This is the accepted version of the article: Llorente, Briardo; Torres Montilla, Salvador; Morelli, Luca; [et al.]. «Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development». Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, Issue 35 (September 2020), p. 21796-21803. DOI 10.1073/pnas.2004405117 This version is available at https://ddd.uab.cat/record/232058 under the terms of the license Supplementary Information for Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development. Briardo LLORENTE*, Salvador TORRES-MONTILLA, Luca MORELLI, Igor FLOREZ- SARASA, José Tomás MATUS, Miguel EZQUERRO, Lucio D’ANDREA, Fakhreddine HOUHOU, Eszter MAJER, Belén PICÓ, Jaime CEBOLLA, Adrian TRONCOSO, Alisdair R. FERNIE, José-Antonio DARÒS, Manuel RODRIGUEZ-CONCEPCION* (*) Corresponding authors: BL, Email: briardo.llorente@mq.edu.au MRC, Email: manuel.r.c@csic.es This PDF file includes: SI Materials and Methods Figures S1 to S6 Tables S1 to S2 SI References Other supplementary materials for this manuscript include the following: Datasets S1 to S4 1 SI Materials and Methods Gene constructs. Viral vectors used in this study have been described (1) with the following exceptions. A plasmid with recombinant clone TEV-GFP was constructed from plasmid pGTEVa (2) and used for Arabidopsis Ler plants. The binary plasmid pGTuMV-UK1 was used for Arabidopsis Col plants. Briefly, a cDNA of the UK1 isolate of Turnip mosaic virus (TuMV-UK1; GenBank accession number NC_002509.2) was cloned in vector pG35Z (3) flanked by the Cauliflower mosaic virus 35S promoter and terminator. To generate the different crtB versions, we amplified by PCR the Pantoea ananatis crtB gene (1). We also amplified cDNA sequences encoding the plastid-targeting peptide of Arabidopsis hydroxymethylbutenyl 4-diphosphate synthase (4), Arabidopsis PSY and tomato PSY2. Primers used for cloning procedures are listed in SI Appendix, Table S2. PCR products were cloned using the Gateway system first into plasmid pDONR-207 and then into plasmid pGWB405 (5) to generate 35S:crtB-pGWB405, 35S:(p)crtB- pGWB405, 35S:(p)crtB-GFP-pGWB405, 35S:AtPSY-pGWB405, and 35S:SlPSY2-pGWB405, or plasmid pGWB506 (5) to generate 35S:GFP-crtB-pGWB506. To generate TuMV-crtB and LMV- crtB, the crtB sequence was cloned by Gibson assembly between positions 8758 and 8759 of TuMV-UK1 and 9034 and 9035 of LMV after amplification with flanking sequences complementing the TuMV-UK1 and LMV nuclear inclusion a protease (NIaPro) cleavage sites, respectively (SI Appendix, Table S2) to mediate the release of the crtB protein from the viral polyproteins. ZYMV-crtB was generated as described (1). Transcript analyses. Total RNA was extracted from leaves with the Maxwell 16 LEV Plant RNA Kit (Promega) and quantified with a NanoDrop (Thermo Scientific) as described (1). For reverse transcription-quantitative PCR (RT-qPCR) analyses, the First Strand cDNA Synthesis Kit (Roche) was used to generate cDNA according to the manufacturer’s instructions, with anchored oligo(dT)18 primers and 1 μg of total RNA. Relative mRNA abundance was evaluated via quantitative PCR using LightCycler 480 SYBR Green I Master Mix (Roche) on a LightCycler 480 real-time PCR system (Roche). Quantification of crtB-encoding transcripts was conducted by RT- qPCR using the ACT gene Niben101Scf03410g03002 for normalization and the primers listed in SI Appendix, Table S2. RNAseq service was performed by Sequentia Biotech SL (Barcelona, Spain). RNA concentration in each sample was assayed with a ND-1000 spectrophotometer (NanoDrop) and its quality assessed with the TapeStation 4200 (Agilent Technologies). Indexed libraries were prepared from 1 μg/ea purified RNA with TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions. Libraries were quantified using the TapeStation 4200 and pooled such that each index-tagged sample was present in equimolar amounts, with final concentration of the pooled samples of 2 nM. The pooled samples were subject to cluster generation and sequencing using a NextSeq 500 System (Illumina) in a 2x75 paired end format at a final concentration of 1.8 pmol. The raw sequence files generated (.fastq