Supplemental Data Figure S1. Alignments of Putative Amino Acids Sequences of Mips Transporting

Supplemental data Figure S1. Alignments of putative amino acids sequences of MIPs transporting non-aqua substrates.

Ammonia Transporters

AtTIP2_1 ------MAG-VAFGSFDDSFSLASLRAYLAEFISTLLFVFAGVGSAIA 41(60)

AtTIP2_3 ------MVK-IEVGSVGDSFSVSSLKAYLSEFIATLLFVFAGVGSAVA 41(60)

TaTIP2_1 ------MPGSIAFGRFDDSFSLASFRAYIAEFISTLIFVFAGVGSVIA 42(60)

TaTIP2_2 ------MPGSIAFGRFDDSFSLASFKAYIAEFISTLIFVFAGVGSAIA 42(60)

GmNOD26 MADYSAGTESQEVVVNVTKNTSETIQRSDSLVSVPFLQKLVAEAVGTYFLIFAGCASLVV 60(60)

. .. .*:. :: ::* :.* :::*** .* :.

AtTIP2_1 YAKLTSDAALDTPGLVAIAVCHGFALFVAVAIGANISGGHVNPAVTFGLAVGGQITVITG 101(120)

AtTIP2_3 FAKLTSDGALDPAGLVAIAIAHAFALFVGVSIAANISGGHLNPAVTLGLAIGGNITLITG 101(120)

TaTIP2_1 YTKVSGGAPLDPSGLIAVAICHGFGLFVAVAIGANISGGHVNPAVTFGLALGGQITILTG 102(120)

TaTIP2_2 YTKVSGGAPLDPSGLIAVAICHGFGLFVAVAIGANISGGHVNPAVTFGLALGGQITILTG 102(120)

GmNOD26 NENYYNMITFP-----GIAIVWGLVLTVLVYTVGHISGGHFNPAVTIAFASTRRFPLIQV 115(120)

: . .: .:*: .: * * * .:*****.*****:.:* .:.::

AtTIP2_1 VFYWIAQLLGSTAACFLLKYVTGGLAVPTHSVAAGLGSIEGVVMEIIITFALVYTVYATA 161(180)

AtTIP2_3 FFYWIAQCLGSIVACLLLVFVTNGKSVPTHGVSAGLGAVEGVVMEIVVTFALVYTVYATA 161(180)

TaTIP2_1 IFYWVAQLLGAIVGAFLVQFCT-GVATPTHGLS-GVGAFEGVVMEIIVTFGLVYTVYATA 160(180)

TaTIP2_2 IFYWVAQLLGAIVGAFLVQFCT-GVATPTHGLS-GVGAFEGVVMEIIVTFGLVYTVYATA 160(180)

GmNOD26 PAYVVAQLLGSILASGTLRLLFMGNHDQFSGTVPNGTNLQAFVFEFIMTFFLMFVICGVA 175(180)

* :** **: .. : * . . .:..*:*:::** *::.: ..*

AtTIP2_1 ADPKKGSLGTIAPLAIGLIVGANILAAGPFSGGSMNPARSFGPAVAAGDFSGHWVYWVGP 221(240)

AtTIP2_3 ADPKKGSLGTIAPIAIGFIVGANILAAGPFSGGSMNPARSFGPAVVSGDLSQIWIYWVGP 221(240)

TaTIP2_1 ADPKKGSLGTIAPIAIGFIVGANILVAGPFSGGSMNPARSFGPAVASGDFTNIWVYWAGP 220(240)

TaTIP2_2 ADPKKGSLGTIAPIAIGFIVGANILVAGPFSGGSMNPARSFGPAVASGDFTNIWIYWAGP 220(240)

GmNOD26 TDNR--AVGEFAGIAIGSTLLLNVIIGGPVTGASMNPARSLGPAFVHGEYEGIWIYLLAP 233(240)

:* : ::* :* :*** : *:: .**.:*.*******:***.. *: *:* .*

AtTIP2_1 LIGGGLAGLIYGNVFMGSSEHVPLASADF------250(278)

AtTIP2_3 LVGGALAGLIYGDVFIGSYEAVETREIRV------250(278)

TaTIP2_1 LIGGGLAGVVYRYLYMCD-DHSSVAGNDY------248(278)

TaTIP2_2 LIGGGLAGVVYRYVYMCD-DHSSVAGNDY------248(278)

GmNOD26 VVGAIAGAWVYNIVRYTDKPLSETTKSASFLKGRAASK 271(278)

::*. .. :* : .

