Journal
PLANT PHYSIOLOGY
Volume 154, Issue 4, Pages 1998-2007Publisher
AMER SOC PLANT BIOLOGISTS
DOI: 10.1104/pp.110.161828
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Funding
- Takeda Science Foundation
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The resinous portions of Aquilaria plants, called agarwood, have been used as medicines and incenses. Agarwood contains a great variety of sesquiterpenes, and a study using cultured cells of Aquilaria showed the production of sesquiterpenes (alpha-guaiene, alpha-humulene, and delta-guaiene) to be induced by treatment with methyl jasmonate (MJ). In this study, the accumulation and production of sesquiterpenes were quantified. The amounts accumulated and produced reached a maximum at 12 h, and the most abundant product was alpha-humulene at 6 h and delta-guaiene after 12 h. However, a headspace analysis of the cells revealed that alpha-humulene is likely to be volatilized; so overall, the most abundant sesquiterpene in the cells was delta-guaiene. A cDNA library from RNA isolated from MJ-treated cells was screened using PCR methodologies to isolate five clones with very similar amino acid sequences. These clones were expressed in Escherichia coli, and enzymatic reactions using farnesyl pyrophosphate revealed that three of the clones yielded the same compounds as extracted from MJ-treated cells, the major product being delta-guaiene. These genes and their encoded enzymes are the first sesquiterpene synthases yielding guaiane-type sesquiterpenes as their major products to be reported. Expression of a fourth terpene synthase gene in bacteria resulted in the accumulation of the protein in insoluble forms. Site-directed mutagenesis of the inactive clone and three-dimensional homology modeling suggested that the structure of the N-terminal domain was important in facilitating proper folding of the protein to form a catalytically active structure.
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