Characterization of an Ancient Clade of Plant Polyamine Uptake Transporters

Characterization of an ancient clade of plant polyamine uptake transporters

(Supplemental Data)

1Vaishali Mulungi, 2,3Marcus C. Chibucos, 1Vipaporn Phuntumart,1Paul F. Morris

1Biological Sciences,Bowling Green State University, Bowling Green, Ohio 43403

2Department of Microbiology and Immunology

3Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201

Table S1 Primer sequences used in this study

Primer / Sequence 5’ to 3’
Primers used for GATEWAY cloning to create entry clone
OsPUT2F / CACCATGACCGGAGCCTGCGAGGC
OsPUT2R / CTAATAATTCTCCTTGCTGACAC
OsPUT3.1F / CACCATGACGAACGCATGGATCTC
OsPUT3.1R / TCAGCACACAAGTGGGATTGTGC
AtPUT1F / CACCATGGGAGACTATAACATGAATG
AtPUT1R / ACGTAACAAAGACTCTTCACAATCC
AtPUT2F / CACCATGCAGAAGCGGAGAATCAT
AtPUT2R / ACGTATTAGAGTTTCTTCGTATTCCCGAG
AtPUT3F / CACCATGACTGAGCTTAGCTCTCCGA
AtPUT3R / CTCCATCAGGTTTGGTAGGTGAGAAC
Primers used for RT-PCR
Primer / Sequence / Number of cycles
RTOsPUT2F / TTCAACTCCATGTTCTGGAACC / 20
RTOsPUT2R / AGAGGAAGTTGAGGAACTCGA
RTOsPUT3.1F / ACGAACGCATGGATCTCGCCGTCGG / 20
RTOsPUT3.1R / GCCACTTTGCCCAGCCTTGCTGAA
OsACT1F / AGGAATGGAAGCTGCGGGTAT / 25
OsACT1R / GCAGGAGGACGGCGATAACA
RTAtPUT1F / CCTCATATTCTACGAAGTCTCAG / 25
RTAtPUT1F / AAATCCATCACAAGCCATCTC
RTAtPUT2F / GAATGTTTATAGCCGAGATGAG / 25
RTAtPUT2R / AGAACTGATGGAGAATTTGACC
RTAtPUT3F / ATCCGATTCTCTTCCTTGAC / 25
RTAtPUT3F / CCAAACTCCTCCTATAACTTTACC
AtACT1F / TATGTGGCTATTCAGGCTGT / 25
AtACT1R / TGGCGGTGCTTCTTCTCTG

Table S2Effect of various amino acids and polyamine precursors on spermidine uptake by PUTs.Spermidine was provided at a concentration of 15 µM (OsPUT1), 2 µM (OsPUT2, OsPUT3.1), 3 µM (AtPUT1) and 1 µM (AtPUT2, AtPUT3) and yeast cells were incubated for 5 mins with the inhibitors (500µM). The numbers represent percent reduction in uptake of [14C]spermidinecompared to the control (when no inhibitor added). Results are means ± SD for three independent assays