Boric Acid Transporters

HvPIP1_3 ------MEGKEEDVRLGANRYSEHQPIGTAAQGG------GADEK 33(60)

HvPIP1_4 ------MEGKEEDVRLGANRYSERQPIGTAAQGG------GADEK 33(60)

ZmPIP1-1 ------MEGKEEDVRLGANKFSERHAIGTAAQG------TDDK 31(60)

AtNIP5_1 MAPPEAEVGAVMVMAPPTPG-TPGTPGGPLITGMRVDSMSFDHRKPTP----RCKCLPVM 55(60)

AtNIP6_1 MDHEEIPS------TPSTPATTPGTPGAPLFGGFEGKRNGHNGRYTPKSLLKSCKCFSVD 54(60)

OsNIP2_1 ------MASNNSRTNSRANYSNEIHDLSTVQNG------TMPTM 32(60)

. . :

HvPIP1_3 DYKEPPPAPFFE----AGELTSWSFYRAGIAEFLATFLFLYISVLTVMGVVGNPSGSKCG 89 (120)

HvPIP1_4 DYKEPPPAPLFE----AEELSSWSFYRAGIAEFLATFLFLYISVLTVMGVVGNPSGSKCG 89 (120)

ZmPIP1-1 DYKEPPPAPLFE----PGELKSWSFYRPGIAEFVATFLFLYISILTVMGVS--KSTSKCA 85 (120)

AtNIP5_1 GSTWGQHDTCFT----DFPSPDVSLTRKLGAEFVGTFILIFTATAGPIVNQ-----KYDG 106(120)

AtNIP6_1 N-EWALEDGRLPPVTCSLPPPNVSLYRKLGAEFVGTLILIFAGTATAIVNQ-----KTDG 108(120)

OsNIP2_1 YYGEKAIADFFP------PHLLKKVVSEVVATFLLVFMTCGAAGISG-----SDLS 77 (120)

: : : :*.:.*::::: . .

HvPIP1_3 TVGIQGIAWSFGGMIFVLVYCTAGISGGHINPAVTFGLFLARKLSLTRAVFYIVMQCLGA 149(180)

HvPIP1_4 TVGIQGIAWSFGGMIFVLVYCTAGISGGHINPAVTFGLFLARKLSLTRAVFYIVMQCLGA 149(180)

ZmPIP1-1 TVGIQGIAWSFGGMILALVYCTAGISG-HINPAVTFGLFLARKLSLTRAVFYIIMQCLGA 144(180)

AtNIP5_1 AETLIGNAACAGLAVMIIILSTGHISGAHLNPSLTIAFAALRHFPWAHVPAYIAAQVSAS 166(180)

AtNIP6_1 AETLIGCAASAGLAVMIVILSTGHISGAHLNPAVTIAFAALKHFPWKHVPVYIGAQVMAS 168(180)

OsNIP2_1 RISQLGQSIAGGLIVTVMIYAVGHISGAHMNPAVTLAFAVFRHFPWIQVPFYWAAQFTGA 137(180)

* : . * : :: ... *** *:**::*:.: :::. :. * * .:

HvPIP1_3 ICGAGVVKGFQTTLYQGNGGGANSVAAGYTKGDGLGAEIVGTFVLVYTVFSATDAKRSAR 209(240)

HvPIP1_4 ICGAGVVKGFQTTLYQGNGGGANSVAAGYTKGDGLGAEIVGTFVLVYTVFSATDAKRSAR 209(240)

ZmPIP1-1 ICGRGVVKGFQQGLYMGNGGRRNVVAPGYTKGDGLGAEIVGTFILVYTVFSATDAKRRAR 204(240)

AtNIP5_1 ICASFALKGVFHPFMSGG-----VTIPSVSLGQAFALEFIITFILLFVVTAVATDTR--- 218(240)

AtNIP6_1 VSAAFALKAVFEPTMSGG-----VTVPTVGLSQAFALEFIISFNLMFVVTAVATDTR--- 220(240)

OsNIP2_1 ICASFVLKAVIHPVDVIG-----TTTPVGPHWHSLVVEVIVTFNMMFVTLAVATDTR--- 189(240)

:.. .:*...... : *.: :* :::.. :.: .*

HvPIP1_3 DSHVPILAPLPIGFAVFLVHLATIPITGTGINPARSLGAAIIYNKKQAWDDHWIFWVGPF 269(300)

HvPIP1_4 DSHVPILAPLPIGFAVFLVHLATIPITGTGINPARSLGAAIIYNKKQAWDDHWIFWVGPF 269(300)

ZmPIP1-1 DSHVPILAPLPIGFAVFLVHLATMGITGTGINPARSLGAAVIYNQHHAWADHWIFWVGPF 264(300)

AtNIP5_1 --AVGELAGIAVGATVMLNILVAGPSTGGSMNPVRTLGPAVASGN---YRSLWVYLVAPT 273(300)

AtNIP6_1 --AVGELAGIAVGATVMLNILIAGPATSASMNPVRTLGPAIAANN---YRAIWVYLTAPI 275(300)

OsNIP2_1 --AVGELAGLAVGSAVCITSIFAGAISGGSMNPARTLGPALASNK---FDGLWIYFLGPV 244(300)

* ** :.:* :* : : : :. .:**.*:**.*: .: : *:: .*

HvPIP1_3 IGAALAAIYHVVVIRAIPFKSRG------292(354)

HvPIP1_4 IGAALAAIYHVVVIRAIPFKSRD------292(354)

ZmPIP1-1 IGAALAAIYHQVIIRAIPFKSRS------287(354)

AtNIP5_1 LGAISGAAVYTGVKLNDSVTDPPRPVRS---FRR------304(354)

AtNIP6_1 LGALIGAGTYTIVKLPE-EDEAPKERRS---FRR------305(354)

OsNIP2_1 MGTLSGAWTYTFIRFEDTPKEGSSQKLSSFKLRRLRSQQSIAADDVDEMENIQV 298(354)

:*: .* : : .

