Nucleotide sequence of an Arabidopsis cDNA for geranylgeranyl pyrophosphate synthase.

GGPS is an enzyme involved in the early steps of isoprenoid synthesis in plants. The bell pepper (Capsicum annuum) GGPS catalyzes the stepwise addition of isopentenyl PPi, first to dimethylallyl PPi, second to geranyl PPi, and fmally to farnesyl PPi to form geranylgeranyl PPi (Kuntz et al., 1992). Geranylgeranyl PPi is utililized by plants for many compounds such as carotenoids, Chls, and phylloquinones and as a precursor for GA3. A plant GGPS cDNA from Capsicum ripening fruit has been cloned and sequenced, but only the deduced amino acid sequence has been published (Kuntz et al., 1992). To obtain a GGPS cDNA, we have cloned, by amplification using the PCR, a gene fragment for GGPS from Arabidopsis using degenerate oligonucleotides based on conservation of amino acids among severa1 GGPSs (Carattoli et al., 1991; Michalowski et al., 1991; Kuntz et al., 1992; Math et al., 1992). An upstream oligonucleotide, 5'-GCIGCITG(T/ C)GCIGTIGA(A/G)ATG-3' (I = inosine), corresponding to the peptide sequence AACAVEM, and a downstream oligonucleotide, 5 '-GGTTACATCCAAGAT(A/G)TCGTC-3', corresponding to the peptide sequence DDILDVT, were used to amplify Arabidopsis genomic DNA. Upon electrophoresis of the resulting PCR products, a single band of about 450 bp was observed; it was purified and subcloned into SmaI-cut pBluescript using a procedure developed in our laboratory. This fragment was used to screen a cDNA Iibrary conshucted from norflurazon-treated Arabidopsis seedlings (Table I). One plaque hybridizing to the probe was purified and the cDNA insert sequenced. Analysis of the sequence revealed an open reading frame encoding a protein of 371 amino acids. Alignment of this protein to GGPSs from Erwinia uredovora and Rhodobacter capsulatus suggests an N-terminal transit peptide of 76 amino acids. Comparison of this protein sequence to the protein sequence of GGPS from pepper showed 84.5% similarity and 73.5% identity between the putative mature peptides and, unsurprisingly, very little similarity or identity between the putative transit peptides. Sequence differences between the cDNA and the genomic fragment indicate the existence of more than one GGPS gene in Arabidopsis. We propose naming this gene GGPSI. The genomic fragment has been mapped to the lower arm of chromosome 2, whereas