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1. 682. phenylalanine ammonia lyase PAL chalcone synthase CHS Fig 2 Sparvoli 5 1994
3. Jaillon et al 2007 PCR
4. EST Fig 3 Berry development Berry ripening eooooooQ0 00 ei ae Anth
5. 3 Jord o et al 2001 De Freitas et al 2000 Mateus 5 2001 3 1 3 3 3 4NR mRNA C
6. ABA NAA Jeong et al 2004 4 ABA 3 6 mg g XH REK 1 9 mg g 1 0 mg g NAA 0 5 mg g NAA AA HI O 7 TR v0 8TH FLS4 NAA Fig I 6A B D E 25 Z7357 w0 Gf rFLS4 ABA
7. MEL 3 ANR LAR Downey 2004 lk z7 56 3
8. 1990 y Ri A A OH B OH HO o NO e g catechin R OH R H HO Ov ws e epicatechin Rye OH R 7H ig C R2 R epigallocatechin R R OH S 3WOH OH H 2 3 trans flavan 3 ols 2 3 cis flavan 3 ols OH Proanthocyanidins Condensed tannins E n OH e g Cyanidin R OH R H e g Quercetin R OH R H Peonidin R OCH R H Kaempferol R R H HO Ox R Delphinidin R R OH Myricetin R R OH p Petunidin R OCH R OH ni Malvidin R R OCH OH O OH Flavonols Anthocyanidins Fig 1 Flavonoids in wine R and R are H or OH
9. cR don mE A 5 2
10. Fig 5B C Fig I 6A D NAA ABA FLS4 Fig I 6B E FLS4 7753 mRNA Fig I 6C F FLS4 Fig IL 6E F FLS5 FLS4 FLSS
11. C 3 3 C 2 3 Fig 1A B QRGS 2R 3R B 3 4 3 BB 3 4 5 3
12. p4M cDNA ANR DNA CF ANR v4NR ORR LM VvANR mRNA 29 Phenylalanine PAL C4H 4CL 4 coumaroyl CoA 3 x malonyl CoA CHS Chalcone CHI Flavanone F3H F3 H F3 5 H Dihydroflavonols DFR amp OH HO Ow Ro OH OH OH Leucoanthocyanidins LAR s OH HO Or ww R OH OH 2 3 trans flavan 3 ols catechin R OH R H gallocatechin R R OH Proanthocyanidins Condensed tannins LDOX Anthocyanins urer R d OH HO R2 OH OH Anthocyanidins ANR R OH HO O
13. flavonol synthase FLS FLS Holton et al 1993 Pelletier et al 1997 3 2003 anthocyanidin reductase ANR CO 2 3 3 Xie et al 2003 leucoanthocyanidin reductase LAR 2 3 3 Tanner et al 2003
14. 80C 2 2 3 ANR DE DNA VWANR PCR CH BAN cDNA VitiA76S VitiGen tt PCR S ACTTCAGATGTCCCC AGCAG 3 CCCTTGGCCTTGAAATACTC 37 PCR 94 30 b52 C30 b72 C90 900 bp pCR2 1 TOPO Invitrogen fk ABI PRISM 3100 Avant Genetic Analyzer Applied Biosystems Universal GenomeWalker Kit BD Biosciences Clontech DNA GENBTYX MAC
15. Price etal 1995 360 nm UV 3 53 mg l 200 mg l Ritchey and Waterhouse 1999
16. Accumulation and Biosynthetic Gene Expression of Flavonols and Flavan 3 ols in Grape Berries RH Akiko Okuda 2008 3 E MH HR HH HP HP HP HP HRNCHCRCHONOHRRHR HIR iii e NERO PREPARED PP 1 1 INE a M RE 8 box pu p condiaoxen docet deren dere deren dece deren dca deren dct ae 11 M c E Y 18 Ww EE cadena 24 M cR RENE 27 2 3 USE LEER 28 22 d pb d co astunsskusstunss ku etus Eu sepu Eu etu eut 31 LE iN EEE 34 DA NN NN 39 AGUA A ucc 41 3 42 3D od INE e quate rant dta std drunk Stet dota tete Le Sah a nate sh 45 p RES Re 48 3
17. VvLAR1 kkk ATTAAGATCGACATT CAAGCCAATCTCTATGATCTATCTGATGTT TGATAAAT CAT GAATGGTGAGTCAA AAGAACTGTAATCGGGCTTATCATGGTCGGTAGCATTTTTTGTAAATGAGTCGTTGAGGCACATGATTTT TTTGTTAGGACGTGTTATGTGAGATGAAATGAAGCTGCTGTGTAGAACTGAGGTTGATEEAAREUNA CCAATACGAATGCGACATGCATTTAAGCATGCGAATABGARRET AAAGCTGAGAAGCAAGACCTTGTGAT TTGATCATTTGAATAAACGAGAGTGAGAAGTGGTGGTAGAGGCGTTGGCTACTTTTCAAGCATGCGGCTT ACCAAACCGCTCGATCACATTTCCCCCAGAGTAGTAGGTGGTGCACACAAACAGGGAAACCCTACGCGTC AGTGACTCATGAGTCTATGACACAGGGCCCAGGGGGACCATCAGTGGGGTTAAGAAGAGACATTTTTTCC ATCGTTATATGGGCACCAAAACACTTGTACTGGAATATTAATAATTTCTAAATTTAGAATTTTTGGCAGA CACGGAACCATATTGGCCTAGATGGGAATTGGAAAGGGAGCGCCACGTTAGGAGTTTACGGTCATGAGCT ACCCTCCGATTGTCTCGGAGGAGTTTGTAGCCGGTGTCAGTGATCTTAGCTATCAGTCAAGGGTGGTAGC CAATGTGACCGTACTCAATGGACAGCGTTTGTTGAAGGGTAGCTGAAATGCCAGGCCCCAATCCCAGCAC TATATGTTCTCATCTCGATGTCAGCTTTAGTACCAAATCTCTTGGGAGACCATTTCATGATCCATAACTC CGTGTGCTGTGTACGATCGATAGAAAACAACATAAAGCAAAAAGAGTAGTAGAGAAGAAGCATCATCAGA VvLAR2 ATCTCCAATTTGATAGTCTTATAAAGCACATGGAGTATATGGGG GTTGGGATTCCATAATTGTCCTTAATTAAGTTGGGCAAGGGAACATTTCAATAAABGTARTIT TAATTTGG GGAAGAAGATACCTATTTATGGGCTCCATAATTTATTATTAATTAATTTATCAAATTAGAAATAATTAAT ATTACCATAGTTGACCTAAGAGAGGAAGTTTATAABGTABBAGCATAAATGTTTTTACAATTATTAAATA TGTAATTATGAATTTATCTTTTCGAGATGGACGACAACTGTACAAAAAGTTGAAAAGCATAAGTCTTTTT CCTTGGACTTGGAGACGAGBGAARET TA
18. Baranac et al 1997 2003 Jeong et al 2004 Kataoka et al Bergqvist 2001 Downey et al 2004 1 FH 4 coumaroyl CoA 3 x Malonyl CoA mRNA 38362
19. 59 Abe H Urao T Ito T Seki M Shinozaki K and Yamaguchi Shinozaki K 2003 Arabidopsis AtMYC2 bHLH and AtMYB2 MYB function as transcriptional activators in abscisic acid signaling Plant Cell 15 63 78 Baranac J M Petranovi N A and Dimitric IMarkovic J M 1997 Spectrophotometric study of anthocyan copigmentation reactions 2 Malvin and the nonglycosidized flavone quercetin J Agric Food Chem 45 1694 1697 Baudry A Heim M A Dubreucq B Caboche M Weisshaar B and Lepiniec L 2004 TT2 TT8 and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana Plant J 39 366 380 Bergqvist J Dokoozlian N and Ebisuda N 2001 Sunlight exposure and temperature effects on berry growth and composition of Cabernet Sauvignon and Grenache in the central San Joaquin Valley of California Am J Enol Vitic 52 1 7 Bogs J Downey M O Harvey J S Ashton A R Tanner G J and Robinson S P 2005 Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves Plant Physiol 139 652 663 Boss P K Davies C and Robinson S P 1996a Anthocyanin composition and anthocyanin pathwa
20. itis vinifera 3 3 5 34 sth 4 3 3 3 Cheynier and Rigaud 1986
21. H 40 mg YX EL hts EE 200 mg E UK BEBE eX o Fig A 1A B ZS D H ze TH R C C B HA REL HT 3
22. PCR ps A 8 a D e S i D 2 5 P d o S O EE m gt 5 E9399 2 2 seps 89 2 x og o CO e wo N eo Fe QN c oOCQN P Q Tr 3 o O0 TO CT 6 SS E 90g EC B Per skin surface area c 8 o o 6 S 4 o 5 2 2 O 0 Q9 2 2 oo 2 2 5 2 2 3 gt 3 l ITA geranrat eg Date C S 40 Per berry skin o 930 Fig I 5 Accumulation of FL mRNA and 20 quercetin in Cabernet Sauvignon A RT PCR in analysis of the mRNA of FLSs accumulated in the 5 leaves flower buds flowers and berry skins B o 0 ETUR TIME quercetin ug cm skin surface area C quercetin es 5S5 9 9 2 amp g i i 3355228 ug berry skin Vertical bars represent the standard g EANA tg deviation n 3 The standard deviation for some Date data is smaller than the thickness of the line 21 1 3 3 RT PCR HPLC
