## 4 Conclusions  
Based on all of the presented results, it is noticeable that most genes of the SGs biosynthetic pathway in _C. erythraea_ are up-regulated upon wounding, whereby the highest increase in the expression level was observed for _CeSLS_, encoding for secologanin synthase, which catalyzes direct cleavage of the cyclopentane ring of loganin to produce secologanin in _C. roseus_[73; 74]. The highest amount of transcripts of the majority of SGs biosynthetic genes is reached 24 and 48 HAW, when the responsive TFs showed the highest expression levels and the SGs amount significantly increased. Among the analyzed TFs, the bHLH type _CeBIS1_ showed concurrent expression pattern as _CeSLS_ and this specific TF, according to the obtained results, has particular significance in wound-induced regulation of SGs biosynthesis in common centaury leaves. In addition, _CeSLS_ has been recognized as an important gene/enzyme that might regulate biosynthetic flux through the SGs pathway, and it is probably directly under the control of _CeBIS1_ TF. The results further indicate the involvement of the JA signaling pathway in the regulation of SGs biosynthesis, as influenced by wounding. Although differences in the expression levels of other analyzed centaury TFs were not so perceptible, their importance should not be neglected. Although much remains to be investigated in the future, the presented results provide first and significant contribution in understanding the regulatory mechanisms shaping the SGs biosynthetic pathway in common centaury in response to environmental factors. This valuable knowledge could be of great significance for further commercialization and the scaling-up of the SGs production in non-model plant, such as _C. erythraea_, which in turn will provide sustainable sources of these bioactive compounds. The following are available online at [[http://www.mdpi.com/2223-7747/8/12/589/s1](http://www.mdpi.com/2223-7747/8/12/589/s1)]([http://www.mdpi.com/2223-7747/8/12/589/s1](http://www.mdpi.com/2223-7747/8/12/589/s1)), Figure S1: SRM (Single Reaction Monitoring) UHPLC-MS2 chromatograms of targeted compounds (loganic acid, loganin, secologanin, sweroside, swertiamarin and gentiopicrin) and their corresponding MS2 spectra, Figure S2: (**A**) Relative expression of _CeS1_ in _C. erythraea_ leaves at determined time points (h) after wounding (HAW). The relative expressions of the _CeS1_ were normalized against _CeEFI_ gene as an internal control; unounded leaves (at 0 h) were set as a calibrator. Since factorial ANOVA revealed that type of leaves does not significantly contribute to the gene expression, only the effect of time was post hoc tested. Bars with different letters are significantly different (\(p<0.05\)) according to post hoc [PERSON]'s test. Abbreviations: IS - iridoid synthase, LL-local leaves, SL-systemic leaves. (**B**) Phylogenetic tree derived from CelS1, CelS2 and selected ISs and P5\(\beta\)Rs. CLC sequence viewer 8.0 was utilized to align protein sequences and a neighbor joining tree was formed from the alignment. The scale bars represents 0.2 substitutions per site. The GenBank accession numbers are as follows: CrS (_Catharantus roseus_ iridoid synthase, AFW98981.1), CrP5 BR4 (_Catharantus roseus_ progesterone-5-\(\beta\)-reductase4, AIW09146.1), GrIS1 (_Centiana rigescens_ iridoid synthase 1, AKI87774.1), GrIS2 (_Centiana rigescens_ iridoid synthase 2, AKI87775.1), DIP5 BR1 (_Digitalis lanta_ progesterone-5-\(\beta\)-redductase 1, AIF73578.1), DIP5 BR2 (_Digitalis lanta_ progesterone-5-\(\beta\)-redductase 2, ADL28122.1), Dp5 BR2 (_Digitalis purpurea_ progesterone-5-\(\beta\)-redductase 2, ACZ66261.1), LjIS (_Lonicera japonica_ iridoid synthase, AMB61018.1) and OeIS (_Olea europaea_ iridoid synthase, ALV83438.1, Table S1: Primer sequences used for the qPCR analysis (for other SGs metabolic pathway genes primer sets please refer to [4]), Table S2: Results of factorial ANOVA on gene expression data in leaves of _C. erythraea_ upon wounding.