 3). Membranes werewashed at maximum stringency in 0.1 M Na+ (suppliedin all hybridization and washing buffers as Na2HPO4,made to pH 7.2 with H3PO4), 1 mM EDTA and 5%(w/v) SDS at 65 �C. Both clones yielded a simplebanding pattern of one to three bands when probedagainst total DNA digested with three different restric-tion enzymes suggesting that there is only one to twocopies of each clone present in the A. thaliana gen-ome. Based on sequence similarity and differences inbanding patterns on Southern blots it can be concludedthat IPP1 and IPP2 are independent members of a genefamily.

Expression of mRNA for IPP1 and IPP2

Hybridization of cDNA derived probes against north-ern blots containing total RNA from shoots and rootsof A. thaliana demonstrated that the expression ofboth cDNAs occurs predominantly in roots (Figure 4).Northern blots were prepared using total RNA isolatedfrom shoots and roots of 40-day old hydroponicallygrown A. thaliana. At the time of harvest the plantswere at the preflowering stage and exhibited fullyexpanded leaves and actively growing roots. Strin-gency washes of the blots were identical to the pro-cedures described for the Southern analysis. Imageanalysis of the films from the northern blots (Molecu-lar Dynamics) determined that the levels of mRNAexpression in roots for IPP2 was about 1.6-fold overthe levels for IPP1. A prolonged exposure of the north-ern blot resulted in the detection of low levels of bothIPP1 and IPP2 mRNA in leaf tissue. The levels ofexpression in roots were 5.8-fold higher for IPP1 and8-fold higher for IPP2 in comparison to levels in leaftissue. This increase in IPP expression in the root sys-tem may be a reflection of the growth stage of theplant at harvest. Previous studies examining isopren-oid biosynthesis in pepper fruits demonstrated that themetabolic step leading to sterol synthesis followingIPP isomerase, specifically the production of farne-syl pyrophosphate, is constitutively expressed [13].The primary end product of the isoprenoid pathway inA. thaliana is likely phytosterol production for mem-brane synthesis. Thus, reduced mRNA levels for IPP1and IPP2 from leaf tissue may be a result of low growthrates in the fully expanded leaf tissue.

Another possible reason for the increase in IPP1and IPP2 expression in roots could be for an increasedproduction of phytoalexins to counter pathogen attackor in response to other stresses associated with therhizosphere.

Protein encoded by the isolated cDNAs exhibit IPPisomerase activity

Since both A. thaliana cDNAs contained ORFs withpotential start codons downstream from the beginningof the sequences, the existing ATG sequences at pos-ition 96 of IPP1 and position 18 of IPP2 were chosenas initiation codons. They were then examined fortheir ability to synthesize recombinant protein with IPPisomerase activity. Unique NdeI and SalI sites wereintroduced into pBluescript derivatives pBSIPP1 andpBSIPP2, containing the A. thaliana cDNAs using the

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Figure 2. Alignment of Arabidopsis IPP1 and IPP2 with IPP homologues from other organisms. From top to bottom: deduced amino acidsequences of Arabidopsis IPP1, Arabidopsis IPP2, Clarkia breweri IPI1, clarkia xantiana IPI2, Schizosaccharomyces pombe IDI1, andRhodobacter capsulatus Rca.

MORPH Site-Specific Plasmid DNA Mutagenesis Kit(5 Prime-3Prime) and the following primers:(1) 50-GCTTTCTCAGCCGTCCATATGACCGATT-CTAAC-30

(2) 50-CCATTCACAAGCTCTGAGTCGACCATAAG-TTTTGGATCCTCCCCTTCC-30

(3) 50-GAAGAAGCAGACATATGTGTTGGTGTGTC-G-30

(4) 50-CTTTAGTATCTGTCATATGGGTACCAGAG-AAAGC-30

(5) 50-GATTGATTTAAAAACTTTGGATCCGTCG-ACTCAGAGTTTGTGGATGG-30

Primers 1 and 2 correspond to the sense strand andwere used for modifying IPP1. Primers 3, 4, and 5were used for the isolation of two NdeI-SalI DNA frag-ments, differing in length, from IPP2 and correspondto the antisense strand. Nucleotides analogous to theIPP1 and IPP2 open reading frames (ORFs), includ-ing start and stop codons, are shown in italics and thenew restriction sites are underlined. NdeI-SalI restric-tions of isolated mutants gave a 0.6 kb DNA fragmentcontaining the IPP1 ORF, a 0.8 kb DNA fragment con-taining the IPP2 ORF, and a 0.6 kb DNA fragment con-

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Figure 3. Southern blots of DNA isolated from A. thaliana probedwith IPP1 and IPP2. Lanes 1, 2, and 3 correspond to digestion oftotal DNA with HindIII, EcoRI, and XhoI respectively.