CO2 Transporters

NtAQP1 MAENKEEDVKLGANKFRETQPLGTAAQTDKDYKEPPPAPLFEPGELSSWSFYRAGIAEFM 60(60)

AtPIP1_2 -MEGKEEDVRVGANKFPERQPIGTSAQSDKDYKEPPPAPLFEPGELASWSFWRAGIAEFI 59(60)

HvPIP2_1 ------MAKDIEAAPQG-GEFSSKDYSDPPPAPIVDFEELTKWSLYRAVIAEFV 47(60)

* * * * . :.***.:*****:.: **:.**::** ****:

NtAQP1 ATFLFLYITILTVMGLKR------SDSLCSSVGIQGVAWAFGGMIFALVYCTAGISGG 112(120)

AtPIP1_2 ATFLFLYITVLTVMGVKR------SPNMCASVGIQGIAWAFGGMIFALVYCTAGISGG 111(120)

HvPIP2_1 ATLLFLYITVATVIGYKHQSDPTVNTTDAACSGVGTLGIAWAFGGMIFVLVYCTAGVSGG 107(120)

**:******: **:* *: : *:.** *:*********.*******:***

NtAQP1 HINPAVTFGLFLARKLSLTRAIFYIVMQCLGAICGAGVVKGFMVGPYQRLGGGANVVNHG 172(180)

AtPIP1_2 HINPAVTFGLFLARKLSLTRAVYYIVMQCLGAICGAGVVKGFQPKQYQALGGGANTIAHG 171(180)

HvPIP2_1 HINPAVTFGLFLARKVSLIRALLYIIAQCLGAICGVGLVKGFQSSYYVRYGGGANELSAG 167(180)

***************:** **: **: ********.*:**** * ***** : *

NtAQP1 YTKGDGLGAEIIGTFVLVYTVFSATDAKRNARDSYVPILAPLPIGFAVFLVHLATIPITG 232(240)

AtPIP1_2 YTKGSGLGAEIIGTFVLVYTVFSATDAKRNARDSHVPILAPLPIGFAVFLVHLATIPITG 231(240)

HvPIP2_1 YSKGTGLAAEIIGTFVLVYTVFSATDPKRNARDSHIPVLAPLPIGFAVFMVHLATIPITG 227(240)

*:** **.******************.*******::*:***********:**********

NtAQP1 TGINPARSLGAAIIYNTDQAWDDHWIFWVGPFIGAALAAVYHQIIIRAIPFHKSS---- 287(299)

AtPIP1_2 TGINPARSLGAAIIFNKDNAWDDHWVFWVGPFIGAALAALYHVIVIRAIPFKSRS---- 286(299)

HvPIP2_1 TGINPARSLGAAVIYNTDKAWDDQWIFWVGPLIGAAIAAAYHQYVLRASAAKLGSYRSN 286(299)

************:*:*.*:****:*:*****:****:** ** ::** . : *

H2O2 Transporters

AtTIP1_1 MPIRNIAIG------RPDEATRPDALKAALAEFISTLIFVVAGSGSG 41(60)

AtTIP1_2 MPTRNIAIGG------VQEEVYHPNALRAALAEFISTLIFVFAGSGSG 42(60)

AtTIP2_3 --MVKIEVG------SVGDSFSVSSLKAYLSEFIATLLFVFAGVGSA 39(60)

AtPIP2_1 -MAKDVEAVPGEGFQTRDYQDPPPAPFIDGAELKKWSFYRAVIAEFVATLLFLYITVLTV 59(60)

AtPIP2_4 -MAKDLDVNESGPPAARDYKDPPPAPFFDMEELRKWPLYRAVIAEFVATLLFLYVSILTV 59(60)

AtNIP1_2 --MAEISGNGGDARDGAVVVNLKEEDEQQQQQQAIHKPLKKQDSLLSISVPFLQKLMAEV 58(60)

.: : : : : :: *:

AtTIP1_1 MA-----FNKLTEN--GATTPSGLVAAAVA--HAFGLFVAVSVGANISGGHVNPAVTFGA 92 (120)

AtTIP1_2 IA-----FNKITDN--GATTPSGLVAAALA--HAFGLFVAVSVGANISGGHVNPAVTFGV 93 (120)

AtTIP2_3 VA-----FAKLTSD--GALDPAGLVAIAIA--HAFALFVGVSIAANISGGHLNPAVTLGL 90 (120)

AtPIP2_1 IG-----YKIQSDTDAGGVDCGGVGILGIAWAFGGMIFILVYCTAGISGGHINPAVTFGL 114(120)

AtPIP2_4 IG-----YKAQTDATAGGVDCGGVGILGIAWAFGGMIFVLVYCTAGISGGHINPAVTVGL 114(120)

AtNIP1_2 LGTYFLIFAGCAAVAVNTQHDKAVTLPGIAIVWGLTVMVLVYSLGHISGAHFNPAVTIAF 118(120)

:. : : . .: .:* .:. ::: * . ***.*.*****..