Amino acid / Polyamine precursor / OsPUT2 / OsPUT3.1 / AtPUT1 / AtPUT2 / AtPUT3
None / 0 / 0 / 0 / 0 / 0
Arginine / 18.2 ± 15 / 5.4 ± 12 / 9.0 ± 5 / 9.3 ± 5 / 12.4 ± 16
Aspartate / 19.3 ± 12 / 3.6 ± 4 / 13.5 ± 0 / 6.5 ± 5 / 14.1 ± 4
Glutamate / 25.8 ± 8 / 8.7 ± 2 / 3.7 ± 10 / 2.5 ± 10 / 12.7 ± 10
Histidine / 25.5 ±2 / 16.2 ± 6 / 1 4.0 ± 6 / 1.4 ± 8 / 22.9 ± 7
Lysine / 17.5 ± 9 / 23.8 ± 2 / 4.5 ± 7 / 11.5 ± 7 / 11.4 ± 7
Glycine / 29.5 ± 3 / 8.3 ± 10 / 14.5 ± 7 / 3.2 ± 1 / 9.5 ± 6
Methionine / 26.5 ± 7 / 3.2 ± 14 / 9.8 ± 8 / 15.4 ± 7 / 34.0 ± 7
Leucine / 34.5 ± 4 / 7.9 ± 2 / 26.4 ± 8 / 19.4 ± 1 / 41.2 ± 4
Asparagine / 14.2 ± 3 / 10.8 ± 1 / 14.8 ± 8 / 1.8 ± 3 / 18.0 ± 1
Tyrosine / 34.2 ± 8 / 20.9 ± 1 / 3.7 ± 6 / 1.4 ± 2 / 8.8 ± 8
Tryptophan / 22.9 ± 0 / 4.0 ± 1 / 10.3 ± 7 / 7.2 ± 8 / 8.5 ± 7
Glutamine / 4.7 ± 17 / 4.7 ± 13 / 10.3 ± 8 / 3.9 ± 8 / 13.1 ± 8
Cadaverine / 23.6 ± 10 / 6.1 ± 0 / 16.9 ± 5 / 12.9 ± 2 / 8.8 ± 5
Agmatine / 26.5 ± 3 / 19.9 ± 1 / 14.0 ± 7 / 5.4 ± 7 / 12.4 ± 7
Ornithine / 5.5 ± 13 / 38.3 ± 1 / 15.0 ± 4 / 4.7 ± 4 / 15.0 ± 4
Spermine / 2.5 ± 6 / 48.4 ± 4 / 25.1 ± 4 / 18.3 ± 3 / 12.4 ± 6

Table S3Hidden Markov model (HMM) match scores for conserved polyamine transporter regions against sequences from diverse species. An HMM generated from conserved regions of polyamine transporters found in Arabidopsis, Oryza, Leishmania, and Trypanosoma best matched proteins from angiosperms, moss, discicristates, and Volvox. Other matches included hetrokont, haptophyte, cryptophyte, rhodophyte, and amebozoan, followed by excavates, fungi, rhizaria, and choanoflagellate. The HMM did not have any significant matches to Candida or alveolate proteins.

