Fig. 3

Cotyledon-VIGS of CrGATA1. (a) A shematic diagram showing vindoline biosynthetic pathway in C. roseus. T16H2, tabersonine 16-hydroxylase 2; 16OMT, 16-hydroxytabersonine O-methyltransferase; T3O, tabersonine 3-oxygenase; T3R, tabersonine 3-reductase; NMT, 3-hydroxy-16-methoxy-2,3-dihydrotabersonine N-methyltransferase; D4H, desacetoxyvindoline-4-hydroxylase; DAT, deacetylvindoline-4-O-acetyltransferase. (b) Relative expression of CrGATA1 in empty vector control (TRV) and CrGATA1-VIGS (TRV-CrGATA1) cotyledons. (c) Relative expression of T3O, T3R and DAT in the control and CrGATA1-VIGS cotyledons. (d) Contents of tabersonine and vindoline in the control and CrGATA1-VIGS cotyledons. Gene expression was measured using RT-qPCR, and the C. roseus RPS9 gene was used as an internal reference gene. Alkaloids were extracted and analyzed by LC-MS/MS, and the concentrations of the alkaloids were estimated based on peak areas compared with standards. DW, dry weight. The values represent means ± SD from three biological replicates. For each biological replicate, entire cotyledons were pooled from 8–9 seedlings (16–17 cotyledons). Statistical significance was calculated using Student’s t test (* P < 0.05 and ** P < 0.01). The black and grey bars represent the TRV (empty vector control) and TRV-CrGATA1, respectively