Figure 3. Phenotype of transplastomic tobacco plants and accumulation of artemisinic compounds.

(A) Transplastomic lines Nt-AO1-1 and Nt-AO3-1 display a slightly pale and growth-delayed phenotype at the juvenile stage. WAT: weeks after transfer from tissue culture to soil; scale bars: 10 cm. (B) Amorpha-4,11-diene is synthesized in all transplastomic lines, but accumulates to lower levels in the lines displaying an altered phenotype (purple bars). (C) Artemisinic alcohol is detected in similar amounts in all transplastomic plants. (D) Accumulation of artemisinic acid correlates with the altered phenotype of Nt-AO1-1 and Nt-AO3-1. Relative accumulation of amorpha-4,11-diene was profiled by GC-MS analysis of volatile organic compounds (VOCs). Relative accumulation of the sum of free and conjugated artemisinic alcohol and artemisinic acid were determined by GC-MS analysis of the soluble metabolite fraction after saponification (see Materials and methods; Figures 6 and 7). In agreement with previous reports (van Herpen et al., 2010), these compounds were found to be present mainly as conjugates. Expanding leaves of 5–6 plants per line were used for each measurement. Error bars represent the SD. Different letters above the bars indicate significant differences as determined by One-way ANOVA (p<0.001) and the Holm-Sidak post-hoc test.

DOI: http://dx.doi.org/10.7554/eLife.13664.005

Figure 3—figure supplement 1. Phenotypes of Nt-AO2-1 and Nt-AO3-1 plants throughout development.

Six plants per line (Nt-wt, Nt-AO2-1 and Nt-AO3-1) were grown under standard greenhouse conditions and photographs were taken of one representative plant per line at different time points: young plants (before flowering, stage 1), flowering plants (stage 2) and old plants (seed capsules formed, stage 3). Light-green leaves and slightly delayed growth of line Nt-AO3-1 are more evident at the young stage. At later stages, all Nt-AO lines display a wild type-like phenotype and produce viable seeds in indistinguishable amounts. y: young leaf; i: expanding (intermediate) leaf; m: fully expanded (mature) leaf.

DOI: http://dx.doi.org/10.7554/eLife.13664.006

Figure 3—figure supplement 2. Isoprenoids levels throughout development in wild-type Nicotiana tabacum plants (Nt-wt) and the transplastomic lines Nt-AO2-1 and Nt-AO3-1.

Plants were grown under standard greenhouse conditions and samples were taken from young (y), expanding (i), and fully expanded (m) leaves at three developmental stages (1–3; cf. Figure 3—figure supplement 1). Metabolite levels were determined by UPLC analysis. The values represent the peak height for each compound divided by 10.000 and normalized to the fresh weight (FW), resulting in the normalized response/FW. Error bars represent the SD (n = 3 plants per line). Different letters above the bars indicate significant differences as determined by One-way ANOVA (p<0.05) and the Holm-Sidak post-test.

DOI: http://dx.doi.org/10.7554/eLife.13664.007