Taxon / Model identifier / Overall sequence
E-value / Best domain
E-value
Leishmania major strain Friedlin / LmjF.14.0320 / 1.50E-262 / 2.00E-262
Leishmania infantum / LinJ.14.0320 / 2.50E-257 / 3.10E-257
Leishmania mexicana / LmxM.14.0320 / 4.60E-253 / 6.00E-253
Leishmania braziliensis / LbrM.14.0320 / 1.00E-245 / 1.30E-245
Trypanosoma cruzi CL Brener non-Esmeraldo-like / Tc00.1047053506985.40 / 1.80E-238 / 2.30E-238
Trypanosoma cruzi CL Brener Esmeraldo-like / Tc00.1047053504213.110 / 1.70E-236 / 2.10E-236
Ricinus communis / 29827.m002595|PACid:16810027 / 1.30E-212 / 1.60E-212
Mimulus guttatus / mgv1a003994m|PACid:17669487 / 6.10E-211 / 7.80E-211
Glycine max / Glyma01g35320.1|PACid:16245238 / 4.30E-210 / 5.40E-210
Trypanosoma vivax / TvY486_0704490 / 1.10E-209 / 1.50E-209
Glycine max / Glyma09g34750.1|PACid:16277197 / 3.20E-209 / 3.90E-209
Mimulus guttatus / mgv1a005577m|PACid:17677922 / 9.10E-208 / 1.20E-207
Ricinus communis / 30170.m014148|PACid:16821982 / 1.40E-206 / 1.70E-206
Mimulus guttatus / mgv1a019424m|PACid:17689435 / 6.50E-203 / 7.60E-203
Glycine max / Glyma20g24020.1|PACid:16316614 / 7.40E-202 / 8.90E-202
Glycine max / Glyma16g17460.1|PACid:16302285 / 1.50E-201 / 1.70E-201
Ricinus communis / 28152.m000900|PACid:16800050 / 1.60E-197 / 1.90E-197
Mimulus guttatus / mgv1a005195m|PACid:17683464 / 2.40E-195 / 3.00E-195
Physcomitrella patens / Pp1s297_16V6.2|PACid:18046500 / 2.50E-193 / 3.10E-193
Physcomitrella patens / Pp1s297_16V6.3|PACid:18046501 / 2.50E-193 / 3.10E-193
Selaginella moellendorffii / 107507|PACid:15421320 / 6.10E-193 / 7.60E-193
Physcomitrella patens / Pp1s240_55V6.2|PACid:18065765 / 9.40E-193 / 1.20E-192
Leishmania major strain Friedlin / LmjF.35.4410 / 9.60E-193 / 1.10E-192
Physcomitrella patens / Pp1s240_55V6.1|PACid:18065766 / 1.80E-192 / 2.40E-192
Leishmania infantum / LinJ.35.4480 / 3.70E-189 / 4.10E-189
Selaginella moellendorffii / 96930|PACid:15411622 / 7.10E-189 / 8.40E-189
Physcomitrella patens / Pp1s312_22V6.1|PACid:18046215 / 1.10E-188 / 1.30E-188
Physcomitrella patens / Pp1s297_16V6.1|PACid:18046502 / 6.10E-187 / 7.40E-187
Leishmania mexicana / LmxM.34.4410 / 8.10E-187 / 9.10E-187
Glycine max / Glyma01g45410.1|PACid:16246417 / 1.20E-185 / 1.40E-185
Selaginella moellendorffii / 418809|PACid:15409692 / 1.10E-178 / 1.40E-178
Leishmania braziliensis / LbrM.34.4390 / 1.10E-175 / 1.30E-175
Trypanosoma cruzi CL Brener Esmeraldo-like / Tc00.1047053509551.20 / 1.90E-157 / 2.60E-157
Trypanosoma brucei TREU927 / Tb09.211.1760 / 6.70E-152 / 7.80E-152
Trypanosoma brucei gambiense / Tbg972.9.6000 / 2.20E-151 / 2.50E-151
Trypanosoma brucei Lister Strain 427 / Tb427tmp.211.1760 / 1.80E-149 / 2.20E-149
