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First published online March 31, 2006; 10.1104/pp.106.077008 Plant Physiology 141:220-231 (2006) © 2006 American Society of Plant Biologists Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity1Department of Biological Sciences, Lehman College, City University of New York, Bronx, New York 10468 (M.C.-C., C.E.G., C.Z., E.T.W.); and the Graduate School and University Center, City University of New York, New York, New York 10016 (M.C.-C., E.T.W.)
Isoprenoids are the most diverse and abundant group of natural products. In plants, farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors to many isoprenoids having essential functions. Terpenoids and sterols are derived from FPP, whereas gibberellins, carotenoids, casbenes, taxenes, and others originate from GGPP. The corresponding synthases (FPP synthase [FPPS] and GGPP synthase [GGPPS]) catalyze, respectively, the addition of two and three isopentenyl diphosphate molecules to dimethylallyl diphosphate. Maize (Zea mays L. cv B73) endosperm cDNAs encoding isoprenoid synthases were isolated by functional complementation of Escherichia coli cells carrying a bacterial gene cluster encoding all pathway enzymes needed for carotenoid biosynthesis, except for GGPPS. This approach indicated that the maize gene products were functional GGPPS enzymes. Yet, the predicted enzyme sequences revealed FPPS motifs and homology with FPPS enzymes. In vitro assays demonstrated that indeed these maize enzymes produced both FPP and GGPP and that the N-terminal sequence affected the ratio of FPP to GGPP. Their functionality in E. coli demonstrated that these maize enzymes can be coupled with a metabolon to provide isoprenoid substrates for pathway use, and suggests that enzyme bifunctionality can be harnessed. The maize cDNAs are encoded by a small gene family whose transcripts are prevalent in endosperm beginning mid development. These maize cDNAs will be valuable tools for assessing the critical structural properties determining prenyl transferase specificity and in metabolic engineering of isoprenoid pathways, especially in cereal crops.
With more than 30,000 identified compounds, isoprenoids are the oldest, most abundant, and structurally diverse natural products (Lange et al., 2000  ; Kharel and Koyama, 2003). In plants, isoprenoids and their derivatives are involved in a number of important processes, including photosynthesis and photoprotection, defense, regulation of growth and development, and attracting animals involved in pollination and seed dispersion (DellaPenna, 1999). Some isoprenoids are substrates for pathways leading to the production of the pharmaceutically relevant taxenes and casbenes. Protein prenylation, a posttranscriptional addition of farnesyl and geranylgeranyl groups, is part of signal transduction, intracellular trafficking, and tissue differentiation in plants (Crowell, 2000). The complexity of isoprenoid biosynthetic pathways is manifest from the scores of genes and proteins reported to date and the large number of genes predicted from analysis of the Arabidopsis (Arabidopsis thaliana) genome (Lange and Ghassemian, 2003).
Isoprenoid biosynthesis proceeds through sequential 1
One approach to isolating genes encoding isoprenoid synthases is to couple genes encoding these enzymes with a downstream pathway that acts as a reporter of functional enzyme activity. A valuable reporter system is the carotenoid biosynthetic pathway that converts isoprenoid precursors to easily detected colored products (Cunningham et al., 1993
Functional Screening to Identify Maize GGPPS cDNAs
As a preliminary step to identify maize GGPPS cDNAs, the E. uredovora carotenoid gene cluster deleted for crtE was used for functional complementation screening of maize B73 inbred endosperm cDNAs. In the absence of GGPPS encoded by crtE, cells were unable to accumulate the pathway end product, zeaxanthin-
To confirm the identity of the yellow pigment in the
Sequence Analysis of Maize cDNAs
The functional GGPPS endosperm cDNAs from maize were sequenced and found to be virtually identical. Figure 3
shows the nucleotide and predicted amino acid sequences for one long (pME14D1184) and one short cDNA (pME14D1268) that were deposited in GenBank (AF330036 and AF330037, respectively). Excluding the vector-encoded N-terminal
Having employed a GGPPS functional complementation strategy to isolate the maize cDNAs, we predicted homology of the isolated clones to other GGPPS genes. Instead, BLAST searches (Altschul et al., 1997 ) produced a surprising homology to a truncated but otherwise identical FPPS clone from maize inbred W64A (GenBank L39789; Li and Larkins, 1996). Furthermore, BLAST searches showed that our maize clones, although enabling the synthesis of zeaxanthin- -D-diglucoside, shared characteristic signatures with other FPPS sequences, including FARM, the chain-length-determining (CLD) region, and the GQ motif. Additionally, the SARM predicted from the maize isoprenoid synthase clones was identical to that in rice (Oryza sativa) and Arabidopsis FPPS enzymes (Fig. 4
). Based on characteristics of the CLD region and the sequence preceding the GQ (Hemmi et al., 2003), the polypeptides encoded by the maize clones corresponded to type I FPPS (Wang and Ohnuma, 1999).