23. III 4C ANR LAR mRNA Fig III 4B C E F H D 3 WANR 4 GT 1 site Flint et al 3 1985 Z7 FE 0D li FIC RIE THES Oe 1 Jeong et al 2004 M FH 55
24. UDP 3 O UDP glucose flavonoid 3 O glucosyl transferase UFGT mRNA Boss et al 1996a 1996b Kobayashi et al 2001 UFGT Kobayashi 5 2002 Myb HO cDNA Z MH v VimybA EIE Kobayashi 5 2004 VIimybAl 1 VvmybA1 vinifera myb41 UFGT 3
25. 26 7 FLS 5 FLS7 FLS5 FLS mRNA i je ELE EK FLS2 FLS4 KU FLS5 mRNA FLS4 mRNA i FLS5 mRNA OAA HO FTIR V 03838 C FELD RSEO XEUX 7 235 7 vi amp or n E FLS4 mRNA FLS5 mRNA 2 2
26. Roche Diagnostics 558 PCR DIG Probe Synthesis Kit Roche Diagnostics FLS2 1 467 5 AGGCACAATGGCGGTAGAGA 3 JR 5 GGATGAAGCAGTGTGTAAAAGAAAAGG 3 DIG Easy Hyb Roche Diagnostics 42 2XSSC 20XSSC 3 M 0 3 M 0 1 SDS DY 5 2 0 1XSSC 0 1 SDS 65C C15 2 DIG CSPD Roche Diagnostics LAS 1000plus Ww
27. 3 3 3 3 40 120 mg l Ritchey and Waterhouse 1999 3 3 4 8 4 6 Fig IC
28. 4 25 50 0 5 1 5 g 1 10 50 mg l x a Hp 9 at at x T Robichaud and Noble 1990 FE
29. p 24 1 14 000 xg 5 3 0 9 g Sigma th C6288 0 55 30 45 5 000 x g 3 4 5 ml 35 STE 3 STE 0 1 M 0 1M pH 8 0 0 002 M EDTA 4 ml STE 2 5 000 x g 3 0 1 3 M
30. ANR PCR VvANR mRNA Lk mRNA VvANR 41 3 3 1 4 Dili 3
31. Kennedy et al lt RIET IE RRE f y epe 3 54 VyvA4NR VvLARI Fig III 4B E H ylLA4R2 Fig III 4C F D VvLAR2 4NR VvLARI mRNA VvANR VvLARI VvLAR2 mRNA 3 VvANR VvLARI Bogs et al 2005
32. Spayd 5 2002 lk AVA Fig I 1 flavonol synthase FLS Holton et al 1993 FLS 3 flavanone 3 hydroxylase FH _ leucoanthocyanidin dioxygenase LDOX 2
33. 8 Q 5 Per gram of seed weight 5 Per gram of skin weight T ie Per berry skin S S 51 5 20 g 10 E 1 0 g 2 10 es gs 8 8 2 0 8 0 S 0 0 25 Jun 10 Jul 24 Jul 18 Sep 25 Jun 10 Jul 24 Jul 18 Sep 8 25 Jun 10 Jul 24 Jul Date Date Date O FI Monomers E FII Oligomers W Fill Polymers Fig Il 6 Accumulation of flavan 3 ols in the berry skins and seeds of Cabernet Sauvignon 2003 Flavan 3 ol concentrations of the monomeric oligomeric and polymeric fractions were quantified by vanillin assay Results are expressed in catechin equivalents per gram of fresh seed or skin weight A B or per berry skin C Each bar represents the mean of triplicate analysis and error bars represent the standard deviation of the mean The data of 18 Sep is missing from C 38 2 4 ANR VvANR IRR LK WANR DIZ YY BOSE 3 VitiA765 O ANR cDNA 4NR VitiA765 ANR cDNA
34. 3 Fig III 3B E here Kennedy et al Mateus et al 2001 2000b Pourcel et al 2005 Downey 2004 2000a 2000b
35. Dral 4 i Fig 1 4 77S zzl FLS 4 77S5 FLS RT PCR FLS2 FLS4 RU FLSS mRNA FLS4 FLS5 mRNA OE L7 Fig I 5A FLS2 FLS3 FLS4 RU FLS5 mRNA AMET BeEICBW CE 5 FLS mRNA Downey 2003 Ik FLS4 VvFLS123 o C x O FLS4 nul
36. DNA 2 3 NAP Pharmacia ft TE 10 mM Ff 1 mM EDTA pH 8 0 DNA DNA 1 mM DTT TE DNA PCR FLS FLS FLS PCR Table I 1 TA pCR2 1 TOPO Invitrogen ABI PRISM 3100 Avant Genetic Analyzer Applied Biosystems
37. FLS M VS cU cepe eT 9 ESTE SE flavonoid 3 hydroxylase F3 H 3 5 flavonoid 3 5 hydroxylase F3 5 H mRNA F3 5 H 35H FYH Jeong 2006 ELS Suzuki etal 2000 FLS 2
38. Downey et al 2003 Cheynier and Rigaud 1986 Downey et al 2003 I Baranac et al 1997 Flint et al 1985 Price 1995
39. 3 Sun et al 1998b 3 Sigma Chemical 3 33 2 9 2 3 1 ANR ANR DFR ANR DER VvDFR GenBank accession no X75964 PCR PCR ANR VvANR AB199315 5 3 678 bp 264 bp VvVANR 5 Arabidopsis thaliana ANR AtANR A
40. 16 5 Brix 2 5 20 i 15 S gt 1 0 d 0 5 O Control 4 Shading 0 0 18 16 B 14 12 X 10 m g 6 4 2 2 oS aaa SE Fig III 2 Effect of shading on berry development Se PE Oo and ripening of Vitis vinifera Cabernet Sauvignon BE ARCU Demos Sr Berry weight A and total soluble solids expressed Date as Brix B are shown 3 32 3 3 Fig 1 3 wy EYE v bkWUe A 7258v 6 4 wct2 k0 4wBVUI WE E 4E Fig III 3A 6 H 21 H Fig III 3B D F H 48 3 8 16 3
41. Escribano Bail6n et al 1995 T KUSZ D D C Souquetetal 1996 3 60 80 3 10 30 Monagas etal 2003 3 OA GAR BIL 2003 Arabidopsis thaliana BANYULS BAN 2 3 3 leucoanthocyanidin reductase LAR Devic etal 1999 g4 O 2 3 3
42. Fig III 1 3 2 3 3 ANR 2 3 3 LAR Tanner et al 2003 Xie et al 2003 3 2 3 2 3 Xie and Dixon 2005 PA ANR 2 3
43. 3 2 1 4 Dili C4 C6 C4 C8 3 4 Ricardo da Silva et al 1992a 1992b 95 Monagas et al 2003 O Prieur et al 1994
44. 1 2 1 2 1 Milli Q Millipore DNA 1 2 2 tis vinifera Cabernet Sauvignon L Merlot 4 2002 1 80C 2000 ABA NAA 4
45. Fig III 4B E H 4NR VvLARI III AC F HAT VvANR mRNA Mo VvLAR2 2 74X2 mRNA ORE VvLARI 2 LAR mRNA Fig VvLARI RAH ANR LAR mRNA Fig III 4B C E F H I VvLAR2 mRNA 1 0 0 5 1 0 A B Skin C Seed 08 0 4 0 8 E 0 6 0 3 0 6 Coal 0 2 04 X Control gt 0 2 0 1 B Shading 02 0 0 0 0 0 0 0 8 0 4 D E Skin
46. Universal GenomeWalker Kit BD Biosciences Clontech DNA GENETYX MAC Table I 1 The first PCR primers for genomic sequencing of grape FLSs Gene name Sequence of forward F and reverse R primers Primer position FLSI F 5 TCAATTACCGGTTCTGGCC 428 to 446 R 5 GGCCATGACATTCTTGTCTT Compliments of 853 to 872 FLS2 F 5 ATCTGTTCCATAAGATTTGGCC 395 to 416 R 5 TCTGCTGGTGGCTCCAAGAA Compliments of 877 to 896 FLS3 F 5 TTAACTATCAGTTCTGGCC 428 to 446 R 5 ACTCTTGTACTTGCCATTGCTCA Compliments of 800 to 822 FLS4 F TAGCAGGGATTGGGGGATGTT 189 to 209 R 5 GGCGAGGACATGGTGGGTA ATA AT Compliments of 611 to 634 FLSS F S AATTATTACCCACCATGTCC 610 to 629 R S ACTCTTGTACTTGCCATTGCTCA Compliments of 800 to 822 Primer positions are corresponding to the base from the start codon of the citrus FLS cDNA GenBank accession no AB011796 The first nucleotide of the start codon was defined as position 1 1 2 4 1 2 3 DNA ug Dral 1 0
47. 3 Monagas 2003 HPLC V RU 3 6 25 7 24 3 mg g Fig II 6A B 3 Fig II 6C 2003