Chlamydomonas reinhardtii / Cre06.g292350.t1.1|PACid:19863756 / 4.50E-149 / 5.60E-149
Glycine max / Glyma14g35520.1|PACid:16296510 / 3.10E-148 / 3.60E-148
Trypanosoma vivax / TvY486_0904620 / 2.70E-147 / 3.20E-147
Ricinus communis / 29835.m000645|PACid:16810254 / 7.10E-147 / 9.00E-147
Glycine max / Glyma02g37200.1|PACid:16249329 / 1.10E-144 / 1.40E-144
Ricinus communis / 29835.m000642|PACid:16810251 / 2.50E-143 / 2.90E-143
Volvox carteri / 42153|PACid:17999007 / 3.10E-141 / 3.50E-141
Mimulus guttatus / mgv1a018405m|PACid:17670984 / 1.10E-140 / 1.40E-140
Thalassiosira pseudonana / jgi|Thaps3|681|fgenesh1_pm.C_chr_11a000006 / 2.40E-120 / 3.20E-120
Trypanosoma cruzi CL Brener Esmeraldo-like / Tc00.1047053506773.90 / 7.90E-114 / 9.40E-114
Trypanosoma cruzi CL Brener non-Esmeraldo-like / Tc00.1047053508799.120 / 1.00E-110 / 1.20E-110
Trypanosoma vivax / TvY486_1107030 / 2.30E-110 / 2.80E-110
Glycine max / Glyma05g28810.1|PACid:16259980 / 3.70E-109 / 4.10E-109
Glycine max / Glyma08g11960.1|PACid:16270829 / 7.10E-109 / 8.20E-109
Aureococcus anophagefferens / jgi|Auran1|32482 / 3.30E-108 / 4.20E-108
Thalassiosira pseudonana / jgi|Thaps3|34333|e_gw1.5.167.1 / 1.10E-107 / 2.60E-57
Leishmania major strain Friedlin / LmjF.11.0520 / 1.60E-103 / 1.90E-103
Trypanosoma brucei Lister Strain 427 / Tb427tmp.02.4520 / 4.10E-103 / 4.90E-103
Trypanosoma brucei gambiense / Tbg972.11.7590 / 6.90E-103 / 8.20E-103
Trypanosoma brucei TREU927 / Tb11.02.4520 / 9.20E-103 / 1.10E-102
Trypanosoma congolense / TcIL3000.11.7240 / 1.20E-101 / 1.40E-101
Trypanosoma congolense / TcIL3000.0.06990 / 3.30E-101 / 4.00E-101
Leishmania mexicana / LmxM.11.0520 / 6.40E-101 / 7.70E-101
Leishmania infantum / LinJ.11.0550 / 7.50E-101 / 8.70E-101
Galdieria sulphuraria / Stig_17 Gs23570.1 / 3.50E-99 / 3.90E-99
Guillardia theta / jgi|Guith1|108589|au.34_g9691 / 4.30E-99 / 5.20E-99
Hyaloperonospora arabidopsidis / OOMYCETE_Hp_810464 / 1.40E-98 / 1.80E-98
Glycine max / Glyma10g42980.1|PACid:16281937 / 4.20E-98 / 3.70E-62
Leishmania braziliensis / LbrM.11.0290 / 1.10E-97 / 1.30E-97
Selaginella moellendorffii / 431542|PACid:15401408 / 1.20E-97 / 2.30E-97
Selaginella moellendorffii / 430321|PACid:15419263 / 3.00E-97 / 4.90E-97
Galdieria sulphuraria / Stig_28 Gs35110.1 / 2.70E-96 / 3.00E-96
Entamoeba histolytica HM-1:IMSS / EHI_100280 / 5.10E-96 / 6.40E-96
Entamoeba dispar SAW760 / EDI_127100 / 2.50E-95 / 3.10E-95
Entamoeba invadens IP1 / EIN_167910 / 3.20E-93 / 4.00E-93
Emiliania huxleyi / jgi|Emihu1|52699|gw1.103.43.1 / 3.60E-93 / 1.30E-92
Ricinus communis / 29835.m000646|PACid:16810255 / 8.50E-92 / 1.70E-70
Guillardia theta / jgi|Guith1|86991|estExt_Genewise1Plus.C_340045 / 5.00E-89 / 6.30E-89