AtTIP1_1 FIGGNITLLRGILYWIAQLLGSVVACLILKFATGGLAVPAFG----LSAGVG---VLNAF 145(180)

AtTIP1_2 LLGGNITLLRGILYWIAQLLGSVAACFLLSFATGGEPIPAFG----LSAGVG---SLNAL 146(180)

AtTIP2_3 AIGGNITLITGFFYWIAQCLGSIVACLLLVFVTNGKSVPTHG----VSAGLG---AVEGV 143(180)

AtPIP2_1 FLARKVSLPRALLYIIAQCLGAICGVGFVKAFQSSYYTRYGGGANSLADGYS---TGTGL 171(180)

AtPIP2_4 FLARKVSLVRTVLYIVAQCLGAICGCGFVKAFQSSYYTRYGGGANELADGYN---KGTGL 171(180)

AtNIP1_2 ASCGRFPLKQVPAYVISQVIGSTLAAATLRLLFGLDQDVCSGKHDVFVGTLPSGSNLQSF 178(180)

...* * ::* :*: . : . * . ..

AtTIP1_1 VFEIVMTFGLVYTVYATAIDPKNGS----LGTIAPIAIGFIVGANILAGGAFSGASMNPA 201(240)

AtTIP1_2 VFEIVMTFGLVYTVYATAVDPKNGS----LGTIAPIAIGFIVGANILAGGAFSGASMNPA 202(240)

AtTIP2_3 VMEIVVTFALVYTVYATAADPKKGS----LGTIAPIAIGFIVGANILAAGPFSGGSMNPA 199(240)

AtPIP2_1 AAEIIGTFVLVYTVFS-ATDPKRSARDSHVPVLAPLPIGFAVFMVHLATIPITGTGINPA 230(240)

AtPIP2_4 GAEIIGTFVLVYTVFS-ATDPKRNARDSHVPVLAPLPIGFAVFMVHLATIPITGTGINPA 230(240)

AtNIP1_2 VIEFIITFYLMFVISGVATDNRAIG------ELAGLAVGSTVLLNVIIAGPVSGASMNPG 232(240)

*:: ** *::.: . * * : . :* :.:* * : ..:* .:**.

AtTIP1_1 VAFGPAVVS---WTWTNHWVYWAGPLVGGGIAGLIYEVF-FINT-THEQLPTTDY----- 251(300)

AtTIP1_2 VAFGPAVVS---WTWTNHWVYWAGPLIGGGLAGIIYDFV-FIDENAHEQLPTTDY----- 253(300)

AtTIP2_3 RSFGPAVVS---GDLSQIWIYWVGPLVGGALAGLIYGDV-FIGS--YEAVETREIRV--- 250(300)

AtPIP2_1 RSFGAAVIYNKSKPWDDHWIFWVGPFIGAAIAAFYHQFV-LRASGSKSLGSFRSAANV-- 287(300)

AtPIP2_4 RSFGAAVIYNNEKAWDDQWIFWVGPMIGAAAAAFYHQFI-LRAAAIKALGSFGSFGSFRS 289(300)

AtNIP1_2 RSLGPAMVY---SCYRGLWIYIVSPIVGAVSGAWVYNMVRYTDKPLREITKSGSFLKTVR 289(300)

::*.*:: *:: ..*::*. .. : . .

AtTIP1_1 -----

AtTIP1_2 -----

AtTIP2_3 -----

AtPIP2_1 -----

AtPIP2_4 FA--- 291(305)

AtNIP1_2 NGSSR 294(305)

Silicic Acid Transporters

HvNIP2_1 --MASN-SRSNSRATFSSEIHDIGTVQNSTTPSMVYYTERSIADYFPPHLLKKVVSEVVS 57(60)

ZmNIP2_1 --MSTN-SRSNSRANFNNEIHDIGTAQNSSMPPTYY--DRSLADIFPPHLLKKVVSEVVS 55(60)

OsNIP2_1 --MASNNSRTNSRANYSNEIHDLSTVQNGTMPTMYYG-EKAIADFFPPHLLKKVVSEVVA 57(60)

OsNIP2_2 MASTTAPSRTNSRVNYSNEIHDLSTVQSVSAVPSVYYPEKSFADIFPPNLLKKVISEVVA 60(60)

ZmNIP2_2 MAAASTTSRTNSRVNYSNEIHDLSTVQSGSVVPTLFYPDKSIADIFPPHLGKKVISEVVA 60(60)

:: **:***..:..****:.*.*. : . : ::::** ***:* ***:****:

HvNIP2_1 TFLLVFVTCGAAAISAHDVTRISQLGQSVAGGLIVVVMIYAVGHISGAHMNPAVTLAFAI 117(120)

ZmNIP2_1 TFLLVFVTCGAAGIYGSDKDRISQLGQSVAGGLIVTVMIYAVGHISGAHMNPAVTLAFAV 115(120)

OsNIP2_1 TFLLVFMTCGAAGISGSDLSRISQLGQSIAGGLIVTVMIYAVGHISGAHMNPAVTLAFAV 117(120)

OsNIP2_2 TFLLVFVTCGAASIYGEDMKRISQLGQSVVGGLIVTVMIYATGHISGAHMNPAVTLSFAF 120(120)

ZmNIP2_2 TFLLVFVTCGAASIYGEDNRRISQLGQSVAGGLIVTVMIYATGHISGAHMNPAVTLSFAC 120(120)

******:*****.* . * ********:.*****.*****.**************:**

HvNIP2_1 FRHFPWIQVPFYWAAQFTGAICASFVLKAVLHPITVIGTTEPVGPHWHALVIEVVVTFNM 177(180)

