The beet R locus encodes a new cytochrome P450 required for red betalain production. Gregory J. Hatlestad, Rasika M. Sunnadeniya, Neda A. Akhavan, Antonio Gonzalez, Irwin L. Goldman, J. Mitchell McGrath, Alan M. Lloyd Supplementary Table 1 Primers used during PCR amplification as indicated in the text and methods. Primer Name Primer sequence 5 to 3 VIGS76AD1F GCTAATCTTGCTAAAATTCACGG VIGS76AD1R TTATGGTGGGCTAATTCCACTG VIGSDODA1F GTTAAACCTACTGTTAATGCTGTC VIGSDODA1R GCTGCCCAAGGTGCAACTCC RTPCRBvACTF TCTATCCTTGCATCTCTCAG RTPCRBvACTR ATCATACTCGCCCTTGGAGA RTPCRDODA1F CATTGGTTCAGGAAGTGCAA RTPCRDODA1R ACGAAGCCATGAATCAAAGG RTPCR76AD1F CTTTTCAGTGGAATTAGCCCACC RTPCR76AD1R CCCAATATCTTCCATAATGTTCCA 76AD1cDNAF ATGGATCATGCAACATTAGCAATG 76AD1cDNAR CCAAAACCAACCACAAACAA PgDODAcDNAF ATGGGTGTTGGGAAGGAAGTG PgDODAcDNAR ATCATATGGAAGTGAACTTGTAAG
Fagales Cucurbitales Rosales Fabales Zygophyllales Celastrales Oxalidales Malpighiales Sapindales Malvales Brassicales Crossosomatales Myrtales Geraniales Saxifragales Lamiales Solanales GenAanales Garryales Asterales Dipsacales Apiales Aquifoliales Cornales Ericales Myrothamnaceae/Gunneraceae Berberidopsidales Santalales Caryophyllales Buxaceae/Didymelaceae Trochodendraceae Proteales Sabiaceae Ranunculales Ceratophyllales Monocots Winterales Laurales Magnoliales Chloranthales Piperales Illiciaceae Schisandraceae Austrobaileyaceae Nymphaeaceae Amborellaceae eurosid I eurosid II euasterid I euasterid II magnoliids rosids asterids eudicots Supplementary Figure 1 Flowering plant phylogenetic relationships redrawn from Soltis et al. s1. Betalain families are restricted to the Caryophyllales. All other angiosperms produce anthocyanin pigments.
Intensity Intensity 25000 20000 15000 10000 5000 25000 20000 15000 10000 5000 Bull s Blood non-silenced 3 methoxytyramine betaxanthin 350 355 360 365 Golden Globe No CYP76AD1 CYP76AD1 silenced in Bull s Blood 350 355 360 365 Golden Globe 35S::CYP76AD1 350 355 360 365 350 355 360 365 Supplementary Figure 2 Mass spec analysis of a yellow betaxanthin. There are a wide variety of betaxanthins produced in beets because betalamic acid will condense with many amino compounds (Fig. 1). This analysis concentrates on a single compound, (arrows point to 3 methoxytyramine betaxanthin peak) and shows that suppression of CYP76AD1 in red beet leads to increased producaon of yellow betaxanthin and that overexpression of CYP76AD1 in yellow beets leads to decreased producaon of yellow betaxanthin, the opposite of what is seen for betacyanin (Fig. 1 and 2).
betanidin cyclo-dopa betalamic acid Supplementary Figure 3 Mass spec analysis of betanidin, cyclo DOPA, and betalamic acid in yeast expressing both DODA and CYP76AD1 but not fed L DOPA shows that L DOPA is required for produceon of these compounds.
