From Golden Rice to astarice: bioengineering astaxanthin biosynthesis in

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1 From Golden Rice to astarice: bioengineering astaxanthin biosynthesis in rice endosperm Supplemental Information Table of Contents Supplemental item Page Supplemental Figure 1. The expanded carotenoid biosynthetic pathways in 2 transgenic plants. Supplemental Figure 2. Heatmap of the expression of the carotenogenic genes and 3 diacylglycerol acyltransferase genes different rice tissues. Supplemental Figure 3. Assembly of the four target genes in the TGSII system for 4 engineering astaxanthin biosynthesis in rice endosperm. Supplemental Figure 4. Assembly of the two or three target genes for engineering 5 β-carotene or ketocarotene biosynthesis in rice endosperm, respectively. Supplemental Figure 5. Grain phenotypes of Golden Rice (GR), Canthaxanthin Rice 6 (CR), and Astaxanthin Rice (AR, or astarice) lines enriched with β-carotene, canthaxanthin, and astaxanthin, respectively, in the endosperm. Supplemental Figure 6. Detection of the transgenes by PCR and the grain colors in 7 the T 1 GR, CR, and AR lines. Supplemental Figure 7. The presence and stable inheritance of the transgenes 8 detected by PCR in the transgenic plants of the T 1 and T 3 generations. Supplemental Figure 8. An example of detection of selection marker-free AR plants. 9 Supplemental Figure 9. Comparison of plant growth and agronomic traits between wild-type HG1 and the T 4 transgenic lines. Supplemental Figure 10. Summary of the contents of astaxanthin and total carotenoids in previously reported transgenic plants and this study for astaxanthin biosynthesis Supplemental Table 1. Carotenoid and astaxanthin biosynthesis-related genes used or analyzed in this study. Supplemental Table 2. Primers used for PCR analysis of genomic DNA (gdna) and expression analysis by qrt-pcr Supplemental references. 14 1

2 Supplemental Figure 1. The expanded carotenoid biosynthetic pathways in putatively engineered plants. The initial precursors of carotenoids are generated via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. It is predicted that in rice endosperm, β-carotene produced by the transgene-encoded enzymes (in blue) phytoene synthase (PSY) and bacterial phytoene desaturase (CrtI) can be catalyzed by additional two transgene-encoded enzymes (in blue) β-carotene hydroxylase (BHY) and β-carotene ketolase (BKT) to generate the target end-product astaxanthin. The other enzymes (in black) are encoded by endogenous carotenogenic genes in plant species. DXS, 1-deoxy-D-xylulose 5-phosphate synthase (DXP); DXR, DXP reductoisomerase; GGPPS, geranylgeranyl diphosphate synthase; PDS, phytoene desaturase; ZISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerase; LCYE, lycopene ε-cyclase; LCYB, lycopene β-cyclase; EHY, ε-carotene hydroxylase; VDE, violaxanthin de-epoxidase; ZEP, zeaxanthin epoxidase. 2

3 Supplemental Figure 2. Heatmap of the expression of the carotenogenic genes and diacylglycerol acyltransferase genes in different rice tissues. The expression data were prepared by the web tool RiceXPro ( using the Agilent 44K microarray of the japonica rice variety Nipponbare. The rice carotenogenic genes are expressed mainly in leaf blade and leaf sheath, probably due to that carotenoids are accessory pigments in photosynthesis. The carotenogenic genes and those genes encoding diacylglycerol acyltransferases for catalyzing hydroxylated esterification of carotenoids with fatty acid were expressed at relatively higher levels in non-endosperm tissues, but were not expressed or showed low expression in endosperm (blue box). 3

4 Supplemental Figure 3. Assembly of the four target genes in the TGSII system for engineering astaxanthin biosynthesis in rice endosperm. (A) The structures of donor constructs in the vectors pyl322d1 (d1) and pyl322d2 (d2) with different target genes and endosperm-specific promoter cassettes. N, Not I site. (B) Not I-restriction analysis of the donor constructs. Arrows indicate the target gene fragment. (C) Not I-restriction analysis of the intermediate constructs and final acceptor construct (pyltac380mf-bhbkpc, see Figure 1A) from different rounds of gene assembly with increasing target genes and the selectable marker/marker excision cassette in the binary acceptor vector (pyltac380gw or 380GW). Arrows indicate the newly stacked gene(s) in each gene assembly cycle. 4

5 Supplemental Figure 4. Assembly of the two or three target genes for engineering β-carotene or ketocarotene biosynthesis in rice endosperm, respectively. (A) The structure of the pyltac380mf-pc construct and Not I-restriction analysis of the intermediate and final binary constructs from different rounds of gene assembly. (B) The structure of the pyltac380mf-bkpc construct and Not I-restriction analysis of the intermediate and final binary constructs from different rounds of gene assembly. 5

6 Supplemental Figure 5. Grain phenotypes of Golden Rice (GR), Canthaxanthin Rice (CR), and Astaxanthin Rice (AR, or astarice) lines enriched with β-carotene, canthaxanthin, and astaxanthin, respectively, in the endosperm. (A) Accumulation of astaxanthin in developing grains on 7, 14, or 21 days after pollination (DAP) of the wild-type indica line HG1 and the transgenic AR-H8 line (T 2 ). (B) Phenotypes of dried seeds and dried brown (unpolished) and polished grains of the parent (wild-type HG1) and the transgenic GR, CR, and AR lines (T 4 ). 6

