Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes.

Pinus densiflora Siebold et Zucc. is the major green canopy species in the mountainous area of Korea. To assess the response of resin acid biosynthetic genes to mechanical and chemical stimuli, we cloned cDNAs of genes encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (1-deoxy-d-xylulose 5-phosphate synthase (PdDXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PdDXR) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (PdHDR)) by the rapid amplification of cDNA ends (RACE) technique. In addition, we cloned the gene encoding abietadiene synthase (PdABS) as a marker for the site of pine resin biosynthesis. PdHDR and PdDXS occurred as two gene families. In the phylogenetic trees, PdDXSs, PdDXR and PdHDRs each formed a separate clade from their respective angiosperm homologs. PdDXS2, PdHDR2 and PdDXR were most actively transcribed in stem wood, whereas PdABS was specifically transcribed. The abundance of PdDXS2 transcripts in wood in the resting state was generally 50-fold higher than the abundance of PdDXS1 transcripts, and PdHDR2 transcripts were more abundant by an order of magnitude in wood than in other tissues, with the ratio of PdHDR2 to PdHDR1 transcripts in wood being about 1. Application of 1 mM methyl jasmonate (MeJA) selectively enhanced the transcript levels of PdDXS2 and PdHDR2 in wood. The ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 reached 900 and 20, respectively, on the second day after MeJA treatment, whereas the transcript level of PdABS increased twofold by 3 days after MeJA treatment. Wounding of the stem differentially enhanced the transcript ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 to 300 and 70, respectively. The increase in the transcript levels of the MEP pathway genes in response to wounding was accompanied by two orders of magnitude increase in PdABS transcripts. These observations indicated that resin acid biosynthesis activity, represented by PdABS transcription, was correlated with the selective transcriptions of PdDXS2 and PdHDR2. Introduction of PdDXS2, PdHDR1 and PdHDR2 rescued their respective knockout Escherichia coli mutants, confirming that at least these three genes were functionally active. Intracellular targeting of the green fluorescent protein fused to the N-terminal 100 amino acid residues of these genes in the Arabidopsis transient expression system showed that the proteins were all targeted to the chloroplasts. Our results suggest that the MEP pathway regulates resin biosynthesis in the wood of P. densiflora by differential transcription of the multiple PdDXS and PdHDR genes.

[1]  Yeon-Bok Kim,et al.  Two copies of 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (CMK) gene in Ginkgo biloba: molecular cloning and functional characterization , 2008, Planta.

[2]  J. Holopainen,et al.  Long‐term effects of exogenous methyl jasmonate application on Scots pine (Pinus sylvestris) needle chemical defence and diprionid sawfly performance , 2008 .

[3]  T. Sharkey,et al.  Molecular cloning and characterization of two cDNAs encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Hevea brasiliensis. , 2008, Journal of plant physiology.

[4]  A. Kobayashi,et al.  1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes in gymnosperms Ginkgo biloba and Pinus taeda , 2007, Planta.

[5]  Jörg Bohlmann,et al.  Functional identification and differential expression of 1-deoxy-d-xylulose 5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies) , 2007, Plant Molecular Biology.

[6]  J. Yeo,et al.  Variation in Susceptibility of Pine Species Seedlings with the Pine Wood Nematode, Bursaphelenchus xylophilus, in Greenhouse , 2007 .

[7]  D. Ro,et al.  Diterpene resin acid biosynthesis in loblolly pine (Pinus taeda): functional characterization of abietadiene/levopimaradiene synthase (PtTPS-LAS) cDNA and subcellular targeting of PtTPS-LAS and abietadienol/abietadienal oxidase (PtAO, CYP720B1). , 2006, Phytochemistry.

[8]  T. Kuzuyama,et al.  Cloning and functional characterization of 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (GbMECT) gene from Ginkgo biloba. , 2006, Phytochemistry.

[9]  T. Kuzuyama,et al.  Cloning and characterization of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MECS) gene from Ginkgo biloba , 2006, Plant Cell Reports.

[10]  T. Kuzuyama,et al.  Identification of class 2 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase genes from Ginkgo biloba and their transcription in embryo culture with respect to ginkgolide biosynthesis. , 2006, Planta medica.

