Volume 27, Issue 19, Pages 3034-3041.e3 (October 2017) Horizontal Transfer of a Synthetic Metabolic Pathway between Plant Species  Yinghong Lu, Sandra Stegemann, Shreya Agrawal, Daniel Karcher, Stephanie Ruf, Ralph Bock  Current Biology  Volume 27, Issue 19, Pages 3034-3041.e3 (October 2017) DOI: 10.1016/j.cub.2017.08.044 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Metabolic Pathway of Astaxanthin Biosynthesis and Its Introduction as a Synthetic Operon Construct into the Tobacco Plastid Genome (A) Astaxanthin pathway and links to other metabolic pathways. The precursor of carotenoids, geranylgeranyl diphosphate (GGDP), is produced in plastids by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here we have used the lycopene β-cyclase from daffodil (orange) to enhance conversion from lycopene to β-carotene [10] and the enzymes CrtW (β-carotene ketolase; blue arrows) and CrtZ (β-carotene hydroxylase; red arrows) from the marine bacterium Brevundimonas to convert β-carotene to astaxanthin. The eight differentially ketolated and/or hydroxylated intermediates are indicated [11, 12]. (B) Physical map of the targeting region in the wild-type tobacco plastid genome (N.t. ptDNA). Genes above the horizontal line are transcribed from left to right, and genes below the line are transcribed from right to left. The location of the two restriction sites used for RFLP analyses and the expected fragment size are indicated. (C) Physical map of the transgenic locus in the plastid genome of transplastomic Nt-AXT lines. The three cistrons in the astaxanthin operon construct are separated by two identical intercistronic expression elements (IEE [9]). The transgene expression cassette is driven by the rRNA operon promoter from tobacco (Nt-Prrn) fused to the 5′ UTR from gene10 of phage T7 and followed by the 3′ UTR from the Chlamydomonas rbcL (Cr-TrbcL) gene. 3′ UTRs of the tobacco rps16 (Nt-Trps16) and rbcL (Nt-rbcL) genes stabilize the processed monocistronic NpLyc and BsCrtW mRNAs. The selectable marker gene aadA is under the control of a chimeric rRNA operon promoter (Nt-Prrn) and the 3′ UTR from the tobacco psbA (Nt-psbA) gene [13]. The aadA is flanked by loxP sites and is therefore excisable [14, 15]. The sequence of the transformation construct is available under the accession number GenBank: MF580999. (D) RFLP analysis of transplastomic tobacco lines expressing the enzymes of the astaxanthin pathway from the synthetic operon construct (Nt-AXT lines). DNA samples were digested with MluI and AgeI, and the relevant restriction fragment was detected by hybridization to a psbZ-specific probe (fragment sizes: 2.15 kb in the wild-type, Nt-WT, and 7.32 kb in Nt-AXT lines). (E) Northern blot analyses to analyze mRNA accumulation in transplastomic lines expressing the synthetic astaxanthin operon. Proper processing of the tricistronic operon transcript is revealed by the appearance of prominent bands corresponding to the three monocistronic mRNAs (smallest band in all three blots). The larger hybridizing bands represent the tricistronic precursor transcript and (dicistronic) processing intermediates that were not further characterized. Current Biology 2017 27, 3034-3041.e3DOI: (10.1016/j.cub.2017.08.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Phenotype of Transplastomic Tobacco Plants Expressing the Synthetic Astaxanthin Operon (A) Primary transplastomic callus tissue (left) appears orange (white arrow), whereas callus of a spontaneous spectinomycin-resistant line (that has arisen from a point mutation in the 16S rRNA gene; right explant [24]) is green. (B) Regenerating orange shoot of a transplastomic line. (C–E) Wild-type (C), heteroplasmic transplastomic (D), and homoplasmic transplastomic (E) tobacco plants growing in tissue culture. (F) A wild-type plant (left), two heteroplasmic transplastomic plants (middle), and a homoplasmic transplastomic plant (right) growing in the greenhouse. (G–I) Stem cross-sections of the wild-type (G), a heteroplasmic transplastomic plant (H), and a homoplasmic transplastomic plant (I). (J and K) A mature flower from the wild-type (J) and a transplastomic plant (K). (L–Q) Examples of seed assays to confirm the homoplasmic state of transplastomic lines. (L–N) Seeds germinated on antibiotic-free medium. Seeds from a wild-type plant (L), a homoplasmic transplastomic plant (M) and a heteroplasmic transplastomic plant (N) were analyzed. (O–Q) Seeds germinated on spectinomycin-containing medium. The same lines as in (L)–(N) were analyzed. Current Biology 2017 27, 3034-3041.e3DOI: (10.1016/j.cub.2017.08.