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.
Kim
Yeon-Bok
YB
Program in Applied Life Chemistry, Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.
Kim
Sang-Min
SM
Kang
Min-Kyoung
MK
Kuzuyama
Tomohisa
T
Lee
Jong Kyu
JK
Park
Seung-Chan
SC
Shin
Sang-Chul
SC
Kim
Soo-Un
SU
D000085:Acetates / Q000494:pharmacology; D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D004896:Erythritol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D054883:Oxylipins / Q000494:pharmacology; D010802:Phylogeny; D028223:Pinus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012116:Resins, Plant / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D015398:Signal Transduction; D013403:Sugar Phosphates / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014166:Transferases / Q000737:chemistry / Q000235:genetics*; D014934:Wood / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
C114232:2-C-methylerythritol 4-phosphate; D000085:Acetates; D003517:Cyclopentanes; D009097:Multienzyme Complexes; D054883:Oxylipins; D010940:Plant Proteins; D012333:RNA, Messenger; D012116:Resins, Plant; D013403:Sugar Phosphates; C072239:methyl jasmonate; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D019747:Aldose-Ketose Isomerases; D004896:Erythritol