In Vitro Isoprenoid Assays
The maize cDNAs shared structural motifs with FPPS enzymes but clearly functioned in the heterologous bacterial system as GGPPS. To directly investigate the reaction products, the novel maize enzymes were further analyzed by in vitro incorporation of [4-14C]IPP in cell-free extracts from transformants of the pBluescript-based constructs. The dephosphorylated, radiolabeled products were identified by reversed-phase thin-layer chromatography (TLC). To ensure that [4-14C]-IPP incorporation results reflected differences in the activities of enzymes tested, but not in their expression levels, cDNAs cloned in vectors with the same promoter [e.g. pBluescript II SK (–) and pUC8] and the same host cells were used when assaying cell extracts. Figure 5
shows that cell extracts containing either the long (1184) or short (1268) maize FPPS cDNAs (lanes 2 and 3, respectively) yielded GGPP (as indicated by the dephosphorylated form, geranylgeraniol [GGOH]), and pathway intermediates GPP and FPP (as indicated by geraniol [GOH] and farnesol [FOH]). Identical products were seen for the bona fide GGPPS from H. brasiliensis (lane 4) but were virtually absent when cells contained only the empty vector, negative control (lane 1). The fact that cell-free extracts containing the maize isoprenoid synthases produced [14C]GGPP (lanes 2 and 3) explains why plasmids pME14D1184 and pME14D1268 could complement
We proceeded to test if the GGPP produced by the maize enzymes was influenced by the 38-residue N-terminal -galactosidase (lacZ) sequence encoded by the cloning vector or by the 41-residue N terminus encoded by the 5'-untranslated region (UTR) in the long clone. To this effect, pME14D1184 was subjected to PCR to amplify either the entire insert containing the 5'-UTR that encodes 41 residues upstream of the open reading frame (ORF), or the ORF only (as it starts from nucleotide 123 in Fig. 3 and contains the nine codons missing from the shorter maize clone). The amplification products were subcloned in the expression vector pQE30, which unlike pBluescript II SK (–), does not encode a -galactosidase (lacZ) N-terminal peptide fusion, but contains instead a 6X-His tag. The 5'-UTR-containing construct was designated pQE1184UTR; the subcloned ORF was named pQE22ORF (see Fig. 6C
for construct cartoons).
