Plant Science 169 (2005) 571578 www.elsevier.

com/locate/plantsci

Molecular cloning and expression of a cDNA encoding 1-deoxy-D-xylulose-5-phosphate synthase from oil palm Elaeis guineensis Jacq.
Sawitri Khemvong, Wallie Suvachittanont *
Biochemistry Department, Faculty of Science, Prince of Songkla University, Hat Yai 90112, Thailand Received 29 November 2004; received in revised form 2 March 2005; accepted 2 May 2005 Available online 25 May 2005

Abstract In higher plants, there are two pathways leading to the formation of isoprenoids: the acetate/mevalonate (MVA) pathway and the recently discovered 2C-methyl-D-erythritol-4-phosphate (MEP) pathway. The initial step on the MEP pathway is the condensation of pyruvate and glyceraldehyde-3-phosphate yielding 1-deoxy-D-xylulose-5-phosphate (DXP). The reaction is catalyzed by 1-deoxy-D-xylulose-5-phosphate synthase (DXS), and is encoded by the dxs gene. In this study, parts of dxs have been cloned from young leaves and mesocarp of oil palm (Elaeis guineensis Jacq., Tenera) The surprising results show that there are at least two dxs genes encoding DXS in different organs of oil palm, dxs1 in leaves and dxs2 in fruits. Cloning of full-length cDNA encoding dxs1 by the RLM-RACE (RNA ligase-mediated rapid 0 0 amplication of 5 and 3 cDNA ends) method demonstrated that the cDNA contained an open reading frame of 2301 bp encoding a deduced peptide of 707 amino acid residues with a predicted molecular mass of 76.4 kDa and isoelectric point of 7.0. Expression of dxs2 and dxr transcript levels in oil palm fruits from each developmental stage were analyzed by a semiquantitative RT-PCR method. The level of dxs2 transcripts increases with the time after fertilization and reaches its maximum after 18 weeks while the expression of dxr is unchanged. The pattern of dxs2 transcripts appears to correlate with b-carotene accumulation during oil palm fruit ripening thus supports the assumed role of the dxs gene in carotenoid biosynthesis by the MEP pathway. # 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: b-Carotene; Carotenoids; 2C-methyl-D-erythritol-4-phosphate; 1-Deoxy-D-xylulose-5-phosphate synthase; 1-Deoxy-D-xylulose-5-phosphate reductoisomerase; Elaeis guineensis Jacq.

1. Introduction Isoprenoids are the largest group of natural products, encompassing over 30,000 known to date [1]. Some isoprenoids have essential functions in the cell, for instance,
Abbreviations: bp, base pairs; CMK, 4-diphosphocytidyl-2C-methyl-Derythritol kinase; CMS, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose5-phosphate; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; IPP, isopentenyl diphosphate; MEP, 2C-methyl-D-erythritol-4-phosphate; MVA, acetate/mevalonate; MECPS, 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase; RLM-RACE, RNA ligase-mediated rapid amplication of 50 and 30 cDNA ends; RT-PCR, reverse transcriptase-polymerase chain reaction * Corresponding author. Tel.: +66 74 288 242; fax: +66 74 446 656. E-mail address: wallie.s@psu.ac.th (W. Suvachittanont).

sterols are required as structural components of membranes, dolichol as a sugar carrier in the glycosylation of proteins, ubiquinone and plastoquinone as electron carriers in photosynthesis and respiration, and abscisic acid, cytokinins, gibberellic acid and sterols as hormones that control physiological processes and development. In spite of the structural and functional diversity of isoprenoids, all are derived from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). IPP and DMAPP are essential intermediates for the synthesis of isoprenoids compounds. Both IPP and DMAPP can be synthesized by two different biosynthetic routes: either via the acetate/ mevalonate (MVA) pathway, which is shared with fungi, yeast and animals [2], occurs in the cytoplasm, or via the mevalonate-independent pathway currently known as the

