Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

1

Supplemental Figure 1. Alignment of Amino Acid Sequences of Proteins with Steroid 5α-Reductase Domain from Human (hSRD5A3), Yeast Saccharomyces cerevisiae (Dfg10p) and 60 Plant Species. Highly conserved amino acids are highlighted. The alignment was made using the ClustalW program. Related to Figure 1.

AtPPRD2_[Arabidopsis_thaliana]/1-343AlPPRD_[Arabidopsis_lyrata]/1-342CrPPRD_[Capsella_rubella]/1-342CsPPRD2_[Camelina_sativa]/1-342EsPPRD1_[Eutrema_salsugineum]/1-339AtPPRD1_[Arabidopsis_thaliana]/1-320BrPPRD2_[Brassica_rapa]/1-345BnPPRD1_[Brassica_napus]/1-349ArPPRD_[Arabis_alpina]/1-339ThPPRD2_[Tarenaya_hassleriana]/1-338EsPPRD2_[Eutrema_salsugineum]/1-337EgPPRD2-like_[Eucalyptus_grandis]/1-338NnPPRD2_[Nelumbo_nucifera]/1-338BnaPPRD2_[Brassica_napus]/1-312BrPPRD2-like[Brassica_rapa]/1-303BvPPRD2_[Beta_vulgaris]/1-341CsPPRD2-like_[Citrus_sinensis]/1-338CcPPRD_[Citrus_clementina]/1-338MnPPRD2_[Morus_notabilis]/1-338PmPPRD2-like_[Prunus_mume]/1-376PpPPRD_[Prunus_persica]/1-333CmPPRD_2_[Cucumis_melo]/1-338PtPPRD1_[Populus_trichocarpa]/1-339VvPPRD2_[Vitis_vinifera]/1-338PtPPRD2_[Populus_trichocarpa]/1-339PePPRD2-like_isoform_X4_[Populus_euphratica]/1-339PePPRD1-like_isoform_X1_[Populus_euphratica]/1-339MdPPRD2_[Malus_domestica]/1-338MdPPRD2-like_[Malus_domestica]/1-338TcPPRD1_[Theobroma_cacao]/1-338PbPPRD2-like[Pyrus_x_bretschneideri]/1-338GaPPRD_[Gossypium_arboreum]/1-484TcPPRD2_[Theobroma_cacao]/1-356FvPPRD2-like_[Fragaria_vesca]/1-342PdPPRD1_[Phoenix_dactylifera]/1-337StPPRD2-like_[Solanum_tuberosum]/1-337EgPPRD1_[Elaeis_guineensis]/1-337PvPPRD_[Phaseolus_vulgaris]/1-340SiPPRD2_[Sesamum_indicum]/1-337NsPPRD2_[Nicotiana_sylvestris]/1-337PePPRD2-like_isoform_X2_[Populus_euphratica]/1-379SlPPRD2_[Solanum_lycopersicum]/1-343CsPPRD2-like_[Cucumis_sativus]/1-338GsPPRD2_[Glycine_soja]/1-333GmPPRD2-like_[Glycine_max]/1-342NtPPRD2_[Nicotiana_tomentosiformis]/1-337GsPPRD1_[Glycine_soja]/1-339JcPPRD_[Jatropha_curcas]/1-336PePPRD1-like_isoform_X2_[Populus_euphratica]/1-316PePPRD2-like_isoform_X3_[Populus_euphratica]/1-359MaPPRD1/1-340MtPPRD2_[Medicago_truncatula]/1-484GaPPRD1_[Gossypium_arboreum]/1-332OsPPRD1_[Oryza_sativa_Japonica_Group]/1-354GaPPRD2_[Gossypium_arboreum]/1-401ZmPPRD1_[Zea_mays]/1-359HvPPRD_[Hordeum_vulgare]_/1-347PePPRD1-like_isoform_X3_[Populus_euphratica]/1-306EgPPRD_[Eucalyptus_grandis]/1-253ObPPRD1-like_[Oryza_brachyantha]/1-351PePPRD2-like_isoform_X1_[Populus_euphratica]/1-399RcDFG10_[Ricinus_communis]/1-342MtPPRD1[Medicago_truncatula]/1-468OsPPRD2_[Oryza_sativa_Japonica_Group]/1-353BdPPRD1_[Brachypodium_distachyon]/1-359OsPPRD2_[Oryza_sativa_Indica_Group]/1-353CaPPRD2-like_[Cicer_arietinum]/1-331SiPPRD1-like_[Setaria_italica]/1-353TuPPRD1_[Triticum_urartu]/1-353ZmPPRD2_[Zea_mays]/1-302ApPPRD2_[Auxenochlorella_protothecoides]/1-273SRD5A3_[Homo_sapiens]_/1-318Dfg10p_[Saccharomyces_cerevisiae_S288c]/1-253

