Two copies of 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (CMK) gene in Ginkgo biloba: molecular cloning and functional characterization

Abstract

4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (CMK or YchB), the fourth enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway, phosphorylates the 2-hydroxyl group of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol in the presence of ATP. Two isogenes encoding CMK (GbCMK1 and GbCMK2) were cloned and characterized from Ginkgo biloba. The activities of both isozymes were confirmed by complementation assay using Escherichia coli NMW29, a ychB knock-out mutant. The transcript profiles of GbCMKs in the radicles and the cotyledons of the cultured Ginkgo biloba embryos demonstrated that the transcript levels of GbCMK1 were similar in both organs, whereas that of GbCMK2 was predominantly high in the ginkgolide-synthesizing radicles. Selective increases in the transcript abundance of GbCMK2 in the radicles, induced by light and methyl jasmonate treatments, were observed. These differential induction patterns of the transcripts imply GbCMK1 and GbCMK2 respectively have high correlations with the primary and the secondary metabolisms. The transit peptides of both isozymes delivered the fused green fluorescent protein (GFP) into the chloroplast in the Arabidopsis and the Nicotiana transient expression systems; interestingly, the transit peptide of GbCMK1 delivered the GFP protein into the cytosol and the nucleus in addition to the chloroplasts.

Developmental pattern of Ginkgo biloba levopimaradiene synthase (GbLPS) as probed by promoter analysis in Arabidopsis thaliana

“…They found that only GbDXS2 among the two isogenes of GbDXS correlated with the ginkgo trilactone biosynthesis, confirming that the trilactone synthesis is regulated at the transcription level . They also found that two other genes in MEP pathway, 4-(cytidine 5 0 -diphospho)-2-C-methyl-D-erythritol kinase (GbCMK) (Kim et al 2008a) and 1 -hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (GbHDR) (Kim et al 2008b) have correlation with ginkgolide biosynthesis occurrence in the roots.…”

Characterization and Transcriptional Profiling of Ginkgo biloba Mevalonate Diphosphate Decarboxylase Gene (GbMVD) Promoter Towards Light and Exogenous Hormone Treatments

“…The upregulation of a gene under the influence of SA and MeJA indicates the involvement of this gene in such responses. The transcripts of GbDXS (Gong et al, 2006), GbCMK2 (Kim et al, 2008b), GbLPS (Kim et al, 2012), and GbIDS2 (Kang et al, 2013) are positively responsive to SA and MeJA treatments, whereas their respective isogenes exhibit either a negative or minimal response. The genes that showed a positive response are possibly involved in the biosynthesis of TTLs.…”

“…However, the presence of IPP cross-talk further complicates the origin of TTL building block in G. biloba (Lichtenthaler et al, 1997;Schwarz and Arigoni, 1999). A few of the structural genes involved in the MVA and MEP pathway have been isolated and well characterized in G. biloba, such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; Shen et al, 2006), 1-deoxy-D-xylulose 5-phosphate synthase (DXS; Gong et al, 2006;Kim et al, 2006a), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; Gong et al, 2005;Kim et al, 2006a), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS; Kim and Kim, 2010), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (IDS; Kim et al, 2008a), 2-C-methyl-Derythritol 4-phosphate cytidyltransferase (MECT; Kim et al, 2006b), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECS; Kim et al, 2006c;Gao et al, 2006), and 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK; Kim et al, 2008b). Ginkgolide content in ginkgo can be increased by upregulating some of these genes (Gong et al, 2006;Kim et al, 2006a;Kim et al, 2008aKim et al, , 2008b.…”

Screening, isolation, and molecular characterization of putative fragrance-related transcripts from Vanda Mimi Palmer

“…The BLASTX and BLASTP analyses (NCBI) showed that the deduced amino acid sequence of VMPCMEK is 66-73% similar to 4-(cytidine 5 0 -diphospho)-2-C-methyl-D-erythritol kinases from other plants such as Hevae brasiliensis (GenBank accession no: BAF98293.1), Solanum lycopersicum (GenBank accession no: AAF87717.1), Catharanthus roseus (GenBank accession no: ABI35992.1) and Ginkgo biloba (GenBank accession no: AAZ80384.1 and AAZ80385.1). Besides that, VMPCMEK also contained the conserved ATP-binding site for functional activity of 4-(cytidine 5 0 -diphospho)-2-C-methyl-D-erythritol kinase (CMEK) as found in the CMEK sequences from other plants (Kim et al 2008) (Fig. 3).…”