Artemisinic compounds and expression levels of the transgenes were measured in young (stage 1), flowering (stage 2) and old plants (stage 3; see Figure 3—figure supplement 1). (A) Amorpha-4,11-diene accumulates to higher levels in line Nt-AO2-1 than in Nt-AO3-1. (B) Artemisinic alcohol is present at similar levels in early (1) and late stages (3) of development in lines Nt-AO2-1 and Nt-AO3-1, but it is slightly higher in the flowering stage (2) of line Nt-AO3-1. (C) Artemisinic acid accumulates to high levels during all developmental stages of line Nt-AO3-1, whereas in line Nt-AO2-1, it is detectable only in mature leaves of young and flowering plants. Relative accumulation of amorpha-4,11-diene and artemisinic alcohol was profiled, the tissue content of artemisinic acid was quantified using an authenticated reference standard (n = 5–6 plants per line; Figures 6 and 7). The sum of free and conjugated artemisinic alcohol and artemisinic acid were determined. y: young leaf; i: expanding (intermediate) leaf; m: fully expanded (mature) leaf. Error bars represent the SD. (D–G) Northern blot analysis of the expression of the four transgenes. Total RNA samples from N. tabacum wild-type (Nt-wt) plants and the transplastomic lines Nt-AO2-1 and Nt-AO3-1 (at the developmental stages 1–3) were separated in denaturing 1.5% agarose gels, blotted and hybridized to strand-specific RNA probes. Below each blot, the rRNA-containing region of the ethidium bromide-stained gel prior to blotting is shown as a control for RNA integrity and equal loading. The Nt-wt sample corresponds to RNA extracted from a fully expanded leaf of a N. tabacum wild-type plant at developmental stage 2. The smallest labeled band in each blot corresponds to the monocistronic mRNA. Larger bands represent unprocessed polycistronic precursor transcripts and read-through transcripts (which are common in plastids; e.g., Elghabi et al., 2011; Lu et al., 2013). CYP transcripts accumulate to higher levels in line Nt-AO3-1, while CPR transcripts accumulate to higher levels in line Nt-AO2-1, resulting in a higher CYP/CPR expression ratio in line Nt-AO3-1.