Lane 1 in Figure 6A shows the 14C-incorporation activity in an extract from cells transformed with the empty pQE30 vector. The remaining lanes display the activities of extracts containing protein products of pME14D1184 (contains lacZ, 41-residue maize UTR, and ORF; lane 2; compare with Fig. 5), pQE1184UTR (contains 6X-His tag, 41-residue UTR, and ORF; lane 3), and pQE22ORF (contains 6X-His tag and ORF; lane 4). Polypeptides with UTR-encoded residues produced significant amounts of GGPP, regardless of an N-terminal -galactosidase fragment (lane 2) or not (lanes 3). This clearly shows that the GGPP production was not a consequence of having a -galactosidase N-terminal fusion but was a property inherent in the maize enzyme itself. Remarkably, the construct (pQE22ORF) without lacZ and the UTR also produced GGPP (lane 4), and FPP at higher levels then the short clone that is truncated by nine additional codons (Fig. 5, lane 3). To further demonstrate that it was the FPPS enzyme alone that was responsible for GGPPS activity, we also tested the purified protein in the in vitro assay. Lane 5 (Fig. 6B) shows the isoprenoid synthase activity of the purified protein product from pQE1184UTR. This protein, without the -galactosidase fragment and devoid of bacterial cytoplasmic background, clearly produced GGPP (in addition to FPP and GPP), a phenotype corroborated by pQE1184UTR complementation of the  crtE lesion in E. coli (data not shown). In summary, these results indicate the GGPPS activity associated with the maize FPPS clones was not due to presence of the 38-residue N-terminal -galactosidase fragment encoded by pBluescript II SK (–). Furthermore, it appears that the 41 amino acids encoded by the 5'-UTR influenced the ratio of FPP/GGPP product associated with the FPPS enzymes. Truncation at the N terminus also interfered with FPPS activity.
To determine whether GGPP production by the maize enzymes was influenced by pH, we used the in vitro assay at varying pH. Typically, neutral to alkaline pH conditions have been used for in vitro prenyl transferase assays, e.g. FPPS from Arabidopsis and yeast (Saccharomyces cerevisiae) has been assayed at pH 7.0 (Cunillera et al., 1996
Hybridization of the maize cDNAs to genomic DNA from several maize lines revealed multiple bands and maize B73 bacterial artificial chromosome genomic DNA clones divided into at least three distinct groups (data not shown). Similarly, chromosome mapping data also indicated that the maize isoprenoid synthase gene mapped to three loci, closely linked with bnl117.20 (8L, 69.8), umc15B (3L, 142.6), and npi280 (6L, 144.7).
Despite evidence for more than one gene copy in maize, a northern blot of total RNA from a developmental series of maize B73 endosperms showed a single major transcript, approximately 1.5 kb in size (Fig. 7A
), a size comparable to that of the endosperm library-derived long cDNA clones. Transcripts were barely detectable until 15 and 20 d after pollination (DAP). The observed transcript levels were consistent with the abundance of cDNA clones isolated from the endosperm cDNA library. Transcript accumulation for Shrunken-1, a gene encoding a starch biosynthetic enzyme, gave a similar profile of temporal expression (Fig. 7B), as previously reported (Wurtzel et al., 1987
The use of a functional complementation strategy as a preliminary screen for plant isoprenoid biosynthesis cDNAs provided new insight into enzyme activity of maize FFPS, which would otherwise not have been predicted from informatic analysis. We demonstrated that the GGPPS activity associated with maize FPPS could be harnessed by coupling to a downstream pathway. While we ruled out the effect of an N-terminal -galactosidase fusion to elicit this unexpected activity, we showed that the FPPS N terminus impacts both its activity and specificity.
