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. 2022 Oct 4;13:5856. doi: 10.1038/s41467-022-33516-1

Fig. 4. Comparison of transplastomic plants expressing fAMP constructs controlled by different promoter systems.

Fig. 4

a Phenotypes of transplastomic plants. Two genotypes were used as recipients for introduction of fAMP constructs: the wild type and a transplastomic line harboring the riboswitch-controlled T7 RNA polymerase (RT7RP). The promoters used to drive fAMP expression is given in each row. Shown are transplastomic plants expressing fAMPs under the control of the Prrn promoter (images from Fig. 2a), transplastomic plants expressing fAMPs under RAmpER control (uninduced), and two PT7-controlled fAMP lines in the wild-type background. Photos of soil-grown plants were taken one month after sowing. Scale bars: 5 cm. 3-M: 3-Molluxubin, 3-N: 3-Novescandin, 3-W: 3-Wamdeposin, 6-MW: 3-M + 3-W, 9-MNW: 3-M + 3-N + 3 W. The numbers 5 or 15 indicate the lengths of the amino acid linkers separating the AMPs (see Supplementary Table 1 and Supplementary Data 2). b Images of the plants in (a) two months after sowing. Scale bars: 5 cm. c Northern blot analysis comparing the fAMP expression levels in Prrn (P), RAmpER (uninduced) (R) and PT7 (T) plants (see Methods). MB: methylene blue staining (loading control). This blot was performed twice with similar results. d Western blot analysis of fAMP accumulation. Strong constitutive expression from Prrn (P) is compared with basal RAmpER expression (in the non-induced state; R) and low-level background expression from PT7 (in the absence of the T7 RNA polymerase; T). Total protein samples (20 µg) were loaded, and fAMPs were immunodetected with an anti-HA antibody. The Coomassie-stained membrane is shown below the immunoblot as loading control. Note that pale and white lines have reduced amounts of the large subunit of RuBisCO (at ∼55 kDa). Two independent blots were performed with similar results. Plant material for northern and western blots was produced in sterile culture to enable inclusion of lines that do not survive on soil. 3M15: 3-Molluxubin-15, 3N5: 3-Novescandin-5, 3W5: 3-Wamdeposin-5, 6MW5: 3M5 + 3W5, 9MNW15: 3M15 + 3N15 + 3W15. Source data are provided as a Source Data file.