Supplementary Material
Figure S1: Alignment of the pfam family PF02458 proteins from rice, Brachpodium and Arabidopsis. Highlighted in color is the HXXXD motif, which is proposed to be the active site for transferase activity. The different colors for this motif are relative to the grouping of proteins into clades (I–V) by Piston et al. (2010).
* 20 * 40 * 60 * 80 * 100
Bradi2g04990.1 M------AAAKK-----SVSRLAQRLVLPAE--PTPSGP------LRLSWLDRYPTQMALIESLHVFKPAPARHAD---AC-- 59
Bradi2g04980.1 M------AAANK-----SVSRLPQRLVLPAE--PTPAGP------LRLSWLDRYPTQMALIESLHVFKPAPAHDG------56
Os01g08380.1 M------AAAK-----SVERLGQRRVVPAE--PTPAGP------LRLSWLDRYPTQMALIESLHVFKPALDRAIGGDDVA-- 61
Os05g08640.1 M------AAAAPDK-----AVERLSQKLVHPSS--PTPSAP------LRLSWLDRYPTQMALIESLHVFKPDPARDAAG--QG-- 62
Bradi2g33980.1 M------AAAAAAKP---VPVERLAQRLVAPAG--PTPEGP------LRLSWLDRYPTQMALIESLHVFKPDMAREGD------61
Os06g39470.1 M------RGSAAAAA---ATVTRVAQRVVAPSA--ATPGGA------LPLSWLDRYPTQRALIESLHVFKGRADAAVA------61
Bradi1g36980.1 M------RSAGAVAA---ATVTRVAQRVVAPSA--PTPGGE------LPLSWLDRYPTQRALIESLHVFKGRAGATEG------61
Os06g39390.1 M------GV---FAVTKVSEGPVRPSA--ATPSET------LPLAWVDRYPTHRGLVESVHIYLRRDDAAVEAPCADGG 62
Os01g09010.1 M------AGTGS---FKVTRISEGAVKPAA--ATPEET------LPLAWVDRYPTHRGLVESMHIFRSGADAAPG------58
Bradi2g05480.1 M------ASPSS---FKVTRISEGPVKPASP-DTPGHT------LPLAWVDRYPTHRGLVESMHIFRSGADAAPG------59
Os05g19910.1 M------VA---VTVMRKSRNFVGPSPPTPPAEIT---T----TLELSSIDRVPGLRHNVRSLHVFRRHKNSGPVVDGDS-- 64
Os01g18744.1 M------G---FAVVRTNREFVRPSAATPPSSGE------LLELSIIDRVVGLRHLVRSLHIFSAAAPSGGDAKPS--- 61
Os05g04584.1 M------G---FTVTRTSRSLVAPSS--PTPAET------LPLSVIDRVAGLRHLVRSLHVFEAGGRNGGGE------55
Bradi2g36910.1 M------EKK---FTVTRTSKSLVPPSSSSPTPAATEDDAPVPVIMRLSTIDRVPGLRHLVLSLHAFDGHGVVAGEDDEERI- 73
Os01g42880.1 M------AA---AAVTKSPPALVPPAG--PTPGGS------LPLSSIDKTAAVRVSVDFIQVFPSSAEAAKDQA----- 57
Bradi2g43520.1 M------A---PTVTKSPPALIPPAG--PTPGGS------LPLSSIDKTAAVRVSVDFIQVFPSSADAPRDQG----- 56
Os01g42870.1 M------AAAAAVAAVAAAAAAAE---PTVSKSAPELVPPAG--PTPRGA------LPLSSIDKTAAVRVSVDFIQVFPPATGGPAADGQE--- 74
Bradi2g43510.1 M------ADAA---PTVSKSPPALVPPVG--PTPRGA------LPLSSIDKTAAVRVSVDFIQVFPPSTDGASAG------58
Os04g09590.1 M------KN---VVITKSSPELVG-LSTKPAPPPPGD------ISLSSFDEALAF-AAFTSFHIFTNGIVEPAM------57
Os04g11810.1 M------S---YIVNKSSPELVGPPTTKPVAPTVADV------INLSSFDKAIGS-YLFTSFHVFDNGIVEPAM------58