Emiliania huxleyi / jgi|Emihu1|73488|e_gw1.234.15.1 / 4.70E-87 / 6.40E-87
Phytophthora sojae / OOMYCETE_Ps_138431 / 1.70E-86 / 2.20E-86
Entamoeba invadens IP1 / EIN_015980 / 2.50E-86 / 3.00E-86
Guillardia theta / jgi|Guith1|49465|gw1.47.53.1 / 3.50E-85 / 3.90E-85
Phytophthora sojae / OOMYCETE_Ps_130864 / 3.60E-85 / 5.70E-85
Entamoeba histolytica HM-1:IMSS / EHI_020320 / 1.00E-83 / 1.20E-83
Thalassiosira pseudonana / jgi|Thaps3|14347|gw1.10.71.1 / 1.60E-83 / 1.40E-41
Entamoeba dispar SAW760 / EDI_070700 / 5.50E-83 / 6.60E-83
Emiliania huxleyi / jgi|Emihu1|454057|estExtDG_Genemark1.C_990157 / 7.90E-81 / 8.60E-45
Trypanosoma congolense / TcIL3000.9.3900 / 2.10E-77 / 2.40E-77
Emiliania huxleyi / jgi|Emihu1|55003|gw1.1505.2.1 / 2.20E-75 / 8.80E-75
Entamoeba invadens IP1 / EIN_093780 / 4.20E-75 / 5.20E-75
Giardia lamblia ATCC 50803 / GL50803_16433 / 1.20E-70 / 5.20E-64
Giardia lamblia P15 / GLP15_4303 / 5.20E-70 / 5.10E-63
Phytophthora sojae / OOMYCETE_Ps_136938 / 1.10E-69 / 3.20E-69
Giardia intestinalis ATCC 50581 / GL50581_1008 / 1.80E-68 / 6.40E-63
Phytophthora ramorum / OOMYCETE_Pr_82397 / 2.80E-65 / 1.20E-64
Phytophthora sojae / OOMYCETE_Ps_136939 / 6.40E-57 / 1.10E-56
Glycine max / Glyma08g36760.1|PACid:16273020 / 6.20E-56 / 8.10E-56
Physcomitrella patens / Pp1s257_92V6.1|PACid:18045893 / 1.00E-49 / 1.30E-49
Emiliania huxleyi / jgi|Emihu1|110614|fgeneshEH_pg.137__39 / 1.20E-46 / 5.00E-28
Emiliania huxleyi / jgi|Emihu1|464278|estExtDG_fgeneshEH_pg.C_430093 / 3.50E-41 / 1.10E-21
Emiliania huxleyi / jgi|Emihu1|252995|gm1.152300001 / 1.90E-36 / 2.80E-34
Emiliania huxleyi / jgi|Emihu1|98964|fgeneshEH_pg.16__190 / 3.30E-34 / 5.30E-33
Bigelowiella natans / jgi|Bigna1|139819|aug1.52_g14527 / 8.50E-32 / 1.00E-31
Emiliania huxleyi / jgi|Emihu1|119926|fgeneshEH_pg.545__7 / 1.10E-29 / 7.20E-29
Bigelowiella natans / jgi|Bigna1|32021|gw1.21.198.1 / 4.40E-25 / 4.70E-25
Cochliobolus heterostrophus / jgi|CocheC5_1|89332|estExt_Genewise1.C_120385 / 2.30E-21 / 4.70E-12
Phytophthora ramorum / OOMYCETE_Pr_75271 / 5.40E-20 / 8.10E-20
Guillardia theta / jgi|Guith1|146664|fgenesh2_pg.131_#_1 / 7.60E-18 / 7.90E-18
Phytophthora ramorum / OOMYCETE_Pr_77806 / 6.50E-17 / 8.50E-17
Phytophthora ramorum / OOMYCETE_Pr_75270 / 8.30E-16 / 1.20E-15
Emiliania huxleyi / jgi|Emihu1|238517|gm1.27200001 / 1.00E-15 / 3.50E-15
Emiliania huxleyi / jgi|Emihu1|246853|gm1.53300002 / 3.40E-15 / 3.90E-15
Phytophthora sojae / OOMYCETE_Ps_145134 / 5.10E-15 / 1.10E-14
Ricinus communis / 27940.m000339|PACid:16799564 / 4.60E-14 / 1.20E-13
Phytophthora ramorum / OOMYCETE_Pr_77804 / 7.20E-14 / 1.10E-13
Mimulus guttatus / mgv1a003403m|PACid:17673697 / 6.40E-12 / 1.10E-11