[11]  J. Bohlmann,et al.  Insect-Induced Conifer Defense. White Pine Weevil and Methyl Jasmonate Induce Traumatic Resinosis, de Novo Formed Volatile Emissions, and Accumulation of Terpenoid Synthase and Putative Octadecanoid Pathway Transcripts in Sitka Spruce1[w] , 2005, Plant Physiology.

[12]  R. Croteau,et al.  DEFENSIVE RESIN BIOSYNTHESIS IN CONIFERS. , 2003, Annual review of plant physiology and plant molecular biology.

[13]  A. Fürholz,et al.  Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Rodríguez-Concepcíon,et al.  Elucidation of the Methylerythritol Phosphate Pathway for Isoprenoid Biosynthesis in Bacteria and Plastids. A Metabolic Milestone Achieved through Genomics1 , 2002, Plant Physiology.

[15]  D. Strack,et al.  Two distantly related genes encoding 1-deoxy-d-xylulose 5-phosphate synthases: differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots. , 2002, The Plant journal : for cell and molecular biology.

[16]  Diane M. Martin,et al.  Methyl Jasmonate Induces Traumatic Resin Ducts, Terpenoid Resin Biosynthesis, and Terpenoid Accumulation in Developing Xylem of Norway Spruce Stems1 , 2002, Plant Physiology.

[17]  J. Chappell.  The genetics and molecular genetics of terpene and sterol origami. , 2002, Current opinion in plant biology.

[18]  R. Croteau,et al.  Genomic organization of plant terpene synthases and molecular evolutionary implications. , 2001, Genetics.

[19]  A. Zekry,et al.  Real‐time reverse transcriptase–polymerase chain reaction (RT–PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I , 2001, Immunology and cell biology.

[20]  Katoh,et al.  Regulation of oleoresinosis in grand fir (Abies grandis). Differential transcriptional control of monoterpene, sesquiterpene, and diterpene synthase genes in response to wounding , 1998, Plant physiology.

[21]  B. M. Lange,et al.  A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. D. de Graaf,et al.  Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  R. Croteau,et al.  Regulation of Oleoresinosis in Grand Fir (Abies grandis) (Coordinate Induction of Monoterpene and Diterpene Cyclases and Two Cytochrome P450-Dependent Diterpenoid Hydroxylases by Stem Wounding) , 1994, Plant physiology.

[24]  A. Theologis,et al.  Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. , 1994, The Plant journal : for cell and molecular biology.

[25]  H. Sahm,et al.  Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. , 1993, The Biochemical journal.

[26]  J. Cairney,et al.  A simple and efficient method for isolating RNA from pine trees , 1993, Plant Molecular Biology Reporter.

[27]  R. Douce,et al.  Biosynthesis of the thiazole moiety of thiamin (vitamin B1) in higher plant chloroplasts. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[28]  J. Goldstein,et al.  Regulation of the mevalonate pathway , 1990, Nature.

[29]  A. Marpeau,et al.  Effects of wounding on the terpene content of twigs of maritime pine (Pinus pinaster Ait.) , 1989, Trees.

[30]  A. Marpeau,et al.  Effects of wounds on the terpene content of twigs of maritime pine (Pinus pinaster Ait.) , 1989, Trees.

[31]  N. Joye,et al.  Resin acid composition of pine oleoresins , 1967 .

[32]  T. Kuzuyama,et al.  Functional Identification of Ginkgo biloba 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) Gene by Using Escherichia coli Disruptants Defective in DXS Gene , 2005 .

[33]  Diane M. Martin,et al.  Traumatic resin defense in Norway spruce (Picea abies): Methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase , 2004, Plant Molecular Biology.

[34]  Wilhelm Gruissem,et al.  Biochemistry & Molecular Biology of Plants , 2002 .

[35]  S. Takahashi,et al.  Construction and characterization of Escherichia coli disruptants defective in the yaeM gene. , 1999, Bioscience, biotechnology, and biochemistry.

[36]  M. Rohmer.  The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. , 1999, Natural product reports.

[37]  N. Fielding,et al.  The pine wood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle (= B. lignicolus Mamiya and Kiyohara): an assessment of the current position , 1996 .