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Accumulation of Carotenoids and Chlorophylls in Transplastomic Nt-AXT Lines (A) HPLC analysis of pigment extracts. Note that peaks for the abundant carotenoids in the wild-type (lutein, β-carotene, and violaxanthin) are completely absent from transplastomic plants and instead a gigantic astaxanthin peak appears. Smaller peaks surrounding the astaxanthin peak most likely represent lowly abundant pathway intermediates (Figure 1A). mAU, milli-absorbance units. (B) Pigment accumulation in an age series of six consecutive leaves (L1 to L6, representing leaves number 2 to 7 from a plant at the nine-leaf stage raised in tissue culture; leaves were counted from the top to the bottom) of the wild-type and two independently generated transplastomic lines. For each plant line, four different plants were measured. Error bars represent the SD. DW, dry weight. (C) Light microscopic image of leaf mesophyll cells from wild-type tobacco and an Nt-AXT plant. Note that the massive accumulation of astaxanthin leads to dark red particles in chloroplasts of the transplastomic leaves. (D) Transmission electron microscopy image of a typical mesophyll chloroplast in wild-type tobacco and two representative images of chloroplasts in Nt-AXT plants. Note the underdeveloped thylakoid network (T) and the high abundance of aggregated plastoglobules (black; P) in the transplastomic chloroplast. CW, cell wall; M, mitochondrion; V, vacuole. See also Figure S1. Current Biology 2017 27, 3034-3041.e3DOI: (10.1016/j.cub.2017.08.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Transfer of the Synthetic Astaxanthin Operon from N. tabacum to N. glauca by Horizontal Genome Transfer (A) Reciprocal grafting of the transplastomic astaxanthin-producing N. tabacum line (Nt-AXT-1) with a kanamycin-resistant N. glauca line (Ng-kan). (B) Detection of horizontal genome transfer on regeneration medium containing kanamycin and spectinomycin. The control explants from the two graft partners (three stem sections and three leaf pieces each; N. glauca: leaves top, stems bottom; N. tabacum: stems top, leaves bottom) are in the two left frames, and the excised graft sites are in the right frame. Growing orange calli (indicated by arrows) from the graft sites indicate horizontal genome transfer events. Note that the control explants from N. glauca are less severely bleached and slightly proliferate on this medium due to the milder growth-inhibitory action of spectinomycin compared to kanamycin. The Nt-AXT-1 control explants lost their orange color due to sensitivity to kanamycin, a potent inhibitor of chloroplast translation. (C) Growth of regenerated transplastomic astaxanthin-synthesizing N. glauca plants (Ng-AXT; right) in comparison to a plant of the Ng-kan line used for grafting (left). (D) An astaxanthin-synthesizing N. glauca plant after transfer to the greenhouse. (E) A flowering branch of a wild-type N. glauca tree. (F) A flowering branch of an astaxanthin-synthesizing N. glauca tree obtained by horizontal genome transfer. (G) Confirmation of horizontal genome transfer by RFLP analysis. Total DNA was digested with the restriction enzymes MluI and AgeI (cf. Figure 1B). RFLP analysis of seven independent horizontal genome transfer lines of N. glauca (Ng-AXT lines) detects the same 7.3 kb fragment that is present in the transplastomic N. tabacum line used for grafting (Nt-AXT-1). Note that line Ng-AXT-8 is heteroplasmic and still contains copies of the resident N. glauca plastid genome, as evidenced by presence of the hybridization signal at 2.15 kb. (H) Flower phenotypes of an N. glauca wild-type plant (upper flower) and an Ng-AXT line (lower flower). See also Figures S2–S4. Current Biology 2017 27, 3034-3041.e3DOI: (10.1016/j.cub.2017.08.044) Copyright © 2017 Elsevier Ltd Terms and Conditions