## 2 Results and Discussion
### Wounding-Induced Alterations in the SGs Content
and displayed major MS\({}^{2}\) fragments [M-C\({}_{6}\)H\({}_{10}\)O\({}_{5}\)-H\({}_{2}\)O-CO\({-}\)24-H]\({}^{-}\) at _m_/_z_ 155 resulting from RDA (Retro-Diels-Alder) cleavage and [M-C\({}_{6}\)H\({}_{10}\)O\({}_{5}\)-H\({}_{2}\)O-CO-C\({}_{2}\)O\({}_{2}\)-H]\({}^{-}\) at _m_/_z_ 123. Pseudomolecular ion [M+CH\({}_{3}\)COOH-H]\({}^{-}\) at _m_/_z_ 417, which was visible as a peak at Rt = 3.00 min., was assigned to swersodic (**4**), and it generated major MS\({}^{2}\) product ions [M-C\({}_{6}\)H\({}_{10}\)O\({}_{5}\)-H]\({}^{-}\) at _m_/_z_ 195 by the loss of a glucose unit and [C\({}_{6}\)H\({}_{12}\)O\({}_{6}\)-H\({}_{2}\)O-H]\({}^{-}\) at _m_/_z_ 161. Swertiamarin (**5**) with pseudomolecular ion [M+CH\({}_{3}\)COOH-H]\({}^{-}\) at _m_/_z_ 433 eluted at Rt = 2.61 min. and its main MS\({}^{2}\) diagnostic fragments were [C\({}_{6}\)H\({}_{12}\)O\({}_{6}\)-H]\({}^{-}\) at _m_/_z_ 179, corresponding to deprotonated glucose, and [M-C\({}_{7}\)H\({}_{12}\)O\({}_{8}\)-H]\({}^{-}\) at _m_/_z_ 149 resulting from the loss of the glucose, H\({}_{2}\)O, and CO\({}_{2}\) moieties. Compound **6** with [M+CH\({}_{3}\)COOH-H]\({}^{-}\) at _m_/_z_ 415, eluting at Rt = 2.90 min., was identified as gentiopicrin (Figure S1). Its MS\({}^{2}\) data showed the fragments [C\({}_{6}\)H\({}_{12}\)O\({}_{6}\)-H]\({}^{-}\) at _m_/_z_ 179 corresponding to deprotonated glucose, and [M-H\(-\)C\({}_{8}\)H\({}_{12}\)O\({}_{8}\)]\({}^{-}\) at _m_/_z_ 119, which resulted from the loss of glucose, H\({}_{2}\)O, and C\({}_{2}\)O\({}_{2}\) moieties. Similar fragmentation patterns of compounds **4-6** were previously described [45]. Figure 1: Wounding-induced changes in the accumulation of secoiridoid glucosides in _C. erythraea._ (**A**) The proposed biosynthetic pathway of secoiridoid glucosides (SGs) in _C. erythraea_ (modified from [4]). Arrow with a solid line indicates the known enzyme, arrow with dashed line designates unknown enzyme. (**B**) Visualization of the wounding procedure within the experimental set-up: wounded (local leaves–LL) and non-wounded leaves (systemic leaves—SL). (**C**) UHPLC/DAD chromatogram of non-wounded leaves at the beginning of experiment; extracted is the enlarged part of the chromatogram  
where iridoids and secoriridoids elute. (**D**) Total iridoids content in control leaves and upon 96 h after wounding (HAW), 1--loganic acid, 2--loganin, 3--secologanin, 4--serexoside, 5--swertiamarin, 6--gentiopicrin. (**E**) Heat maps based on the scaled values presenting the relationships between LL and SL of _C. erythraea_ at determined time points (h) after wounding (HAW). Scaling was performed for each compound independently. The values are represented by the variation from green (min concentration) to red color (max concentration), as indicated on the color scale. For the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article. Abbreviations: GPPS--geranyl diphosphate synthase, GES--geraniol synthase, G8O--geraniol-8-oxidase, 8 HG0-8-hydrohygeraniol oxidoreductase, IS--iridoid synthase, IO--iridoid oxidase, 7 DLGT--7-deoxylogane acid glucosyltransferaseerase, 7 DLH--7-deoxyloganic acid hydrolase, LAMT--loganic acid O-methyltransferasease and, SLS--secologanin synthase. The highest increase in total iridoids content was detected at 96 HAW in the SL leaves (Figure 1). The most abundant compound among analyzed iridoids was **4**, which reached 2753 \(\upmu\)g 100 mg\({}^{-1}\) FW (Figure 1). The second most abundant SG in analyzed samples was **6** (\(\sim\)2000 \(\upmu\)g 100 mg\({}^{-1}\) FW), followed by **5** (\(\sim\)200 \(\upmu\)g 100 mg\({}^{-1}\) FW) and **3** (up to 123 \(\upmu\)g 100 mg\({}^{-1}\) FW). Iridoids **1** and **2** reached up to 60 and 4\(\upmu\)g 100 mg\({}^{-1}\) FW, respectively. The content of **1** and four SGs (3-6) in _C. erythraea_ leaves was increased after wounding in a time-dependent manner, showing similar accumulation patterns (Figure 1, heat maps).