1
(a) ATGAAGAAGCGCTTAACCACTTCCACTTGTTCTTCTTCTCCATCTTCCTCTGTTTCTTCTTCTACTACTACTTCCTCTCCTATTCAGTCGGAGG CTCCAAGGCCTAAACGAGCCAAAAGGGCTAAGAAATCTTCTCCTTCTGGTGATAAATCTCATAACCCGACAAGCCCTGCTTCTACCCGACGCAG CTCTATCTACAGAGGAGTCACTAG(g/a;wri1-1)tttttatttttttggaaattaaatgattggttgttgagattggatttgggttttgtct taaaactgcatttgtaagattgcatgttgttttgtgggattttgcagacatagatggactgggagattcgaggctcatctttgggacaaaagct CTTGGAATTCGATTCAGAACAAGAAAGGCAAACAAGgtttcgtcttcttctttttttcttcgtaactcatgattttgtttgttttaataaagat ctggactttaactgataaatttggtttctttgatctgttgtttgatctcaacttcgtcacaacttcaccagtttatctgggtaagctctaattc tctgaaacaaaagatcaattgttttttactaaattgaaagagaaaaaaaaagagagagtggatttggtcagaagaatctcaactgctttcacgc gtaattgcaggagcatatgacagtgaagaagcagcagcacatacgtacgatctggctgctctcaagtactggggacccgacaccatcttgaatt TTCCGgtaaaccaaaaaacaaaaatcagattgttttgatatgcatgtttgtgattttggaatctggattataattaaaaaaaaaaatggggaaa atcaggcagagacgtacacaaaggaattggaagaaatgcagagagtgacaaaggaagaatatttggcttctctccgccgccagagcagtggttt CTCCAGAGGCGTCTCTAAATATCGCGGCGTCGCTAGg( wri1-3)ttcctttttttctctctttctttttttaatttctttagatttattttt taaatttcggaatttactaccaaattgagaaatgatttttcctattttcggatatctgaataacagaattaattattaggaaaaaatctgatca tgaaaattttgcttttagaatattctctttttcttaaaaaaaatcataaattatgtttttttcagcactgctaaagtttatggattcaatagtt tggtcattttattcttaaaaataggattatttt( wri1-5)tgttcataaaaaca(g/a;wri1-6)gcatcaccacaacggaagatgggagg CTCGGATCGGAAGAGTGTTTGGGAACAAGTACTTGTACCTCGGCACCTATAgtacgttatctcctttcccttttttcttctagtaatttttaga aaaaaatagatatgtactcttggttaatttaaaataattagcgtaattattgacttttttataacttaccgggcataacggatcctttttacct gttatgctttataatatataattttgtaagtataaaatagagtgtgataatgtttagactgttttttgttgttgtttataagagtgatttaaga atattaatttgtttagtgagacaattagaataatataatggggaagcagtggcagtggggtttgaattttacacacactgactcacgtgaggcg agagttttgacatcatgtccctttaattgatttttatcttttaatcaaatcaactttttttcttcttcttttttaattatctgatccctctgca taattaccttttaaattctgcattttttgtggatccgatactctgaatacaaaaattgagaactctgcagaagggaatattaacaacaactctt ttactgaaaagtaatcccatttttttttattgtttttgctgactcttaccgggtccttagatttatttaaggacctcctaatcttcaacaaatc tcaaatttttgaaaattagatttttttaaaaaagtaatacaaa(wri1-4 )ttgagatttgcaaaaatataggcattgttgttctataacaaa gatactttatttataccaaaaaaaagaaaagagttgtcaagagcataattacaaaaaaaaaaattgaaataagtagtagttgtgaaattttgta tagaaaaataatgtaggttacaagtgtaaaggcgcgtgtagcgcgcgtaggtcacgtgataacactctcacttcataaaaggacaaaatagttc agagaggctttaggaccaaacccgaggtcgatctggtttgctcttgtttttttggtttattaaattggattaattagttagagttgagacttgc tctgagtaggagtcacgagccctcacgtgcactgctcatctctctctatctctctaccatatctttcatctttgtcc(wri1-10)tccgaaca aaatctggtctaactttatcttcttttcttttaataattgtcttctctactttaccattatttttctctagattattctggtaccaaacttata acttaatagttgattagtgcttagagttgacactaggttggtgttttaattattgttaattaactcagtaatcgtacgttcgtgtattaattat atacacaaattgttgcgatgacttaaattaagtcacaagtttggactctttagtgtttagagcggcgcagtggaggagaaatggtctttgtaca cgcctcacatctccacacaaatcgtgtaaccctagttgtccctacaaaaacacgtcaccaaattctattgattctttgtctttattaggtatca taaattctctaatttaaatatgaaacgacaaagaagaaactcttctttttaacacttgtttggtcttatttggttatagccacttaccaggtaa tgtgaaagttaacaacaagttgcgttaactttcaaaacactgactttgtttgcattgtcttgatttagaagctttttagctaacacagttgtct atttattggttttacagatacgcaggaggaagctgctgcagcatatgacatggctgcgattgagtatcgaggcgcaaacgcggttactaatttc GACATTAGTAATTACATTGACCGGTTAAAGAAGAAAGGTGTTTTCCCGTTCCCTGTGAACCAAGCTAACCATCAAGAGGGTATTCTTGTTGAAG CCAAACAAGAAGTTGAAACGAGAGAAGCGAAGGAAGAGCCTAGAGAAGAAGTGAAACAACAGTACGTGGAAGAACCACCGCAAGAAGAAGAAGA GAAGGAAGAAGAGAAAGCAGAGCAACAAGAAGCAGAGATTGTAGGATATTCAGAAGAAGCAGCAGTGGTCAATTGCTGCATAGACTCTTCAACC ATAATGGAAATGGATCGTTGTGGGGACAACAATGAGCTGGCTTGGAACTTCTGTATGATGGATACAGGGTTTTCTCCGTTTTTGACTGATCAGA ATCTCGCGAATGAGAATCCCATAGAGTATCCGGAGCTATTCAATGAGTTAGCATTTGAGGACAACATCGACTTCATGTTCGATGATGGGAAGCA