## 2 Results and Discussion
### Secoriridoids-Related Biosynthetic Genes Expression Profiles in Response to Wounding  
The production, accumulation, and distribution of specialized metabolites within the plant are usually related to the expression levels of the biosynthetic genes. We have recently revealed potential candidates for 10 genes encoding for enzymes responsible for the synthesis of secologanin (**3**) in _C. erythraea_, including _CeGPPS_, _CeGES_, _CeG8O_, _Ce8 HGO_, _Ce1S_, _Ce1O_, _Ce7 DLGT_, _Ce7 DLH_, _CeLAMT_, and _CeSLS_[4]. The expression of these genes is well correlated with the iridoids content and it is organ-specific, genotype-dependent, and mainly MeJA-inducible [4]. Leaves have been highlighted as a major site of SGs biosynthesis and accumulation, and the transcriptional regulation of iridoids biosynthesis has been proposed [4]. We have investigated the time-dependent expression patterns of SGs biosynthetic genes, in both LL and SL, to address transcriptional control of increased SGs accumulation in response to wounding in _C. erythraea. CeLAMT_ was omitted from the analysis due to its extremely low expression level. Quantitative real-time PCR (qRT-PCR) analysis revealed that nine out of the 10 targeted transcripts showed an increasing trend of expression, not only in LL, but also in the SL (Figure 2). _CeIS1_ was the only gene showing no increasing trend of expression after wounding (Figure S2A). In our previous work [4], this gene showed no transcriptional response to the MeJA treatment either. Phylogenetic analysis was performed in order to investigate the evolutionary relationship of _C. erythraea_ ISs (CeIS1 and CeIS2) with some of the characterized ISs and progesterone-5-\(\beta\)-reductases (P5 BRs) from other species (Figure S2B). CeIS1 evidently subclades with the known P5 BRs, while CeIS2 is positioned within the distinct branch of ISs (Figure S2B). Previous studies have shown that _CrIS_ in _C. roseus_ is co-expressed with the other genes of the MIA pathway, while _CrP5 BRs_ do not follow the same pattern of expression [52; 53]. Thus, _CeIS2_ has shown to be a promising candidate based on high sequence similarity to _CrIS_ and its co-expression pattern in response to wounding, which looks to be similar to those of the other SGs biosynthetic genes. Although the expression of _CeIS1_ is neither MeJA- nor wound-induced, its involvement in constitutive SGs biosynthesis in _C. erythraea_ cannot be neglected. Significantly increased expression levels of the majority of analyzed SGs biosynthetic genes in _C. erythraea_ leaves were detected 24 HAW (Figure 2). The exception was observed in the expression pattern  
Figure 2: Gene expression of secoiridoid glucosides (SGs) metabolic pathway genes in _C. erythraea_ leaves at determined time points (h) after wounding (HAW). The relative expressions of the genes were normalized against _CeEF1_ gene as an internal control; non-wounded leaves at 0 HAW 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. In all cases, bars with different letters are significantly different (\(p<0.05\)) according to post hoc [PERSON]’s test. Abbreviations: GPPS–geranyl diphosphate synthase, GES—geraniol synthase, GSO—geraniol-8-oxidase, 8 HGO—8– hydrohyograniol oxidoreductase, IS2—iridoid synthase, IO—iridoid oxidase, 7 DLGT—7- deoxyloganetic acid glucosyltransferase, 7 DLH2—7-deoxyloganic acid hydrolase, SLS—secologanin synthase, EF1—elongation factor 1, LL—local leaves, and SL—systemic leaves. of _CeGPPS_ which encodes the enzyme that catalyzes the condensation of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) to geranyl diphosphate (GPP), which is the key precursor of monoterpene biosynthesis. In common centaury, the enhanced _CeGPPS_ expression was noticed 2 and 4 HAW. Significantly higher expression levels of _CeGES_, _CeG80_, _Ce8 HGO_, _Ce8S2_, _CeIO_, _Ce7 DLGT_, and _Ce7 DLH2_ were recorded 24 and 48 HAW.