A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas : Cloning, characterization and subcellular localization

Background:Jatrophacurcas isarichreservoirofpharmaceuticallyactiveterpenoids.Morethan25terpenoidshavebeen isolatedfromthisplant,andtheiractivitiesareanti-bacterial,anti-fungal,anti-cancer,insecticidal,rodenticidal,cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas . IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results: Afull-length JcIPI cDNAconsistingof1355bpwascloned.Itencodedaproteinof305aminoacids.Analysisof deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a signi ﬁ cant evolutionary relatedness with Ricinus communis . Southern blot analysis showed the presence of an IPI multigene family in J. curcas . Comparative expression analysis of tissue speci ﬁ c JcIPI demonstrated the highest transcript level in ﬂ owers. Abiotic factors could induce the expression of JcIPI . Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion: This is the ﬁ rst report of cloning and characterization of IPI from J. curcas . Our study will be of signi ﬁ cant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further con ﬁ rmation.

or decreasing the toxic ones could enhance the comprehensive utilization of J. curcas.
Recent interest generated in the regulation of terpenoid content has prompted many workers to elucidate the biosynthetic pathway. As the biogenesis of terpenoids is yet to be fully understood in J. curcas, it is imperative to identify, clone and characterize key pathway genes to understand the regulatory role of various enzymes. Up to now, several key genes involved in terpenoid biosynthesis pathway have been isolated and characterized from J. curcas. However, there is little information about the cloning and characterization of Jatropha IPI.
The present investigation was aimed at molecular characterization of IPI from J. curcas. Using degenerate primers and RACE PCR strategy, we have cloned a full-length cDNA encoding JcIPI. The gene was analyzed in detail, expression analysis was performed in specific tissues or under stress conditions, and southern-blot analysis was carried out. JcIPI heterologous expression in E. coli and subcellular localization in tobacco were also included in this study.

Plant materials
The J. curcas mature seeds, roots, stems, leaves, flowers, barks and yellow fruits were collected from a five-year old plant in Sichuan Province, China, then snap frozen in liquid nitrogen and transferred to a -80°C freezer till further use for comparative tissue specific expression profile. Young seedlings of J. curcas germinated from the collected mature seeds were cultivated in a culture room maintained at 26 ± 2°C under a 16/8 h (light/dark) photoperiod. Nicotiana tabacum cultivar growing in soil was used for protoplast isolation and transient expression analysis.

RNA isolation and cDNA synthesis
Total RNA was isolated from the leaves of J. curcas using RNAprep pure Plant Kit (Tiangen, China). The quality and quantity of RNA were checked by agarose gel electrophoresis and by spectrophotometry (NanoVue ND-1000, China) respectively. RNA samples having A260/ 280 in the range of 1.8 to 2.0 and A260/230 N 2 were selected for cDNA synthesis and were stored at -80°C prior to RT-PCR and RACE analysis. First strand cDNA was synthesized using the Primescript™ RT reagent Kit (Takara, China).

Cloning of full length JcIPI
Two degenerate primers (DP1 and DP2, Table 1) designed on the basis of the conserved amino-acid regions of published IPIs from other plants were used for the amplification of the core cDNA fragment of JcIPI. Using cDNA as the template, PCR was performed under the following conditions: 95°C for 4 min, 35 cycles of 94°C for 30 s, 53.5°C for 30 s and 72°C for 50 s followed by a final extension of 72°C for 10 min. A 590 bp DNA fragment was recovered, cloned into the pMD19-T vector (Takara, China) and sequenced (Invitrogen, China). This fragment was subsequently used for designing gene specific primers for the cloning of 3′ and 5′ ends of JcIPI by RACE-PCR.
To obtain the 3′and 5′-ends of JcIPI, the anchored-RACE extension method was used as described by Newton et al. [17]. The initial 3′ RACE PCR was carried out using 3P1 and P1 primers. The primary PCR product was used as the template for nested 3′ RACE PCR reaction in which 3P2 primer was used along with P2 primer. Both initial and nested PCR reactions were carried out under the following conditions: 4 min at 95°C, 35 cycles (30 s at 94°C, 30 s at 55°C and 1 min at 72°C) and 10 min at 72°C. For 5′ RACE PCR, a polyA tail was added to the 3′ end of the cDNA using the terminal deoxynucleotidyl transferase according to the manufacturer instructions (Takara, China). Then similar reactions were carried out for 5′ RACE-PCR in which 5P1 and P0 primers were used for the initial reaction while 5P2 and P2 primers for the nested PCR reaction. Thermo profile was as follows: 4 min at 95°C, 35 cycles (30 s at 94°C, 30 s at 59°C and 1 min at 72°C) and 10 min at 72°C. The two nested amplified fragments of both 3′ and 5′ RACE were cloned into pMD19-T vector and sequenced. After assembling the sequences of 3′ and 5′ RACE products, the full length cDNA sequence of JcIPI was obtained.