7 Supplemental Figure 6. Detection of the transgenes by PCR and the grain colors in the independent T 1 lines. (A) Golden Rice line GR-H1~H6; (B) Canthaxanthin Rice lines CR-H1~H5; (C) Astaxanthin Rice AR-H1~H13. The letter H used in the lines is derived from the receptor parent HG1. + indicates the positive (present) results for PCR detection of the genes. The primers used are given in Supplemental Table 2. 7

8 Supplemental Figure 7. The presence and stable inheritance of the transgenes detected by PCR in the transgenic plants of different generations (T 1 and T 3 ). (A, B) the GR lines; (C, D) the CR lines; (E, F) the AR lines. 8

9 Supplemental Figure 8. An example of detection of selection marker-free AR plants. (A) The locations of the three primers and the sizes of the amplification products in the T-DNA of pyltac380mf-bhbkpc used for the PCR assay of selection marker-free transgenic plants. In the T 1 heterozygotes for the Cre/loxP-mediated marker-gene excision, the two fragments (0.55 kb and 0.4 kb) could be amplified with the three primers (the 5.5-kb fragment is too large to be amplified with a 30-s extension). loxp1r is the mutant loxp site for gene assembly (5), which is omitted in Fig. 1A for simplification. (B) PCR analysis of the T 2 AR-H8 plants derived from a marker-excised T 1 heterozygote using the three primers. Two plants (indicated with red arrows) with only the 0.4-kb band were homozygous for the marker-excision. (C) The marker-free AR T 2 plants were confirmed by sequencing of the 0.4-kb PCR fragment. 9

10 Supplemental Figure 9. Comparison of plant growth and agronomic traits between wild-type HG1 and the T 4 lines. (A) whole plants; (B) plant height; (C) seed-setting rate; (D) tiller number; (E) spike length; (F) spike branch number; (G) grain length; (H) grain width; (I) grain thickness; (J) 1000-grain weight; (K) seed germination rate. Thirty plants for each line were investigated, and no significant differences were detected in all traits. 10

11 Supplemental Figure 10. Summary of the contents of astaxanthin and total carotenoids in previously reported transgenic plants and this study for astaxanthin biosynthesis. Asterisks indicate fresh weight. Idi, bacterial IPP isomerase gene; CrtB, bacterial phytoene synthase gene; CrtE, bacterial GGPP synthase gene; CrtI, bacterial phytoene desaturase gene; CrtO, bacterial β-carotene ketolase gene; CrtW, bacterial β-carotene ketolase gene; CrtY, bacterial lycopene β-cyclase gene; CrtZ, bacterial β-carotene hydroxylase gene; OR, ORANGE gene; ZmLYCE, Zea may lycopene ε-cyclase gene; LYCB, lycopene β-cyclase gene; szmpsy1, synthetic Zea may phytoene synthase gene for rice codon optimization; spacrti, synthetic Pantoea ananatis phytoene desaturase gene for rice codon optimization; shpbhy, synthetic Haematococcus pluvialis β-carotene hydroxylase gene for rice codon optimization; scrbkt, synthetic Chlamydomonas reinhardtii β-carotene ketolase gene for rice codon optimization. 11

12 Supplemental Table 1. Carotenoid and astaxanthin biosynthesis-related genes used or analyzed in this study. Protein Species Gene Locus ID or Reference Accession No. Phytoene synthase Phytoene desaturase β-carotene hydroxylase β-carotene ketolase Maize (Zea mays) Pantoea ananatis Haematococcus pluvialis Chlamydomonas szmpsy1 spacrti shpbhy scrbkt MG MG MG MG This study This study This study In this study reinhardtii Phytoene synthase Phytoene desaturase ζ-carotene isomerase ζ-carotene desaturase Carotenoid isomerase Lycopene β-cyclase Lycopene ε-cyclas β-carotene hydroxylase ε-carotene hydroxylase Diacylglycerol acyltransferase 1 Diacylglycerol acyltransferase 2 Diacylglycerol acyltransferase 3 Diacylglycerol acyltransferase 4 Rice (Oryza sativa) OsPSY OsPDS OsZISO OsZDS OsCRTISO OsLCYB OsLCYE OsBHY OsEHY OsDGAT1 OsDGAT2 OsDGAT3 OsDGAT4 Os12g Os03g Os12g Os07g Os11g Os02g Os01g Os10g Os03g Os01g a Os09g a Os01g a Os01g a Schaub et al., 2005 Chen et al., 2010 Schaub et al., 2005 Schaub et al., 2005 Schaub et al., 2005 Du et al., 2010In Schaub et al., 2005 Schaub et al., 2005 Schaub et al., 2005 In this study In this study In this study In this study a These genes were identified based on sequence similarity with orthologous genes from Arabidopsis PES1 and PES2 (Lippold et al., 2012). 12