48. FLS ANR LAR 1 FLS FLS FLS 2 ANR ANR 3 3 ANR LAR c Gat 3 x Malonyl CoA Flavonols HO OW 2 3 trans flavan 3 ols OH OH HO OW ws k 2 3 cis flavan 3 ols E OH OH Phenylalanine Cinnamic acid
49. CE Pi A 7 Fig 4 2 3 Fig 1D Fig lE 1 3 C Fig 1A B B 4 3 80 90 4 10
50. 4 1 HPLC Fig I SB 7 25 Fig I 5C 8 7 Bs 1 3 4 22m 7 V0 SBE FLS4 00 55 00 NAA Eis A ORK ABA NAA
51. Table II 1 ANR DFR NADPH NADH VvANR ANR DFR 5 GXXGXXG A Xie et al 2004 Rossman NADPH NADH 34 Ww VvANR ANR A DFR G Table II 2 VvANR ANR ANR Table II 2 Amino acid sequence conservation between ANRs and DFRs at the 5 amino terminal regions including the Rossmann dinucleotide NADPH NADH binding domain GenBank protein id Species Enzyme Sequence NP 176365 1 At Col ANR 12 KACVIGGTGNLASILIKHLL AAN77735 1 MY ANR 13 KACVIGGTGFVASLLIKOLL BAD89742 1 Vv CS ANR 11 TACVVGGTGFVASLLVKLLL BAA85261 1 At Le DFR 7 TVCVTGASGFIGSWLVMRLL AAR27014 1 Mt DFRI 7 TVCVTGASGFIGSWLVMRLM AAR27015 1 MY DFR2 7 TVCVTGASGFIGSWLVMRLM CAAS3578 1 Vv LF DFR 7 TVCVTGASGFIGSWLVMRLL Consensus ae OK x NADPH NADH binding domain GXXGXXG A Arabidopsis thaliana ecotype Columbia Medicago truncatula Vitis vinifera Cabernet Sauv
52. VvFLSI 7757 VvFLS2 77S7 77S7 mRNA mRNA 24 Fig L5B C 5 0 FLS FFLS mRNA FLS mRNA Downey et al 2003
53. 9 18 H Fig II 5 ANR mRNA A B Seed Skin 2 0 15 x pe 1 0 E os 3 0 0 25 Jun 10 Jul 24 Jul 18 Sep 25 Jun 10 Jul 24 Jul 18 Sep Date Date Fig II 5 mRNA accumulation of the ANR gene VvANR in berry seeds A and skins B of Cabernet Sauvignon 2003 The mRNA level of VvANR was measured by real time quantitative PCR analysis Results are expressed as the molar ratio of the mRNA level of VvANR to that of the ubiquitin gene Each bar represents the mean of triplicate analysis and error bars represent the standard deviation of the mean 37 2 3 3 3 Fig II 6 3
54. Fig III 3F 1 EX 3 VvANR VvLARI KU VvLAR2 mRNA PCR Fig III 4 Fig III 4A VvLARI mRNA V XE mRNA Fig III 4G Fig III B C VvANR mRNA Fig III 4D VvANR mRNA VvLAR2 VvLARI VvANR Fig III 4E F VvLARI mRNA VvANR mRNA VvLARI Fig III 4H Fig III 41 RAIS VY C VvANR VvLARI RO VvLAR2
55. DNA TaKaRa Ex Tag pCR2 1 TOPO Invitrogen fE Genetic Analyzer Applied Biosystems f DNE TA ABI PRISM 3100 Avant E DNA PLACE http www dna affrc go jp PLACE signalscan html 47 3 9 3 3 1 Fig III 2A LY UH 8 16 DBE EEK OREO BRAMWELL Ror WM 10 12 10 Fig III 2B
56. 2 Koyama et al 2007 Peyrot des Gachons and Kennedy 2003 Koyama 2007
57. 3 Xie ez al 2003 2006 60 90 2 3 3 Kennedy et al 2000a 2000b Bogs 2005 lk Z7 ANR cDNA VvANR LAR cDNA VvLARI VvLAR2 ANR LAR VvANR mRNA 2 3 42
58. GT 1 site GRWAAW I box GATAA TGACG motif Terzaghi and Cashmore 1995 GATA box Gilmartin et al 1990 53 3 4 Fig III 2 Fig III 3B D WAC koa C IRSE OD KA zt 3 Fig III 3B D E H 3 3 3 seg see Ete x ELE WAR Mesue ve EA 2 Fig Il 6
59. anthocyanidin reductase ANR 28 Xie etal 2003 ANR Medicago Xie et aL 2004 Desmodium LAR Tanner et al 2003 ANR LAR 4 dihydroflavonol 4 reductase DFR ANR LAR DER NADPH NADH Devic et al 1999 Tanner et al 2003 Xie et al 2004 3 Fig II 1 3 4
60. 610 629 5 CCCTTGGCCTTGAA ATACTC 3 982 1001 OFA HEA i amp DIG Easy Hyb Roche Diagnostics 40 FOR AV 2XSSC 20XSSC 3 M 0 3 M 0 1 SDS 3 2 0 1XSSC 0 1 SDS 13 2 DIG CSPD Roche Diagnostics LAS 1000plus 2 2 6 RNA mRNA Qeuna 1998 RNA 260 nm RNA QuantiTect SYBR Green PCR Kit Qiagen tt GeneAmp 5700 PCR Applied Biosystems PCR amp fTV VWANR mRNA
61. TIGR EST ANR TC45542 EST TC45542 VvANR Fig II 3 VvANR 7 VvANR Myc Myb Myc Myb _ lt Dooner et al 1991 Roth et al 1991 Myb MYB Ph3 Solano et al 1995 Myb Kobayashi ez al 2002 Baudry 2004 4 thaliana J Myc Myb BAN
62. 2 2 4 ANR LAR DFR ANR LAR DFR DDBJ CLUSTALW version 1 83 31 Roderic Page TreeView version 1 6 6 CLUSTALW Kimura correction gap 2 2 5 ODNA 10 ng gg7IIT gzHI Saci RO Dral 1 0 Roche Diagnostics 392 PCR DIG Probe Synthesis Kit Roche Diagnostics ViiA765 S ACTTCAGATGTCCCCAGCAG 3
63. C VvANR 2 3 3 PAL LDOX Fig II 1 mRNA Boss et al 1996c Kobayashi et al 2001 PAL LDOX mRNA mRNA 2 3 3 2003 Tanner 2 3
64. Sav 1 3 4 8 12 1000 mg l ABA Sigma A1049 200 mg l NAA Sigma N640 RRIAZ L ABA NAA 1N Tween 80 0 1 w v 3 18 20 CH 2004 1 2 3 DNA FLS Couch Fritz 1990 DNA 2 5 g 11 VUCI BMEUL BK 25 ml A 0 1 M 30 mM EDTA pH 7
65. 3 Desmodium O 3 40 2 5 XH 3 3 C ANR ts vinifera ANR VvANR VvANR ANR
66. Pelletier et al 1997 FLS Petunia Holton et al 1993 Arabidopsis Pelletier et al 1997 Solanum Van Eldik et al 1997 Citrus Moriguchi et al 2002 4 coumaroyl CoA Quercetin R OH R H Kaempferol R R H 3 x malonyl CoA R Myricetin Ri R OH R 2i ox HO OV ww Chalcone R2 R CHI Y OH 5 F3H F3 H F3 5 H OH O FLS 9H O Flavanone 9 Dihydroflavonols Flavonols HO R R DFR Y OH R N Ri LAR oH OH Leucoanthocyanidins gt 2 3 trans flavan 3 ols a HO 0 ES LDOX Y EJ Re UFGT ANR P ud occ amp Anthocyanidins gt 2 3 cis flavan 3 ols H Anthocyanins Q Proanthocyanidins Condensed tannins Fig I 1 Schematic representation of the biosynthetic pathway of flavonoids Enzyme names are abbreviated as follows CHS chalcone synthase CHI chalcone isomerase F3H flavanone 3 hydroxylase F3 H flavonoid 3 hydroxylase F3 5 H flavonoid 3 5 hydroxylase FLS flavonol synthase DFR dihydroflavonol 4 reductase LAR leucoanthocyanidin reductase LDOX leucoanthocyanidin dioxygenase ANR anthocyanidin reductase UFGT UDP glucose flavonoid 3 O glucosyl transferase Ri and Ro are H or OH 2003 z27 2 FLS cDNA Downey tk FLS is 00t
67. Sep Pak Plus tCig Cis Waters fE 3 FI FID FID 3 3 45 Sun etal 1998b REN Sigma Chemical 3 3 2 4 RNA mRNA Loulakakis 1996 DE RNA 260 nm RNA I am QuantiTect SYBR Green PCR Kit Qiagen fE GeneAmp 5700 PCR Applied Biosystems PCR amp Twv
68. VvANR VvLARI VvLAR2 mRNA 3 mRNA 44 3 2 3 2 1 Milli Q 7K Millipore DNA 3 2 2 4 2004 FICE HRO 1 40 6 7 4 i OIG 9f E E RR