Experimental conversions between FPPS and GGPPS have been achieved using genes from several microorganisms and usually they have been generated by changes in amino acids in the CLD or other regions. The conversion of archaeal GGPPS to FPPS by site-directed mutagenesis identified key residues involved in determining product length specificity, a result that in turn served as the basis for a hypothesis on the evolution of eubacterial and eukaryotic FPPS types from an ancestral GGPPS (Ohnuma et al., 1997
Quaternary structure is another factor influencing isoprenoid synthase specificity. For example, the M. piperita GPPS heterotetramer has two regulatory small subunits (GPPS.ssu) and two large catalytic subunits (GGPPS.lsu). When GPPS.ssu is coexpressed with conifer GGPPS in E. coli, it causes the latter to produce GPP instead of GGPP, apparently by constricting the active site. However, GPPS.ssu does not seem to affect FPPS specificity (Burke and Croteau, 2002
It is conceivable that metabolic context may also impact the maize FPPS enzyme activity, whether the enzyme is expressed by an endogenous gene or as a transgene. Metabolic context has been shown to affect enzyme regiospecificity when subcellular location was altered as in the case of a fatty acid desaturase (Heilmann et al., 2004
Secondary reactions catalyzed by certain enzymes in addition to synthesizing their main products are not a rare occurrence. The term catalytic promiscuity has been applied to this phenomenon (Copley, 2003
Based on similarity to FPPS2, an Arabidopsis cytoplasmic form (GenBank Q43315) and absence of signatures associated with subcellular targeting (including plastid, mitochondrion, and endoplasmic reticulum; Emanuelsson et al., 2000
The existence of a multigene family, with at least three members, is inferred from genomic DNA analysis and chromosome mapping data. Gene families for isoprenoid biosynthetic enzymes have been reported in other plants. Two cDNAs corresponding to two fpps genes have been reported for Arabidopsis (Lange and Ghassemian, 2003
To date, metabolic engineering of isoprenoids has sometimes led to unexpected results due to insufficient understanding of metabolic networks (Jørgensen et al., 2005
Enzymes and Chemicals
All reagents were analytical or HPLC grade. Organic reagents and solvents were purchased from Sigma-Aldrich. Salts and buffers were obtained from Fisher Scientific. Molecular biology reagents, including restriction enzymes, were bought from Invitrogen. Perkin-Elmer was the source of [
Maize (Zea mays L. cv B73) inbred plants were grown in the field (Pelham Bay, Bronx, NY) and cross pollinated. Endosperms were dissected at various DAP. Tissue was quickly frozen in liquid nitrogen and stored at –80°C until used.
Cell growth, bacterial transformation, and other methods were performed according to standard protocols (Sambrook et al., 1989
Total RNA was isolated from maize B73 endosperm at 14 DAP for production of a unidirectional cDNA library contained in pBluescript II SK (–) (Gallagher et al., 2003
The maize B73 endosperm cDNA library was screened by functional complementation of the Erwinia carotenogenic cluster
Plasmid DNAs were extracted from the double transformants, after growth in LB medium plus antibiotics, by using the Wizard Midikit (Promega) and separated by agarose gel electrophoresis. Library-derived plasmids were isolated from the agarose gel, purified using Geneclean II (Bio101), and utilized to retransform E. coli TOP10F' cells containing pACCAR25
Both strands of cDNAs were initially sequenced using standard primers for pBluescript II SK (–), then by walking, using internal primers, in an automated Applied Biosystems 3730XL capillary apparatus at the DNA Sequencing Facility of the University of Chicago Cancer Research Center. Sequence assembly and analysis were done using BioImage software (Millipore) and Vector NTI Suite v. 8 (InforMax).
E. coli TOP10F' cells containing plasmid pACCAR25
E. coli TOP10F' was used to propagate expression plasmids pME14D1030, pME14D1268, pBS-FPS, pHBGG6 (Takaya et al., 2003
A protocol to assay isoprenoid synthase activity was developed from a combination of the procedures of Cunillera et al. (1996)
The pME14D1184 insert was amplified using Promega's Master Mix in a Touchgene Gradient thermocycler (Techne). Primers for amplification of insert including the 5'-UTR were 5'-ACTGCAGTCCCTCCCTCCTTCCTTCCTTC-3' (sense) and 5'-GAAGCTTATCCAAGAGCACCCTACTTCTG-3' (antisense), which introduced PstI and HindIII restriction sites (underlined) at the product's 5'- and 3'-ends, respectively. To limit amplification to the ORF, the oligonucleotide 5'-ACTGCAGTGGCGGCGGGCGGGAATGG-3' (sense) was designed. This primer also contains a PstI site (underlined) but the initiation codon was changed to GTG (italicized), as the ATG-containing amplification product did not direct the synthesis of recombinant protein. PCR for both sets was performed with an initial denaturation step at 94°C for 3 min followed by 30 cycles of 45 s at 95°C, 45 s at 60°C, and extension for 2 min at 72°C, and a final extension for 10 min at 72°C. PCR products were digested with PstI and HindIII, gel purified using the Mini-Elute PCR Purification kit (Qiagen), and ligated into the dephosphorylated corresponding sites of expression vector pQE30 (Qiagen). The resulting sequence-verified constructs, respectively named pQE1184UTR and pQE22ORF, were used to transform E. coli M15 cells. The recombinant protein expressed from these pQE30-derived constructs contains an N-terminal 6XHis tag for purification by affinity chromatography.