13 Supplemental Table 2. Primers used for PCR analysis of genomic DNA (gdna) and expression analysis by qrt-pcr. Primer Sequence (5-3 ) Purpose F- PSY381 R- PSY381 F- CrtI282 R-CrtI282 F- BHY322 R -BHY322 F- BKT261 R- BKT261 Fm-1 R1 Rmas F-PSY80 R-PSY80 F-CrtI189 R-CrtI189 F-BHY116 R-BHY116 F-BKT196 R-BKT196 F-rPSY176 R-rPSY176 F-rPDS112 R-rPDS112 F-rZISO124 R-rZISO124 F-rZDS137 R-rZDS137 F-rCRTISO158 R-rCRTISO158 F-rLCYB147 R-rLCYB147 F-rLCYE144 R-rLCYE144 F-rBHY130 R-rBHY130 GATGAGCTTGCACAGGCAGG CAATGAACATGGGAGCAGTAGC TCGCATACCTCGAGCAGCAC TCAGGTCCTCCAACATCAGG GGCTATGCTGCTCTGCACG CCTCTTACTCCAGTCCAACTC CTCCACTCGCTAACCAGCTG CGCGAGCTATCTGTCTGCAC AATTAATTCCTAGGCCACCATGTTG GTCTGGACCGATGGCTGTGTAG GAATTGAAGACATGCTCCTGTC GAGGGCGTATGTTGGTAAAGGGAAGAA ATGAACATGGGAGCAGTAGCGATTTT CCGCGATCAACTCAATGCTTACCA CCCGATTGTGAGGACGAAACCAG GGCCCATCAATTGCACCACAGT GAACCAAGCGCTCCACCTCCTCT TCTTCATGACTGCCGCGCCGATACTCT TAAGGCCACCGGTGATGTTCCCAATGA ACAGTACCGGTGATGGGGAT AGACCTGCCTCTGCCAATTC ATCCCGGACTGTGAACCTTG GAACTGCACCCTCCATCGAA GGTGCTTAGCCCACACGTTA TCACCATAGCGTGATGCCAG CATGCCATTGCCGAATGAGG CTGGTGACTCGCGGTACAAT TCAAGGAGGTAGGCTCACCA AAAACAGCTGTCGCCAACAC TCCTGTCGTCGAGGCTCTTC CTCTGTCCTGGATGAGGTTG GTGGCGAGGATTCCTTGGTT TTGATCATCGTTGAGCCGGT GGGATTACGCTGTTCGGGAT TGTGATGTATCTGGTGGGCG PCR detection of gdna for szmpsy1 PCR detection of gdna for spacrti PCR detection of gdna for shpbhy PCR detection of gdna for scrbkt PCR detection of marker-free with Fm-1/R1 for assembly and Fm-1/Rmas for deletion szmpsy1 spacrti shpbhy scrbkt OsPSY OsPDS OsZISO OsZDS OsCRTISO OsLCYB OsLCYE OsBHY 13

14 F- rehy143 R -rehy143 F-OsActin1 R-OsActin1 CTCAACGACGTCTTCGCCAT ACGAACATGTACGCCATCCC CAACACCCCTGCTATGTACG ATCACCAGAGTCCAACACAA OsEHY OsActin1 gene as a reference for qrt-pcr Supplemental references: Bai, C., Berman, J., Farre, G., Capell, T., Sandmann, G., Christou, P. and Zhu, C. (2017). Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity. Transgenic Res. 26: Campbell, R., Morris, W.L., Mortimer, C.L., Misawa, N., Ducreux, L.J., Morris, J.A., Hedley, P.E., Fraser, P.D. and Taylor, M.A. (2015). Optimising ketocarotenoid production in potato tubers: effect of genetic background, transgene combinations and environment. Plant Sci. 234: Chen, Y., Li, F. and Wurtzel, E.T. (2010). Isolation and characterization of the Z-ISO gene encoding a missing component of carotenoid biosynthesis in plants. Plant Physiol.153: Du, H. Wang, N., Cui, F., Li, X., Xiao, J. and Xiong, L. (2010). Characterization of the β-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice. Plant Physiol. 154: Farré, G., Perez-Fons, L., Decourcelle, M., Breitenbach, J., Hem, S., Zhu, C., Capell, T., Christou, P., Fraser, P.D. and Sandmann, G. (2016). Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid. Transgenic Res. 25: Fujisawa, M., Takita, E., Harada, H., Sakurai, N., Suzuki, H., Ohyama, K., Shibata, D. and Misawa, N. (2009). Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation. J. Exp. Bot. 60: Gerjets, T., and Sandmann, G. (2006). Ketocarotenoid formation in transgenic potato. J. Exp. Bot. 57: Harada, H., Maoka, T., Osawa, A., Hattan, J.I., Kanamoto, H., Shindo, K., Otomatsu, T. and Misawa, N. (2014). Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids. Transgenic Res. 23: Hasunuma, T., Miyazaw, S., Yoshimura, S., Shinzaki, Y., Tomizawa, K., Shindo, K., Choi, S.K., Misawa, N. and Miyake, C. (2008). Biosynthesis of astaxanthin in tobacco leaves by 14

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