69. Fluka Chemie Fluka Chemie cm 2002 3 1 3 1 3 1 FLS PCR 5 FLS GenBank EMBL DDBJ AB086055 FLSI AB086056 FLS2 AB213565 FLS3 AB092591 FLS4 AB213566 FLS5 FLSI FLS4 0 A KRIZ 1005 bp FLS5 9 bp 996 bp Fig 1 2 2 472772ops s thaliana FLS AtFLS AB006697 REGION 71532 to 72698 467 bp 328bp 210bp FLS1 e L H Lr
70. pH5 2 13 SDS 14 000 x g 4C 10 2 30 60 DHA 14 000 x gs 4C 10 2ml 70 400 pl ORAKEL 100 ul 10 M 30 C 3 14 000 x g 4C 10 70 50 1mM 14 pH6 4 260 nm RNA FLS FLS mRNA RNA RT PCR TaKaRa RNA PCR Kit AMV Ver 3 0 transcriptase XL 0 125 yM qdT 5 ngil O RNA RNA cDNA
71. 1 2 5 RNA RT PCR mRNA Geuna 1998 3 gt n p 13 RNA 1 g EL 6 ml RNA BEB 4 ml BSA s BSA Sigma A8022 10 mg ml Sigma H9399 BEB 2 ml PVPP BEB 0 1 M 0 001 M EDTA 2 SDS 1 N 200 mM HE M pH 9 5 DTT 16 mM 1 ATA Sigma A1895 PVPP Sigma P6755 2 BEB Eid I UI 400 pl 10 mg m K tt 37C 20 14 000 x g 3
72. 4MA Myc Myb gg IL BamHI KU Sacl 1 AY Dral 2 Gig 1 4 Dral 2 ANR 1 EST ANR VvANR ANR ANR PCR 4NR mRNA Fig II 3 39 3 7w b7yuyvkvyr vvioBEZf mVvci20 E85 Fig IT 6
73. Fig HI 3B D 3 10 12 H Fig III 3B 3 Fig III 3D E Fill Polymers Flavan 3 ols and PAs mg g FW N w e e eo gt Large leaf imi Flower bud i Flower HEN E FII Oligomers E FI Monomers S amp 9 2 a 5 5 10 10 lt B Skin control C Skin shading D Seed control E Seed shading 4 4r 8 8r Es Veraison p F 4 5 3 3 1 6 6 92 2 2 2 4 4f C S A 1 2 2 i o a LL 0 o m 0 0 25 25 X F Skin control G Skin shading 80 H Seed control so Seed shading S20 20 SZ 60 60 TE 15 15 o o c amp 10 10 a e c E o s 5 20 20 iL 0 0 0 0 gt oa qne o e ES ood Dom un E 85323838 332388 5238 23238 ya e2s ya ees sego ya ees Date Date Date Date Fig III 3 Accumulation of flavan 3 ol monomers and proanthocyanidins PAs in the leaves flower buds and flowers A berry skins B C F G and seeds D E H I of Cabernet Sauvignon
74. YR H VvLAR2 D B S vix VvLARI A VvANR VvLARI RU VvLAR2 mRNA mRNA JE DEEN 3 ANR LAR jim VvLARI VvLAR2 00 F u e 4 Fig III 5 1 G box CACGTG Terzaghi and Cashmore 1995 1 1I box 4 GATA box Fig II 3 DEEN Ja Fig III 4B E H Fig E JE 2 v4NR
75. 13 94 1 HH 57 C30 5 72 C30 fb zc 45 DNA 1 2 5 PCR RNA 3 VvANR mRNA Ubiquitin 2 2 7 3 Bourzeix 1986 80 20 v v 50 50 v v ARK KRU 75 25 viv Sun D 1998a Sep Pak Plus tCis Cig Waters th 3 FD Zu FII FI
76. ABA Spayd 2002 Noda et aL 2004 5 FLS
77. Phenylpropanoid rm 4 Coumaric acid Pathway O cy COOH HC o FA nm 4 Coumaroyl CoA COSCoA O CHS OH HO OH Q Q Chalcone OH O cH v OH Flavanone OH O F3H F3 H F3 5 H y R OH FLS Ho OW ws Dihydroflavonols OH Flavonoid o9 Pathway DFR y R OH LAR HO OW us lt Re Leucoanthocyanidins OH H OH Lpox yy R OH ANR HO Os J R lt Anthocyanidins OH Anthocyanins Fig 2 Schematic representation of the anthocyanin biosynthetic pathway Enzyme names are abbreviated as follows PAL phenylalanine ammonia lyase C4H cinnamate 4 hydroxylase 4CL 4 coumarate CoA ligase CHS chalcone synthase CHI chalcone isomerase F3H flavanone 3 hydroxylase F3 H flavonoid 3 hydroxylase F3 5 H flavonoid 3 5 hydroxylase FLS flavonol synthase DFR dihydroflavonol 4 reductase LAR leucoanthocyanidin reductase LDOX leucoanthocyanidin dioxygenase ANR anthocyanidin reductase UFGT UDP glucose flavonoid 3 O glucosyl transferase Ri and Ro are H or OH 1 dd dq un br d 1 1 Dili
78. FLS zh Wellmann et al 2002 5 FLS Fg I 3 F3H X75965 30 LDOX X75966 42 Table I 3 ENS I 3 3 FLS FNS I EE 2 FLS x 19 FLS1 68 GMFOIVNH 204 NYYPPCPRPDLALGVVSHTD FLS2 68 GMFOIVNH 204 NYYPPCPRPDLALGVVAHTD FLS3 68 GIFOIVNH 204 NYYPPCPRPDLALGVVAHTD FLS4 68 GMFOIVNH 204 NYYPPCPRPDLALGVVAHTD FLS5 65 GFFLLKDH 201 NMYPPCPOPOLALGVEPHTD AtFLS 68 GLFQVVNH 204 NYYPPCPRPDLALGVPAHTD CuFLS 68 GIFOVTNH 204 NYYPPCPRPDLALGVVAHTD MdFLS 66 GMYQIVNH 206 NYYPPCPRPDLALGVVAHTD FLS1 261 GDQLEILSNGKYKSVLHRTTVKKDMTRMSWPVFLEP FLS2 261 GDQLEILSNGKYKSVLHRTTVTKEITRMSWPVFLEP FLS3 261 GDQLEILSNGKYKSVLHRTTVNKEMTRMSWPVFLEP FLS4 261 GDQLEILSNGKYKAVLHRTTVNKEKTRMSWPV
79. FLS4 FLS FLSS FLSS FLSS ABA 22 FLS A B C abernet Sauvignon abernet Sauvignon abernet Sauvignon Cab t Sauvi i Cab t Sauvi j Cab t Sauvi T 5 4 25 E x 8 23 zs ES 6 S EE 2 8 2 ns 23 4 2 uu d E 5 O M x 0 o a 2 4 2 4 2 4 Weeks after veraison Weeks after veraison Weeks after veraison D E F Merlot Merlot Merlot 5 4 80 4 X 3 lt X 60 a3 es S E ES2 E E 40 g 22 3 3 4 u 3 1 U 5 20 0 T 0 T 0 2 5 2 5 2 5 Weeks after veraison Weeks after veraison Weeks after veraison BABA LINAA W Shading Control Fig I 6 Quercetin concentrations and mRNA levels of FLSs in the skins of control berries and berries treated with ABA NAA and shading A and D quercetin ug cm skin surface area B and E the mRNA level of FLS4 C and F the mRNA level of FLS5 For these analyses Cabernet Sauvignon A C and Merlot D F were used Results of a single measurement without replication were shown in A and D Vertical bars represent the standard deviation n 3 in B C E
80. 5 BE oe a ey 3 ANR LAR ANR LAR 57
81. AMV reverse transcriptase XL 0 125 uM qT 0 5 ng ul RNA RNA cDNA 32 PCR S GCTGCTGTTACCATCAATCA 3 1774 1793 3 5 GCAGGATAG CCCCAAGTAGG 3 1867 1993 3 4 113 bp 7 Downey 2003 Ubiquitin PCR UCHR Le Ubiquitin TIGR EST http www tigr org tigr scripts tgi T_index cgi species grape TC38636 S TCTGAGGCTTCGTGGTGGTA 3 5 AGGCGTGCATAACATTTGCG 3 99 bp PCR 1 0 u OF CDNA 0 3 uM 20 pl 9
82. F Seed g 0 6 0 3 0 1 04 02 S gt 0 2 0 1 7 Ni d wl 0 0 0 0 in ji cf mi 0 0 Cs Fi sm E fm mm 0 6 0 2 3 G H Skin Seed lt 2 z m l QC 04 Veraison 2 d 1 N 0 1 amp 02 1 d j i 1 Fl em ii 0 0 0 0 o m 9933 8232252392388d 2823232922353 Sere NE 3 vacet ES odo 39 20 3222442406 9 s 5 amp ot lt 8 uo rc wo Kr A Qa on A de Se Date Date Fig III 4 mRNA levels of VvANR A C VvLARI D F and VvLAR2 G I in the leaves flower buds and flowers A D G berry skins B E H and seeds C F I of Cabernet Sauvignon The mRNA levels were measured by real time quantitative PCR analysis The results are expressed as the molar ratio of the mRNA level of VvANR VvLARI or VvLAR2 to that of the ubiquitin gene The vertical bars represent the standard deviation n 3 3 3 4 LAR LAR WLARI VvLAR2 GenBank EMBL DDBJ AB262457 AB262458 Bogs 2005 7 0 VvLARI VvLAR2 60 51 77 2
