Expressed sequence tags from Cryptomeria japonica sapwood during the drying process

Secondary metabolites called norlignans are produced in the xylem of Cryptomeria japonica D. Don. Several norlignans have roles in the defense of sapwood against microbial invasion and in the coloration of heartwood. The biosynthetic pathway of norlignans is largely unknown. Norlignans have been reported to accumulate in the sapwood during the drying of C. japonica logs. To search for genes encoding enzymes that catalyze the synthesis of norlignans, we carried out suppression subtractive hybridization using the fresh sapwood of a felled log and the drying sapwood in which a norlignan, agatharesinol, accumulated. A total of 1050 expressed sequence tags were obtained from the subtracted cDNA library, and these were assembled into 146 contigs and 361 singletons. Of these 507 unique sequences, 263 were functionally classified into 12 categories. "Metabolism" was the largest category, with 23% (61) of classified sequences. Twenty-six sequences that encode 16 enzymes were assigned to "secondary metabolism." Expression analysis of 15 genes related to "secondary metabolism" revealed that 12 of these genes had transcripts that were induced during the sapwood drying process. Of the 12 genes, 10 encoded enzymes that use aromatic compounds as substrates. In addition, 58 sequences representing 22 defense-related proteins were found. Our subtraction library should be a useful source for isolating genes encoding proteins involved in secondary metabolism including norlignan biosynthesis and defense in C. japonica xylem.