ZmNIP2_1 FRHFPWIQVPFYWAAQFTGSICASFVLKAVLHPIAVLGTTTPTGPHWHSLVIEIIVTFNM 175(180)

OsNIP2_1 FRHFPWIQVPFYWAAQFTGAICASFVLKAVIHPVDVIGTTTPVGPHWHSLVVEVIVTFNM 177(180)

OsNIP2_2 FRHFPWIQVPFYWAAQFTGAMCAAFVLRAVLYPIEVLGTTTPTGPHWHALVIEIVVTFNM 180(180)

ZmNIP2_2 FRHFPWIQVPFYWAAQFTGAMCAAFVLKAVLHPIAVIGTTTPSGPHWHALLIEIVVTFNM 180(180)

*******************::**:***:**::*: *:*** * *****:*::*::*****

HvNIP2_1 MFVTLAVATDTRAVGELAGLAVGSSVCITSIFAGAVSGGSMNPARTLGPALASNRYPGLW 237(240)

ZmNIP2_1 MFVTLAVATDTRAVGELAGLAVGSAVCITSIFAGAVSGGSMNPARTLGPALASNLYTGLW 235(240)

OsNIP2_1 MFVTLAVATDTRAVGELAGLAVGSAVCITSIFAGAISGGSMNPARTLGPALASNKFDGLW 237(240)

OsNIP2_2 MFVTCAVATDSRAVGELAGLAVGSAVCITSIFAGPVSGGSMNPARTLAPAVASNVYTGLW 240(240)

ZmNIP2_2 MFVTCAVATDSRAVGELAGLAVGSAVCITSIFAGPVSGGSMNPARTLAPAVASNVFTGLW 240(240)

**** *****:*************:*********.:***********.**:*** : ***

HvNIP2_1 LYFLGPVLGTLSGAWTYTYIRFEDPP--KDA--PQKLSSFKLRRLQSQ-SVAADD-DELD 291(300)

ZmNIP2_1 IYFLGPVLGTLSGAWTYTYIRFEEAPSHKDM--SQKLSSFKLRRLQSQ-SVAVDD-DELD 291(300)

OsNIP2_1 IYFLGPVMGTLSGAWTYTFIRFEDTP-KEGS--SQKLSSFKLRRLRSQQSIAADDVDEME 294(300)

OsNIP2_2 IYFLGPVVGTLSGAWVYTYIRFEEAPAAAGGAAPQKLSSFKLRRLQSQ-SMAADEFDNV- 298(300)

ZmNIP2_2 IYFLGPVIGTLSGAWVYTYIRFEEAPAAKD---TQRLSSFKLRRMQSQ--LAADEFDTV- 294(300)

:******:*******.**:****:.* . .*:********::** :*.*: * :

HvNIP2_1 HIPV 295(304)

ZmNIP2_1 HIQV 295(304)

OsNIP2_1 NIQV 298(304)

OsNIP2_2 ----

ZmNIP2_2 ----

Urea Transporters

NtAQP1 --MAENKEEDVKLGANKFRETQPLGTAAQ--TDKDYKEPPPAPLFEPGELSSWS------50(60)

ZmPIP1-5 ---MEGKEEDVRLGANRYSERQPIGTAAQGTEEKDYKEPPPAPLFEAEELTSWS------51(60)

NtTIPa ------MAKIAVG------SSREAIQPD------16(60)

AtTIP4_1 ------MKKIELG------HHSEAAKPD------16(60)

AtTIP1_1 -----MPIRNIAIG------RPDEATRPD------18(60)

AtTIP1_2 -----MPTRNIAIGG------VQEEVYHPN------19(60)

AtTIP1_3 -----MPINRIAIG------TPGEASRPD------18(60)

AtTIP2_1 ------MAGVAFG------SFDDSFSLA------16(60)

AtTIP5_1 ----MRRMIPTSFSS------KFQGVLSMN------20(60)

CpNIP1 -----MSSSQDPQLVQQ------QSVVDVEEFVSVENPDSKR 31(60)

OsNIP2_1 -----MASNNSRTNSRA------NYSNEIHDLSTVQNGTMPT 31(60)

AtNIP6_1 MDHEEIPSTPSTPATTPGTPGAPLFGGFEGKRNGHNGRYTPKSLLKSCKCFSVDNEWALE 60(60)

NtAQP1 ------FYRAGIAEFMATFLFLYITILTVMGLK--RSDSLCSSVGIQGV 91 (120)

ZmPIP1-5 ------FYRAGIAEFVATFLFLYISILTVMGVS--KSSSKCATVGIQGI 92 (120)

NtTIPa ------CIQALIVEFICTFLFVFAGVGSAMAANKLNGD---PLVSLFFV 56 (120)

AtTIP4_1 ------CIKALIVEFITTFLFVFAGVGSAMATDSLVGN---TLVGLFAV 56 (120)

AtTIP1_1 ------ALKAALAEFISTLIFVVAGSGSGMAFNKLTENGATTPSGLVAA 61 (120)

AtTIP1_2 ------ALRAALAEFISTLIFVFAGSGSGIAFNKITDNGATTPSGLVAA 62 (120)