0168-9452/$ see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.05.001

572

S. Khemvong, W. Suvachittanont / Plant Science 169 (2005) 571578

2C-methyl-D-erythritol-4-phosphate (MEP) pathway, which is found in plastids. The MEP pathway is also found in protozoa, most bacteria [35], green algae [6,7] and higher plants [8,9]. The initial step of MEP pathway involves the condensation of pyruvate and glyceraldehydes-3-phosphate [10,11] forming 1-deoxy-D-xylulose-5-phosphate (DXP) as the rst intermediate. This reaction is catalysed by 1-deoxy-Dxylulose-5-phosphate synthase (DXS), a novel type of transketolase [1214], which is encoded by dxs gene. In plants and Escherichia coli, the DXP produced by this reaction is utilized in plastid for IPP biosynthesis as well as in the production of thiamin and pyridoxol [15,16]. The cloning and characterization of dxs gene were rst described for Escherichia coli [13,14]. Homologous genes were subsequently identied in other bacteria [17,18] as well as in higher plants [12,1921]. The enzyme requires thiamin diphosphate and divalent cations such as Mg2+ or Mn2+ for its activity. In the second step, DXP is converted to 2C-methyl-D-erythritol-4-phosphate (MEP) by the enzyme DXP reductoisomerase (DXR) [2224], encoded by dxr gene. MEP is subsequently converted into 2C-methyl-Derythritol-2,4-cyclodiphosphate by consecutive activities of three independent enzymes, 4-diphosphocytidyl-2Cmethyl-D-erythritol synthase (CMS) [25,26], 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (CMK) [27,28], and 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (MECPS) [29], respectively. The nal steps leading to IPP are still unknown. Carotenoids, both carotenes and xanthophylls, are colored pigments common to all photosynthetic organisms. In higher plants, carotenoids play an essential role in photosynthesis in green tissues. They have structural and functional roles in light-harvesting in photosynthetic membranes and in protecting their photosynthetic apparatuses from excess light energy by quenching triplet chlorophylls and singlet oxygen [30,31]. In certain nonphotosynthetic organ of plants, as, for instance, in some ripening fruits, carotenoids accumulate in large amounts in chromoplast and are involved in attraction of animals which facilitate pollination or seed dispersal. Furthermore, dietary carotenoids are essential requirements for human nutrition [32,33]. For example, b-carotene is a precursor of Vitamin A and has been proposed to act as an anti-cancer agent. Deciency of it leads to blindness and premature death [34]. Because of the importance of carotenoids, the gene encoding enzymes of carotenoids biosynthesis in higher plants are a potential target to increase the biosynthesis of carotenoids. Carotenoids are isoprenoids that synthesized in plastids via the MEP pathway. Higher expression of genes from this pathway may be directly related to higher carotenoids in plants as it has been reported that DXS is a rate limiting enzyme in the pathway [35,36] Oil palm trees (Elaeis guineensis Jacq., Tenera) are cultivated for commercial palm oil. Their fruits are a good source of b-carotene. The aim of this work is to clone dxs

gene from young leaves and investigate the expression of dxs and dxr genes in relation to the amount of carotenoids (bcarotene) in mesocarp of oil palm fruits at various developmental stages. This may explain the role of dxs gene in biosynthesis by the MEP pathway of isoprenoids, particularly of carotenoids.

2. Materials and methods 2.1. Plant materials Young leaves and mesocarp of oil palm fruits (E. guineensis Jacq., Tenera) were excised from plants; especially the oil palm fruits were harvested at eight stages of development (approximately 8, 10, 12, 14, 16, 18, 20 and 22 weeks after fertilization) for RNA extraction. These tissue samples were frozen in liquid nitrogen and stored at 80 8C until required. Pigment analysis from the tissues used for RNA extraction was carried out immediately. 2.2. Total RNA isolation Total RNA was isolated from 5 g of young leaves and mesocarp of oil palm fruits using of 20 ml extraction buffer containing 1 M TrisHCl pH 8.0, 0.5 M EGTA pH 8.0, 1 M NaCl, 10% SDS, phenol pH 8.0 and b-mercaptoethanol. The mixtures were incubated at 65 8C for 5 min and further extracted twice with 1 volume phenol/chloroform (1:1) and then twice with 1 volume chloroform/isoamyl alcohol (24:1). Total RNA was precipitated in 8 M LiCl at 20 8C for 2 h, followed by centrifugation at 16,230 g for 20 min at 4 8C. The RNA pellets were successively washed with 2 M LiCl, and 70% ethanol. After a short drying at room temperature, the pellets were resuspended in DEPC water and reprecipitated with 300 mM sodium acetate, pH 5.2 and 2.5 volume of absolute ethanol. Total RNA obtained was used as template for cDNA synthesis. 2.3. Primer design and RT-PCR Degenerate dxs oligonucleotides were derived from the highly conserved amino acid sequences among the plant species in Capsicum annuum, Lycopersicon esculentum, Arabidopsis thaliana and Medicago truncatula DXS-homo0 logous. The forward primer FDXS 5 -TGGGATGTTGGT0 0 CATCAG-3 and the reverse primer RDXS 5 -ACTGC(A/ 0 G)TGTTGTTC(C/T)GCTAT-3 were derived from the amino acid sequences WDVGHQS and IAEQHAV, respectively. These primers were used to amplify a 938 base pairs fragment by one-step reverse transcriptase-polymerase chain reaction (RT-PCR). A 50 ml reaction was carried out according to the Qiagen OneStep RT-PCR kit protocol. The thermal prole consisted of reverse transcription at 50 8C for 30 min, initial PCR activation step at 95 8C for 15 8C and was followed by 40 cycles of 1 min denaturation at 94 8C, 1 min annealing at