AT5G41040.1 MVAENNKNKDVTLSASMDNNNNNIKGTNIHLEVHQKEPALVKPES--ETRKG------LYFLSNLDQNIAV--IVRTIYCFKS--EERGNEE----- 80
Bradi5g01240.1 MVAAEQENG---SSVAMAAGNMNSKAQ--LVSVKRGEPSLVAPAE--PTPTGE------HYYLSNLDQNIAV--IVQTVYCFKCPPSGRGHEAG---- 79
* 120 * 140 * 160 * 180 * 200
Bradi2g04990.1 ------PGPAR-TIERALAQALVRYYPLAGRLAFTD---DGG-Q------SHVDCGGP---RSGVWFTEAEAACGLEDVD 119
Bradi2g04980.1 ------ADPAR-TIERALAQALVRYYPLAGRLAFTD---DGG-Q------SHVDCGGP---GSGVWFTEAAAACGLEDVD 116
Os01g08380.1 ------VGPAR-TIERALARALVHYYPLAGRLAFSD---SG--E------VCVDCG-----DAGVWFTEAEASCSLEDVD 118
Os05g08640.1 ------LAPAR-AIETALARALVEYYPLAGRLAVSR---DSG-E------LQVDCCGGAGGHGGVWFIEAAVPCRLEDVD 125
Bradi2g33980.1 ------SPAR-AVERALARALVDYYPLAGRLAVS----DAG-E------LQVDCR-----DGGVWFIEAAVRCRLEDVD 117
Os06g39470.1 ------PAA-AIERALAAALVSYYPIAGRLAERG---DGG-E------LVVDCTG-----EGVWFIEATASCSLEDVD 117
Bradi1g36980.1 ------PVK-AIERALAAALVSYYPLAGRLAVPA---DGG-E------LVVDCTG-----EG------DVD 104
Os06g39390.1 VIVEGKKKNNKPAAAVVRGALADALVHYYPFAGRIVEDE---RSPGRP------AVLCSG-----EGVYFVEAAANCTLADVN 131
Os01g09010.1 ------VIRDALARALVFFYPLAGRIVEPE---AGSPA------IRCTA-----DGVYFAEAAADCSLEDVR 110
Bradi2g05480.1 ------VIRDALAKALVFFYPLAGRIVEPE---KEAAAAAAAAAAGENGDGEKKAPAASFPLGVRCAG-----DGVYFAEAEAECSLEDVR 136
Os05g19910.1 ------RRPAA-VIRAALARALADYPAFAGRFVGSL---LAG-D------ACVACTG-----EGAWFVEAAADCSLDDVN 122
Os01g18744.1 ------PAR-VIKEALGKALVDYYPFAGRFVDGG---GGPGS------ARVECTG-----EGAWFVEAAAGCSLDDVN 118
Os05g04584.1 ------PARVVIREALGKALVEYHPFAGRFVEGD---GGG-E------VAVACTG-----EGAWFVEATAACSLEEVK 112
Bradi2g36910.1 ------RWPAR-VVREALGKALVDYYPFAGRFVVDE---EG--E------VGVKCSG-----EGAWFVEAKAECSLEEAR 130
Os01g42880.1 ------ASVAAMREGFARALVHYYPVAGRIAEPV---PGE------PEIDCTG-----EGVWFIEAEASCSLEEAR 113
Bradi2g43520.1 ------ASVATMREGFAKALVHYYPVAGRIAEPV---QGE------PEVECTG-----EGVWFVEAEASCTLEDAR 112
Os01g42870.1 ------DAVARMRDGFARALVPYYPVAGRIAEPA---PGD------VVVDCTG-----EGVWFVEATASCTLADVN 130
Bradi2g43510.1 ------DQVAAMRDGFARALVPYYPVAGRIAEPT---PGD------LVVDCTG-----EGVWFVEAAASCSLADVN 114
Os04g09590.1 ------A--IKRALSQALVYYYPIAGRANFA----AGERR------LRISCTG-----EGVGFVAATASCALDDVK 110
Os04g11810.1 ------T--IKGALSQALVYYYPIAGRLVITGAADGGGDQ------LCVSCTG-----EGVAFVSATASCALDDVK 115
AT5G41040.1 ------AVQVIKKALSQVLVHYYPLAGRLTISP---EGK------LTVDCTE-----EGVVFVEAEANCKMDEIG 135