AtTIP1_3 ------AIRAAFAEFFSMVIFVFAGQGSGMAYGKLTGDGPATPAGLVAA 61 (120)

AtTIP2_1 ------SLRAYLAEFISTLLFVFAGVGSAIAYAKLTSDAALDTPGLVAI 59 (120)

AtTIP5_1 ------ALRCYVSEFISTFFFVLAAVGSVMSSRKLMAGDVSGPFGVLIP 63 (120)

CpNIP1 SQFGS-LFKNHYPP--GFSRKLVAEVIATYLLVFVTCGAAALNGS-----DAQRVSQLGA 83 (120)

OsNIP2_1 MYYGEKAIADFFPP--HLLKKVVSEVVATFLLVFMTCGAAGISGS-----DLSRISQLGQ 84 (120)

AtNIP6_1 DGRLPPVTCSLPPPNVSLYRKLGAEFVGTLILIFAGTATAIVNQK-----TDGAETLIGC 115(120)

: *.. ::: :

NtAQP1 AWAFGGMIFALVYCTAGISGGHINPAVTFGLFLARKLSLTRAIFYIVMQCLGAICGAGVV 151(180)

ZmPIP1-5 AWSFGGMIFALVYCTAGISGGHINPAVTFGLFLARKLSLTRALFYMVMQCLGAICGAGVV 152(180)

NtTIPa AMAHALVVAVTISAGFRISGGHLNPAVTLGLCMGGHITVFRSILYWIDQLLASVAACALL 116(180)

AtTIP4_1 AVAHAFVVAVMISAG-HISGGHLNPAVTLGLLLGGHISVFRAFLYWIDQLLASSAACFLL 115(180)

AtTIP1_1 AVAHAFGLFVAVSVGANISGGHVNPAVTFGAFIGGNITLLRGILYWIAQLLGSVVACLIL 121(180)

AtTIP1_2 ALAHAFGLFVAVSVGANISGGHVNPAVTFGVLLGGNITLLRGILYWIAQLLGSVAACFLL 122(180)

AtTIP1_3 SLSHAFALFVAVSVGANVSGGHVNPAVTFGAFIGGNITLLRAILYWIAQLLGAVVACLLL 121(180)

AtTIP2_1 AVCHGFALFVAVAIGANISGGHVNPAVTFGLAVGGQITVITGVFYWIAQLLGSTAACFLL 119(180)

AtTIP5_1 AIANALALSSSVYISWNVSGGHVNPAVTFAMAVAGRISVPTAMFYWTSQMIASVMACLVL 123(180)

CpNIP1 SVAGGLIVTVMIYAVGHISGAHMNPAVTTAFAATRHFPWKQVPLYAAAQLSGATCAAFTL 143(180)

OsNIP2_1 SIAGGLIVTVMIYAVGHISGAHMNPAVTLAFAVFRHFPWIQVPFYWAAQFTGAICASFVL 144(180)

AtNIP6_1 AASAGLAVMIVILSTGHISGAHLNPAVTIAFAALKHFPWKHVPVYIGAQVMASVSAAFAL 175(180)

: . . : : :**.*:***** . .:. .* * .: .. :

NtAQP1 KGFMVGPYQRLGGGANVVNHGYTKGDGLGAEIIGTFVLVYTVFSATDAKRNARDSYVPIL 211(240)

ZmPIP1-5 KGFQEGLYMGAGGGANAVNPGYTKGDGLGAEIVGTFVLVYTVFSATDAKRSARDSHVPIL 212(240)

NtTIPa NYLTA----GLETPVHTLANGVSYGQGIIMEVILTFSLLFTVYTTIVDPK---KGILEGM 169(240)

AtTIP4_1 SYLTG----GMGTPVHTLASGVSYTQGIIWEIILTFSLLFTVYATIVDPK---KGSLDGF 168(240)

AtTIP1_1 KFATG----GLAVPAFGLSAGVGVLNAFVFEIVMTFGLVYTVYATAIDPK---NGSLGTI 174(240)

AtTIP1_2 SFATG----GEPIPAFGLSAGVGSLNALVFEIVMTFGLVYTVYATAVDPK---NGSLGTI 175(240)

AtTIP1_3 KVSTG----GMETAAFSLSYGVTPWNAVVFEIVMTFGLVYTVYATAVDPK---KGDIGII 174(240)

AtTIP2_1 KYVTG----GLAVPTHSVAAGLGSIEGVVMEIIITFALVYTVYATAADPK---KGSLGTI 172(240)

AtTIP5_1 KVTVM----EQHVPIYKIAGEMTGFGASVLEGVLAFVLVYTVFT-ASDPR---RGLPLAV 175(240)

CpNIP1 RLLLHP-----IKHLGTTTPSGSDLQALVMEIVVTFSMMFVTCAVATDTK-----AVGEL 193(240)

OsNIP2_1 KAVIHP-----VDVIGTTTPVGPHWHSLVVEVIVTFNMMFVTLAVATDTR-----AVGEL 194(240)

AtNIP6_1 KAVFEP-----TMSGGVTVPTVGLSQAFALEFIISFNLMFVVTAVATDTR-----AVGEL 225(240)

. * : :* :::.. : : .