S. Khemvong, W. Suvachittanont / Plant Science 169 (2005) 571578

573

55 8C and 1 min elongation at 72 8C and a nal extension at 72 8C for 10 min in the last cycle. 2.4. Vector and bacterial strains used for cloning PCR amplication products were puried by using QIAquick PCR purication kit (Qiagen) and then ligated to pGEM-T Easy vector (Promega). The ligation mixture containing product from RT-PCR was transformed into 0 Escherichia coli XL1-blue MRF competent cells (Stratagene), using a heat shock method described by Sambrook et al. [37]. Transformants were selected by ampicillin LBagar plates and the presence of the pGEM-T Easy containing the insert was determined by screening for blue/white colonies using IPTG and X-Gal. Recombinant plasmids were extracted by using QIAprep spin miniprep kit (Qiagen) according to the suppliers instructions and analyzed to conrm the insert by restriction enzyme (EcoRI) digestion and DNA sequencing. 2.5. 5 and 3 RACE procedures Invitrogen GeneRacer kit was used for both 5 and 3 RLM0 0 RACE (RNA ligase-mediated rapid amplication of 5 and 3 0 0 cDNA ends). RNA for 5 and 3 RACE was prepared according to the GeneRacer protocol. Five micrograms of total oil palm leaf RNA (including mRNA, truncated RNA and non-mRNA) was treated with calf intestinal alkaline phosphate to remove 0 the 5 -terminal phosphatase from any RNA that is not full length mRNA. These RNA were then treated with tobacco 0 acid pyrophosphatase to remove the 5 -cap structure of the 0 full-length mRNA thus leaving a 5 -PO4 only on full-length mRNA. RNA ligase was then used to ligate the GeneRacer RNA-oligo primer to only the full length mRNA. First-strand cDNA synthesis was accomplished with SuperScript III Reverse Transcriptase, GeneRacer oligo (dT) primer, and RNA treated as described above as template in 20 ml reaction according to the manufacturers protocol. Two microliters of this reverse transcriptase reaction mixture was used as templates for second-stranded cDNA synthesis and amplication. This was accomplished with high delity Platinum 0 Taq DNA polymerase (Invitrogen), GeneRacerTM 5 Nested 0 0 primer with 5 gene specic primer 5RDXS1 (5 -CAACAGC0 0 CATCCCAAGGGCCGCCGAGAT-3 ) for 5 RACE, and 0 TM 0 GeneRacer 3 Nested primer with 3 gene specic primer 0 3FDXS1 (5 -CTTTCTTCGCCGCTTTCCGACAAGGTGT0 0 3 ) for 3 RACE in a touchdown PCR protocol as described by the manufacturer. Proofreading polymerase was used, and a nal 10 min incubation at 72 8C with Taq polymerase was 0 performed to add a 3 A-overhang. 2.6. Semiquantitative RT-PCR This was performed by conducting parallel reactions on each RNA sample: one using specic primers for dxs2 and dxr and the other using primer for a house-keeping gene (18S
0 0 0 0

rRNA). Total RNA of the oil palm mesocarp at each developmental stage was extracted as described above and used as templates to synthesize the rst-strand cDNA with random primers (Promega) in 25 ml reaction. Two microgram of total RNA was incubated with 1 mg of the primers mentioned above, 1 U/ml of RNasin ribonuclease inhibitor (Promega) and AMV reverse transcriptase buffer (250 mM TrisHCl pH 8.0, 250 mM KCl, 50 mM MgCl2, 50 mM DTT and 2.5 mM spermidine) at 70 8C for 5 min. After the addition of 10 mM dNTPs and 30 U of AMV reverse transcriptase (Promega), the reaction mixture was incubated at 37 8C for 1 h. Second-strand cDNA amplications were performed with primers and the corresponding products are as follows: 0 0 0 5 -TGGGATGTTGGTCATCAGTC-3 and 5 -ACTGCGTGT 0 TGTCTGCTAT-3 were used to amplify a 938 base pair 0 fragment of dxs2 cDNA; 5 -GGCTCTATTGGAACTCA0 0 0 GAC-3 and 5 -ACCCGCATACGGGCCAACC-3 were used 0 to amplify a 773 base pair fragment of dxr cDNA; 5 0 0 CAAAGCAAGCCTACGCTCTG-3 plus 5 -CGCTCCAC0 CAACTAAGAACG-3 were used to amplify a 530 base pair fragment of 18S rRNA cDNA. Conditions for PCR reaction were as follows: 1 cycle at 95 8C for 5 min; 35 cycles at 95 8C for 1 min, 55 8C for 1 min, and 72 8C for 1 min; 1 cycle at 72 8C for 10 min. PCR product be from various experiments had conrmed that it was in the linear range. PCR products were separated by agarose gel electrophoresis and bands intensity was measured by densitometric analysis. Expressions of dxs2 and dxr transcripts at various stages of oil palm fruits were analyzed and compared by using the intensity ratio of the dxs2 and dxr bands with 18S rRNA. 2.7. Sequencing and sequence analysis The cDNA insert in pGEM-T Easy vector was bidirectionally sequenced by the dideoxynucleotide chaintermination method of Sanger et al. [38] using the automated DNA sequencer (ABI Prism 377 DNA Sequencer) according to the manufacturers instructions. Nucleotide and amino acid sequences were compared with databases using the BLAST and FASTA network service. Protein alignment was performed using the ClustalX software [39]. Prediction of protein catalytic and TPP binding sites as well as signal transit peptide was carried out using the SignalP [40,41] and ChloroP [42] program, respectively. The phylogenetic tree was constructed using the Progressive Alignment program [43]. 2.8. Extraction and determination of b-carotene Frozen powder 100 mg for ripe fruits and 200 mg for immature fruits from the oil palm mesocarp tissue used for RNA extraction was disrupted with mortar and pestle and mixed with 1 ml of chloroform:methanol (2:1). The homogenate were centrifuged at 5000 g for 10 min at room temperature. The lower phase was recovered then distilled water 0.2 volume of the extract was added in glass tubes and centrifuged at 5000 g for 10 min to be completely separate