files) underwent quality control analysis using FastQC (http://www.bioinformatics. babraham.ac.uk/projects/fastqc/). Data analysis was performed with the online platform AIR (www.transcriptomics.cloud) using the SolGenomics Network (https://solgenomics.net/) N. benthamiana 1.01 (Niben v101) reference genome (6). Metabolite analyses. Primary metabolites were extracted as described previously (7) using approximately 20 mg of lyophilized leaf tissue. Derivatization and gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) analyses were carried out as described previously (7). Metabolites were identified manually using the TagFinder software in combination with the reference library mass spectra and retention indices from the Golm Metabolome Database, http://gmd.mpimp-golm.mpg.de. The parameters used for the peak annotation of the 52 2 metabolites can be found in SI Appendix, Dataset S4, which follows recommended standards on the report of metabolite data (8). Data were normalized to the mean value of the GFP control samples (i.e., the value of all metabolites for GFP control samples was set to 1). The means and standard errors of five to six replicates at 96 hpi are presented in SI Appendix, Dataset S5. Photosynthetic measurements. Photosynthetic efficiencies were assessed by measuring chlorophyll a fluorescence with a MAXI-PAM fluorometer (Heinz Walz GmbH). Leaves were placed under the camera and effective quantum yield (ΔF/Fm’) was measured as (Fm’−Fs)/Fm’, where Fm’ and Fs are, respectively, the maximum and the minimum fluorescence of light- exposed plants. The light intensity chosen was 21 PAR (actinic light, AL=2) as the last value able to generate a response in the (p)crtB-infiltrated areas before having a null photosynthetic activity. Each value is the average result of three biological replicates and three different AOI for each replicate. NPQ was also measured using the MAXI-PAM unit. All recordings were performed every day at the same time slot, but the order of the samples was randomized to reduce the bias related to the length of the light stress recovery protocol. Plants were dark-adapted for 30 min before measurement and then submitted to a continuous 801 PAR light (AL=17) for 10 min. After this period plants were left recovering in darkness for 40 min. During the whole protocol, Ft was monitored and Fm’ values were estimated with a saturating pulse (SAT) every 60 seconds. NPQ and its relative components qE, qZ and qI were calculated as described (9) with some modifications. Briefly: NPQ was calculated as (Fm-Fm0)/Fm, where Fm and Fm 0 are the maximum fluorescence after the dark acclimation and after the light stress, respectively. The relative contributions of qE, qZ and qI to NPQ were estimated by monitoring NPQ relaxation kinetics in the dark: following the 10 min exposure to saturating light used to measure NPQ, leaves were left in darkness, and Fm 0 was measured again after 10 and 40 min. The qE component of NPQ relaxes within 10 min of a leaf being placed in darkness such that NPQ persisting after 10 min in the dark consists of qZ + qI. The qZ component of NPQ relaxes within tens of minutes so that NPQ persisting after 40 min in the dark (when the Ft value is linear) consists of qI, which is either irreversible in the dark or requires several hours to relax. Consequently, (qI+qZ) was calculated as (Fm-Fm1)/Fm1, where Fm1 is the value of Fm measured after 10 min in the dark following NPQ measurement. qZ was calculated as (Fm-Fm2)/Fm2, where Fm2 is the value of Fm measured after 40 min in the dark following measurement of NPQmax. qE was calculated as NPQ–(qI+qZ) and qI was calculated as (qI+qZ)-qZ. For the calculation of the de-epoxidation state (DES), agroinfiltrated leaf areas were exposed for 10 min to a continuous 801 PAR light (AL=17) in the MAXI-PAM unit, sampled under the same light and immediately frozen before pigment extraction and quantification. The operation of the xanthophyll cycle, comprising the sequential de-epoxidation of the pigments violaxanthin (Vx) to antheraxanthin (Ax) and zeaxanthin (Zx) was followed by calculating DES as (Zx+0.5×Ax)/(Zx+Ax+Vx), where Zx, Ax