CGAGTGCTTGAACTTGGAAAATCTGGATTGTTGCGTGGTGGGAAGAGAGAGCCCACCCTCTTCTTCTTCACCATTGTCTTGCTTATCTACTGAC TCTGCTTCATCAACAACAACAACAACAACCTCGGTTTCTTGTAACTATTTGgtctgagagagagagctttgccttctagtttgaatttctattt cttccgcttcttcttcttttttttcttttgttgggttctgcttagggtttgtatttcagtttcagggcttgttcgttggttctgaataa (b) Allele Background Mutation type Position Reference wri1-1 Col-2 EMS (G-to-A) 20,115,021 Cernac & Benning (2004) wri1-2 Col-2 EMS (?) - Focks & Benning (1998) wri1-3 Col-0 T-DNA (SALK_085693) 20,115,774 Baud et al. (2007) wri1-4 Col-0 T-DNA (SALK_008559) 20,116,785 Baud et al. (2007) wri1-5 Ws-0 T-DNA (FLAG_158E08) 20,116,043 Baud et al. (2007) wri1-6 Col-0 EMS (G-to-A) 20,116,058 this study wri1-10 Col-0 T-DNA (GK109D06) 20,117,186 Maeo et al. (2009) 2
3
4
5
6
2.12 2.14 0.87 0.86 0.86 1.25 1.57 6.38 5.11 4.20 2.70 2.68 2.67 2.66 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm) 1.98 1.77 1.61 1.69 1.39 1.30 1.10 0.85 19.63 146.16 145.72 130.96 125.08 125.79 121.55 118.30 112.09 75.45 39.98 37.44 37.39 37.29 37.08 32.66 32.43 31.35 25.73 25.57 24.38 24.08 21.00 17.63 11.90 11.87 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 Chemical Shift (ppm) 7
32.67 125.08 112.09 77.21 32.43 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 Chemical Shift (ppm) 125.08 112.09 77.21 32.43 32.67 25.74 24.08 19.64 17.64 11.91 11.87 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 Chemical Shift (ppm) 37.44 39.97 37.39 37.08 31.34 25.57 24.38 22.30 21.00 8
8 16 24 32 40 48 56 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shift (ppm) 64 72 80 88 96 104 112 120 128 136 144 F1 Chemical Shift (ppm) 9
m/z--> Abundance 388 1200000 1000000 800000 151 600000 400000 200000 191 193 430 55 69 81 95 107 122 135 164 177 233 344 m/z--> 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 Abundance 388 250000 200000 150000 151 100000 50000 191 193 55 430 69 81 95 107 121 135 177 207 233 344 360 m/z--> 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 10
11
Abundance 21000 α-tocomonoenol mass spectrum (TMS ether) - rapeseed oil 500 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 237 8000 7000 73 6000 5000 4000 3000 2000 436 55 85 221 1000 277 193 113 133 148 163 205 96 178 249 262 295 326 341 374 401 416 0 m/z--> 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 Abundance γ-tocomonoenol mass spectrum (TMS ether) - rapeseed oil 486 65000 60000 55000 50000 45000 40000 35000 223 30000 25000 20000 73 15000 10000 5000 263 55 179 194 209 83 97 109 124 135 151 163 0 233 248 284 302 313 332 346 360 374 402 416 430 447 471 m/z--> 40 50 60 70 80 90 100110120130140150160170180190200210220230240250260270280290300310320330340350360370380390400410420430440450460470480490500510520 12
Abundance 42000 γ-tocomonoenol mass spectrum (TMS ether) - linseed oil 486 40000 38000 36000 34000 32000 30000 28000 26000 24000 22000 223 20000 18000 16000 14000 73 2000 209 85 179 95 105 193 236 248 416 115 124 135 152 163 282 311 332 346 360 381 402 430 446 471 0 m/z--> 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 12000 10000 8000 6000 4000 55 263 Abundance α-tocomonoenol mass spectrum (TMS ether) - sunflower oil 500 190000 180000 170000 160000 150000 140000 130000 120000 110000 237 100000 90000 80000 70000 73 60000 50000 40000 30000 20000 55 277 10000 223 83 95 193 208 109 119 133 149 165 177 247 262 291 320329 339 360 374 388 416 430 444 471 485 0 m/z--> 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 13
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15