574

S. Khemvong, W. Suvachittanont / Plant Science 169 (2005) 571578

chloroform from watermethanol fractions. To saponify chlorophylls and lipids in these fractions, chloroform were evaporated to dryness and dissolved in 4 ml of methanol. Then, 360 ml of 1 M aqueous solution of KOH was added, and the solution was incubated at room temperature in darkness for 12 h. Unsaponied lipids (carotenoids) were extracted with hexane and then completely evaporated. Subsequently, the residue was dissolved in chloroform and quantied the carotenoids (b-carotene) using spectrophotometer as described by Solovchenko et al. [44]. 2.9. Accession number The nucleotide and deduced amino acid sequences of the full length dxs1 cDNA and partial fragment of dxs2 gene described in this report have been submitted to GenBank and the assigned accession number are AY583783 and AY611205, respectively.

codon TAG at position 2122. In addition, the coding region 0 0 is anked by 5 and 3 untranslated sequences of 45 and 135 bp, respectively. The protein encoded by this cDNA has 707 amino acid residues (accession number AY583783) with a predicted molecular mass of 76.4 kDa and a deduced isoelectric point of 7.0. 3.2. Comparison of the amino acid sequences of DXS from different species and analysis of conserved residues Alignment of the deduced amino acid sequence of DXS from oil palm leaves with the amino acid sequences of several representative DXS from other plants shows that more differences were found in the N-terminal domain than in the C-terminal region. Despite the difference, the sequence comparison reveals considerably high identities, namely 86% with C. annuum (078328). The sequences of DXS from Tagetes erecta (AAG10432), Narcissus pseudonarcissus (CAC08458), M. truncatula2 (CAD22531), Catharanthus roseus (CAA09804), Stevia rebaudiana (CAD22155), Oryza sativa (BAC84616), Morinda citrifolia (AAL32062), A. thaliana (NP_193291), Triticum aestivum (BT009346), M. truncatula (CAD22530), Artemisia annua (AAD56390), Andrographis paniculata (AY254390) and L. esculentum (AAD38941) and that of oil palm share similarities between 67% and 85%, and DXS from oil palm shows also a 50% similarity to bacterial DXS, such as Escherichia coli. Plant DXS proteins contain an N-terminal domain which is not present in the bacterial enzyme. This domain is poorly conserved but shows the general features of plastidial targeting sequences, both of an abundance of the hydroxylated residues serine and threonine including a shortage of the acidic residues aspartic acid and glutamic acid [46], that direct them to the chloroplast stroma and cleaved off by stromal processing peptidase. In agreement with this, the ChloroP predictor suggested a chloroplast transit peptide at the N-terminal of 44 amino acids sequences. Thus, the mature oil palms DXS consists of 663 amino acids. In these chloroplast DXS proteins, the amino acid sequences in the transit peptide parts varied more than the corresponding mature amino acid sequences. Based on the consensus sequence for the chloroplast transit peptide cleavage site (V/I-X-A/C#A; X denotes a basic amino acid) [47], the oil palm DXS transit peptide has the cleavage site at amino acids [4447] (V#CAS). In addition, the DXS sequences contain a highly conserved domain referred to as the thiamin diphosphate (TPP) binding site, present in all enzymes what require TPP as a cofactor. To identify specic TPP binding site and key residues for DXS activity, the DXS amino sequences of algae, oil palm and other plants were compared with Escherichia coli DXS. The result suggests that the common TPP sequence motif begins with the highly conserved sequence -GDG- and concludes with the highly conserved sequence -LNDN- (GDGAMTAGQAYEAMN NAGYLDSDMIVILNDN, amino acids 204234). Amino acids residues between G211, A213, E215, A216, N218 and