83. OF RNA cDNA Y PCR 1 pl cDNA 0 3 uM 20 pl 9 5 13 94 1 5 30 72 30 ME 45 PCR RT PCR Ee BEEF VIR Ubiquitin mRNA PCR Downey ez al 2003 Ubiquitin The Institute for Genomic Research TIGR EST http www tigr org tigr scripts tgi T_index cgi species grape Release 4 0 September 21 2004 TC38636 15 Table 1 2 PCR DNA PCR E Z N A Cycle Pure Kit O
84. Yamamoto N Shindo H Kakuta T Koizumi T and Hashizume K 2005 Anthocyanidin reductase gene expression and accumulation of flavan 3 ols in grape berry Am J Enol Vitic 56 336 342 3 Fujita A Soma N Goto Yamamoto N Mizuno A Kiso K and Hashizume K 2007 Effect of shading on proanthocyanidin biosynthesis in the grape berry J Japan Soc Hort Sci 76 112 119 67
85. grape skins Phytochemistry 43 509 512 Sparvoli F Martin C Scienza A Gavazzi G and Tonelli C 1994 Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape Vitis vinifera L Plant Mol Biol 24 743 755 Spayd S E Tarara J M Mee D L and Ferguson J C 2002 Separation of sunlight and temperature effects on the composition of Vitis vinifera cv Merlot berries Am J Enol Vitic 53 171 182 Sun B S Leandro M C Ricardo da Silva J M and Spranger M I 1998a Separation of grape and wine proanthocyanidins according to their degree of polymerization J Agric Food Chem 46 1390 1396 Sun B S Ricardo da Silva J M and Spranger M I 1998b Critical factors of vanillin assay for catechins and proanthocyanidins J Agric Food Chem 46 4267 4274 Suzuki K Tsuda S Fukui Y Fukuchi Mizutani M Yonekura Sakakibara K Tanaka Y and Kusumi T 2000 Molecular characterization of rose flavonoid biosynthesis genes and their application in petunia Biotechnol Biotechnol Eq 14 56 62 Tanner G J Francki K T Abrahams S Watson J M Larkin P J and Ashton A R 2003 Proanthocyanidin biosynthesis in plants J Biol Chem 278 31647 31656 Terzaghi W B and Cashmore A R 1995 Light regulated transcription Annu Rev Plant Physiol Plant Mol Biol 46 445 474 Van Eldik G J Reijnen W H Ruiter R K Van Herpen M M A Schrauwe
86. o VvFLSI Z7S7 FLS VvFLS2 VvFLSI w FLS 5 FLSI FLSS 2 2 67 VvFLS2 FLS mRNA WELZ Ee MAM ORRICK S FLS mRNA abscisic acid ABA 1 1 naphthaleneacetic acid NAA
87. s Ro OH OH 2 3 cis flavan 3 ols epicatechin R OH R H epigallocatechin R R OH Fig II 1 Schematic representation of the biosynthetic pathway of proanthocyanidin and anthocyanin Enzyme abbreviations PAL phenylalanine ammonia lyase C4H cinnamate 4 hydroxylase 4CL 4 coumarate CoA ligase CHS chalcone synthase CHI chalcone isomerase F3H flavanone 3 hydroxylase F3 H flavonoid 3 hydroxylase F3 5 H flavonoid 3 5 hydroxylase DFR dhydroflavonol 4 reductase LDOX leucoanthocyanidin dioxygenase UFGT UDP glucose flavonoid 3 O glucosyl transferase LAR leucoanthocyanidin reductase ANR anthocyanidin reductase Ri and R are H or OH 30 2 2 2 2 1 Milli Q Millipore DNA 2 2 2 4 2003
88. to the transcription start site Several putative cis acting regulatory elements in the VvANR promoter region are indicated 36 ANR ANR cDNA 3 DER cDNA pg II gzHI Sgcl 1 AY Dral 2 Fig II 4 T SSBRE 242 2 emo Roc a 3 5 d Fig II 4 Southern blot analysis of the ANR gene 0 8 from the grape genome Each lane shows 10 ug of DNA of Cabernet Sauvignon digested with Bgill BamHI Sacl and Dral Positions of the molecular size markers are shown at left 2 3 2 VvANR mRNA OBE PCR VvANR mRNA VvANR mRNA
89. 0 1 MtANR Medicago truncatula ANR AAN77735 1 PcANR Phaseolus coccineus ANR CAD91909 1 VvANR Vitis vinifera Cabernet Sauvignon ANR BAD89742 1 DuLAR Desmodium uncinatum LAR CAD79341 1 LuLAR Lotus uliginosus LAR AAU45392 1 VVLARI and VvLAR2 V vinifera Shiraz LARs CAI26310 1 and CAI26308 1 CsDFR Camellia sinensis DFR BAA84939 1 AtDFR A thaliana ecotype Landsberg erecta DFR BAA85261 1 VVDFR V vinifera Lambrusco Foglia Frastagliata DFR CAA53578 1 Grape ANR LARs and DFR are underlined ATATACATGCTTCCTATATCTCAGCCACCTAATACACCTGGGCAGGTA 631 TAAGGATATTTTATCATTCCCTTGTGGAAAGGGGATTATTTTCGAATIBIBEAABAAGTCGTAAGAAGTCTG 561 TAGTGGGTGGCTCTTAATAACGTCGGCTTCCAAAATTTCTGTAATTTTAAAAGCATATATGAAGGGTTGA 491 CCCAAAAAAG AMA A AGHA AAT CTTGGTATGAATTTATGTTAGTTGGGAACCATCIGTTAAA 421 GGATTCAACCCATCTTCATAGCCAAACTCAAGCTTAAAGGGTTGAGCTTAGGTTGAACCCAGTATCTTCC 351 ATATTCAGTAATAGAATTTTTTTATTTTTATATGAATCCTTGAGGACTACATATCTGGTATATAAAGAGT 281 TATATATTCATACAGCTCCTCTGTGTATICAGATGTGGACTGCTGTTGAGATATGCAGTCAAACAGGGCTT 211 TGCTAGCAGCAACGTTGGAGTTGTTTGTTGAGCAGCCTENCGTGCTCACCTAABABABNTCACCTTCTCC 141 TCTTCTTTTCAATGGTTCAACGTACAATCTGAGGTCTATACCCTCTCTATATAAGAGACGCAGAGAGCGT 71 TATCCAAGATACAAATTACTTGCATTAATTAAGAGAACATCGGTTTTTGCGAATTTGGAAGTGAGGGCAT 1 G box DOX CC Myc recognition site EN GT site KEKE GATA box Myb binding site Fig II 3 Nucleotide sequence of the grape ANR gene VvANR between 678 and 1 relative
90. 0020 54 n c IURE 57 58 at a SE 60 67 HHH ZN 68 ii dcr ABA AMV ANR ATA BSA cDNA CHS DDBJ DFR DNA DTT EDTA EMBL EST F3H F3 H F3 S H FLS FNS FW HPLC LAR LDOX mRNA NAA NADH NADPH FE abscisic acid avian myeloblastosis virus anthocyanidin reductase aurintricarboxylic acid bovine serum albumin complementary deoxyribonucreic acid chalcone synthase DNA Data Bank of Japan dihydroflavonol 4 reductase deoxyribonucreic acid dithiothreitol ethylenediaminetetraacetic acid European Molecular Biology Laboratory expressed sequence tag flavanone 3 hydroxylase flavonoid 3 hydroxylase flavonoid 3 5 hydroxylase flavonol synthase flavone synthase fresh weight high performance liquid chromatography leucoanthocyanidin reductase leucoanthocyanidin dioxygenase messenger ribonucleic acid 1 naphthaleneacetic acid reduced nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide phosphate iii PAL PCR PTFE PVPP RNA rRNA RT PCR SDS SSC TIGR UDP UFGT phenylalanine ammonia lyase polymerase chain reaction poly tetrafluoroethylene poly vinylpolypyrrolidone ribonucleic acid ribosomal ribonucleic acid reverse transcription polymerase chain reaction sodium dodecyl sulfate sodium chloride sodium citrate The Institute for Genomic Research uridine diphosph