The Arabidopsis EMS-mutagenized plants screened for seed tocochromanol were originally produced to perform a screen on plant defences and carry a T-DNA with the promat1g51850:dao1 construct in the Col-0 accession. The cauliflower 35S terminator sequence from paeq-hyg was cloned into the XbaI/SacI sites of pgreen0229 (http://www.pgreen.ac.uk/jit/jit_fr.htm), yielding plasmid pgreen-t35. The dao1 sequence (Erikson et al., 2004) was amplified from plasmid prlm208qcz (BASF Plant Science, Limburgerhof, Germany) using the primers DaoF-EcoRI-F (5 - AATTGAATTCATGCACTCGCAGAAGCGCGTC-3 ) and DaoR-Xbal-F (5 - AATTTCTAGACTACAACTTCGACTCCCGCGCC-3 ) introducing restriction sites for EcoRI and XbaI. The PCR-fragment was cloned into the EcoRI/XbaI sites of pgreen-t35 to create the vector pgreen-dao1:t35. Subsequently, 853 bp of the promoter region of the PAMPresponsive gene At1g51850 were amplified using primers At1g51850promF-Clal (5 - AATTATCGATATGTGATTTTATGGGAAAGCAATCTTGTT-3 ) and At1g51850promR-E5 (5 -AATTGATATCTGTTCTCCTTACTGTCCACAGGAGAGC-3 ) and cloned into the ClaI and HindIII sites of pgreen-dao1:t35, fusing the promoter to the dao1 coding sequence. The construct together with helper plasmid psoup were transformed into Agrobacterium tumefaciens GV3101 pmp90 via electroporation. Arabidopsis thaliana Columbia-0 plants were transformed using the floral dip method described and transformed seedlings were selected after BASTA selection. Homozygous line 9.3.3 containing a single copy of the dao1-cassette was selected based on segregation on BASTA-containing half-strength MS medium and southern blot analysis. For Southern blot analysis, genomic DNA was digested with EcoRV and probed with the dao1-fragment obtained via PCR from prlm208qcz using primers DaoF-EcoRI-F and DaoR-Xbal-F. 16
Almeida J, Azevedo MdS, Spicher L, Glauser G, vom Dorp K, Guyer L, Carranza AdV, Asis R, de Souza AP, Buckeridge M et al. 2016. Down-regulation of tomato PHYTOL KINASE strongly impairs tocopherol biosynthesis and affects prenyllipid metabolism in an organ-specific manner. Journal of Experimental Botany 67:919-934. Dwiyanti MS, Yamada T, Sato M, Abe J, Kitamura K. 2011. Genetic variation of tocopherol methyltransferase gene contributes to elevated -tocopherol content in soybean seeds. BMC Plant Biology 11:152-168. Fritsche S, Wang X, Li J, Stich B, Kopisch-Obuch FJ, Endrigkeit J, Leckband G, Dreyer F, Friedt W, Meng J et al. 2012. A candidate gene-based association study of tocopherol content and composition in rapeseed (Brassica napus). Frontiers in Plant Science 3:129. Gilliland LU, Magallanes-Lundback M, Hemming C, Supplee A, Koornneef M, Bentsink L, DellaPenna D. 2006. Genetic basis for natural variation in seed vitamin E levels in Arabidopsis thaliana. Proceedings of the National Academy of Sciences U.S.A. 103:18834-18841. Hass CG, Tang S, Leonard S, Traber MG, Miller JF, Knapp SJ. 2006. Three nonallelic epistatically interacting methyltransferase mutations produce novel tocopherol (vitamin E) profiles in sunflower. Theoretical and Applied Genetics 113:767-782. Li Q, Yang X, Xu S, Cai Y, Zhang D, Han Y, Li L, Zhang Z, Gao S, Li J et al. 2012. Genome-wide association studies identified three independent polymorphisms associated with -tocopherol content in maize kernels. PLoS ONE 7:e36807. Lipka AE, Gore MA, Magallanes-Lundback M, Mesberg A, Lin H, Tiede T, Chen C, Buell CR, Buckler ES, Rocheford T et al. 2013. Genome-wide association study and pathway-level analysis of tocochromanol levels in maize grain. G3 Genes Genomes Genetics 3:1287-1299. Marwede V, Gül MK, Becker HC, Ecke W. 2005. Mapping of QTL controlling tocopherol content in winter oilseed rape. Plant Breeding 124:20-26. 17