3. Results and discussion 3.1. Cloning and DNA sequencing of a full-length cDNA encoding for DXS At the beginning of this work, the partial sequence of oil palm leaves and fruit mesocarp dxs were amplied by one step RT-PCR using degenerated primers designed from highly conserved amino acid sequence from C. annuum (accession number 078328), L. esculentum (accession number AAD38941), A. thaliana (accession number NP_193291) and M. truncatula (accession number CAD22530) DXS-homologous. Following PCR reactions, PCR products were amplied and sequenced. The sequence analysis shows that the PCR products from both leaves and mesocarp are 938 base pairs fragments encoding for proteins with deduced peptide sequence of 312 amino acids, which have high sequence homology to the dxs gene member from C. annuum 80% and 84% identity, respectively. Surprisingly, a preliminary comparison of both nucleotide and amino acid sequences from two different sources were not the same, they differed 19.40% in nucleotide and 8.65% in amino acid sequences. The result shows that there are at least two dxs genes encoding DXS in distinct organs of oil palm, dxs1 in the leaf and dxs2 in the fruit. This is consistent with the reports that the dxs gene from Morinda citrifolia probably contains up to four genes [21] as well as the dxs gene in Rhodobacter capsulatus [18]. In tomato, however, the presence of a single copy gene has been reported [45]. Based on the sequence of the cDNA fragment, two sets of 0 0 gene specic primers were designed to obtain the 5 and 3 0 0 ends followed by 5 and 3 RACE protocol as described in materials and methods. The full-length cDNA of dxs1 gene revealed to be 2301 bp nucleotides. This sequence had an open reading frame of 2121 bp starting with an initiation codon ATG at the position 46 and ending with a termination

S. Khemvong, W. Suvachittanont / Plant Science 169 (2005) 571578

575

G221 are perfectly conserved in all members of DXS family. In addition, N-terminal domain of oil palm shows a good match with a histidine residue (H49), which has been reported to be involved in DXS catalysis [48]. The histidine residue in oil palm DXS corresponding to Escherichia coli DXS H49 is localized at position 102 (H102). 3.3. Phylogenetic analysis Phylogenetic analysis of DXS enzymes from different species revealed three grouping; higher plants, algae and bacteria. In particular, plant DXS can be divided into two groups (Fig. 1); group1 consists of C. annuum, L. esculentum, M. truncatula1, Artemisia annua, A. thaliana, Triticum aestivum, Andrographis paniculata and E. guineensis and the other group comprises of Catharanthus roseus, S. rebaudiana, M. truncatula2, O. sativa, N. pseudonarcissus, Morinda citrifolia and T. erecta. Mutation and duplication events of the

ancestral dxs gene took place prior the separation of species into different genera, Furthermore, plant DXS tends to branch with the homologue form of a-proteobacteria, for instance, R. capsulatus and algae Chlamydomonas reinhardtii, respectively. The similarities of DXS provide a reasonably support that the nuclear encoded enzyme was acquired through gene transfer to the nucleus in the process of the endosymbiotic origin of plastids. 3.4. Correlation of dxs2 and dxr transcript levels and b-carotene biosynthesis Strong correlation between the temporal patterns of dxs2 and dxr expression and carotenoids accumulation suggested that DXS or DXR activity might be a limiting factor for carotenoid biosynthesis during oil palm fruit ripening. To analyse the effect of dxs2 and dxr transcripts on b-carotene accumulation, the oil palm fruit at various developmental

Fig. 1. Phylogenetic relationship of the deduced amino acid sequences of DXS family from different species. Cluster analysis was done using the Progressive Alignment program [43]. The species and corresponding accession number are as follows. CATH (Catharanthus roseus; CAA09804); STEV (S. rebaudiana; CAD22155); NARC (N. pseudonarcissus; CAC08458); MED2 (M. truncatula2; CAD22531); ORYS (O. sativa; BAC84616); TAGE (T. erecta; AAG10432); MORI (Morinda citrifolia; AAL32062); MED2 (M. truncatula; CAD22530); ANDR (Andrographis paniculata; AY254390); CAPS (C. annuum; 078328); LYCO (L. esculentum; AAD38941); ARAB (A. thaliana; NP_193291); ARTE (Artemisia annua; AAD56390); PALM (E. guineensis; AY583783); TRIT (Triticum aestivum; BT009346); CHLA (C. reinhardtii; T08140); ESCH (Escherichia coli; NP286162); AGRO (Agrobacterium tumefaciens STR. C58; NP353769); SINO (Sinorhizobium meliloti; NP384986); MESO (Mesorhizobium loti; NP107784); BRUC (Brucella suis 1330; NP697464); NOVO (Novosphingobium aromaticivorous; ZP00096461); RHOD (Rhodospirillum rubrum; ZP0013656); RHOC (R. capsulatus; P26242); CHRO (Chromobacterium violaceum; ATCC 12472; NP902362); NITR (Nitrosomonas europaea ATCC 19718; NP841218).