and Vx are the concentrations of the corresponding xanthophylls. Respiration measurements. Before respiration measurements, N. benthamiana plants were placed in the dark for about 30 min to avoid light-enhanced dark respiration. Four leaf discs of 3.8 2 cm each were harvested from leaf sections of two different plants infiltrated with the (p)crtB construct, weighted and placed into the respiration cuvette containing the respiration buffer (30 mM MES pH 6.2, 0.2 mM CaCl2). Oxygen uptake rates were measured in darkness using a liquid-phase Clark-type oxygen electrode (Rank Brothers) at a constant temperature of 25ºC. Dry weights from leaf discs were determined after drying for 2 days at 60ºC. 3 SI Figures Figure S1. Localization of GFP-tagged proteins. A, Subcellular localization of the indicated constructs in agroinfiltrated N. benthamiana leaf cells by confocal laser scanning microscopy. Pictures were taken 3 days after agroinfiltration (72 hpi). Bars, 20 µm. B, N. benthamiana leaf agroinfiltrated with constructs to express a plastid-targeted version of GFP (P-GFP) or the untargeted GFP protein (upper picture). At 72 hpi, GFP fluorescence was examined in agroinfiltrated tissue to confirm the localization of P-GFP in chloroplasts and GFP in the cytosol (middle pictures). Effective quantum yield of PSII was quantified at 96 hpi (lower plot). 4 Figure S2. Chromoplast differentiation triggered by crtB in N. benthamiana leaves resembles that associated to ripening in tomato fruit. Heatmap represents GO term enrichment values (Z-scores) calculated from log2FC values in N. benthamiana leaves 4 days after agroinfiltration with (p)crtB compared to GFP (lane NbB). Data publicly available from tomato fruit (light ripe vs. mature green, SlO, and red ripe vs. mature green, SlR) and Arabidopsis leaves (senescent -30D- vs. controls -16D-, AtS) are also shown. Only GO terms with a significant enrichment in the N. benthamiana experiment are shown. 5 Figure S3. Level of transcripts for carotenoid-related proteins. Heatmap represents log2FC values in N. benthamiana leaves 4 days after agroinfiltration with (p)crtB compared to GFP (lane NbB). Data publicly available from tomato fruit (light ripe vs. mature green, SlO, and red ripe vs. mature green, SlR) and Arabidopsis leaves (senescent -30D- vs. controls -16D-, AtS) are also shown. The position of enzymes in pathways and/or the biological function of the proteins selected are indicated in the cartoon and represented by colors. Stripped cells mark missing isogenes and their colors correspond to the closest homolog in the genome. Asterisks mark differentially expressed genes in the NbB lane (DESeq2, FDR ≤ 0.05). 6 Figure S4. Chromoplast differentiation induces glycolytic and oxidative energy metabolism. Heatmap represents statistically significant FC (fold-change) values of metabolite levels in N. benthamiana leaves 4 days after agroinfiltration with (p)crtB relative to those in GFP controls. Inset represents respiration rates. Mean and standard deviation values of n = 3 independent samples are shown. Asterisk represents statistical significance (t test, P < 0.05). 7 Figure S5. Chromoplast differentiation in leaves reduced NPQ and impairs the xanthophyll cycle. A, Non-photochemical quenching (NPQ) components in N. benthamiana leaf sections agroinfiltrated with (p)crtB. B, Leaves producing GFP or (p)crtB were treated as shown in the left panel and then collected to quantify their carotenoid levels. De-epoxidation state (DES) was calculated as (Zx+0.5×Ax)/(Zx+Ax+Vx), where Zx, Ax and Vx are the concentrations of zeaxanthin, antheraxanthin, and violaxanthin, respectively. 