Sequence analysis
The nucleotide sequence of JcIPI obtained was translated using Translate tool (http://www.expasy.ch/tools/dna.html) and the properties of deduced amino acid sequence were estimated using ProtParam (http://www.expasy.ch/tools/protparam.html). Structural and functional regions were identified in deduced protein sequence by conserved domain database (CDD) at the National Center for Biotechnology Information (NCBI). Secondary structure was determined by SOPMA (http://npsa-pbil.ibcp.fr) program.

Phylogenetic analysis and prediction of three-dimensional structure of JcIPI
Protein sequences were retrieved from the GenBank through the BLAST p algorithm at NCBI using JcIPI sequence as a query and several IPIs with the highest score from different plants were selected. Sequences were aligned using the DNAman software package (Version 5.2.9, Canada) using default parameters. Phylogenetic analysis of deduced amino acid alignments was generated using the neighbor-joining method with MEGA 5.1 software [18]. The three-dimensional structure of JcIPI was predicted using Phyre 2 web server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi? id=inde%20x).

Southern blot analysis
Genomic DNA was isolated from the leaves of J. curcas using Plant Genomic DNA Kit (Tiangen, China). About 10 μg genomic DNA was completely digested with Kpn I, Xba I, BamH I and EcoR I/BamH I respectively, separated by electrophoresis on a 0.8% (W/V) agarose gel and then transferred to a Hybond-N + nylon membrane. DNA hybridization was performed according to the standard procedures [19]. The membrane was washed twice at 20°C for 5 min in 2 × SSC, 0.1% (W/V) SDS and then twice in 0.5 × SSC, 0.1% SDS at 65°C for 15 min. DNA probe was the PCR product amplified with QJcIPI1 and QJcIPI2 primers (ORF sequence from 700 to 899, Table 1), labeled using DIG High Prime DNA Labeling and detected using Detection Starter Kit II (Roche, Germany).

Tissue specific expression analysis by quantitative real-time PCR (qRT-PCR)
Tissue specific expression of JcIPI was analyzed in mature seeds, roots, stems, leaves, flowers, barks and yellow fruits. Total RNA was extracted from the tissues mentioned above, purified by using DNase 1 from DNA contamination and reversely transcribed to the first-strand cDNA by oligo (dT) 18 primer. QJcIPI1, QJcIPI2, 18SrRNA1 and 18SrRNA2 primers ( Table 1) were designed close to the 3′-end sequence of JcIPI and 18S rRNA (an internal control, GenBank accession no. FJ489608.1) respectively. The primers were tested for linearity by constructing standard curves on five serial 10-fold dilutions in order to ensure maximum specificity and efficiency during qRT-PCR. QRT-PCR was performed using a Bio-Rad iCycler MyiQ Real-Time PCR System in a 20 μL reaction volume containing 1 μL appropriate diluted cDNA from each sample, 10 μL SsoFast™ EvaGreen® Superemix (Bio-Rad, USA), 1 μL forward primer, 1 μL reverse primer and 7 μL ddH 2 O. PCR conditions were: 95°C for 30 s, 40 cycles of 95°C for 5 s and 50°C for 10 s, followed by a final melt curve profile (65-95°C). Quantification was determined with the comparative C T method and each datum represented the average of three parallel samples.

Gene expression of JcIPI under stress conditions by qRT-PCR
In order to investigate the gene expression under stress conditions including low temperature (4°C), salinity (NaCl), drought (mannitol) and methyl jasmonate (MeJA), the seedlings of J. curcas were cultured on MS solid mediums with the method described by Attaya et al. [20] with a slight modification. The procedures involved the decoating of the seeds, subsequent soaking in distilled water for 4 h at room temperature, surface sterilizing with 70% (v/v) ethanol, immersing in 0.1% (w/v) mercuric chloride (HgCl 2 ) solution and rinsing by sterile distilled water (four times). These surface sterilized seeds germinated on MS mediums. After 3 weeks, the seedlings were transferred to MS liquid mediums under stress conditions mentioned above for 24 h in the culture room. Leaves were collected, immediately frozen in liquid nitrogen and stored at -80°C before RNA isolation. The method of qRT-PCR was the same with the section of tissue specific expression analysis.