MDHATLAMILAI---WFISFHFIKLLFSQQTTKL--LPPGPKPLPIIGNILEVGKKPHRS 55 MDNATLAMILTI---WFISINFIKMFFYHQNTKLS-LPPGPKPLPIIGNILEVGKKPHRS 56 MDFLTLVMILSI---IFFFYNLLKMKFTTHSDAQ--LPPGPKPMPIFGNIFELGEKPHRS 55 MDYTPLVLLLLLPCFVWLCFHFLILGSTHRKSFQARLPPGPRPLPIIGNLLELGDKPHQS 60 **.*.::* : :: ::: : :. *****:*:**:**::*:*.***:* FANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNSVTAGDHHKLTM 115 FANLAKIHGPLISLRLGSVTTIVVSSAEVAKEMFLKKDQPLSNRNVPNSVTAGDHHKLTM 116 FANLAKTHGPLMSLRLGSVTTIVVSSAEVAKEMFLKNDQSLADRSVPNSVTAGDHHKLTM 115 FTTLSKTYGPLMSLKLGSTTTIVISSPKTAQEVLNKKDQAFASRTVLNAIQIQDHHKFSM 120 *:.*:* :***:**:***.****:**...*:*:: *:*:.::.*.: *:: ****::* SWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECAQKGQAVDIGKAA 175 SWLPVSPKWRNFRKITAVHLLSPLRLDACQSLRHAKVQQLYQYVQECALKGQSVDIGKAA 176 SWLPVSPKWKNFRKITAVHLLSPQRLDACHALRHAKVKQLYEYVQECALKGEAVDIGKAA 175 VFLPASAHWRNLRKICSMQIFSPQRVEASQDLRRKVVQQLLEHARESCNSGRAVDVGRAA 180 :**.*.:*:*:*** :::::** *::*.: :*: *:** ::.:*...*.:**:*:** FTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADYFPILGCVDPSGIR 235 FTTSLNLLSKLFFSKELACHKSHESQELKQLIWNIMEDIGKPNYADYFPILGCIDPLGIR 236 FTTSLNLLSNLFFSVELANHTSNTSQEFKQLIWDIMEDIGKPNYADYFPLLKYVDPSGIR 235 FTTTLNLLSNTFFSVDLAHYDSNLSQEFKDLIWSIMVEAGKPNLADFFPGLRLVDPQGIQ 240 ***:*****: *** :** : *: ***:*:***.** : **** **:** * :** **: RRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFKQN--ELTMGEINHL 293 RRLAANFDKLISVFQTIISERLENDINSN--ATTNDVLDVLLQLYKQK--ELSMGEINHL 292 RRLAANFDKLIDVFQSFISKRLSSAYSSA--TSLDDVLDVLLKLLKEK--ELNMGEINHL 291 KRMTVYFNKLLDVFDGFINQRLPLKASSP----DNDVLDALLNLNKQHDHELSSNDIRHL 296 :*:: *:**: **: :* :**.: :****.**:* *:: **..:*.** LVDIFDAGTDTTSSTFEWVMTELIRNPEMMEKAQEEIKQVLGKD-KQIQESDIINLPYLQ 352 LVDIFDAGTDTTSSTFEWVMAELIRNPKMMEKAQQEIHEVLGKD-RQIQESDIIKLPYLQ 351 LVDIFDAGTDTTSNTFEWAMAELMRNPIMMKRAQNEIALVLGKDNATIQESDIANMPYLQ 351 LTDLFSAGTDTISSTIEWAMAELLNNPKAMAKAQDELSQVVGKD-RIVEESDVTKLPYLQ 355 *.*:*.***** *.*:**.*:**:.** * :**:*: *:*** ::***: ::**** AIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGRDPNAWQNADIFSP 412 ALIKETLRLHPPTVFLLPRKADMDVELYGYVVPKDAQILVNLWAIGRDSQVWEKPNVFLP 411 AIIKETLRLHPPTVFLLPRKAITNVKLYGYIVPKNAQILVNLWAIGRDPKVWKNPNEFLP 411 AVVKETFRLHPPAPFLVPRKAEMDSEILGYAVPKNAQVLVNVWAIGRDSRTWSNPNSFVP 415 *::***:*****: **:**** : :: ** ***:**:***:******...*.:.: * * ERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQFFNWKLEGDISPKD 472 ERFLGSDVDVKGRDFGLLPFGAGKRICPGMNLAIRMLTLMLATLLQFFNWKLEDGMNPQD 471 DRFLNSDIDVKGRDFGLLPFGAGRRICPGMNLAYRMLTLMLATLLQSFDWKLPHRNSPLD 471 ERFLECQIDVKGRDFQLIPFGAGRRICPGLLLGHRMVHLMLASLLHSFDWKLEDSMRPED 475 :**:.::******* *:*****:*****: *. **: ****:**: *:*** * * LDMDEKFGIALQKTKPLKLIPIPRY 497 LDMDEKFGIALQKNKPLEIIPSLRH 496 LDMDEKFGIALQKTKPLEIIPLIKY 496 MDMSEKFGFTLRKAQPLRAVPTKP- 499 :**.****::*:* :**. :* Supplementary Figure 4 ClustalW alignment of CYP76AD1 from Beta vulgaris, CYP76AD2 from Amaranthus cruentus, CYP76AD3 from Mirabilis jalapa, CYP76T21 from Vi7s vinifera. The cytochrome P450 conserved heme binding domain is underlined. Small arrowhead points at the last naeve amino acid predicted to be in the yellow sugar beet allele.
Supplementary reference. S1. Soltis, P.S., Soltis, D.E., & Chase, M.W. Angiosperm phylogeny inferred from multiple genes as a tool for comparative biology. Nature 402, 402-404 (1999).