Bradi5g01240.1 ------AADALRDALSRVLVHYHPLAGRLAISP---EMK------LAVDCTG-----EGVVFVEADAACDLADMG 134
* 220 * 240 * 260 * 280 * 300
Bradi2g04990.1 YLEHP---MMISKDELLP-PTP---AAEEGDERRLVLLVQVTSFACGGFVVGFRFSHAVADGPGAAQFMAAVGDLARGAESLSMEPQWGRD-AVP--DPA 209 Clade III
Bradi2g04980.1 YLEHP---MMIPKDALLP-PTPH--AAEEGDERRLVLLVQVTSFACGGFVVGFRFSHAVADGPGAAQFMAAVGDLARGAESLSMEPQWGRD-AVP--DPA 207 Clade III
Os01g08380.1 YLEYP---MMVPKDELLP-PTP---AGEE--ERELVLLVQVTAFACGGFVVGFRFSHAVADGPGAAQFMAAVGELARGAGGVSVDPVWGRD-AIP--DPA 206 Clade III
Os05g08640.1 YLEYP---LAISKDELLPHPRPR--PTRD-EEDKLILLVQVTTFACGGFVVGFRFSHAVADGPGAAQFMGAVGELARGGERITVAPSWGRD-AVP--DPA 216 Clade III
Bradi2g33980.1 YLEYP---LAVDKDELLPHPRPK--PTHE-EESKLILLVQVTTFDCGGFVVGFRFSHAVADGPGAAQFMGAVGELARGAGRISVPPAWGRD-AIP--DPA 208 Clade III
Os06g39470.1 YLEYP---LMVDKDELLPHPTYP--ASESHPEDSLILLVQVTQFACGGFVVGFRFSHAVADGPGAAQFMTAVGEIARGRAAPALAPAWGRD-AIPC-PPS 210 Clade III
Bradi1g36980.1 YLEYP---LMMPKDELLPHPTYP--AADPLPEDSFILLVQVTQFACGGFVVGFRFSHAVADGPGAAQFMTAVGEIARGRAGPSVKPAWGRE-AIPS-PPA 197 Clade III
Os06g39390.1 HLERP---LLLSKEDLVPCPTPEQWPVEP---HNSLAMIQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPEPRVKPIWARD-RFP--DPD 222 Clade IV
Os01g09010.1 FLERP---LLLPKEDLVPYPGDDRWGVEP---HNTIMMMQITKFTCGGFVMGLRFNHASADGMGAAQFINAVGDMARGLPEPRVKPVWDRE-KFP--NPS 201 Clade IV
Bradi2g05480.1 FLERP---LLLPKEDLVPYPGPDKWSVEP---HNTIMMMQITKFTCGGFVMGLRFNHASADGMGAAQFIKAVGDMARGLPEPAVKPVWDRE-KFP--NPS 227 Clade IV
Os05g19910.1 GLEYP---LMISEEELLPAPED---GVDP---TSIPVMMQVTEFTCGGFILGLVAVHTLADGLGAAQFITAVAELARGMDKLRVAPVWDRS-LIP--NPP 210 Clade V
Os01g18744.1 GLDHP---LMIPEDDLLPDAAP---GVHP---LDLPLMMQVTEFSCGGFVVGLISVHTMADGLGAGQFINAVGDYARGLDRPRVSPVWARE-AIP--SPP 206 Clade V
Os05g04584.1 LLDHP---MVIPKEELLPEPAP---DVQP---LDIPLMMQVTEFTCGGFVVGLISVHTIADGLGAGQFINAVADYARGLAKPRVSPVWARD-AIP--DPP 200 Clade V
Bradi2g36910.1 HLDGNPMEMVIPKEDLLPEPIP---GVDP---LDIPLIMQVTEFTCGGFVVGLISVHTIADGLGAGQFINAVADYARGLPKPRVSPVWARD-LVP--DPP 221 Clade V
Os01g42880.1 NLERP---LCIPKEELLPRPPP---EVRV---EDTVLLAQITKFTCGGFAVGICFSHLVFDGQGAAQFLKAVGEMARGLPEPSLKPIWARD-AIP--NPP 201 Clade I
Bradi2g43520.1 NLERP---LCIPKEELLPRPPP---EVRL---EDTVLLAQITKFTCGGFSVGICFSHLVFDGQGAAQFLKAVGEMARGLPEPSLKPIWSRD-AIP--NPP 200 Clade I
Os01g42870.1 NLERP---LLIAKEHLLPRPPP---EEKL---EDLILMAQVTKFTCGGFAVGICFSHLVFDGQGAAQFLKAAGEMARGLPAPSVPPVWDRD-AIP--DPP 218 Clade I
