Glycyrrhiza glabra is an important medicinal plant throughout the world. Glycyrrhizin is a triterpenoid that is among the most important secondary metabolites produced by liquorice. Drought stress is proposed to enhance the levels of secondary metabolites. In this study, the effect of drought stress on the expression of important genes involved in the glycyrrhizin biosynthetic pathway was examined. Drought stress at the seedling stage was applied to 8-day-old plants using polyethylene glycol. Subsequently, the samples were collected 0, 4, 8 or 24 h post-treatment. At the adult plant stage, 10-month-old plants were subjected to drought stress by discontinuing irrigation. Subsequently, samples were collected at 2, 16 and 28 days after drought imposition (S(2d), S(16d) and S(28d), respectively). We performed semi-quantitative RT-PCR assays to evaluate the gene expression levels of sequalene synthase (SQS), β-amyrin synthase (bAS), lupeol synthase (LUS) and cycloartenol synthase (CAS) during stress. Finally, the glycyrrhizin content of stolons was determined via HPLC. The results revealed that due to osmotic stress, the gene expression levels of SQS and bAS were increased, whereas those of CAS were relatively unchanged at the seedling stage. At the adult plant stage, the expression levels of SQS and bAS were increased under drought stress conditions, whereas the gene expression level of CAS remained relatively constant. The glycyrrhizin content in stolons was increased only under severe drought stress conditions (S(28d)). Our results indicate that application of controlled drought stress up-regulates the expression of key genes involved in the biosynthesis of triterpenoid saponins and directly enhances the production of secondary metabolites, including glycyrrhizin, in liquorice plants.

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The fungal organism Colletotrichum gloeosporioides is the causative agent of anthracnose disease of Citrus fruits. It is recently introduced as a potential producer of anticancer metabolite paclitaxel. Here, we introduce the optimal conditions for growth and sporulation of C. gloeosporioides. We have considered four fungal culture media, that is potato dextrose agar (PDA), carnation leaf agar (CLA), potato carrot agar (PCA) and water agar (WA), based on which sporulation inducers like Watman or Fabriano filter papers could be added, and evaluated both for vegetative growth and sporulation. Three light regimens, i.e. continuous light, 16/8 hrs light/darkness, and continuous darkness were applied in combination with the culture media. All experiments were tracked on 7th, 15th, 21st, and 30th day after incubation. At 28°C, PDA and PCA culture media, under continuous light, provided the best condition for C. gloeosporioides maximal growth. Decreasing light periods decreased the fungal growth. Furthermore, fungal sporulation showed a high dependence on light, temperature and culture medium in use. Under 16/8 h light/darkness interval at the same temperature C. gloeosporioides sporulation was at its maximum on Fabriano paper placed on PDA medium. At a lower temperature, that is 22°C, C. gloeosporioides sporulation on the same culture media was highly defected. Furthermore, aging generally increased the fungal sporulation.