Statistical analysis

Three main experiments with transgenic plants (transplastomic and RNAi lines) growing under greenhouse or fully controlled conditions (phytochamber) were performed in this work. Transgenic lines were compared in two groups (transplastomic and RNAi lines) due to the high number of lines and biological replicates. Thus, transplastomic and RNAi lines were always grown and compared in two different groups with their own wild-type control. The same leaf tissue (fourth leaf bottom to top) from 6-week-old tobacco was used to perform all the molecular analysis. The first experiment included a set of tobacco plants used for molecular analysis such as qPCR (n=3 and three technical replicates), pigment quantification via UPLC (n=5), and hormone (n=5 and three technical replicates) quantification. In addition, for primary and secondary metabolites (n=6) statistical analysis was performed in R (R Core Team, 2018) using an unpaired Wilcoxon test with the default parameters within the function compare_means of ggpubr package (Kassambara, 2018). The second main experiment consisted of tobacco plants used for physiological parameters and biomass quantification (n=5) of tobacco plants grown in the greenhouse. Due to the high number of transgenic lines and biological replicates, the experiments were performed with 3 d of difference, starting with the transplastomic lines and then the RNAi lines (each transgenic group with their own wild type). The third main experiment consisted of a set of tobacco plants used for photosynthetic analysis (n=3–8). Plants were grown in a controlled-environment chamber in a consecutive manner due to space constrains (first transplastomic and then RNAi lines, each group with its own wild-type control). In a fourth experiment, tobacco seedlings grown in solid MS medium were used. Germination rate (n=20), root length (n=6), and biomass (n=6) experiments were performed in 10-day-old tobacco seedlings. These experiments were repeated three times in different Petri dishes. For the last (fifth) experiment, corresponding to the hormone and hormone inhibitors experiments, 10-day-old tobacco seedlings growing on half-strength solid MS medium were transferred to a 24-well plate. Each row consisted of six wild type, and six replicates of the three transplastomic or RNAi lines, and therefore the plants within a plate were always compared with each other (these plants came from the same Petri dish where they grew for 10 d). Due to the experimental design for the five different data sets of plants, an unpaired, two-tailed Student’s t-test was performed to compare each set of transgenic lines with the wild type (unless otherwise stated). GraphPad Prism 5.0 software was used to prepare the figures and perform the t-tests.