Conservation

Quality

Consensus

AtPPRD2_[Arabidopsis_thaliana]/1-343AlPPRD_[Arabidopsis_lyrata]/1-342CrPPRD_[Capsella_rubella]/1-342CsPPRD2_[Camelina_sativa]/1-342EsPPRD1_[Eutrema_salsugineum]/1-339AtPPRD1_[Arabidopsis_thaliana]/1-320BrPPRD2_[Brassica_rapa]/1-345BnPPRD1_[Brassica_napus]/1-349ArPPRD_[Arabis_alpina]/1-339ThPPRD2_[Tarenaya_hassleriana]/1-338EsPPRD2_[Eutrema_salsugineum]/1-337EgPPRD2-like_[Eucalyptus_grandis]/1-338NnPPRD2_[Nelumbo_nucifera]/1-338BnaPPRD2_[Brassica_napus]/1-312BrPPRD2-like[Brassica_rapa]/1-303BvPPRD2_[Beta_vulgaris]/1-341CsPPRD2-like_[Citrus_sinensis]/1-338CcPPRD_[Citrus_clementina]/1-338MnPPRD2_[Morus_notabilis]/1-338PmPPRD2-like_[Prunus_mume]/1-376PpPPRD_[Prunus_persica]/1-333CmPPRD_2_[Cucumis_melo]/1-338PtPPRD1_[Populus_trichocarpa]/1-339VvPPRD2_[Vitis_vinifera]/1-338PtPPRD2_[Populus_trichocarpa]/1-339PePPRD2-like_isoform_X4_[Populus_euphratica]/1-339PePPRD1-like_isoform_X1_[Populus_euphratica]/1-339MdPPRD2_[Malus_domestica]/1-338MdPPRD2-like_[Malus_domestica]/1-338TcPPRD1_[Theobroma_cacao]/1-338PbPPRD2-like[Pyrus_x_bretschneideri]/1-338GaPPRD_[Gossypium_arboreum]/1-484TcPPRD2_[Theobroma_cacao]/1-356FvPPRD2-like_[Fragaria_vesca]/1-342PdPPRD1_[Phoenix_dactylifera]/1-337StPPRD2-like_[Solanum_tuberosum]/1-337EgPPRD1_[Elaeis_guineensis]/1-337PvPPRD_[Phaseolus_vulgaris]/1-340SiPPRD2_[Sesamum_indicum]/1-337NsPPRD2_[Nicotiana_sylvestris]/1-337PePPRD2-like_isoform_X2_[Populus_euphratica]/1-379SlPPRD2_[Solanum_lycopersicum]/1-343CsPPRD2-like_[Cucumis_sativus]/1-338GsPPRD2_[Glycine_soja]/1-333GmPPRD2-like_[Glycine_max]/1-342NtPPRD2_[Nicotiana_tomentosiformis]/1-337GsPPRD1_[Glycine_soja]/1-339JcPPRD_[Jatropha_curcas]/1-336PePPRD1-like_isoform_X2_[Populus_euphratica]/1-316PePPRD2-like_isoform_X3_[Populus_euphratica]/1-359MaPPRD1/1-340MtPPRD2_[Medicago_truncatula]/1-484GaPPRD1_[Gossypium_arboreum]/1-332OsPPRD1_[Oryza_sativa_Japonica_Group]/1-354GaPPRD2_[Gossypium_arboreum]/1-401ZmPPRD1_[Zea_mays]/1-359HvPPRD_[Hordeum_vulgare]_/1-347PePPRD1-like_isoform_X3_[Populus_euphratica]/1-306EgPPRD_[Eucalyptus_grandis]/1-253ObPPRD1-like_[Oryza_brachyantha]/1-351PePPRD2-like_isoform_X1_[Populus_euphratica]/1-399RcDFG10_[Ricinus_communis]/1-342MtPPRD1[Medicago_truncatula]/1-468OsPPRD2_[Oryza_sativa_Japonica_Group]/1-353BdPPRD1_[Brachypodium_distachyon]/1-359OsPPRD2_[Oryza_sativa_Indica_Group]/1-353CaPPRD2-like_[Cicer_arietinum]/1-331SiPPRD1-like_[Setaria_italica]/1-353TuPPRD1_[Triticum_urartu]/1-353ZmPPRD2_[Zea_mays]/1-302ApPPRD2_[Auxenochlorella_protothecoides]/1-273SRD5A3_[Homo_sapiens]_/1-318Dfg10p_[Saccharomyces_cerevisiae_S288c]/1-253