Bradi2g43510.1 GLERP---LLIPKGELIPRPPP---EEKL---EDLILMAQVTKFTCGGFAVGICFSHLVFDGQGAAQFLKAAGELARGLPAPSVAPVWDRD-AIP--DPP 202 Clade I
Os04g09590.1 LFDPP--FAAVLKELAVDYPAEG--CGED----DPLLLMQVTEFACGGFVVGVTWNHVVADGLGIAQFLQAVGDLARGLPRPSVFPVSCGDGSLPA-LPP 201 Clade II
Os04g11810.1 LFDPP--FAALLKELAVAHPAAG--EAEA----DPLLLMQVTEFACGGFVVGMTWNHVVADGKGIAQFLRAVGELARGLPRPSVLPVSCGDDSLPE-LPP 206 Clade II
AT5G41040.1 DITK------PDPETLGKLVYDVVDAKN-ILEIPPVTAQVTKFKCGGFVLGLCMNHCMFDGIGAMEFVNSWGQVARG-LPLTTPPFSDR-TILNARNPP 225
Bradi5g01240.1 DLSTT------PDPAALGQLVYSIPGAKN-ILQMPPITAQVTRFKCGGFSLGLAMNHCMFDGLGAMEFVNSWAEMARGATELTVPPYLDRAAVLRARDPP 227
* 20 * 40 * 60 * 80 * 400
Bradi2g04990.1 GAV------VGALPDPAGAKRLEYLAMDISADYIDHFKAQYNSTNNG---G-ARCSAFEVLVAKAWQSRTRAA-GFDPSTT------VHLCF 284
Bradi2g04980.1 GAV------VGALPDPAGAKRLEYLAMDISADYIDHFKSQYNSSNNG---GGARCSAFEVLVAKAWQSRTRAA-GFDPSAT------VHLCF 283
Os01g08380.1 AAV------IGSLPDPAGAKRLEYLAVDISADYINHFKNQYNAEAHAAAAGVARCSAFEVLIAKAWRSRTRAA-GFEPDTT------VNLCF 285
Os05g08640.1 GAM------VGALPEPAGASRLEYLAIDISADYINHFKSQFAAATGG-----ARCSAFEVLIAKAWQSRTRAA-AFDPSTP------INLSF 290
Bradi2g33980.1 GAL------VGRLPEPAGAKRLEYLAIDISADYINHFKAQFAAATGG-----ARCSAFEVLIAKAWQSRTRAA-GFDEESP------VHLSF 282
Os06g39470.1 AA------VGPLPVPT-ELRLQYLAMDISTDYIDHFKARFLEQTGH------RCSAFEVLIAKAWQSRTRAA-GFAPGSP------VHVCF 281
Bradi1g36980.1 AAP------VGPLPVPT-ELRLQYLAMDISTDYIEHFKARFLEQAGH------RCSAFEVLIAKAWQARTRAA-RFAPGTP------VHVCF 269
Os06g39390.1 IKP------GPLPELP-VLPLQYIAFDFPAAYLGKLKAQYAATAGA----SKICSAFDIVIAKLWQCRTRAI-AADPAAA------VKLCF 295
Os01g09010.1 IKP------GPLPGLP-VLALDYIVLDFPTGYIDGLKAQYKAHSG------KFCSGFDVLTAKLWQCRTRAL-NLEPGAT------VKLCF 272
Bradi2g05480.1 IKP------GPLPELP-VLALDYIVLDFPTGYIDGLKAQYKAHSG------KFCSGFDVLTAKLWQCRTRAL-NLEPGAT------VKLCF 298
Os05g19910.1 KLP------PGPPPSFQ-SFGFQHFSTDVTSDRIAHVKAEYFQTFGQ------YCSTFDVATAKVWQARTRAV-GYKPEIQ------VHVCF 282
Os01g18744.1 KLP------PGPPPELK-MFQLRHVTADLSLDSINKAKSAYFAATGH------RCSTFDVAIAKTWQARTRAL-RLPEPTSR------VNLCF 279
Os05g04584.1 RMP------APPPRLE-LLDLRYFTVDLSPDHIAKVKSAFFESTGH------RCSAFDVCVAKTWQARTRAL-VAAAAAAGDDDQERRTVRVCF 280
Bradi2g36910.1 KMP------APPPKLE-LLDLRHFTVDLSPDHIAKVKSQYFASTGH------RCSAFDVVVAVTWQSRTRAL-RLAGAGY---DD----VHVCF 294
Os01g42880.1 KPP------LGPPPSFT-AFNFEKSVIEISLDSIKRVKDQVASET------NQKCSTFDVVTAMMFKSRTLAI-DFAPDAD------VRLGF 273
