1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes in gymnosperms Ginkgo biloba and Pinus taeda

Abstract

Isoprenoids are synthesized through the condensation of five-carbon intermediates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), derived from two distinct biosynthetic routes: cytosolic mevalonate (MVA) and plastidial 2-C-methyl-D: -erythritol 4-phosphate (MEP) pathways. 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS; EC 1.17.1.2), which catalyzes the last step of MEP pathway, was cloned as a multicopy gene from gymnosperms Ginkgo biloba (GbIDS1, GbIDS2, and GbIDS2-1) and Pinus taeda (PtIDS1 and PtIDS2), and characterized. Phylogenetic tree constructed with other plant IDSs demonstrated gymnosperm IDSs were distinctively different from angiosperm IDSs. The gymnosperm IDS clade contained two subclades, one composed of GbIDS1 and PtIDS1, and the other composed of GbIDS2, GbIDS2-1, and PtIDS2. G. biloba IDSs, except GbIDS2-1, successfully complemented Escherichia coli DLYT1, a lytB disruptant, confirming the in vivo competency of isozymes. During the 4 weeks study period, although transcript levels of GbIDS1s were similar both in roots and leaves of cultured G. biloba embryo, the transcripts of GbIDS2 predominantly occurred in the embryo roots, where diterpene ginkgolides are biosynthesized. Levels of PtIDS2 transcripts in the diterpenoid resin-producing wood were 4-5 times higher than those in other tissues. Higher levels of GbIDS1 transcripts were induced by light, whereas those of GbIDS2 were increased by methyl jasmonate treatment. These results strongly imply GbIDS2 and PtIDS2 have high correlation with secondary metabolism. In Arabidopsis transient expression system, N-terminal 100 amino acid residues of GbIDS1 delivered fused GFP protein into chloroplast as well as cytosol and nucleus, whereas those of GbIDS2, GbIDS2-1, and two PtIDSs delivered GFP only into chloroplast.

Cloning and Characterization of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) Pathway Genes of a Natural-Rubber Producing Plant,Hevea brasiliensis

“…In contrast, HDR from G. biloba complemented the HDR gene-disrupted E. coli mutant DLYT1(pMMV22S). 18) These results suggest that the reducing shuttle system for catalytic activity in HDR is different between gymnosperm and angiosperm.…”

“…25) Biosynthesis is induced by external stimuli such as wounding, fungal attack, and the methyl jasmonate treatment. 18,25) Our results in the gene expression analysis of HbDXSs also indicate that HbDXS2 which belongs to DXS2 is dominantly expressed in specialized tissues such as laticifer. However, the existence of a plastid transit peptide in HbDXS2 suggests dominant expression of HbDXS2 in plastids, such as Frey-Wyssling particles in the laticifer.…”

“…To perform complementation assays of the MEP pathway genes, MEP pathway gene-disrupted E. coli mutants were used as follows: DXM3 for DXS, 12) DYM1 for DXR, 13) NMW33 (pTMV20KM) for CMS, 14) NMW29(pTMV20KM) for CMK, 15) NMW25(pTMV20KM) for MCS, 16) NMW18 (pTMV20KM) for HDS, 17) and DLYT1(pMMV22S) for HDR. 18) pTMV20KM is a pTMV20 19) derivative with 1.4-kb kan r cassette (a kanamycin-resistant gene, aphII) from pKC7 19) inserted at the ScaI site in the amp r gene of pTMV20. The MEP pathway genes of E. coli, A. thaliana, and H. brasiliensis were cloned into a vector, pTTQ18, 19) for the HDR gene, and into another vector, pMW118, (Nippon Gene, Tokyo) for the other genes.…”

The genetic basis of foliar terpene yield: Implications for breeding and profitability of Australian essential oil crops

“…For example, multiple copies of dxs have been found in Medicago truncatula (Walter et al 2002), Arabidopsis thaliana (Araki et al 2000;Estévez et al 2000), Oryza sativa (Kim et al 2005), Picea abies (Phillips et al 2007), Ginkgo biloba (Kim et al 2008a), Eucalyptus grandis (Külheim et al 2011) and Melaleuca alternifolia (Webb et al 2013). Other genes including cmk (Kim et al 2008a), hdr (Kim et al 2008b) and dxr (Seetang-Nun et al 2008) can occur as multi-copy genes (in Ginkgo biloba, Pinus taeda and Hevea brasiliensis, respectively), although all are present as single copies in Arabidopsis thaliana (Cordoba et al 2009), so unlike dxs, multiple copies of these genes are not universal amongst plants (Table 1). Very little work has been done on copy number variation within the Myrtaceae family.…”

“…Of the genes in the MVA pathway, all genes can occur as multi-copy genes (Table 1). In Hevea brasiliensis there are two copies of hmgs and four copies of hmgr while the other genes in the pathway are single copy (Kim et al 2008b), in Medicago truncatula there are five copies of hmgr (Kevei et al 2007). All the reactions within the pathway have been characterized in plants and some work has focused on the control of metabolite flux through the MVA pathway in plants.…”