Heterologous expression of JcIPI in E. coli
Using first strand cDNA as the template, the entire ORF containing the start codon and the stop codon was amplified by two gene-specific primers (SP1 and SP2, Table 1) containing BamH I and EcoR I restriction sites respectively. The amplified product was double digested with BamH I and EcoR I enzymes and ligated into pET-32a vector digested with the same two restriction enzymes. The pET-JcIPI construct was transformed into E. coli BL-21 (DE3) cells (Merck, Germany).
An overnight culture of E. coli BL-21 (DE3) cells containing pET-JcIPI construct was grown at 37°C in Luria broth medium (LB). 1% of the   overnight culture was used to inoculate fresh LB medium to obtain exponentially growing culture (OD of 0.5-0.6). 0.5 mM IPTG (Isopropyl-β-D-1-thiogalactopyranoside) was added to exponentially growing cells. Induced culture at 16°C was harvested after 12 h. The harvest 1 mL culture aliquots were centrifuged at 12,000 rpm for 1 min at 4°C and the pellet was resuspended in 1 mL extraction buffer (50 mM Tris-HCl, pH 8.0, 10 M MgCl 2 , 20% glycerol and 5 mM 2-mercaptoethanol). Cells were disrupted by sonication on ice for 15 to 20 s burst time period. The lysate was centrifuged at 12,000 rpm for 1 min at 4°C The supernatant, precipitate and overall lysate were boiled with 2 × SDS loading dye for 10 min and loaded on (15%) SDS-PAGE gel.

Protoplast isolation and transient expression
The ORF of JcIPI without the stop codon was amplified by SSP1 and SSP2 primers containing Xba I and BamH I restriction sites respectively. The amplified product was double digested with Xba I and BamH I enzymes and ligated into PBI121-GFP vector digested with the same two restriction enzymes. This JcIPI-GFP construct was used for subcellular localization. The isolation and transfection of N. tabacum leaf mesophyll protoplasts were conducted as described by Yoo et al. [21]. Briefly, protoplasts were isolated from well-expanded leaves of N. tabacum plants. Volumes of JcIPI-GFP plastids (10 to 20 μg) were transfected into 150 μL protoplasts using a PEG-calcium transfection solution. Protoplasts were incubated overnight at 22 ± 2°C under white light to allow expression of the introduced gene. The GFP fluorescence was examined and photographed using a Leica SP5 confocal microscope (Leica, Germany).

Cloning and sequence analysis of JcIPI
A full length gene designated as JcIPI (GenBank accession no. KJ908689) was isolated from J. curcas leaves. Based on the conserved amino acid domains of IPIs from other plants, degenerate primers were designed to amplify an initial core fragment of 590 bp. RACE technique was used to obtain the 3′ and 5′ ends of JcIPI. RACE amplicons of 3′ and 5′ ends were amplified, sequenced and assembled, thus a full length gene sequence of 1355 bp was generated containing an ORF of 918 bp encoding a protein of 305 amino acids. The 5′ and 3′ untranslated region (UTR) sequences were of 154 and 283 bp respectively. The comparison of the full-length cDNA and the genomic DNA of JcIPI revealed that five introns were present in the genomic DNA (Fig. 2).

Analysis of the deduced protein sequence
Multiple sequence alignments showed a high degree of conservation in the functional region (aa 90-270). It also revealed that JcIPI had a significant similarity with many IPIs from higher plants at the amino acid level such as Hevea brasiliensis (92%), Populus trichocarpa (90%), Medicago truncatula (85%), Ricinus communis (84%), Theobroma cacao (82%), Gossypium barbadense (78%), Vitis vinifera (77%) and Corylus avellana (76%), indicating that JcIPI belonged to the IPI superfamily (Fig. 3). In order to investigate the evolutionary position of JcIPI among the phylogenetic tree of various IPIs, aphylogenetic tree of IPIs was constructed using MEGA 5.1 software as shown in Fig. 4. Among IPIs, JcIPI and RcIPI showed the closest relationship because they both belonged to the family Euphorbiaceae.
IPIs have a typical region called NUDIX motif associating with their catalytic function. These NUDIX enzymes classically catalyze the hydrolysis of Nucleoside DIphosphate linked to another moiety (X) by nucleophilic substitution in presence of a divalent cation [22]. CDD search at NCBI indicated the presence of conserved active sites, metal binding sites and NUDIX motif in JcIPI, suggesting that the protein may encode for a functional enzyme. The secondary structure analysis of JcIPI showed that predicted JcIPI protein  consisted of 42.95% alpha helix, 11.80% extended strand, 2.30% beta turn and 42.95% random coil. The three dimensional structure of was predicted by Phyre 2 web server (Fig. 5) using Homo sapiens isopentenyl diphosphate delt-isomerase 2 as a template. The model covered 76% JcIPI sequence with 100% confidence. It showed that JcIPI was a compact globular protein and belonged to the class of α/β protein. It had two typical structure domains with Cys-159 and Glu-188 active sites respectively, which was consistent to other IPIs. Metal irons are crucial for catalysis and engaged in the interactions with the diphosphate moiety through bindings with the Cys-159 and Glu-188.