Bradi2g43520.1 KPP------LGPPPSFT-AFNFEKSVVEISLDSIKRVKDQVASET------SQKCSTFDVVTAIIFKCRALAI-GFAPEAD------VRLGF 272
Os01g42870.1 KPPP------RGPPPSFT-AFNFVTQVVDVSPESIARIKEDFSASPTG---GGQACSTFDAVTAVVFRCRALAASSLPDDAE------VRLGF 295
Bradi2g43510.1 KLP------RGPPPSFT-AFNFVTQVVEISPENIARIKEDFKAAT-----GGETCSTFDAVTAVVFKCRALAV-ELPDTAE------VRLGF 275
Os04g09590.1 LVAA------IEKTMLSLETKRFAYLDITIPSTMIERVKAEYAAAAGNVDSG-EPCTVFEAVTAALWRSRTRAVISS--DDPD------APAPLVF 282
Os04g11810.1 LVAA------MEKAMLTQESKQFAYLDVTIPSSVIGRVKAAFDDVGDVARSGGEPCTVFEAVAAVLWRSRTRAVLISGDSDAD------TPAPLVF 290
AT5G41040.1 KIENLHQEFEEIEDKSNINSLYTKEPTLYRSFCFDPEKIKKLKLQATENSESL--LGNSCTSFEALSAFVWRARTKSLKMLSDQKT------KLLF 313
Bradi5g01240.1 LISFPHREFEEIPDVSGMAALYGDQDLTYRSFCFGPDRLERVRGLAQSQAQ----A--SFTTFEALSGLVWRARTAALGLAPEQQT------KLLF 311
* 20 * 40 * 60 * 80 * 500
Bradi2g04990.1 AMNARPLLHAS--LPSAGAG------FYGNCYYIMRVSAPAGKVSGSSVPEVVKIIKDGKRRMPAEFARWASGEAGAGG----EDPYRITSDYRTLLVSD 372
Bradi2g04980.1 AMNARPLLHAS--LPSAGAG------FYGNCYYIMRVSAPAGKVSGSSVPEVVKIIKDGKRRMPAEFARWASGEAGAGG----EDPYRITSDYRTLLVSD 371
Os01g08380.1 AMNARPLLHAS--LPRGGAG------FYGNCYYIMRVSAPAGKVAGSSVTEVVKIIKDGKRRMPSEFSRWAAGDMAGG------DPYQITSDYRTLLVSD 371
Os05g08640.1 AMNARPLL-----LPRGGAG------FYGNCYYIMRVASTAGRVATASVTDVVRMIREGKKRLPSEFARWAAGEMAG------VDPYQITSDYRTLLVSD 373
Bradi2g33980.1 AMNARPLLHAR--LPSGGAG------FYGNCYYIMRVSSTAGKVASSSMADVVKIIKEGKKRLPSEFARWAAGEMAG------VDPYQITSDYRTLLVSD 368
Os06g39470.1 AMNARPVLR-----RALPDG------FYGNCYYIMRVTAAAGAVADASVNDVVRLIREGKKRLPGEFARWSGGGGGGE-----DDPYRITSDYRTLLVSD 365
Bradi1g36980.1 AMNARSALPQ---PRAVPDG------FYGNCYYIMRVSAPAEAVSDAPLHEVVRLIREGKKRLPSEFARWSRGEMNG------DPYRITSDCRTLLVSD 353
Os06g39390.1 FASARQVLG------LETG------YWGNAIFPVKVSAAAGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGG------ADPFQMTFDYESVYVSD 376
Os01g09010.1 FASVRHLLK------LDRG------YYGNSIFPVKMSAPSETVLSSSVMEVVDMIRQAKERMAVEFFQFAKEETE------QDPFQMTFNYESIYVSD 352
Bradi2g05480.1 FASVRHLLK------LDPG------YYGNSIFPVKMSAPSEKVLGSSVMEVIDMIREAKQRMAVEFFQFAKEETK------QDPFQMSFDYESIYVSD 378
Os05g19910.1 FANTRHLLTQV---LPKDGG------YYGNCFYPVTVTAIAEDVATKELLDVIKIIRDGKARLPMEFAKWASGDVKV------DPYALTFEHNVLFVSD 366
Os01g18744.1 FANTRHLMAGAAAWPAPAAGGNGGNGFYGNCFYPVSVVAESGAVEAADVAGVVGMIREAKARLPADFARWAVADFRE------DPYELSFTYDSLFVSD 372
Os05g04584.1 FANTRHLMLKGDGAAAAATG------FYGNCFYPVAAVASGGEVAGADIVDVVRIVRDAKARLAADVARWAVGGFEE------DPYELTFTYDSLFVSD 367