576

S. Khemvong, W. Suvachittanont / Plant Science 169 (2005) 571578

palm fruits while dxr did not. Therefore, dxs2 transcripts accumulation in oil palm fruit is controlled by organ-specic and development signals. The increase in dxs2 transcripts accumulation correlates with the transition from young to mature fruits, therefore, dxs2 expression is associated with the activation of carotenoid biosynthesis at the onset of ripening. This is consistent with the report that the dxs downregulation correlated with a lower accumulation of photosynthesis-related isoprenoids such as carotenoids and chlorophylls [49]. In contrast, a strong induction of dxs expression was observed during fruit ripening at the stages of high rate of carotenoids accumulation, although the dxs transcript levels decreased in ripe fruit [45] and also up regulated of dxs in correlation with carotenoids accumulation in pepper fruit is reported [19]. Notably, the carotenoid biosynthesis is a highly controlled process in chromoplastcontaining tissues, suggested that a specic regulation of genes and enzymes is involved in carotenoid biosynthesis at each plastid developmental stage. These ndings suggest that dxs gene might play a regulation role in carotenoid biosynthesis, particularly in the synthesis of b-carotene by the MEP pathway.

Acknowledgements This work was supported in part by the Graduate School and the University Academic Excellence Strengthening Program from Prince of Songkla University, Thailand. The authors would like to thanks Dr. Nualpun Sirinupong for her generosity in providing extra information and her technical assistance in various aspects of this work. Thanks also go to S.A. Hempenius for reading the manuscript.

Fig. 2. Semiquantitative RT-PCR analysis of dxs2 and dxr expression during oil palm fruit development. (A) Pattern of dxs2 and dxr transcripts. The cycle number for RT-PCR was selected to allow linear amplication of the cDNA under study. 18S rRNA was amplied as a cDNA loading control and ethidium bromide-stained rRNA gel is shown as uniform integrity of RNA sample. (B) Relative intensity of dxs2 and dxr transcripts indicated as a ratio of the intensity of both genes versus 18S rRNA. (C) b-carotene concentrations during oil palm fruits ripening following weeks after fertilization.

References
[1] J.C. Sacchettini, C.D. Poulter, Creating isoprenoid diversity, Science 277 (1997) 17881789. [2] J.L. Goldstein, M.S. Brown, Regulation of the mevalonate pathway, Nature 343 (1990) 425430. [3] M. Rohmer, M. Knani, P. Simonin, B. Sutter, H. Sahn, Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate, Biochem. J. 295 (1993) 517524. n, S. Sauret-gueto, F. Gallego, L. [4] N. Campos, M. Rodr guez-Concepcio Lois, A. Boronat, Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for genetic analysis of the 2C-methyl-D-erythritol-4phosphate pathway for isoprenoid biosynthesis, Biochem. J. 353 (2001) 5967. [5] B.M. Lange, T. Rujan, W. Martin, R. Croteau, Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 1317113177. ller, H.K. Lichtenthaler, M. Rohmer, [6] A. Disch, J. Schwender, C. Mu Distribution of the mevalonate and glyceraldehydes phosphate/pyruvate pathways for isoprenoid biosynthesis in cellular algae and the cyanobacterium Synechocystis PCC 6714, Biochem. J. 333 (1998) 381388.

stages were used for RNA analysis performed by a semiquantitative RT-PCR method as well as for measurement of b-carotene content by a spectrophotometric method. The results showed that dxs2 transcript levels increased continuously from 8 to 18 weeks after fertilization and then declined (Fig. 2). The dxs2 transcript levels at 18 weeks are about 2.7 times higher than 8 weeks after fertilization, while the expression of dxr is unchanged. b-carotene content in oil palm mesocarp at various developmental stages also increased with time after fertilization and reached a maximum at 16 weeks corresponding with the dxs2 expression. The relationship of relative intensity between dxs2 and dxr genes and the amount of b-carotene, signicantly showed that high level of dxs2 expression correlated very well with the amount of b-carotene at all the stages of oil

