PR4 was isolated as an endophyte from the rhizome of Picrorhiza kurroa. Picrorhiza kurroa Royle ex. Benth (Plantaginaceae) is a perennial herb endemic to the north western alpine Himalayas. The endophyte PR4 was grown on PDA and in PDB at 26 ℃ for 15 days with constant shaking at 200 rpm in the latter case.

The two candidate NR-PKSs (PKS_3671 and PKS_4063) show differences in their domain organizations. PKS_3671 possesses two ACP-domains. Apart from that, only PKS_3671 contains a SAT-domain . These domains provide the first building block in the polyketide assembly, which usually is different from the extender unit malonyl-CoA (also known as the 'starter unit effect'). The ACA-synthesis however is believed to involve merely malonyl-CoA molecules. Even though the ACA-producing PKSs MdpG, ACAS, EncA, AptA and ClaG contain SAT-domains, an amino acid sequence alignment of these domains revealed that they all lack the active-site cysteine in the GXCXG motif and therefore most likely have no acyl transferase activity. Instead, all malonate building blocks are assumed to be loaded by the MAT. Under this aspect, the SAT-domain of PKS_3671 (that includes the correct GXCXG motif) likely incorporates a starter unit different from malonyl-CoA indicating that this enzyme is not involved in the biosynthesis of ACA. Therefore, the ACA-synthesizing PKS in C. asteris would rather be PKS_4063 that misses the SAT-domain .In the monodictyphenone and cladofulvin pathways, the cluster-encoded gene products MdpH and ClaH are crucial enzymes pushing the biosynthesis towards emodin. These EthD-domain-containing enzymes are suggested to catalyze the decarboxylation of ACA (3) into atrochrysone (4). Surprisingly, no such EthD-domain is encoded in the whole C. asteris genome. On the other hand, four genes directly attached to the putative ACA-synthase-coding gene pks_4063 show high similarity to genes of non-investigated PKS clusters in other fungi , which indicates an involvement in tailoring reactions of the respective polyketide pathways. According to InterProScan and BLASTp analyses, the genes sky_4060-62 encode a dehydratase and two dehydrogenases potentially catalyzing the multistep conversion of ACA (3) into emodin (1). Gene sky_4059 codes for a monooxygenase that putatively can connect two emodin molecules to the final product skyrin (2) in the style of the monooxygenase ClaM involved in the dimerization of the bisanthraquinone cladofulvin. Thus, the presence of these genes in the gene cluster gives further support to the hypothesis that PKS_4063 is the ACA-synthase in C. asteris. Mutational studies will be done in order to confirm these assumptions after a gene transfer system for this strain has been developed.

PR4 was isolated as an endophyte from the rhizome of Picrorhiza kurroa. Picrorhiza kurroa Royle ex. Benth (Plantaginaceae) is a perennial herb endemic to the north western alpine Himalayas. The endophyte PR4 was grown on PDA and in PDB at 26 ℃ for 15 days with constant shaking at 200 rpm in the latter case.

The two candidate NR-PKSs (PKS_3671 and PKS_4063) show differences in their domain organizations. PKS_3671 possesses two ACP-domains. Apart from that, only PKS_3671 contains a SAT-domain . These domains provide the first building block in the polyketide assembly, which usually is different from the extender unit malonyl-CoA (also known as the 'starter unit effect'). The ACA-synthesis however is believed to involve merely malonyl-CoA molecules. Even though the ACA-producing PKSs MdpG, ACAS, EncA, AptA and ClaG contain SAT-domains, an amino acid sequence alignment of these domains revealed that they all lack the active-site cysteine in the GXCXG motif and therefore most likely have no acyl transferase activity. Instead, all malonate building blocks are assumed to be loaded by the MAT. Under this aspect, the SAT-domain of PKS_3671 (that includes the correct GXCXG motif) likely incorporates a starter unit different from malonyl-CoA indicating that this enzyme is not involved in the biosynthesis of ACA. Therefore, the ACA-synthesizing PKS in C. asteris would rather be PKS_4063 that misses the SAT-domain .In the monodictyphenone and cladofulvin pathways, the cluster-encoded gene products MdpH and ClaH are crucial enzymes pushing the biosynthesis towards emodin. These EthD-domain-containing enzymes are suggested to catalyze the decarboxylation of ACA (3) into atrochrysone (4). Surprisingly, no such EthD-domain is encoded in the whole C. asteris genome. On the other hand, four genes directly attached to the putative ACA-synthase-coding gene pks_4063 show high similarity to genes of non-investigated PKS clusters in other fungi , which indicates an involvement in tailoring reactions of the respective polyketide pathways. According to InterProScan and BLASTp analyses, the genes sky_4060-62 encode a dehydratase and two dehydrogenases potentially catalyzing the multistep conversion of ACA (3) into emodin (1). Gene sky_4059 codes for a monooxygenase that putatively can connect two emodin molecules to the final product skyrin (2) in the style of the monooxygenase ClaM involved in the dimerization of the bisanthraquinone cladofulvin. Thus, the presence of these genes in the gene cluster gives further support to the hypothesis that PKS_4063 is the ACA-synthase in C. asteris. Mutational studies will be done in order to confirm these assumptions after a gene transfer system for this strain has been developed.