Bradi2g36910.1 FANTRHLMLHG---GAGAAG------FYGNCFYPVRATCGSAEVASADVAGVVKVVRDAKARLAGDVARWAVGGFEQ------DPYELTFTYDSLFVSD 378
Os01g42880.1 AASTRHLLNN---VLPSVDG------YYGNCVYPGGLAKTSQEVKDASLVEIVTAIREAKDVLSTRFLDWMSGGAKE------NHYNVSLDYGTLVVTD 357
Bradi2g43520.1 AAGTRHLLNN---VLPSVEG------YYGNCVYPGGLAKTSQDVKEASLVEIVTAIREAKDALSTRFLDWMSGGAKE------NHYNVSLDYGTLIVTD 356
Os01g42870.1 AASTRHLLAG---VLPAVDG------YYGNCVYPVGIARHRGAVRGAALAEVVGVVREAKEALAARFTDWLGGGGGG------DHYDVPLDYGTVTVSD 379
Bradi2g43510.1 AASTRHLLQG---VLPSVDG------YYGNCVYPVGITRSSKTIREAALTEVVGVMREAKEALTVRFTDWMRGGAKD------DHYNVPLDYGTVTVSD 359
Os04g09590.1 AANARKHVG------AKEG------YYGNCVTSQVAVPTSDEVANGDLKHVVRLIRRAKEEIPLQFKNAGGGGMNG---KRVKQLAGVLFGYSAFYVAS 366
Os04g11810.1 AANVHKHVG------AKHG------YYGNCVTSQVVAATSGEVANGDANDVVKLIRRAKELIPAQFEN-GGVAMNGAAARVERQLMSALFGYNAFYVAS 376
AT5G41040.1 AVDGRAKFEP-----QLPKG------YFGNGIVLTNSICEAGELIEKPLSFAVGLVREAIKMVTDGYMRSAIDYFEV------TRARPSLSSTLLITT 394
Bradi5g01240.1 AVDARRRLSP-----PLPRG------YFGNGIVLTNALATAGDLLASPVSSAAAMVQDAVRMVTDEYVRSAVDYFEA------TRARPSLASTLLITT 392
* 20 * 40 * 60 *
Bradi2g04990.1 WTRLGFAEVDYGWGPPAHVVPLTN-LDYIATCILVKPWAHKPGARLITQCVTPDRVAAFHQGMLDMN*----- 438
Bradi2g04980.1 WTRLGFAEVDYGWGPPAHVVPLTN-LDYIATCILVKPWAHKPGARLITQCVTPDRVAAFHQGMLDMN*----- 437
Os01g08380.1 WTRLGFAEVDYGWGPPAHVVPLTN-LDYIATCILVRPWAHKPGARLITQCVTPDRVAAFHEGLLDLN*----- 437
Os05g08640.1 WTRLGFAEVDYGWGPPGHVVPLTN-LDYIATCILVKPWAHKPGARLITQCVTPDRVTAFHDAMVDIN*----- 439
Bradi2g33980.1 WTRLGFAEVDYGWGPPAHVVPLTN-LDYIATCILVKPWAHKPGARLITQCVTPDRVAAFHDAMVDTN*----- 434
Os06g39470.1 WSRLGFAEVDYGWGAPVHVVPLTN-LDYIATCILVRPSAHKPGARLITQCVAADAVDAFHNDMMRLD*----- 431
Bradi1g36980.1 WSRLGFAEVDYGWGAPVHVVPLTN-LDYIATCILVRPSAHKPGARLITQCVAADGVDEFHRDMMRLD*----- 419
Os06g39390.1 WSKLGFNDVDYGYGAPSAAGPLVN-CDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARMREDLV*---- 443
Os01g09010.1 WSKLGFAEVDYGFGPPKFAGPLVN-NDFIASVVILKAPLPLDGTRMLASCVTKEHSEEFVRGMKEDLP*---- 419
Bradi2g05480.1 WSKLGFSDVDYGFGPPMFAGPLVN-NDFIASVVILKAPLPLDGTRMLASCVTKEHSDEFVRGMKEDMP*---- 445
Os05g19910.1 WTRLGFFEVDYGWGTPNHIIPFTY-ADYMAVAVLGAPPMPKKGTRIMTQCVENKCIKEFQDEMKAFI*----- 432
Os01g18744.1 WTRLGFLEADYGWGPPSHVIPFAY-YPFMAVAIIGAPPVPKTGARIMTQCVEDDHLPAFKEEIKAFDK*---- 439
Os05g04584.1 WTRLGFLDADYGWGTPSHVVPFSY-HPFMAVAVIGAPPAPKLGARVMTMCVEEAHLPEFRDQMNAFAAAN*-- 436