91. 3 Downey 2004 A r1 EORR EEA NATOR O IE 373 HO CHS R2 Z Chalcone OGlc OH CHI Y Anthocyanins k Flavanone Ri UFGT Ry F3H F3 H F3 5 H Y rs DFR Dihydroflavonols gt OH OF HO 0 LDOX T3 R Q OH RS Y OH OH R Leucoanthocyanidins ia LAR Y A ANR Y Ri Ry OH LW OF HO OS R ad OH 2 Pu cis flavan 3 ols e g 2 3 trans flavan 3 ols e g catechin R OH R H OH epicatechin R OH R H Proanthocyanidins Condensed tannins Fig III I Schematic representation of the biosynthetic pathways of flavonoids The condensation reactions indicated with broken arrows are speculative Enzyme names are abbreviated as follows CHS chalcone synthase CHI chalcone isomerase F3H flavanone 3 hydroxylase F3 H flavonoid 3 hydroxylase F3 S5 H flavonoid 3 5 hydroxylase FLS flavonol synthase DFR dihydroflavonol 4 reductase LAR leucoanthocyanidin reductase LDOX leucoanthocyanidin dioxygenase ANR anthocyanidin reductase UFGT UDP glucose flavonoid 3 O glucosyltransferase R and R are H or OH 43 3
92. 8 0 35M 2 3 WC 2 Sigma 40 000 0 7 BSA Sigma ft fraction V 10 mM 2 CMA CHE L 7 200 rpm 4 10 3 A 3 ml ORB OIM 50 mM EDTA pH 7 8 2 N 2 3 N V 2 Sigma tt 40 000 0 7 BSA Sigma fraction V 10 mM 2 HE 3 ml B 2 6 ml mlX0 9 g OF ERY OAL BEL 3 3 ml Beckman 10 mg ml 80 100 000 rpm 20C
93. AAAAAGTGCAGCTGAGTATTCATCTGAACATAAMAGTGATGG ATATAATTATAAAAAAAATTTAAAAAAAAAAAAAAATTGCTACCTTTAAGAAATCGGAAGGATTCCAGAG TTGAGTTGACTCGGAGGAATCCATCCGAGTTGCTGAGTCGGGGAATGCAACAGAGTCATCTTCAACTACC AGTCCAACTAGGGCATAACCTAACAATCAGAACAGTCAAACGCGCTAAAATACGCGGGGGGTGTGCACAC TATCCTCCACCCTTTCCACCTTAAACTGCCTCTATTTAATGGTGACGACCCTGGTCATTTAGCCACCGCC ACCACCCTTTTCCCCCCCTCCTGCACACCCTCTTCTTCTTTTTCTTCTTTCTCABBBERET AACACCGAT EE GT 1 site m DOX TGACG motif GATA box 841 771 701 631 561 491 421 351 281 211 141 71 701 631 561 491 421 351 281 211 141 71 Fig III 5 Nucleotide sequences of VvLARI between 910 and 1 and VvLAR2 between 744 and 1 relative to the transcription start sites Several putative cis acting regulatory elements conserved in light regulated genes are indicated in the VvLARI and VvLAR2 promoter regions The VvLARI and VvLAR2 sequences were obtained from the genomic DNA of Cabernet Sauvignon GenBank accession nos AB262457 and AB262458 except that a sequence between 66 and 1 in VvLARI is a cDNA sequence of Cabernet Sauvignon CF514437 52 PLACE E Hw7f DEEN Ja VvLARI VvLAR2 Fig III
94. ACATTTGCG Compliments of 289 to 308 Primer positions indicate the base from the start codon The first nucleotide of the start codon was defined as position l Ubiquitin primers were designed in the sequence TC38636 in an EST database of grape TIGR http www tigr org tigr scripts tgi T_index cgi species grape Release 4 0 September 21 2004 1 2 6 20C Hakkinen Auriola 1998 1 2 M 20 mg l Merck 1 gll 7eg7g 16 Sigma Aldrich Chemie th 50 200 mg ORK 85C 2 HPLC 0 45 um PTFE DISMIC 13HP Hewlett Packard HPLC Agilen
95. C005882 REGION complement 43221 to 44652 yDFR VvANR KUS L 3 VitiA763 98 8 UL VvANR ANR 84 BN000166 Tanner et al 2003 cDNA 99 8 VvANR 338 VitiA765 ANR 98 5 99 7 Table II 1 Homology of deduced amino acid sequences of VvANR Vitis vinifera Cabernet Sauvignon to other anthocyanidin reductase ANR and dihydroflavonol 4 reductase DFR VvANR AtANR MtANR AtDFR VvANR mE 652 74 7 45 5 VvDFR 45 9 39 8 42 0 75 9 Arabidopsis thaliana ecotype Columbia ANR GenBank protein id NP_176365 1 i Medicago truncatula ANR AAN77735 1 A thaliana ecotype Landsberg erecta DFR BAA85261 1 V vinifera Lambrusco Foglia Frastagliata DFR CAA53578 1 VvANR A thaliana Medicago truncatula ANR 657759805 fi DFR
96. FLEP FLS5 258 GDQIEVLSNGKYKSVLHRSTVNKERTRMSWAVFCAP AtFLS 261 GDQILRLSNGRYKNVLHRTTVDKEKTRMSWPVFLEP CuFLS 261 GDQIEILSNGKYKAVLHRTTVNKDKTRMSWPVFLEP MdFLS 263 GDQMEIMSNGKYTSVLHRTTVNKDKTRISWPVFLEP Fig I 3 Comparison of the deduced amino acid sequences of five grape FLSs and other FLSs Three regions of high similarity among 2 oxoglutarate dependent dioxygenases Wellmann et al 2002 are shown Six strictly conserved amino acid residues two histidines an aspartic acid an arginine and two glycines are marked by asterisks FLS1 to FLSS Vitis vinifera Cabernet Sauvignon FLSs GenBank accession nos AB086055 AB086056 AB213565 AB092591 AB213566 AtFLS Arabidopsis thaliana FLS AB006697 CuFLS Citrus unshiu FLS AB011796 MdFLS Malus domestica FLS AF119095 ELS BRFOALP RERETZE OIC 7ZS2 1 558 Dral 4 Fig I 4 kbp Dral 212 5 1 20 14 0 95 79 Fig I 4 Southern blot analysis of the FLS gene fro
97. Intron 1 Intron 2 467 bp 328 bp 210 bp FLS2 tees Intron 1 Intron 2 467 bp 328 bp 210 bp FLS3 es Intron 1 Intron 2 467 bp 328 bp 210 bp FLS4 Intron 1 Intron 2 458 bp 328 bp 210 bp FLS5 Intron 1 Intron 2 Fig I 2 Genomic structures of five FLSs obtained in this study The open boxes and solid lines indicate the coding and non coding regions respectively L cDNA VvFLS2 AY257979 VvFLSI AY257978 Downey et al 2003 98 0 98 9 FLSI FLS4 WFLS2 VvFLSI FLSI E FLS4 DU ER 95 1 100 FLS1 FLS4 335 Y 3 7 E83 25 0 bk 80 FLS AtFLS Citrus FLS AB011796 Malus 18 FLS AF119095 60 70 Table I 3 FLSS 4 D FLS Table I 3 Identity of the deduced amino acid sequences of FLSs of Vitis vinifera Cabernet Sauvignon to the FLSs of other plants grape F3H and
98. PCR 20 RT PCR AMV reverse wl X 1 pl OFFA cDNA 0 2 uM Table L2 PCR FLS RT PCR 18SrRNA 106 bp S TTGGCCTTCGGGATCGGAGT 3 5 TTCGCAGTTGTTCGTCTTTC 3 Vitis ED 188 rRNA GenBank accession no AF207053 PCR 94 C15 FLS 60C rRNA 56 C 15 572 C10 PCR 5 3 u 2 0 QuantiTect SYBR Green PCR Kit Qiagen GeneAmp 5700 PCR Applied Biosystems PCR 77S2 FLS5 mRNA 50 ng l