S. Khemvong, W. Suvachittanont / Plant Science 169 (2005) 571578 [7] J. Schwender, M. Seemann, H.K. Lichtenthaler, M. Rohmer, Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehydes-3phosphate non-mevalonate pathway in the green algae Scenedesmus obliquus, Biochemistry 316 (1996) 7380. [8] H.K. Lichtenthaler, J. Schwender, A. Disch, M. Rohmer, Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonateindependent pathway, FEBS Lett. 400 (1997) 271274. ner, C. Mu ller, M. Focke, S. Braun, [9] J. Schwender, J. Zeidler, R. Gro F.W. Lichtenthaler, H.K. Lichtenthaler, Incorporation of 1-deoxy-Dxylulose into isoprene and phytol by higher plants and algae, FEBS Lett. 414 (1997) 129134. [10] D. Arigoni, S. Sagner, C. Latzel, W. Eisenreich, A. Bacher, M.H. Zenk, Terpenoid biosynthesis from of 1-deoxy-D-xylulose in higher plants by intramolecular skeleton rearrangement, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 1060010605. [11] M. Rohmer, M. Seemann, S. Horbach, S. Bringer-Meyer, H. Sahm, Glyceraldehydes-3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis, J. Am. Chem. Soc. 118 (1996) 2564 2566. [12] B.M. Lange, M.R. Wildung, D. McCaskill, R. Croteau, A family of transketolase that directs isoprenoid biosynthesis via a mevalonateindependent pathway, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 2100 2104. [13] L.M. Lois, N. Campos, S. Rosa Poutra, K. Danielsen, M. Rohmer, A. Boronat, Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D1-deoxyxylulose-5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 21052110. rken, T. Wiegert, S. Grolle, A.A. De Graaf, [14] G.A. Sprenger, U. Scho S.V. Taylor, T.P. Begley, S. Bringer-Meyer, H. Sahm, Identication of a thiamin-dependent synthesis in Escherichia coli required for the formation of the 1-deoxy-D-xylulose-5-phosphate precursor to isoprenoids, thiamin, and pyridoxol, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 1285712862. [15] J.H. Julliard, R. Douce, Biosynthesis of thiazole moiety of thiamin (Vitamin B1) in higher plant chloroplasts, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 20422045. [16] R.E. Hill, K. Himmeldrik, I.A. Kennedy, R.M. Paulosky, B.G. Sanger, E. Wolf, I.D. Spenser, The biogenetic anatomy of vitamin B6: a 13C NMR investigation of the biosynthesis of pyridoxol in Escherichia coli, J. Biol. Chem. 271 (1996) 3042630435. [17] T. Kuzuyama, M. Takagi, S. Takahashi, H. Seto, Cloning and characterization of 1-deoxy-D-xylulose-5-phosphate synthase from Streptomyces sp. Strain CL190, which uses both the mevalonate and nonmevalonate pathway for isopentenyl diphosphate biosynthesis, J. Bacteriol. 182 (2000) 891897. [18] F.M. Hahn, L.M. Eubanks, C.A. Testa, B.S.J. Blagg, J.A. Baker, C.D. Paulter, 1-deoxy-D-xylulose-5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus, J. Bacterial. 183 (2001) 111. [19] F. Bouvier, A. dHarlingue, C. Suire, R.A. Backhaus, B. Camara, Dedicated roles of plastid transketolases during the early onset of isoprenoid biogenesis in pepper fruits, Plant Physiol. 117 (1998) 14231431. [20] K. Chahed, A. Oudin, N. Guivarch, S. Hamdi, J.C. Chenieux, M. Rideau, M. Clastre, 1-deoxy-D-xylulose-5-phosphate synthase from periwinkle: cDNA identication and induced gene expression in terpenoid indole alkaloid-producing cells, Plant Physiol. Biochem. 38 (2000) 559566. [21] Y.S. Han, S. Roytrakul, M.C. Verberne, R.V.D. Heijden, H.J.M. Linthorst, R. Verpoorte, Cloning of a cDNA encoding 1-deoxy-Dxylulose-5-phosphate synthase from Morinda citrifolia and analysis of its expression in relation to anthraquinone accumulation, Plant Sci. 164 (2003) 911917.

