• 한국균학회소식:학술대회논문집
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• 2016.05a
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• pp.19-20
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• 2016

Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.13
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• pp.1-40
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• 1973

These studies were aimed at clarification of genetic and ecological variation in culm length, panicle length and plant height of the $\textrm{F}_2$ plants in some selected crosses made between semi-dwarf rice varieties and tall Japonica ones. One Indica semi-dwarf, Taichung Native 1, one Indica $\times$ Japonica hybrid, IE51 and one Japonica semi-dwarf, Tankanbaekmang were used as short-gene donors while two of medium maturity varieties, Jinheung and Kwanok and one late veriety, Palkweng were used as the corresponding counterpart of respective dwarf varieties in a series of crosses. Five different crosses, Kwanok $\times$ Tankanbaekmang, Palkweng $\times$ Tankanbaekmang, Jinheung $\times$ T(N)1, Kwanok $\times$ T(N)1 and Kwanok $\times$ IE51, were made among the above six varieties. The $\textrm{F}_2$ plants of these crosses together with the concerned parental varieties were grown under several different conditions including three levels of each nitrogen and planting space, three planting seasons and three locations in 1968, to investigate variation in length of culm and panicle, and plant height. On the other hand, the F$_3$ progenies which were derived from the shortest 10 percent of the plants of three $\textrm{F}_2$ populations, Kwanok $\times$ T(N)1, Jinheung $\times$ T(N) 1 and Kwanok $\times$ IE51 grown in the previous year, were compared each other on the basis of selection efficiency in culm length. The experimental results could be summarized as follows; 1. Genetic behavior A. It was revealed that Tankanbaekmang, one of Japonica dwarf has a simple recessive gene responsible for short culm expression, showing a typical segregation ratio of three tall to one short culm plants in $\textrm{F}_2$ generation of the crosses either with Kwanok or Palkweng. B. In the both combinations, segregation pattern of the panicle length was exactly same as that of culm length. It seems that the same gene controls both culm length and panicle length. C. No difference between segregation of culm length and plant height in the above crosses was observed. D. T(N)1, one of Indica semi-dwarf did not show such a simple genetic behavior as detected from the crosses with Tankanbaekmang in segregation of culm length but formed a continuous and normal distribution curve. Therefore, some nonallelic genic actions might be involved in expression of culm length of the counterpart varieties of T(N)1. In particular, a transgressive segregation appeared toward the direction of longer culm length in case of Jinheung $\times$ T(N)1. The genetic behavior of panicle length and plant height generally coincided with that of culm length in all the cases. E. IE51 demonstrated exactly the same genetic behavior as that of T(N)1 when this variety was crossed with Kwanok. It was clearly clarified that the simple recessive gene controlling dwarfism from T(N)1 was well incorporated into this variety. 2. Ecological variation A. In general, there was a decreasing tendency in culm length and plant height of rice plant as seeding delayed while it was not so noticeable in panicle length. The decreasing magnitude varied from variety to variety and from cross to cross. Genetic behavior of the culm length and related characters of these materials was not disturbed by the variation of seeding season, nitrogen level, planting space and experimental location. E. The elongation mode of the upper three internodes was very similar to the segregation mode of culm length, panicle length and plant height in $\textrm{F}_2$ populations of . all the crosses investigated in this study. Accordingly, this result confirmed that the roles of the upper three internodes are very important in manifesting plant stature in rice. C. The effect of nitrogen on culm length and the related other two characters seemed to be meager. However, it was true to show an increasing tendency of those characters as nitrogen level got increased from 4 kg to 12kg per l0a, with different magnitude depending upon variety or cross. D. Also, the effect of planting space on culm length, panicle length and plant height was relatively small in all the cases. Those characters varied again depending upon variety or cross. However, a general increasing tendency was detected in manifestation of those traits under denser planting space condition. E. All the parental varieties produced shorter culm, panicle and plant height when they were grown at the lower latitude locations. It might be attributed to the fact that their reproductive growth accelerated with increased temperature prevailing at the lower latitude locations such as Iri and Mi1yang. On the countrary, $\textrm{F}_2$ population reacted differently to the different locations from the parental varieties. All the $\textrm{F}_2$ plants produced the longest culm, panicle and plant at Milyang. 3. Selection efficiency A. The heritability of culm length in Kwanok $\times$ T(N)1, Kwanok $\times$ IE51 and Jinheung$\times$T(N)1 was 92 percent, 74 percent and 55 percent, respectively. B. The actual genetic advance for culm length obtained from the progeny lines of the selected plants(10 precent) from the $\textrm{F}_2$ generation, was comparable to the expected advance calculated from the original $\textrm{F}_2$ populations. As compared with the $\textrm{F}_2$ population, the $\textrm{F}_3$ plants of Kwanok $\times$ T(N)l shortened on the average by 20.8cm, those of Kwanok $\times$ IE51 did 8.7cm and those of Jinheung$\times$T(N)1 20.0cm, respectively. C. Panicle length of the populations was differently affected from one cross to another by the selection based upon culm length in $\textrm{F}_2$ Kwanok $\times$ T(N)1 did not show any noticeable shortening of its culm length due to the selection pressure. On the other hand, both Kwanok $\times$ IE51 and Jinheung $\times$ T(N)1 showed a considerable shortening of their panicles in case of selection for culm length. Based upon the above results, it could be concluded that the ecological variation in culm length, panicle length and plant height was relatively small and fallen within the range of genetic variation. Considering from the fact that the simple recessive gene governing short height of Tankanbaekmang always accompanied with some undesirable characters such as short panicle and extremely small grain, the short gene of T(N)1 seemed to be more useful as dwarf gene source since it did not carry short gene together with such undesirable traits.