Dry leaves of Menlba piperita L. 'Kliment-63' and 'Zefir' of 1997 crop were used.

The oil yield from 'Zefir' was 0.97% and that from 'Kliment-63' was 0.54%. The oil from 'Zefir' was found to be rich in menthol (46.2-50.2%) and menthyl acetate (16.8-22.5%). In the oil from 'Kliment-63,' the content of these components was lower, while the menthone content was higher (20.0-23.1%).

The aerial parts of flowering Mentha pulegium plants (cut at ground level) and individual M. pulegium plants were collected in the summer (July, 2003) from three wild populations located in the Municipality of Laganas, Zakynthos, W. Greece. Location 1 (N 37° 41′ 29″, E 20° 50′ 25″; map datum WGS 84; altitude 3 m; 14/07/03) was close to Keri Beach (Limni Keriou), Location 2 (N 37° 43′ 34″, E 20° 50′ 41″; altitude 3 m; 13/07/03) was near the village of Kalamaki and Location 3 (N 37° 39′ 39″, E 20° 48′ 44″; altitude 160 m; 17/07/03) was near the village of Vasilikos. The three locations are within the mainland limits of the protected area of the National Marine Park of Zakynthos (NATURA 2000 Network, site GR 2210002; 14).

The composition of the inflorescence (I), leaf (L) and stem (S) essential oils of wild Mentha pulegium plants from three populations (1-3) on the island of Zakynthos were examined. Pulegone (32.8 %, S1 to 75.8 %, I3) was the major constituent of all of the oils. The other main constituents were piperitenone (5.1 %, L3 to 35 %, I2), isomenthone (4.3 %, I2 to 28.6 %, L3) and piperitone (0.5 %, I3 to 5.2 %, L2). In total, C-3-oxygenated p-menthane compounds constituted from 73.0 % (S1) to 96.2 % (I2) of the oils. The piperitenone content of the inforescence oils was up to 2.4 (Loc 3) times higher than that of the leaf oils. Correspondingly, the isomenthone content of the leaf oils was up to 4.2 (Loc 2) times higher than that of the inforescence oils. The ratio of the isomenthone:piperitenone content of the oils (n=3) was markedly different for the inforescence (mean 0.31), leaf (mean 2.91) and stem (mean 1.12) oils.

The aerial parts of flowering Mentha spicata plants (cut at ground level) and individual M. spicata plants were collected in the summer (July, 2003) from three wild populations located in the Municipality of Laganas, Zakynthos, W. Greece. Location 1 (N 37° 39′ 39″, E 20° 48′ 44″; map datum WGS 84; altitude 160 m; 14/07/03) was near the village of Keri, Location 2 (N 37° 41′ 29″, E 20° 50′ 25″; altitude 3 m; 14/07/03) was close to Keri Beach (Limni Keriou) and Location 3 (N 37° 43′ 34″, E 20° 50′ 41″; altitude 35 m; 14/07/03) was near the village of Pandocratoras. The three locations are within the mainland limits of the protected area of the National Marine Park of Zakynthos (NATURA 2000 Network, site GR 2210002; 8).

The main oil constituents were trans-piperitone oxide, piperitenone oxide and 1,8-cineole. On a whole plant basis (aerial parts) the trans-piperitone oxide content ranged from 1.4 % location (Loc 1) to 32.5% (Loc 3) and appeared to have an inverse relationship with the 1,8-cineole content which ranged from 10.8 % (Loc 3) to 37.9 % (Loc 1). 1,8-cineole was the major oil constituent (37.9 %) of M. spicata plants from Loc 1. The major constituent of the inflorescence oils was piperitenone oxide which ranged from 32.4 % (Loc 3) to 46.3 % of the oil (Loc 1). The major constituent of the leaf oils was 1,8-cineole (40.5 %) in plants from Loc 1 and trans-piperitone oxide in plants from Loc 2 (19.8 %) and Loc 3 (33.5 %). This is the first report for wild populations in Greece of a M. spicata oil in which 1,8-cineole is the major constituent. The observed variation in essential oil composition between locations and plant organs in July would not appear to be directly related to the climatic conditions.