Figure 4. Localization of IPP expression in Arabidopsis plants. TotalRNA was isolated from leaves (L) and roots (R) of hydroponicallygrown plants and probed with IPP1 and IPP2. Blots were stripped andreprobed with tubulin to determine equal loading of RNA betweensamples (data not shown). Quantification of the blots using phosphorimage analysis (Molecular Dynamics) revealed relative levels ofmRNA; IPP1 (L)=1.0, IPP1 (R)=5.8, IPP2 (L)=1.2, IPP2 (R)=9.5.

taining a truncated version of the IPP2 ORF startingat the ATG codon at position 171 of IPP2. The threeNdeI-SalI DNA fragments were ligated into E. coliexpression vector pARC306N to create pFMH16(IPP1), pFMH17 (IPP2), and pFMH18 (truncated-IPP2). After sequence analysis of the ORFs containedin the three bacterial expression constructs, E. coliJM101 was transformed with pFMH16, pFMH17and pFMH18 to allow the synthesis of recombin-ant protein by induction with naladixic acid [11].

Figure 5. SDS-polyacrylamide gel of recombinant A. thaliana IPPisomerases. Lane M, molecular weight standards; lane A, super-natant from cell-free extract of pARC306N/JM101; lane B, super-natant from cell-free extract of pFMH16/JM101 (IPP1); lane C,supernatant from cell-free extract of pFMH17/JM101 (IPP2); laneD, supernatant from cell-free extract of pFMH18/JM101 (truncated-IPP2). The percentages of IPP isomerase in lanes B, C, and D weredetermined using ImageMaster VDS software (Hoefer PharmaciaBiotech).

Cell paste from JM101/pFMH16, JM101/pFMH17,JM101/pFMH18, and JM101/pARC306N was sus-pended in 10 mM potassium phosphate, 10 mM 2-mercaptoethanol (BME), 1 mM phenylmethylsulfonylfluoride, pH 7.0, and distrupted by sonication. The res-ulting homogenates were subjected to centrifugationat 23 700� g to remove cellular debris. The clarifiedcrude extracts were assayed for IPP isomerase activityby the acid lability procedure [20] and protein con-tent was determined by the method of Bradford [6].Cell-free homogenates from each of the three recom-binant strains contained IPP isomerase activity that waswell above background levels as defined by cell-freehomogenates from JM101/pARC306N. Specific activ-ities, in the cell-free extracts from E. coli transform-ants containing pFMH16, pFMH17, pFMH18, andpARC306N were 1.2, 0.2, 0.6 and 0.0 �mol min�1

mg�1 respectively. Shifts in mobility between IPP1,IPP2 and the truncated IPP2 protein products are notdetectable with the gel system presented. Interestingly,activity of soluble recombinant protein produced bythe longer ORF contained in pFMH17 was signific-antly lower than either of the shorter ORFs contained inpFMH16 and pFMH18. An analysis of the SDS-PAGEgel (see Figure 5) showed that the recombinant pro-tein resulting from plasmids pFMH16, pFMH17, andpFMH18 was 50%, 15%, and 32% of the total proteinpresent respectively. Thus, the observed differences inactivity closely approximates differences in the levelof recombinant protein present in the cell-free extracts.The removal of 56 amino-terminal residues from the

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IPP2 protein does not appear to substantially alter thecatalytic activity of the enzyme and may reflect a formof the protein that is present in vivo after removal of alocalization sequence. An experiment in which 56 N-terminal amino acids were removed from S. cerevisiaeIPP isomerase gave a protein that was able to com-plement the disrupted yeast isomerase gene and createa viable strain [10]. Clearly a much smaller portionof these proteins is all that is necessary for enzymaticactivity and may constitute a catalytic core that is simil-ar in size to the 176 amino acid Rhodobacter capsulatusIPP isomerase [11]. The amino and/or carboxy termin-al extensions present in some IPP isomerase homo-logues may serve for other functions such as signaland transit sequences.

Acknowledgements

This project was supported in part by a grant from theNational Institute of Health (GM 25521 CDP).

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