Background: Agarwood is an expensive resinous heartwood derived fromAquilariaplants that is widely used in traditional medicines, incense and perfume. Only wounded trees can produce agarwood, and the huge demand for the agarwood products has led allAquilariaspp. being endangered and listed in the Appendix II of the CITES (http://www.cites.org). The major components of agarwood are sesquiterpenes and phenylethyl chromones. Owing to a lack of genomic information, the molecular basis of wound-induced sesquiterpenes biosynthesis and agarwood formation remains unknown.Results: To identify the primary genes that maybe related to agarwood formation, we sequenced 2 cDNA libraries generated from healthy and woundedA. sinensis(Lour.) Gilg. A total of 89,137 unigenes with an average length of 678.65 bp were obtained, and they were annotated in detail at bioinformatics levels. Of those associated with agarwood formation, 30 putatively encoded enzymes in the sesquiterpene biosynthesis pathway, and a handful of transcription factors and protein kinases were related to wound signal transduction. Three full-length cDNAs of sesquiterpene synthases (ASS1-3) were cloned and expressed inEscherichia coli, and enzyme assays revealed that they are active enzymes, with the major products being δ-guaiene. A methyl jasmonate (MJ) induction experiment revealed that the expression ofASSwas significantly induced by MJ, and the production of sesquiterpenes was elevated accordingly. The expression of some transcription factors and protein kinases, especiallyMYB4,WRKY4,MPKK2andMAPK2, was also induced by MJ and coordinated withASSexpression, suggesting they maybe positive regulators ofASS.Conclusions: This study provides extensive transcriptome information forAquilariaspp.and valuable clues for elucidating the mechanism of wound-induced agarwood sesquiterpenes biosynthesis and their regulation.

Background: Bupleurum chinenseDC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents ofB. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway.Results: One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). Ade novoassembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from theBupleurumgenus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novelBupleurumgenes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7P450s and 7 uridine diphosphateGTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. TwoP450s and threeUGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression withβ-ASin methyl jasmonate-treated adventitious roots and on their similar expression patterns withβ-ASin variousB. chinensetissues.Conclusions: A collection of high-quality ESTs forB. chinenseobtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics ofB. chinenseand otherBupleurumspecies. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially theP450s andUGTs, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.

As widely recognized, agarwood formation in Aquilaria trees is induced by external wounding. Because agarwood usually harbors specific microbes, the function of microbes in agarwood formation has been debated for almost a century. In this study, two wounding methods, the burning-chisel-drilling method (BCD) and the whole-tree agarwood-inducing method (Agar-Wit), were used under the non-contamination of environmental microorganisms. After pyrosequencing the small rRNA subunits of the wounds induced by the BCD and Agar-Wit, no substantial variation was observed either in fungal and bacterial enrichment and diversity or in the relative abundances of taxa. By contrast, significant variations in fungal and bacterial communities were detected following the partial tree pruning (PTP)-wounding. The wound-induced sesquiterpene biosynthesis and vessel-occlusion formation, however, were found to be similar in all types of wounded trunks. We thus infer that wounding in the absence of variations in microbial communities may induce agarwood formation. This result does not support the long-standing notion that agarwood formation depends on microbes.

Dalbergia odorifera, a critically endangered tree species, produces heartwood containing a vast variety of flavonoids. This heartwood, also known as Chinese rosewood, has high economic and medicinal value, but its formation takes several decades. In this study, we showed that discolored wood induced by pruning displays similar color, structure, and flavonoids content to those of natural heartwood, suggesting that wounding is an efficient method for inducing flavonoid production in D. odorifera. Transcriptome analysis was performed to investigate the mechanism underlying wounding-induced flavonoids production in D. odorifera heartwood. Wounding upregulated the expression of 90 unigenes, which covered 19 gene families of the phenylpropanoid and flavonoid pathways, including PAL, C4H, 4CL, CHS, CHI, 6DCS, F3’5’H, F3H, FMO, GT, PMAT, CHOMT, IFS, HI4’OMT, HID, IOMT, I2’H, IFR, and I3’H. Furthermore, 47 upregulated unigenes were mapped to the biosynthesis pathways for five signal molecules (ET, JA, ABA, ROS, and SA). Exogenous application of these signal molecules resulted in the accumulation of flavonoids in cell suspensions of D. odorifera, supporting their role in wounding-induced flavonoid production. Insights from this study will help develop new methods for rapidly inducing the formation of heartwood with enhanced medicinal value.

Aquilaria sinensis is an important non-timber tree species for producing high-value agarwood, which is widely used as a traditional medicine and incense. Agarwood is the product of Aquilaria trees in response to injury and fungal infection. The APETALA2/ethylene responsive factor (AP2/ERF) transcription factors (TFs) play important roles in plant stress responses and metabolite biosynthesis. In this study, 119 AsAP2/ERF genes were identified from the A. sinensis genome and divided into ERF, AP2, RAV, and Soloist subfamilies. Their conserved motif, gene structure, chromosomal localization, and subcellular localization were characterized. A stress/defense-related ERF-associated amphiphilic repression (EAR) motif and an EDLL motif were identified. Moreover, 11 genes that were highly expressed in the agarwood layer in response to whole-tree agarwood induction technique (Agar-Wit) treatment were chosen, and their expression levels in response to methyl jasmonate (MeJA), salicylic acid (SA), or salt treatment were further analyzed using the quantitative real time PCR (qRT-PCR). Among the 11 genes, eight belonged to subgroup B-3. All 11 genes were significantly upregulated under salt treatment, while eight genes were significantly induced by both MeJA and SA. In addition, the gene clusters containing these upregulated genes on chromosomes were observed. The results obtained from this research not only provide useful information for understanding the functions of AP2/ERF genes in A. sinensis but also identify candidate genes and gene clusters to dissect their regulatory roles in agarwood formation for future research.