Yoshimi Yamamura

Cloning and Expression of Putative Rac/Rop GTPase Genes, Am-rac1 and Am-rac2, Involved in Methyl Jasmonate-Induced Transcriptional Activation of Farnesyl Diphosphate Synthase in Cell Cultures of Aquilaria microcarpa

A homology-based cloning strategy yielded two cDNA clones presumably encoding Rac/Rop GTPases, Am-rac1 and Am-rac2, from callus cultures of Aquilaria microcarpa. Transcriptional levels of Am-rac1, measured using reverse transcription polymerase chain reaction, remained steady, while those of Am-rac2 increased dramatically following treatment of cultured cells with either a yeast extract or methyl jasmonate. These results suggested that Am-rac1 was a constitutively expressed homologue, while Am-rac2 was induced in A. microcarpa cells. Moreover, a farnesyl diphosphate synthase gene of A. microcarpa, Am-FaPS1, was transcriptionally activated in A. microcarpa cells grown in the presence of methyl jasmonate; however, expression levels were markedly lowered in the presence of various signal transduction-related inhibitors involved in Ca2+-, Rap/Rop GTPase-, or ubiquitin-dependent signaling processes. Whereas, expression of Am-FaPS1 was markedly increased, even in the absence of methyl jasmonate in A. microcarpa cells over-expressing Am-rac2. These findings suggested that Rac/Rop GTPase proteins played important roles in jasmonate-induced enhancement of terpenoid metabolism in A. microcarpa.

Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis.

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis.

Transcriptional activation of a geranylgeranyl diphosphate synthase gene, GGPPS2, isolated from Scoparia dulcis by treatment with methyl jasmonate and yeast extract

A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca2+ ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells.

Enhanced accumulation of atropine in Atropa belladonna transformed by Rac GTPase gene isolated from Scoparia dulcis

Leaf tissues of Atropa belladonna were transformed by Sdrac2, a Rac GTPase gene, that is isolated from Scoparia dulcis, and the change in atropine concentration of the transformants was examined. Re-differentiated A. belladonna overexpressing Sdrac2 accumulated considerable concentration of atropine in the leaf tissues, whereas the leaves of plants transformed by an empty vector accumulated only a very low concentration of the compound. A. belladonna transformed by CASdrac2, a modified Sdrac2 of which translate was expected to bind guanosine triphosphate (GTP) permanently, accumulated very high concentrations of atropine (approximately 2.4-fold excess to those found in the wild-type plant in its natural habitat). In sharp contrast, the atropine concentration in transformed A. belladonna prepared with negatively modified Sdrac2, DNSdrac2, expected to bind guanosine diphosphate instead of GTP, was very low. These results suggested that Rac GTPases play an important role in the regulation of secondary metabolism in plant cells and that overexpression of the gene(s) may be capable of enhancing the production of natural products accumulated in higher plant cells.

Expression of specific calmodulin genes isolated from tissue cultured cells of Aquilaria microcarpa in response to methyl jasmonate and yeast extract

Agarwood is formed in wood tissues of Aquilaria, Gonystulus, andGyrinops species in response to mechanical wounding and/ or microbial infection, but is not formed in nonstressed tissues of these trees (Ng et al. 1997; Ito and Honda 2005). Artificial induction of agarwood formation has been very difficult. Okudera and Ito (2009) reported that biosynthesis of several sesquiterpenoids such as α-guaiene, α-humulene, and δguaiene is induced in suspension cultured cells of Aquilaria crassna and Aquilaria sinensis in response to treatment with 100 μM methyl jasmonate (MJ). The accumlation of a spirovetivane-type sesquiterpene has been observed in cells of Aquilaria microcarpa, as well as in other Aquilaria species, when the wood tissues were transformed to agarwood (Ueda et al. 2006). In addition, we have shown (Kenmotsu et al. 2011) that treatment of cell cultures of A. microcarpa with either MJ or yeast extract (YE) results in the marked enhancement of the transcription of a farnesyl diphosphate synthase gene, AmFaPS-1, which is involved in the sesquiterpene biosynthesis. Little is known about the biochemical mechanisms involved in the modulation of plant secondary metabolism by MJ (Creelman and Mullet 1997), but we have shown (Kenmotsu et al. 2011) that the expression activity of Am-FaPS-1 is appreciably elevated in cells treated with Ca-ionophore A23187, MJ, or YE. These results strongly suggested that Ca functions as an important messenger molecule in the activation of plant secondary metabolism evoked by MJ and YE. We have also demonstrated (Kasidimoko et al. 2005, Saitoh et al. 2007) that calmodulin (CAM), a well-known Ca-binding protein, functions as the key mediator and plays a central role in MJ-induced terpenoid biosynthesis in higher plant cells. In animal cells, CAM genes are constitutively expressed (Klee and Vanaman 1982), but recent studies (Takezawa et al. 1995; Yamakawa et al. 2001) suggested that plant CAMs were composed of several isoforms and the expression of specific CAM genes were altered following treatment with appropriate stimuli or under stress conditions. To study the molecular basis of MJand YE-induced terpene biosynthesis in plant cells, we previously isolated two CAM genes, Am-cam1 and Am-cam2 (GenBank accession nos. HM237343 and HM237344, respectively), from cultured tissues of A. microcarpa, and examined transcription in response to treatment with MJ and YE (Kenmotsu et al. 2010). However, the possibility remained that additional Am-cam gene, other thanAm-cam1 and 2, might play important roles. In addition, we found that the early cellular events which lead to the activation of secondary metabolism in appropriately stimulated plant cells, such as gene expression of monomeric GTP-binding proteins and their intracellular translocation, occur very rapidly, and are sometimes terminated within 30 min after the stimulation (Mitamura et al. 2011). In the present study, we isolated two new CAM genes, Am-cam3 and Am-cam4, and systematically examined the transcription patterns of Am-cam1, Am-cam2, Am-cam3, and Am-cam4, in callus of A. microcarpa when treated with MJ, YE, or Ca-ionophore A23187.

Characterization of ent -kaurene synthase and kaurene oxidase involved in gibberellin biosynthesis from Scoparia dulcis

Gibberellins (GAs) are ubiquitous diterpenoids in higher plants, whereas some higher plants produce unique species-specific diterpenoids. In GA biosynthesis, ent-kaurene synthase (KS) and ent-kaurene oxidase (KO) are key players which catalyze early step(s) of the cyclization and oxidation reactions. We have studied the functional characterization of gene products of a KS (SdKS) and two KOs (SdKO1 and SdKO2) involved in GA biosynthesis in Scoparia dulcis. Using an in vivo heterologous expression system of Escherichia coli, we found that SdKS catalyzed a cyclization reaction from ent-CPP to ent-kaurene and that the SdKOs oxidized ent-kaurene to ent-kaurenoic acid after modification of the N-terminal region for adaptation to the E. coli expression system. The real-time PCR results showed that the SdKS, SdKO1 and SdKO2 genes were mainly expressed in the root and lateral root systems, which are elongating tissues. Based on these results, we suggest that these three genes may be responsible for the metabolism of GAs in S. dulcis.