Emiliania huxleyi / jgi|Emihu1|249501|gm1.78800005 / 2.90E-11 / 3.40E-11
Physcomitrella patens / Pp1s77_308V6.1|PACid:18050252 / 4.40E-11 / 5.20E-10
Cochliobolus heterostrophus / jgi|CocheC5_1|97924|estExt_Genewise1Plus.C_11331 / 1.60E-10 / 2.30E-09
Glycine max / Glyma17g15840.1|PACid:16305723 / 5.10E-10 / 3.00E-09
Physcomitrella patens / Pp1s23_303V6.1|PACid:18065660 / 6.50E-10 / 5.70E-09
Selaginella moellendorffii / 409080|PACid:15421732 / 9.10E-10 / 9.70E-09
Chlamydomonas reinhardtii / Cre01.g041050.t1.2|PACid:19866009 / 1.70E-09 / 4.60E-08
SGDID:S000003045 / YGL077C / 2.20E-09 / 3.40E-09
Glycine max / Glyma05g05510.1|PACid:16258425 / 2.70E-09 / 2.70E-09
Ricinus communis / 29932.m000609|PACid:16814094 / 2.80E-09 / 3.80E-09
Glycine max / Glyma09g05580.1|PACid:16274885 / 5.80E-09 / 2.00E-08
Bigelowiella natans / jgi|Bigna1|126621|aug1.3_g1329 / 8.20E-09 / 1.30E-08
Bigelowiella natans / jgi|Bigna1|138372|aug1.44_g13080 / 9.30E-09 / 1.10E-05
Physcomitrella patens / Pp1s49_168V6.1|PACid:18041351 / 1.40E-08 / 7.50E-08
Glycine max / Glyma08g13640.1|PACid:16271033 / 1.70E-08 / 2.80E-08
Mimulus guttatus / mgv1a003219m|PACid:17696622 / 5.80E-08 / 2.20E-07
Physcomitrella patens / Pp1s119_47V6.1|PACid:18062847 / 6.50E-08 / 6.50E-08
Physcomitrella patens / Pp1s9_272V6.1|PACid:18047827 / 1.00E-07 / 5.80E-07
Physcomitrella patens / Pp1s119_47V6.2|PACid:18062846 / 1.10E-07 / 1.10E-07
Mimulus guttatus / mgv1a002919m|PACid:17683581 / 1.30E-07 / 1.30E-07
Glycine max / Glyma09g05540.1|PACid:16274881 / 1.70E-07 / 6.60E-07
Glycine max / Glyma05g30510.1|PACid:16260181 / 3.40E-07 / 1.60E-06
SGDID:S000005214 / YNL270C / 3.70E-07 / 3.80E-03
Physcomitrella patens / Pp1s230_19V6.1|PACid:18073031 / 4.90E-07 / 4.90E-07
Physcomitrella patens / Pp1s230_19V6.2|PACid:18073032 / 4.90E-07 / 4.90E-07
Guillardia theta / jgi|Guith1|88618|estExt_Genewise1Plus.C_640050 / 1.10E-06 / 3.60E-05
Ricinus communis / 29801.m003214|PACid:16809064 / 1.30E-06 / 4.20E-06
Physcomitrella patens / Pp1s360_36V6.1|PACid:18043056 / 1.50E-06 / 3.30E-06
Ricinus communis / 29932.m000608|PACid:16814093 / 1.90E-06 / 7.00E-06
Glycine max / Glyma08g13660.1|PACid:16271035 / 2.10E-06 / 6.60E-06
Glycine max / Glyma09g10300.1|PACid:16275384 / 3.30E-06 / 9.90E-06
Cochliobolus heterostrophus / jgi|CocheC5_1|107499|estExt_Genewise1Plus.C_120021 / 3.70E-06 / 5.90E-06
Glycine max / Glyma09g21070.1|PACid:16275935 / 6.80E-06 / 1.60E-05
Monosiga brevicollis / jgi|Monbr1|27450|fgenesh2_pg.scaffold_19000104 / 6.60E-06 / 3.50E-05
Glycine max / Glyma09g01230.1|PACid:16274386 / 7.80E-06 / 1.50E-05
Ricinus communis / 29917.m001948|PACid:16813552 / 9.70E-06 / 9.70E-06