577

[22] B.M. Lange, R. Croteu, Isoprenoid biosynthesis via a mevalonateindependent pathway in plants: cloning and expression of 1-deoxy-Dxylulose-5-phosphate reductoisomerase from peppermint, Arch. Biochem. Biophys. 365 (1999) 170174. ller, J. Zeidler, H.K. Lichtenthaler, Cloning and [23] J. Schwender, C. Mu heterologous expression of a cDNA encoding 1-deoxy-D-xylulose-5phosphate reductoisomerase of Arabidopsis thaliana, FEBS Lett. 455 (1999) 140144. [24] S. Takahashi, T. Kuzuyama, H. Watanabe, H. Seto, A 1-deoxy-Dxylulose-5-phosphate reductoisomerase catalyzing the formation of 2C-methyl-D-erythritol-4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 98799884. [25] F. Rohdich, J. Wungsintaweekul, W. Eisenreich, G. Rochter, C.A. Schuhr, S. Hecht, M.H. Zenk, A. Bacher, Biosynthessis of terpenoids: 4-diphosphocytidyl-2C-methyl-D-erythritol synthase of Arabidopsis thaliana, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 64516456. [26] F. Rohdich, J. Wungsintaweekul, M. Fellermeier, S. Sagner, S. Herz, K. Kis, W. Eisenreich, A. Bacher, M.H. Zenk, Cytidine-5-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2Cmethyl erythritol, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 11758 11763. ttgen, M. Fischer, W. Eisen[27] F. Rohdich, J. Wungsintaweekul, H. Lu reich, C.A. Schuhr, M. Fellermeier, N. Schramek, M.H. Zenk, A. Bacher, Biosynthessis of terpenoids: 4-diphosphocytidyl-2C-methylD-erythritol kinase from tomato, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 82518256. ttgen, F. Rohdich, S. Herz, J. Wungsintaweekul, S. Hecht, C.A. [28] H. Lu Schuhr, M. Fellermeier, S. Sagner, M.H. Zenk, A. Bacher, W. Eisenreich, Biosynthessis of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of diphosphocytidyl-2C-methylD-erythritol, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 10621067. ttgen, S. [29] S. Herz, J. Wungsintaweekul, C.A. Schuhr, S. Hecht, H. Lu Sagner, M. Fellermeier, W. Eisenreich, M.H. Zenk, A. Bacher, F. Rohdich, Biosynthessis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphos phate, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 24862490. [30] D. Siefermann-Harms, The light-harvesting and protective functions of carotenoids in photosynthetic membranes, Physiol. Plant 69 (1987) 561568. [31] B. Demmimg-Adam, W.W. Adams, The role of xanthophylls cycle carotenoids in the protection of photosynthesis, Trends Plant Sci. 1 (1996) 2126. [32] D. DellaPenna, Nutrition genomics: manipulating plant micronutrients to improve human health, Science 285 (1999) 375379. [33] J. Hirschberg, Production of high-value compounds: carotenoids and Vitamin E, Curr. Opin. Biotechnol. 10 (1999) 186192. [34] S.T. Mayne, Beta-carotene, carotenoids, and disease prevention in humans, FESEB J. 10 (1996) 690701. vez, A. Cantero, A. Reindl, S. Reichler, P. Leo n, 1-deoxy-D[35] J.M. Este xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants, J. Biol. Chem. 276 (2001) 22901 22909. [36] H.P. Affek, D. Yakir, Natural abundance carbon isotope composition of isoprene reects incomplete coupling between isoprene synthesis and photosynthetic carbon ow, Plant Physiol. 131 (2003) 17271736. [37] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, USA, 1989. [38] F. Sanger, S. Nicklen, A.R. Coulson, DNA sequencing with chain termination inhibitors, Proc. Natl. Acad. Sci. U.S.A. 74 (1977) 5463 5467. [39] J.D. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, The clustalX windows interface: exible strategies for multiple sequence alignment aided by guality analysis tools, Nucl. Acids Res. 24 (1997) 48764882.

578

S. Khemvong, W. Suvachittanont / Plant Science 169 (2005) 571578 regulatory role of 1-deoxy-D-xylulose-5-phosphate synthase, Plant J. 22 (2000) 503513. sgen, J. Steppuhn, B. Bruce, K. G. von Heijne, T. Hirai, R.B. Klo Keegstra, R.G. Herrmann, CHLPEP: a database of chloroplast transit peptides, Plant Mol. Biol. 9 (1991) 104126. Y. Gavel, G. von Heijne, A conserved cleavage-site motif in chloroplast transit peptides, FEBS Lett. 261 (1990) 455458. n, A. Boronat, S. Imperial, EssenJ. Querol, M. Rodr guez-Concepcio tial role of residue H49 for activity of Escherichia coli 1-deoxy-Dxylulose-5-phosphate synthase, the enzyme catalyzing the rst step of the 2-C-methyl-D-erythritol-4-phosphate pathway for isoprenoid synthesis, Biochem. Biophys. Res. Commun. 289 (2001) 155 160. G. Giuliano, G.E. Bartley, P.A. Scolnik, Regulation of carotenoid biosynthesis during tomato development, Plant Cell 5 (1993) 379 387.

[40] H. Nielsen, J. Engelbrectht, S. Brunak, A neural network method for identication of prokaryotic and eukaryotic signal peptidase and prediction of their cleavage site, Int. J. Neurol. Syst. 8 (1997) 581591. [41] H. Nielsen, S. Brunak, G. von Heijne, Machine learning approaches for the prediction of signal peptides and other protein sorting signals, Prot. Eng. 12 (1999) 39. [42] O. Emanuelsson, H. Nielsen, G. von Heijne, ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites, Protein Sci. 8 (1999) 978984. [43] D.F. Feng, R.F. Doolittle, Alignment and phylogenetic tree of protein sequences, Meth. Enzymol. 1863 (1990) 375381. [44] A.E. Solovchenko, O.B. Chivkunova, M.N. Merzlyak, I.V. Reshetnikova, A spectrophotometric analysis of pigments in apples, Russ. J. Plant Physiol. 48 (2001) 801808. n, F. Gallego, N. Campos, A. [45] L.M. Lois, M. Rodr guez-Concepcio Boronat, Carotenoid biosynthesis during tomato fruit development:

[46]

[47] [48]

[49]