8 Figure S6. Plant PSY enzymes do not induce chromoplast differentiation in leaves. A, Phenotypes of N. benthamiana leaf sections producing the indicated proteins at 96 hpi. B, Phytoene content in the leaf sections shown in A. 9 SI Tables Table S1. Relative metabolite levels in N. benthamiana leaf sections infiltrated with GFP and (p)crtB constructs. Data is presented as means ± SE of five to six biological replicates normalized to the mean level of the control GFP samples. Metabolite GFP crtB Metabolite GFP crtB Pyruvate 1±0.09 1.05±0.04 2-oxo-Glutarate 1±0.13 1.42±0.11 Valine 1±0.08 1.33±0.03 Fucose 1±0.08 1.19±0.06 Glycerol 1±0.10 0.8±0.12 Phenyalanine 1±0.10 1.03±0.08 Isoleucine 1±0.11 1.38±0.1 Asparagine 1±0.16 1.43±0.18 Glycine 1±0.34 0.88±0.2 3-deoxy-Glucosone 1±0.08 0.87±0.04 Phosporic acid 1±0.08 1.2±0.08 Glycerol-3-P 1±0.10 0.89±0.08 Proline 1±0.18 1.04±0.26 Lysine 1±0.15 0.99±0.05 GABA 1±0.16 0.68±0.1 Dehydroascorbate 1±0.11 0.7±0.08 Alanine 1±0.15 1.41±0.49 Tyramine 1±0.08 1.02±0.05 Serine 1±0.14 1.01±0.11 Ascorbate 1±0.02 0.77±0.07 Succinate 1±0.10 1.1±0.11 myo-Inositol 1±0.07 0.83±0.1 Threonine 1±0.06 1.24±0.11 Tyrosine 1±0.13 1.53±0.15 Fumarate 1±0.11 0.85±0.07 trans-Caffeate 1±0.32 1.44±0.06 Maleate 1±0.12 1.28±0.12 Glucose-6-P 1±0.06 1.1±0.04 Nicotinate 1±0.07 1.08±0.1 Tryptophan 1±0.28 2.1±0.51 beta-alanine 1±0.07 1.22±0.08 Maltose 1±0.10 0.92±0.09 Erythritol 1±0.02 1.24±0.03 Trehalose 1±0.09 0.95±0.07 2-methyl-Malate 1±0.04 1.09±0.04 Glycerate 1±0.16 0.72±0.11 Aspartate 1±0.03 1.35±0.02 Malate 1±0.09 1.33±0.1 Methionine 1±0.14 1.03±0.12 Threonate 1±0.11 1.24±0.09 Ornithine 1±0.18 0.91±0.06 Sucrose 1±0.11 1.14±0.06 Glutamine 1±0.21 1.66±0.7 Quinic acid, 3-caffeoyl-, cis- 1±0.07 1.07±0.07 Xylose 1±0.09 1.13±0.04 Quinic acid, 3-caffeoyl-, trans- 1±0.08 1.02±0.06 Glutamate 1±0.15 1.55±0.17 Fructose 1±0.10 0.56±0.04 Rhamnose 1±0.07 1.17±0.05 Glucose 1±0.06 0.74±0.07 Putrescine 1±0.11 1.81±0.12 Citrate 1±0.09 2.28±0.31 10 Table S2. Primers used in this work. Name Sequence PacrtB_N-tag_attB1-F 5´-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGAATAATCCGTCGTTACTCAATC-3´ PacrtB_C-tag_attB2-R 5´-GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGAGCGGGCGCTGCCAGAGATG-3´ PacrtB_STOP_attB2-R 5´-GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGAGCGGGCGCTGCCAGAGATG-3´ AtHDS_N-tag_attB1-F 5´-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGACTGGAGTATTGCCAGCTC-3´ crtB-5’-AtHDS-3’-R 5´-GATTGAGTAACGACGGATTATTCATATTCCGGATAACCGAAACTCTTCTC-3´ AtPSY_N-tag_attB1-F 5´-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCAAGCTTAGTAGCAAGTCCTTC-3´ AtPSY_STOP_attB2-R 5´-GGGGACCACTTTGTACAAGAAAGCTGGGTTCATATCGATAGTCTTGAACTTGAAG-3´ SlPSY2_N-tag_attB1-F 5´-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTCTGTTGCTTTGTTGTGGGTTG-3´ SlPSY2_STOP_attB2-R 5´-GGGGACCACTTTGTACAAGAAAGCTGGGTTCATGTCTTTGCTAGTGGGGAAGAAG-3´ PacrtE_N-tag_attB1-F 5´-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGACGGTCTGCGCAAAAAAACAC-3´ PacrtE_STOP_attB2-R 5´-GGGGACCACTTTGTACAAGAAAGCTGGGTTTATACTGACGGCAGCGAGTTTTTTGTC-3´ TuMV-crtB-flank-5' 5'-GCAGGAGAA-3' TuMV-crtB-flank-3' 5'-GCTGAAGCTTGCGTTTACCACCAA-3' LMV-crtB-flank-5' 5'-GTGGACACA-3' LMV-crtB-flank-3' 5'-GACATGGACGAAGTTTATCACCAA-3' crtB_qPCR-F 5'-TATTGCTCGCGATATTGTGG-3' crtB_qPCR-R 5'-TTTCAGGTGCCGCATAATTC-3' ACT_qPCR-F 5'-TAAGGTTGTTGCACCACCAG-3' ACT_qPCR-R 5'-ACATCTGCTGGAATGTGCTG-3' 11 SI References 1. 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Bioscience, biotechnology, and biochemistry 71, 2095-2100 (2007). 6. A. Bombarely et al., A draft genome sequence of Nicotiana benthamiana to enhancemolecular plant-microbe biology research. Mol. Plant Microbe Interact. 25,1523- 1530 (2012). 7. J. Lisec, N. Schauer, J. Kopka, L. Willmitzer, A. R. Fernie, Gas chromatography mass spectrometry-based metabolite profiling in plants. Nature protocols 1, 387-396 (2006). 8. A. R. Fernie et al., Recommendations for reporting metabolite data. The Plant cell 23, 2477- 2482 (2011). 9. J. E. Coate, A. F. Powell, T. G. Owens, J. J. Doyle, Transgressive physiological and transcriptomic responses to light stress in allopolyploid Glycine dolichocarpa (Leguminosae). Heredity 110, 160-170 (2013). 12 SI Datasets Dataset S1 (separate file). Genes UP-REGULATED and DOWN-REGULATED in (p)crtB vs GFP agroinfiltrated leaves of N. benthamiana collected at 96 hpi (DESeq2, log2FC ≥ 0.585, FDR ≤ 0.05). Dataset S2 (separate file). Parametric Analysis of Gene Set Enrichment (PAGE) by AgriGO. p value ≤ 0.05 ; q value ≤ 0.05 Dataset S3 (separate file). Expression values (log2FC) of carotenoid-related genes. Dataset S4 (separate file). Metabolite Reporting (Checklist and Overview) 13