An extract of E. coli M15 cells containing pQEM1184UTR was prepared as described above, except that cells were washed with and concentrated (40x) in lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole) before sonication. The His-tagged isoprenoid synthase was purified by chromatography of the cell extract in a Ni-NTA Agarose Superflow column, under native conditions, as per the manufacturer's instructions (Qiagen). Protein concentration in column fractions was determined by the bicinchoninic acid method (BCA, Pierce) using BSA dissolved in the corresponding buffers. Concentration of the BSA stock was determined by A280 using an
Activity of the purified recombinant isoprenoid synthase was determined as described above, but using 1 µg of protein.
RNA from maize B73 endosperms at 0, 5, 10, 15, and 20 DAP was isolated as previously described (Burr and Burr, 1981 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF330036 and AF330037.
We are grateful to Dr. Norihiko Misawa of the Kamaishi Laboratories, Japan, for supplying the E. uredovora crt constructs. We appreciate suggestions from Dr. Núria Cunillera (Universitat de Barcelona, Spain), who also provided strains and related plasmids, and from Dr. Kazunori Okada (Tokyo Gakugei University, Japan) who provided Arabidopsis GGPPS plasmids. We thank Dr. Tanetoshi Koyama (Tokohu University, Japan) for the Hevea GGPPS plasmid and Dr. Gerhard Sandmann (University of Frankfurt, Germany) for Gentiana cDNAs. We thank Dr. Ben Burr (Brookhaven National Laboratory) for mapping data and the following staff from Lehman College of the City University of New York: Dr. Dwight Kincaid for advice on statistical analysis, David Cain for assistance with maize propagation, and Christina Murillo for technical support. Received January 12, 2006; returned for revision March 3, 2006; accepted March 22, 2006.
1 This work was supported by grants from the National Institutes of Health (grant no. S06 GM08225 to E.T.W. and M.C.C.), the City University of New York Collaborative Incentive Program (to E.T.W. and M.C.C.), and the Professional Staff Congress of the City University of New York (to E.T.W.). 
2 Present address: Pfizer, Inc., 235 East 42nd St., New York, NY 10017.
3 Present address: Departament de Produccio Vegetal i Ciencia Forestal, Universitat de Lleida, Av. Alcalde Rovira Roure 177, E–25198 Lleida, Spain. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eleanore T. Wurtzel (wurtzel{at}lehman.cuny.edu). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.077008. * Corresponding author; e-mail wurtzel{at}lehman.cuny.edu; fax 718–960–7348.
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ABSTRACT
RESULTS
4' condensations of isopentenyl diphosphate (IPP; C5) with an allylic acceptor, the first of which is dimethylallyl diphosphate (DMAPP; C5). This reaction is catalyzed by highly conserved prenyl transferases and the elongation stops at discrete chain lengths, depending on enzyme specificity (Ogura and Koyama, 1998
max for peaks 1 (zeaxanthin-
-selinene synthase and 
-humulene synthase in Abies grandis (Steele et al., 1998
-32P]dATP (111TBq/mmol) and [4-14C]IPP (2 mBq/mmol). 
% = 6.6. Purification to >90% was estimated by SDS-PAGE of samples from the chromatographic steps (data not shown).