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. 2020 Dec 29;185(3):836–856. doi: 10.1093/plphys/kiaa075

Figure 8.

Figure 8

shSGD negatively impacts SGD deglycosylation activity. A, Relative expression of SGD and shSGD in leaves of plants transformed by empty vector (EV, black bar) or by the pTRV2-shSGD silencing construct (shSGD, gray bar) measured by RT-qPCR. B, Relative means of strictosidine (stricto), strictosidine aglycone (agly), tabersonine (tab), vindoline (vind), and catharanthine (cath) contents in leaves of EV (black bar) and shSGD (gray bar) transformed plants. Data represent means ± se of three technical replicates performed on four plants transformed with EV or shSGD (see Supplemental Material and Methods). Asterisks denote statistical significance (*P < 0.005; **P < 0.001; by Student’s t test). C, Coomassie blue staining and immunoblot analysis of crude protein extracts (15 µg per lane) from C. roseus leaves agroinfiltrated with pEAQ-HT:GFP-6His and either pEAQ-HT:shSGD-6His or empty vector (EV). D, Strictosidine concentration in infiltrated leaves (three independent biological replicates consisting of three plants transformed with EV, six plants transformed with shSGD construct, and for each plant four infiltrated leaves except for one sample of shSGD were analyzed after 7 d, thus totaling 12 EV samples and 23 shSGD samples). Points correspond to individual samples. Asterisks denote statistical significance in difference between the means (Wilcoxon rank sum test, P < 0.001). E and F, Metabolite correlation networks in control and shSGD overexpressing plants. Correlations among metabolite concentrations were calculated over 87 and 96 samples, respectively, from 10 plants and at least 8 infiltrated leaves for each construct at 7 d (E) and 16 d (F) post-infiltration (dpi). Nodes correspond to metabolites: 1, secologanin; 2, strictosidine; 3, tabersonine; 4, tabersonine imine alcohol; 5, 16-hydroxytabersonine; 6, 16-methoxytabersonine; 7, 16-methoxytabersonine epoxide; 8, 16-methoxy-2,3-dihydrotabersonine; 9, desacetoxyvindoline; 10, desacetoxyvindorosine; 11, desacetylvindoline; 12, vindoline; 13, demethylvindorosine; 14, catharanthine; 15, vinblastine; 16, ajmalicine; 17, serpentine. Node pairs connected by black lines indicate a Spearman rho >0.6 (E) and rho >0.7 (F) for these pairs, the higher the correlation, the shorter the line.