Southern blot analysis
IPI genes have been shown to be duplicated in mammals, many plants and algae [23]. However, as shown in Fig. 6, four bands were detected when the genomic DNA was digested with BamH I. The result suggested that JcIPI was encoded by multiple genes. It revealed the presence of an IPI gene family in J. curcas genomic DNA. These different members in gene family may be regulated in different manners and the specific role of individual needed to be investigated further.

Comparative tissue specific expression of JcIPI
Relative transcript analysis of JcIPI from different tissues (mature seeds, roots, stems, leaves, flowers, barks and yellow fruits) was carried out using quantitative real-time PCR. The expression level in all tissues was normalized against the normal transcript level of 18S rRNA, which was taken as a control in this experiment. Results showed that JcIPI expression could be detected only in flowers, leaves and seed kernels (Fig. 7), suggesting that it is not a constitutively expressed gene in various tissues. JcIPI expression in flowers was very high and the expression profile was similar to Arabidopsis thaliana [4] and Catharanthus roseus [24]. Intriguingly, Wang and Tang [25] found that expression levels of IPI from C. avellana were significantly higher in roots than in either stems or leaves. The reason we considered was that flowers had vigorous life activities and high levels of secondary metabolic activities. Also, the difference of subcellular localization between JcIPI and others may lead to the different expression levels.

JcIPI mRNA expression under stress conditions
Terpenoids in plants have an important role in defense against abiotic stresses [26]. The expressions of many enzyme genes in the biosynthetic pathway can be induced by some environmental factors. To examine the influence of stress conditions like low temperature, salinity, drought and MeJA on IPI expression in J. curcas, the total RNA of leaves from treated plants was subjected to qRT-PCR analysis. The results showed that JcIPI gene transcripts of those plants exposed to stress conditions were obviously higher than those of the control plants (Fig. 8). Abiotic factors like salinity, low temperature and drought correlated with JA response. Microassay in Arabidopsis revealed the detailed changes in gene expressions [27,28]. Under salt and cold stresses, the expression of JcIPI was increased by 147% and 273%, respectively. With the increasing degree of drought, the accumulation of JcIPI mRNA was enhanced too. It appeared that JcIPI was involved in the response to those stresses.

Bacterial expression of JcIPI
In order to observe the expression of JcIPI in E. coli, the entire ORF of JcIPI was cloned into the expression vector pET-32a, containing a functionally T7 promoter and a Trx tag (about 18 kDa). It was a fusion tag to facilitate subsequent purification of the recombinant protein.
The pET-JcIPI construct was sequenced to check for in-frame fusion and then transformed into E. coli BL-21 cells. The resultant recombinant JcIPI fusion protein with an approximate size of 50 kDa (including the Trx tag) was observed on the 15% SDS-PAGE gel (Fig. 9, L-4). This was consistent with the predicted protein mass of 34.36 kDa. The induced E. coli cells transformed with pET-32a did not show recombinant protein bands at the corresponding point (Fig. 9,  L-3). The results showed that JcIPI could highly and correctly expressed in E. coli and provided an experimental basis for future tests of enzyme activity.

Subcellular localization of JcIPI
Transient expression analysis of JcIPI-GFP construct in tobacco protoplasts was used to determine the subcellular localization of JcIPI. Plant cells have two pathways producing IPP/DMAPP: the mevalonic acid (MVA) pathway in cytosols and the methyl-erythritol-phosphate (MEP) pathway in plastids [21]. Correspondingly, there are two IPI isoforms localized in cytosols and plastids respectively. Furthermore, IPI is also present in other organelles such as peroxisomes [29,30] and mitochondria [4,31]. Terpenoids synthesized in different organelles were different, thus the study on subcellular localization was of great importance. Based on Fig. 10, JcIPI-GFP was localized in chloroplasts. Therefore, this JcIPI we obtained is involved in the MEP pathway in J. curcas. It would regulate the subsequent metabolites including mono-, diter-and poly-terpenoids.

Conclusions
Pathway laxity and modulation using metabolic engineering are interesting prospects for regulating the terpenoid production in J. curcas. Rate-limiting enzymes have been found to play important roles in regulating the metabolic flux in several plants [32]. Based on this backdrop, a new chloroplastic isopentenyl diphosphate isomerase (JcIPI) gene was cloned from J. curcas. JcIPI showed more than 75% homology to IPIs from other plants, with active sites of Cys159 and Glu188. Expression levels of JcIPI were highest in flowers of Jatropha plants. An IPI gene family was present in Jatropha genomic DNA. Transient expression revealed that JcIPI was localized in chloroplasts and participated in the MEP pathway. These findings will further our fundamental knowledge of key genes involved in the terpenoid biosynthetic pathway of J. curcas, and provide a foundation for terpenoid metabolic engineering using the JcIPI gene.