99. The concentrations of flavan 3 ols and PAs in the monomeric oligomeric and polymeric fractions were quantified by a vanillin assay The results are expressed in catechin equivalents per gram fresh weight A F I or per berry skin or seeds B E The vertical bars represent the standard deviation n 3 49 150512439 3 Fig III 3B C 10 12 13 E 9 Fig III 3D E 9 13 10 3 3 3 ANR LAR
100. VvANR VvLARI KRU VvLAR2 mRNA AMV reverse transcriptase XL 0 125 uM dqdT 5 ng ul OF RNA RNA cDNA PCR 0 1 ul cDNA 0 3 uM 20 pl 95 C15 94 15 30 gt 72 C30 45 WUbiquitin mRNA V PCR 2 2 6 Table III 1 DNA 1 2 5 PCR RNA 3 VvANR VvLARI RU VvLAR2 mRNA VvUbiquitin 46 Table III 1 PCR primers and annealing temperature
101. Watson B T 1995 Cluster sun exposure and quercetin in Pinot noir grapes and wine Am J Enol Vitic 46 187 194 Prieur C Rigaud J Cheynier V and Moutounet M 1994 Oligomeric and polymeric procyanidins from grape seeds Phytochemistry 36 781 784 Ricardo da Silva J M Belchior A P Spranger M I and Bourzeix M 1992a Oligomeric procyanidins of three grapevine varieties and wines from Portugal Sci Aliments 12 223 237 Ricardo da Silva J M Rosec J P Bourzeix M Mourgues J and Moutounet M 1992b Dimer and trimer procyanidins in Carignan and Mourv dre grapes and red wines Vitis 31 55 63 Ritchey J G and Waterhouse A L 1999 A standard red wine monomeric phenolic analysis of commercial Cabernet Sauvignon wines Am J Enol Vitic 50 91 100 Robichaud J L and Noble A C 1990 Astringency and bitterness of selected phenolics in wine J Sci Food Agric 53 343 353 Roth B A Goff S A Klein T M and Fromm M E 1991 C and R dependent expression of the maize Bz gene requires sequences with homology to mammalian myb and myc binding sites Plant Cell 3 317 325 64 Solano R Nieto C Avila J Ca as L Diaz I and Paz Ares J 1995 Dual DNA binding specificity of a petal epidermis specific MYB transcription factor MYB Ph3 from Petunia hybrida EMBO J 14 1773 1784 Souquet J M Cheynier V Brossaud F and Moutounet M 1996 Polymeric proanthocyanidins from
102. and F The standard deviation for some data 1s smaller than the thickness of the line 23 1 4 AR 5 FLS FLSI FLS5 FLSI FLS4 cDNA VvFLS2 VvFLSI 7ZS7 FLS4 VvFLS2 VvFLSI FLS4 FLS5 TIGR EST FLS TC46143 TC46972 FLSI FLS5 FLS 2 Fig L3 274 X FLS Table IL 3 DNA 3 FLS
103. ate UDP glucose flavonoid 3 O glucosyl transferase iv m 3 A epos ami un 1
104. en R A 1991 Genetic and developmental control of anthocyanin biosynthesis Annu Rev Genet 25 173 199 Downey M O Harvey J S and Robinson S P 2003 Synthesis of flavonols and expression of flavonol synthase genes in the developing grape berries of Shiraz and Chardonnay Vitis vinifera L Aust J Grape Wine Res 9 110 121 Downey M O Harvey J S and Robinson S P 2004 The effect of bunch shading on berry development and flavonoid accumulation in Shiraz grapes Aust J Grape Wine Res 10 55 73 Escribano Bail n M T Guerra M T Rivas Gonzalo J C and Santos Buelga C 1995 Proanthocyanidins in skins from different grape varieties Z Lebensm Unters Forsch 200 221 224 Flint S D Jordan P W and Caldwell M M 1985 Plant protective response to enhanced UV B radiation under field conditions leaf optical properties and photosynthesis Photochem Photobiol 41 95 99 Geuna F Hartings H and Scienza A 1998 A new method for rapid extraction of high quality RNA from recalcitrant tissues of grapevine Plant Mol Biol Rep 16 61 67 Gilmartin P M Sarokin L Memelink J and Chua N H 1990 Molecular light switches for plant genes Plant Cell 2 369 378 61 Hakkinen S and Auriola S 1998 High performance liquid chromatography with electrospray ionization mass spectrometry and diode array ultraviolet detection in the identification of flavonol aglycones and glycosides in berries J Ch
105. for real time quantitative PCR iud GenBank Sequence pios and reverse Primer position temperature accession no C VvANR F 5 GCTGCTGTTACCATCAATCA 1774 to 1793 57 AB199315 R SGCAGGATAGCCCCAAGTAGG Compliments of 1867 to 1993 VvLARI F S AAATGAACTCGCATCTGTGT 690 to 709 57 AJ865336 R S CTGTGGGATGATGTTTTCTC Compliments of 779 to 798 VvLAR2 F 5 GGTGATGGCTCTGTCAAAGC 392 to 611 59 AJ865334 R S CTGGGAGGTCGGAAATGAAG Compliments of 688 to 707 VvUbiquitin F 5 TCTGAGGCTICGTGGTGGTA 210 to 229 60 R SAGGCGTGCATAACATTTGCG Compliments of 289 to 308 The primer positions indicate the base from the start codon The first nucleotide of the start codon was defined as position 1 Ubiquitin primers were designed in the sequence TC38636 in an EST database of grape TIGR http www tigr org tigr scripts tgi T_index cgi species grape Release 4 0 September 21 2004 3 2 5 LAR LAR Biosciences Clontech 1 DEEN Universal GenomeWalker Kit BD tL DNA PCR cDNAZ u yY VvLARI GenBank accession no AJ865336 VvLAR2 AJ865334
106. grape LDOX FLS2 FLS3 FLS4 FLSS AtFLS CuFLS MdFLS F3H LDOX FLSI 78 2 83 3 77 3 49 6 62 6 66 6 67 6 30 2 44 7 FLS2 83 3 80 0 52 2 64 7 69 0 70 0 33 0 44 7 FLS3 81 8 52 5 65 0 71 3 70 8 31 7 46 3 FLS4 51 6 63 5 72 5 71 7 32 0 46 9 FLSS 50 6 55 5 50 6 31 0 46 6 Arabidopsis thaliana ecotype Columbia FLS GenBank accession no AB006697 Citrus unshiu FLS AB011796 Malus domestica FLS AF119095 V vinifera Lambrusco Foglia Frastagliata F3H X75965 V vinifera Lambrusco Foglia Frastagliata LDOX X75966 FLS F3H I flavone synthase I FNS D KU LDOX 2 4 2 2 1 Fe 1 2 Lukaein and Britsch 1997 Citrus unshiu FLS 2 EE
107. ignon A thaliana ecotype Landsberg erecta V vinifera Lambrusco Foglia Frastagliata VvANR ANR LAR DFR Fig II 2 PLACE http ww w dna affrc go jp PLACE signalscan html DNA y4NR OF Fig I 3 1 G box CACGTG 4 GT 1 site GRWAAW 1 I box GATAA Terzaghi and Cashmore 1995 KO 4 GATA box Gilmartin et al 1990 VvANR 4NR 4 Myc CANNTG Abe et al 2003 2 Myb CNGTTR 35 GaANR MtANR AtDFR CsDFR VvDFR VvLAR1 VvLAR2 0 1 bootstrap values 1000 Fig II 2 Phylogenic tree based on the deduced amino acid sequences of ANRs LARs and DFRs AtANR Arabidopsis thaliana ecotype Columbia ANR GenBank protein id NP 176365 1 GaANR Gossypium arboreum ANR CAD9191