Bradi2g36910.1 WTRLGFLEADYGWGPPAHVVPFSY-HPFMAVAVIGAPPKPKLGSRVMTMCVEEDHLPEFRDQMNAFAFTAGK* 449
Os01g42880.1 WSHVGFNEVDYGFGEPSYVFTLNDDVNIVPSVVYLKPPKPKQGIRLVLQCVEAQHSKVFGEELQKLA*----- 424
Bradi2g43520.1 WSHVGFNEVDYGFGEPSYVFTMNDDVNIVPSVVYLKPPKPKQGIRLVLQCVEEQHSAVFREELQKLA*----- 423
Os01g42870.1 WSRVGFNEVDYGFGEPGYVFTLNDDVNIVASVIYLRPPAPKRGIRLMLRCVEEPHAAAFADELAKFA*----- 446
Bradi2g43510.1 WSRVGFNEVDYGFGEPGYVFTLNDDVNIVASVIYLKPPTPKRGIRLMLRCVEEPHAAVFADEIAKYA*----- 426
Os04g09590.1 WRNIGFEAPDFGGGRAARVMCHFE--PTGVPSCVACLPRDGGGASVLSLCVRDEHVDAFLAELATLG*----- 431
Os04g11810.1 WRNIGFEAVDFGGGRPARVMCHVG--PTAVPSCVACLPRDSGGASVLLLCVKEEHVDAFLAELESFK*----- 441
AT5G41040.1 WSRLGFHTTDFGWGEPILSGPVALP---EKEVTLFLSHGEQRRSINVLLGLPATAMDVFQEQFLQI*------457
Bradi5g01240.1 WSRLAFDGADFGWGQPAMSGPVTLP---EKEVILFLAHGQERKSINVLLGLPASAMDAFQELMDEI*------455
Figure S2: Content of p-coumaric acid in isolated cell walls (AIR) of leaves + stems of RNAi::BdAT1 transformants derived from (a) nine T2 plants generated from T0 transformation event 3-4, (b) five T2 plants generated from T0 transformation event 4-11, (c) five T2 plants generated from T0 transformation event 2-3, and corresponding control plants. CONT = control. Mean ± SEM of duplicate determinations from from 3-month-old plants. Asterisks indicate significant differences from the control (Tukey’s, α = 0.05).
Figure S3: Content of p-coumaric acid in isolated cell walls (AIR) of leaves + stems of RNAi::BdAT7 transformants derived from (a) six T2 plants generated from T0 transformation event 2_6, (b) five T 2 plants generated from T0 transformation event 4_11, and corresponding control plants. Mean ± SEM of duplicate determinations from from 3-month-old plants. Asterisks indicate significant differences from the control (Tukey’s, α = 0.05).
Figure S4: Content of p-coumaric acid in isolated cell walls (AIR) of leaves (a, c, e) and stems (b, d, f) of 35S::BdAT1 transformants derived from (a, b) eight T1 plants generated from T0 transformation event 18-19, (c, d) six T1 plants generated from T0 transformation event 18-8, (e, f) eight T1 plants generated from T0 transformation event 18-9, and corresponding control plants. Mean ± SEM of duplicate determinations from 3-month-old plants. Asterisks indicate significant differences from the control (Tukey’s, α = 0.05).