NtAQP1 APLPIGFAVFLVHLATIPITGTGINPARSLGAAIIYNTDQAWDDHWIFWVGPFIGAALAA 271(300)

ZmPIP1-5 APLPIGFAVFLVHLATIPITGTGINPARSLGAAIVYNRSHAWNDHWIFWVGPFIGAALAA 272(300)

NtTIPa GPLLTGLVVGANIMAGGPFSGASMNPARSFGPAFVS---GIWTDHWVYWVGPLIGGGLAG 226(300)

AtTIP4_1 GPLLTGFVVGANILAGGAFSGASMNPARSFGPALVS---GNWTDHWVYWVGPLIGGGLAG 225(300)

AtTIP1_1 APIAIGFIVGANILAGGAFSGASMNPAVAFGPAVVS---WTWTNHWVYWAGPLVGGGIAG 231(300)

AtTIP1_2 APIAIGFIVGANILAGGAFSGASMNPAVAFGPAVVS---WTWTNHWVYWAGPLIGGGLAG 232(300)

AtTIP1_3 APLAIGLIVGANILVGGAFDGASMNPAVSFGPAVVS---WIWTNHWVYWVGPFIGAAIAA 231(300)

AtTIP2_1 APLAIGLIVGANILAAGPFSGGSMNPARSFGPAVAA---GDFSGHWVYWVGPLIGGGLAG 229(300)

AtTIP5_1 GPIFIGFVAGANVLAAGPFSGGSMNPACAFGSAMVY---GSFKNQAVYWVGPLLGGATAA 232(300)

CpNIP1 AGLAVGSAVCITSILAGPVSGGSMNPVRTLGPAMAS---DNYKGLWVYFVGPVTGTLLGA 250(300)

OsNIP2_1 AGLAVGSAVCITSIFAGAISGGSMNPARTLGPALAS---NKFDGLWIYFLGPVMGTLSGA 251(300)

AtNIP6_1 AGIAVGATVMLNILIAGPATSASMNPVRTLGPAIAA---NNYRAIWVYLTAPILGALIGA 282(300)

. : * . : . . .:**. ::*.*. : :: .*. * ..

NtAQP1 VYHQIIIRAIPFHKSS------287(347)

ZmPIP1-5 IYHVVIIRALPFKSRD------288(347)

NtTIPa FICENFFIVR-THVPLPSDESF------247(347)

AtTIP4_1 FIYENVLIDR-PHVPVADDEQPLLN------249(347)

AtTIP1_1 LIYEVFFINT-THEQLPTTDY------251(347)

AtTIP1_2 IIYDFVFIDENAHEQLPTTDY------253(347)

AtTIP1_3 IVYDTIFIGSNGHEPLPSNDF------252(347)

AtTIP2_1 LIYGNVFMGSSEHVPLASADF------250(347)

AtTIP5_1 LVYDNVVVPVEDDRGSSTGDAIGV------256(347)

CpNIP1 WSYKFIRASDKPVHLISPHSFSLKLRRMSR------SDVGEGER--- 288(347)

OsNIP2_1 WTYTFIRFEDTPKEGSSQKLSSFKLRRLRSQQSIAADDVDEMENIQV 298(347)

AtNIP6_1 GTYTIVKLPEEDEAPKERRSFRR------305(347)

.

Supplemental data Figure S1. Alignments of putative amino acids sequences of MIPs transporting non-aqua substrates.

Multiple alignments were performed using ClustalW2 ( Trans-membrane helices (TM1-TM6) and the two short helices forming the two NPAs (HB and HE) (shaded), P1-P5 residues (Froger et al., 1998) (shown in blue), NPA motifs (shown in red), ar/R selectivity filter residues (shown in green) and specificity determining positions (SDPs; highlighted in yellow) are also indicated. The positions corresponding to S115 and S274 in SoPIP2;1 (Tornroth-Horsefiled et al., 2006) are highlighted in green and those predicted to be phosphorylation sites shown in Table S4. The residues corresponding to S262 in GmNOD26 (Guenther et al., 2003) are shown in bold and the ones predicted to be phosphorylation sites are shown in Supplemental data Table S4. The residues at the position corresponding to L197 from SoPIP2;1, determined to be the key residue involved in gating (Tornroth-Horsefield et al., 2006)(occurring just before TM5) are underlined. The numbers at the end of each line indicate amino acid position excluding the introduced gaps (number without parentheses) or including the introduced gaps (number in parentheses).

Supplemental data Figure S2. Logoplots of the amino acids making the AEFXXT motif (left), surrounding the first (middle) and second (right) NPA motifs of MIPs transporting different substrates.

The loplots were made, based on the alignments in Fig.S1. The amino acids are coloured according to their chemical properties; polar (green), acidic (red), basic (blue), non-polar and hydrophobic (black) and asparagine (N) (purple).

A. B.

Supplemental Data Figure S3.

A. Superimposition of the ar/R filter residues of the ammonia transporters GmNOD26 (W-V-A-R) and TaTIP2;1 (H-I-G-R).

GmNOD26 residues labelled and shown in CPK colours: white for carbon, red for oxygen, blue for nitrogen, and yellow for sulphur. TaTIP2;1 residues shown in blue.