108. m the grape genome The total DNA 5 ug of Cabernet Sauvignon was digested with Dral and blotted Positions of the molecular size markers are shown at left 1 3 2 5 FLS 5 20 FLS RT PCR mRNA SOO FLS Fig I 5A IZ FLS2 FLS3 RU FLS5 mRNA FLS4 mRNA 20 BEIT 43v TIL 5 OD FLS mRNA FLS2 FLS4 KU FLS5 mRNA 6 28 BH 7 25 8 7 9 18 A FLS4 FLSS mRNA RT PCR
109. mega Bio Tek DNA DyNA Quant 200 Amersham Bioscience DNA PCR RNA 3 FLS4 FLSS mRNA Ubiquitin Table I 2 PCR primers for RT PCR and real time quantitative PCR and annealing temperature for real time quantitative PCR hos GenBank Pegience or pic and Teveise m Primer position temperature accession no CC FLSI F 5 AATCCTCCTTCTTACAGGGA 451 to 752 AB086055 R S AGCCCTAACCCTACCGACAA Compliments of 811 to 830 FLS2 F 5 AACCCACCTTCGTACAGGGC 451 to 1085 AB086056 R S CCTAACCCTAATGACAGCAA Compliments of 1141 to 1160 FLS3 F 5 ACCATCTTACAGGGATGCTA 456to 1137 AB213565 R STCGCCACCCACACTCTTCTT Compliments of 1227 to 1246 FLS4 F 5 AAACCACCTACTTACAGAGC 451 to 1632 60 AB092591 R 5 ACCTAACCCCAGTGACAGAC Compliments of 1689 to 1708 FLS5 F 5 AACCAAGATGACTAAGAACC 339 to 358 55 AB213566 R S CTTCTGTGACTICCCTGTAG Compliments of 453 to 951 Ubiquitin F 5 TCTGAGGCTTCGTGGTGGTA 210to229 57 5 AGGCGTGCATA
110. n J A M and Wullems G J 1997 Regulation of flavonol biosynthesis during anther and pistil development and during pollen tube growth in Solanum tuberosum Plant J 11 105 113 65 Wellmann F Luka in R Moriguchi T Britsch L Schiltz E and Matern U 2002 Functional expression and mutational analysis of flavonol synthase from Citrus unshiu Eur J Biochem 269 4134 4142 Xie D Y and Dixon R A 2005 Proanthocyanidin biosynthesis still more questions than answers Phytochemistry 66 2127 2144 Xie D Y Sharma S B Paiva N L Ferreira D and Dixon R A 2003 Role of anthocyanidin reductase encoded by BANYULS in plant flavonoid biosynthesis Science 299 396 399 Xie D Y Sharma S B and Dixon R A 2004 Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana Arch Biochem Biophys 422 91 102 Xie D Y Sharma S B Wright E Wang Z Y and Dixon R A 2006 Metabolic engineering of proanthocyanidins through co expression of anthocyanidin reductase and the PAPI MYB transcription factor Plant J 45 895 907 66 E att si x A Ic lm Fujita A Goto Yamamoto N Aramaki I and Hashizume K 2006 Organ specific transcription of putative flavonol synthase genes of grapevine and effects of plant hormones and shading on flavonol biosynthesis in grape berry skins Biosci Biotechnol Biochem 70 632 638 2 Fujita A Soma N Goto
111. ocyanins Flavonols A a A Flowering Veraison Harvest Fig 3 Accumulation patterns of flavonoids in grape berry skins ABA 58 ABA ice ABA ABA
112. ones G P 2000b Development of seed polyphenols in berries from Vitis vinifera L cv Shiraz Aust J Grape Wine Res 6 244 254 Kobayashi S Ishimaru M Ding C K Yakushiji H and Goto N 2001 Comparison of UDP glucose flavonoid 3 O glucosyltransferase UFGT gene 62 sequences between white grapes Vitis vinifera and their sports with red skin Plant Sci 160 543 550 Kobayashi S Ishimaru M Hiraoka K and Honda C 2002 Myb related genes of the Kyoho grape Vitis labruscana regulate anthocyanin biosynthesis Planta 215 924 933 Kobayashi S Goto Yamamoto N and Hirochika H 2004 Retrotransposon induced mutations in grape skin color Science 304 982 Koyama K Goto Yamamoto N and Hashizume K 2007 Influence of maceration temperature in red wine vinification on extraction of phenolics from berry skins and seeds of grape Vitis vinifera Biosci Biotechnol Biochem 71 958 965 Loulakakis K A Roubelakis Angelakis K A and Kanellis A K 1996 Isolation of functional RNA from grapevine tissues poor in nucleic acid content Am J Enol Vitic 47 181 185 Lukatin R and Britsch L 1997 Identification of strictly conserved histidine and arginine residues as part of the active site in Petunia hybrida flavanone 3p hydroxylase Eur J Biochem 249 748 757 Mateus N Marques S Goncalves A C Machado J M and De Freitas V 2001 Proanthocyanidin composition of red Vitis vinifera va
113. pe OD 9 EBL MADE BC CTA200 1 Kit RED 80C Brix 1 De 3 2 3 3 Bourzeix 1986 80 20 viv 50 50 v v 75 25 v v Sun 19983
114. rieties from the Douro valley during ripening infruence of cultivation altitude Am J Enol Vitic 52 115 121 Monagas M G mez Cordov s C Bartolom B Laureano O and Ricardo da Silva J M 2003 Monomeric oligomeric and polymeric flavan 3 0l composition of wines and grapes from Vitis vinifera L cv Graciano Tempranillo and Cabernet Sauvignon J Agric Food Chem 51 6475 6481 Moriguchi T Kita M Ogawa K Tomono Y Endo T and Omura M 2002 Flavonol synthase gene expression during citrus fruit development Physiol Plant 114 251 258 Noda N Kanno Y Kato N Kazuma K and Suzuki M 2004 Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal 63 development in lisianthus Eustoma grandiflorum Physiol Plant 122 305 313 Pelletier M K Murrell J R and Shirley B W 1997 Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis Plant Physiol 113 1437 1445 Peyrot des Gachons C and Kennedy J A 2003 Direct method for determining seed and skin proanthocyanidin extraction into red wine J Agric Food Chem 51 5877 5881 Pourcel L Routaboul J M Kerhoas L Caboche M Lepiniec L and Debeaujon I 2005 TRANSPARENT TESTAIO encodes a laccase like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat Plant Cell 17 2966 2980 Price S F Breen P J Valladao M and
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116. t Technologies fL 1100 ChemStation Zorbax StableBond 12 5x2 VAT mm I D SB C18 5 um Agilent Technologies Zorbax StableBond 150X2 1 mm LD SB C18 5 um Agilent Technologies ot TLT IBIE ATL 1 B AS A 90 WEB 10 B 10 20 35 39 70 407 45 10 0 3 ml 5 1 365 nm Sigma Chemical
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