Table S1: Primer and probe sequences designed for cloning for vector construction and qRT-PCR experiments
Gene specific primers
BdAT1-1F AAGTTCACCTGCGGTGGATTCTCT 60.1°C
BdAT1-R AAAGTTGAAAGCGGTGAACGACGG 59.9°C
BdAT7-5F ACGTCGACTATCTGGAGTACCCTCT 59.8°C
BdAT7-1R AGATCTCGATGGCGAGGTACT 60.1°C
Cloning for RNAi vector
BdAT1-2F CACCAAGTTCACCTGCGGTGGATTCTCT 62.8°C
BdAT1-1R CGAAAGTGGAACATTTCTGGCTGGTC 59.8°C
BdAT7-F1 CACCAAACTGGTAGTGAAGAAAGGCATC 62.0°C
BdAT7-R TCTTACATGGACAGAACAACACCT 59.9°C
Cloning for Over-expression vector
BdAT1-5F ACACATCCTCCTCCGGAGCTC 60.8°C
BdAT1-5R1 ACAGTCTAGAGACACGGCCAA 60.5°C
qRT-PCR
BdAT1-F CAACCCTCCTAAGCCACCTCTT
BdAT7-F AAGGCGCCACCGACTAGTCT
SamDC-F GAGGAGGATGGCGTGCTGTA
UBC18-F CCTCCAAAGGTGGGTGATTG
BdAT1-R GCTGGTCTCACTCGCAACCT
BdAT7-R AAAAGTCTATACACCTTGCGGGTTT
SamDC-R GGCTGGCTGCTTCACAGAATA
UBC18-R GGGATAATGCTCGGGGAAGT
Probes for qRT-PCR
BdAT1 ACCGTCGTTCACTGCT
BdAT7 CCTTCCGTCAGCGTAT
SamDC CCCGTGCAATTTG
UBC18 CCTCTACGCTGGTGAGAC
Primers for RT-PCR Notes
BdAT9-F TCGTGGAGCCGGAGAAAGAGG Product size is 2274 bp
BdAT9-R GGAAGTCCAGCACGATGTAGTCCA
BdAT1-F TCCGTCGACTTCATCCAGGTCTT Product size is 1493 bp
BdAT1-R AAAGTTGAAAGCGGTGAACGACGG
BdAT8-F ATCGAGTCCCTCCACGTCTTCAA Product size is 575 bp
BdAT8-R ATCGAGTCCCTCCACGTCTTCAA
BdAT21-F TGCAGACGGTCTACTGCTTCAAGT Primers designed across
intron. Product size is 379 bp
BdAT21-R AACATGCAGTGGTTCATCGCCA
BdAT2-F CACTACAATGTGCCTTTGGACTAC Product size is 313 bp
BdAT2-R TAGATACTGCAAGAGCGGTTACAC
BdAT6a-F TTCTACGGCAACTGCTACTACATC Product size is 387 bp
BdAT6a-R GTGGAACGCAGCTACTCTGTC
BdAT6b-F GAGTCGCTCCACGTCTTCA Product size is 460 bp
BdAT6b-R GCTCCATTGACAGGGACTCT
BdAT7-F AAACTGGTAGTGAAGAAAGGCATC Product size is 341 bp
BdAT7-R TCTTACATGGACAGAACAACACCT
BdAT3-F CATCTTCGTCTTCCCCAACA Product size is 383 bp
BdAT3-R CTCCTTGGGGATCACCATCT
GAPDH-F AGAAGGTTGTCATCTCTGCACCCA Product size is 391 bp
GAPDH-R ACAGTGAGATCACCAACCGACACA
Table S2: Sequences of theRNAi fragments
BdAT1 RNAi sequence (301 bp)
1AAGTTCACCTGCGGTGGATTCTCTGTGGGTATTTGCTTCAGCCACCTGGTGTTTGATGGG
61CAGGGCGCAGCCCAGTTTCTGAAAGCAGTCGGCGAGATGGCACGAGGGCTCCCTGAGCCA
121TCACTTAAGCCCATCTGGTCCCGTGACGCCATTCCCAACCCTCCTAAGCCACCTCTTGGA
181CCACCACCGTCGTTCACCGCTTTCAACTTTGAGAAGTCAGTGGTTGAGATCTCTCTTGAC
241AGCATCAAGCGTGTCAAAGACCAGGTTGCGAGTGAGACCAGCCAGAAATGTTCCACTTTC
301G
BdAT7 RNAi sequence (341 bp)
1AAACTGGTAGTGAAGAAAGGCATCAAAGAAGGACGGAAGAAAGAAGAAGAAGACTAGAAC
61AGAACATAACGATATGGGGGACAGGTAGACACAGCTAAATGGCGGTTGGCAATAGTGGAA
121TTGGTATTGGATTCCTTTGGACGTTCTCGGTGGTGTTTGGTGAAGACTGTGTACGACAAG
181GGGGTTCTAGTTGAAGGATCAAGGCGCCACCGACTAGTCTCCCCTTCCGTCAGCGTATTG
241TACTATTCAATTTTATTTTTTATTTTTTAAAATATAAACCCGCAAGGTGTATAGACTTTT
301CTGTAGTTGATATCTTCAGGTGTTGTTCTGTCCATGTAAGA
Table S3: Summary of plant transformations
Construct / Transformations / Calli on regeneration medium / Plants regenerated / Plants that survived / Regeneration percentageRNAi::BdAT1 / 2 / 340 / 38 / 19 / 5.6
RNAi::BdAT7 / 2 / 120 / 19 / 11 / 9.2
35S::BdAT1 / 4 / 696 / 45 / 16 / 2.3
Table S4: Results for Blastn of the primer, probe and RNAi trigger sequences for BdAT1 and BdAT7 against the Brachypodium genome assembly v2.1 and Brachypodium sequences in the GenBank Nucleotide Collection.