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Tree Physiology Advance Access originally published online on February 2, 2009
Tree Physiology 2009 29(5):737-749; doi:10.1093/treephys/tpp002
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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

Yeon-Bok Kim1, Sang-Min Kim1, Min-Kyoung Kang1, Tomohisa Kuzuyama3, Jong Kyu Lee4, Seung-Chan Park5, Sang-chul Shin6 and Soo-Un Kim1,2,7

1 Program in Applied Life Chemistry, Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea
2 Plant Metabolism Research Center, Kyung Hee University, Yongin 449-701, Korea
3 Laboratory of Cell Biotechnology, Biotechnology Research Center, University of Tokyo, Tokyo 113-8657, Japan
4 College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Korea
5 Division of Forest Resources and Landscape Architecture, Chonnam National University, Kwangju 500-757, Korea
6 Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul 130-702, Korea
7 Corresponding author (soounkim{at}plaza.snu.ac.kr)


  Abstract

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.

Keywords: 1-deoxy-D-xylulose 5-phosphate reductoisomerase, resin biosynthesis

Received August 20, 2008; Accepted December 18, 2008

This version includes a corrected version of Figure 4.

Supplementary Data

Supplementary data for this article are available at Tree Physiology Online.


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