B. Superimposition of the ar/R filter residues of the boron transportersAtNIP5;1 (A-I-G-R) and HvPIP1;3 (F-H-T-R).

HvPIP1;3 residues labelled and shown in CPK colours: white for carbon, red for oxygen, blue for nitrogen, and yellow for sulphur. AtNIP5;1 residues shown in blue.

Supplemental dataFigure S4. Relationship of the AEFXXT motif with the NPA motif and the ar/R selectivity filter.

The AEFXXT motif shown in ball and stick model, E25 and T29 labelled, the two NPA motifs shown in space-fill and the ar/R filter residues (H-I-G-R) shown in ball and stick and labelled.

AtTIP2_3 ------VTNGKSVPTHGVSAGLG 17

AtTIP2_1 ------VTGGLAVPTHSVAAGLG 17

NtTIPa ------LTAGLETPVHTLANGVS 17

AtTIP4_1 ------LTGGMGTPVHTLASGVS 17

AtTIP1_1 ------ATGGLAVPAFGLSAGVG 17

AtTIP1_2 ------ATGGEPIPAFGLSAGVG 17

AtTIP1_3 ------STGGMETAAFSLSYGVT 17

TaTIP2_1 ------CTGVATPTHGLS-GVG 15

TaTIP2_2 ------CTGVATPTHGLS-GVG 15

HvPIP1_3 -FQ------TTLYQGNGGGANSVAAGYT 21

HvPIP1_4 -FQ------TTLYQGNGGGANSVAAGYT 21

ZmPIP1_1 -FQ------QGLYMGNGGRRNVVAPGYT 21

ZmPIP1_5 -FQ------EGLYMGAGGGANAVNPGYT 21

NtAQP1 -FM------VGPYQRLGGGANVVNHGYT 21

AtPIP1_2 -FQ------PKQYQALGGGANTIAHGYT 21

AtPIP2_4 -FQ------SSYYTRYGGGANELADGYN 21

AtPIP2_1 -FQ------SSYYTRYGGGANSLADGYS 21

HvPIP2_1 -FQ------SSYYVRYGGGANELSAGYS 21

AtNIP1_2 LFGLDQDVCSGKHDVFVGTLP---SGS- 24

GmNOD26 LF------MGNHDQFSGTVP---NGT- 17

OsNIP2_2 VL------YPIEVLGTTTP---TGP- 16

ZmNIP2_1 VL------HPIAVLGTTTP---TGP- 16

HvNIP2_1 VL------HPITVIGTTEP---VGP- 16

ZmNIP2_2 VL------HPIAVIGTTTP---SGP- 16

OsNIP2_1 VI------HPVDVIGTTTP---VGP- 16

CpNIP1 LL------HPIKHLGTTTP---SGS- 16

AtNIP5_1 VF------HPFMSGGVTIP--SVS-- 16

AtNIP6_1 VF------EPTMSGGVTVP--TVG-- 16

AtTIP5_1 ------TVMEQHVPIYKIAGEMT 17

Supplemental data Figure S5. Alignments of the loop C residues of MIPs with non-aqua transport substrates.

Multiple alignments were conducted using ClustalW2( The G residues are shown in bold and underlined.

AtNIP1_2 --TDNRA----IGEL 9

GmNOD26 --TDNRA----VGEF 9

ZmNIP2_2 --TDSRA----VGEL 9

ZmNIP2_1 --TDTRA----VGEL 9

HvNIP2_1 --TDTRA----VGEL 9

AtNIP5_1 --TDTRA----VGEL 9

AtNIP6_1 --TDTRA----VGEL 9

OsNIP2_1 --TDTRA----VGEL 9

OsNIP2_2 --TDSRA----VGEL 9

CpNIP1 --TDTKA----VGEL 9

AtTIP1_1 IDPKNGS----LGTI 11

AtTIP1_2 VDPKNGS----LGTI 11

AtTIP2_3 ADPKKGS----LGTI 11

AtTIP2_1 ADPKKGS----LGTI 11

TaTIP2_1 ADPKKGS----LGTI 11

TaTIP2_2 ADPKKGS----LGTI 11

AtTIP1_3 VDPKKGD----IGII 11

AtTIP4_1 VDPKKGS----LDGF 11

NtTIPa VDPKKGI----LEGM 11

AtPIP2_4 TDPKRNARDSHVPVL 15

HvPIP2_1 -DPKRNARDSHIPVL 14

NtAQP1 -DAKRNARDSYVPIL 14

AtPIP1_2 -DAKRNARDSHVPIL 14

ZmPIP1_5 -DAKRSARDSHVPIL 14

HvPIP1_3 -DAKRSARDSHVPIL 14

HvPIP1_4 -DAKRSARDSHVPIL 14

ZmPIP1_1 -DAKRRARDSHVPIL 14

SoPIP2_1 -DPKRSARDSHVPIL 14

AtPIP2_1 TDPKRSARDSHVPVL 15

AtTIP5_1 SDPRRGLP---LAV- 11

.

Supplemental data Figure S6. Alignments of loop D residues of MIPs with non-aqua transport substrates.

Alignments were conducted using ClustalW2 ( The arrow shows the position corresponding to L197 from SoPIP2;1 (shaded) determined by Tornroth-Horsefield et al. (2006) to be the key residue in aquaporin gating and the residues corresponding to the protonated H193 in SoPIP2;1 are underlined.