Fig. 1.

Introduction of the Daucus carota lycopene β-cyclase1 (DcLCYB1) gene into the plastid genome by stable transformation. (A) Schematic representation for isoprenoid-derived pathways (e.g. carotenoids, GA, ABA) of the changes caused upon expression of DcLCYB1 in nuclear tobacco lines. Up-regulated genes are marked in red; genes shown in black were not changed; and genes shown in grey were not measured. Red stars indicate increased metabolite content. ABA, abscisic acid; β-car, β-carotene; Chl, chlorophyll; DMAPP, dimethylallyl diphosphate; GA, gibberellins; IPP, isopentenyl diphosphate; Lut, lutein; Pchlide, protochlorophillidae; Phy-PP, phytyl diphosphate; Viol, violaxanthin; Zea, zeaxanthin. Gene abbreviation, protein name, and a brief description of each gene measured by qPCR can be found in Moreno et al. (2020). (B) Physical map of the plastid genome region (ptDNA) used as a site for integration, and of the transgenic loci in the generated transplastomic tobacco lines (Nt-pJM37, Nt-pJM36). Native plastid genes are shown in black; introduced elements are shown in pale grey and orange. Genes above the line are transcribed from left to right; genes below the line are transcribed right to left. Promoter (P) and terminator (T) sequence from Chlamydomonas reinhardtii (Cr) are shown in pale grey. T7g10L is the gene 10 leader sequence from the T7 phage, which has been shown to be highly efficient at facilitating high-level translation in green plastids (Oey et al., 2009; Elghabi et al., 2011). The aadA gene encodes the AMINOGLYCOSIDE 3′-ADENYLYLTRANSFERASE enzyme, which confers resistance to the aminoglycoside-type antibiotics spectinomycin and streptomycin and as such serves as a selectable marker for transformed chloroplasts. The black box under psaB indicates the probe used for RFLP analysis. (C) RFLP analysis of transplastomic plants. Total DNA from wild-type and transformed lines was digested with BamHI producing fragments of the sizes indicated in (A). A single, independently isolated transplastomic line is shown for Nt-pJM36; six lines are shown for Nt-pJM37. (D) Inheritance assay of transplastomic DcLCYB1 plants. Germination of the T₁ generation of plastid transformants in the presence of spectinomycin (+spec) revealed the homoplasmic stage for the integrated selectable marker gene aadA. Numbering of the lines refers to the nomenclature use in (B). spec, spectinomycin; WT, wild type.