Conservation

Quality

Consensus

AtPPRD2_[Arabidopsis_thaliana]/1-343AlPPRD_[Arabidopsis_lyrata]/1-342CrPPRD_[Capsella_rubella]/1-342CsPPRD2_[Camelina_sativa]/1-342EsPPRD1_[Eutrema_salsugineum]/1-339AtPPRD1_[Arabidopsis_thaliana]/1-320BrPPRD2_[Brassica_rapa]/1-345BnPPRD1_[Brassica_napus]/1-349ArPPRD_[Arabis_alpina]/1-339ThPPRD2_[Tarenaya_hassleriana]/1-338EsPPRD2_[Eutrema_salsugineum]/1-337EgPPRD2-like_[Eucalyptus_grandis]/1-338NnPPRD2_[Nelumbo_nucifera]/1-338BnaPPRD2_[Brassica_napus]/1-312BrPPRD2-like[Brassica_rapa]/1-303BvPPRD2_[Beta_vulgaris]/1-341CsPPRD2-like_[Citrus_sinensis]/1-338CcPPRD_[Citrus_clementina]/1-338MnPPRD2_[Morus_notabilis]/1-338PmPPRD2-like_[Prunus_mume]/1-376PpPPRD_[Prunus_persica]/1-333CmPPRD_2_[Cucumis_melo]/1-338PtPPRD1_[Populus_trichocarpa]/1-339VvPPRD2_[Vitis_vinifera]/1-338PtPPRD2_[Populus_trichocarpa]/1-339PePPRD2-like_isoform_X4_[Populus_euphratica]/1-339PePPRD1-like_isoform_X1_[Populus_euphratica]/1-339MdPPRD2_[Malus_domestica]/1-338MdPPRD2-like_[Malus_domestica]/1-338TcPPRD1_[Theobroma_cacao]/1-338PbPPRD2-like[Pyrus_x_bretschneideri]/1-338GaPPRD_[Gossypium_arboreum]/1-484TcPPRD2_[Theobroma_cacao]/1-356FvPPRD2-like_[Fragaria_vesca]/1-342PdPPRD1_[Phoenix_dactylifera]/1-337StPPRD2-like_[Solanum_tuberosum]/1-337EgPPRD1_[Elaeis_guineensis]/1-337PvPPRD_[Phaseolus_vulgaris]/1-340SiPPRD2_[Sesamum_indicum]/1-337NsPPRD2_[Nicotiana_sylvestris]/1-337PePPRD2-like_isoform_X2_[Populus_euphratica]/1-379SlPPRD2_[Solanum_lycopersicum]/1-343CsPPRD2-like_[Cucumis_sativus]/1-338GsPPRD2_[Glycine_soja]/1-333GmPPRD2-like_[Glycine_max]/1-342NtPPRD2_[Nicotiana_tomentosiformis]/1-337GsPPRD1_[Glycine_soja]/1-339JcPPRD_[Jatropha_curcas]/1-336PePPRD1-like_isoform_X2_[Populus_euphratica]/1-316PePPRD2-like_isoform_X3_[Populus_euphratica]/1-359MaPPRD1/1-340MtPPRD2_[Medicago_truncatula]/1-484GaPPRD1_[Gossypium_arboreum]/1-332OsPPRD1_[Oryza_sativa_Japonica_Group]/1-354GaPPRD2_[Gossypium_arboreum]/1-401ZmPPRD1_[Zea_mays]/1-359HvPPRD_[Hordeum_vulgare]_/1-347PePPRD1-like_isoform_X3_[Populus_euphratica]/1-306EgPPRD_[Eucalyptus_grandis]/1-253ObPPRD1-like_[Oryza_brachyantha]/1-351PePPRD2-like_isoform_X1_[Populus_euphratica]/1-399RcDFG10_[Ricinus_communis]/1-342MtPPRD1[Medicago_truncatula]/1-468OsPPRD2_[Oryza_sativa_Japonica_Group]/1-353BdPPRD1_[Brachypodium_distachyon]/1-359OsPPRD2_[Oryza_sativa_Indica_Group]/1-353CaPPRD2-like_[Cicer_arietinum]/1-331SiPPRD1-like_[Setaria_italica]/1-353TuPPRD1_[Triticum_urartu]/1-353ZmPPRD2_[Zea_mays]/1-302ApPPRD2_[Auxenochlorella_protothecoides]/1-273SRD5A3_[Homo_sapiens]_/1-318Dfg10p_[Saccharomyces_cerevisiae_S288c]/1-253

Conservation

Quality

Consensus

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- VWL I R - - - - AAWL AG I SP I L I AS I PSPKL SK - - - F RGF I VGF AGRG - - KT L R - - - SSS - - - - - - - - - - - - - - KKF T VPQKF F GHF Y I VGVAWT T L L L VT T WRYAY - ET APL SSEKL H - F PDVASHL T GG - SHVF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL G I - - VWL VR - - - - AAWI AA I L PMV I AS I P I SKL RS - - - F HEL VMGF ARRG - - K I L Q - - - PSSS - - - - - - - - - - - - - QKWT VPQRF F AHF YV I GVAWT T L L L AT T WMYA I - K I APL SPEKL Q - SSE I ASHL T RG - SKVF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ME I EL - - VGL L R - - - - VAWI AG I L PL L VASL PF RRL SR - - - L HEAL MGF AKRG - - K I MH - - - SSS - - - - - - - - - - - - - - SKF T VPQRF F T HF YAL AV I WT GL L L AAT WI YAY - KMAPMASEPF L - YSS I ASHL T GG - SHPF T L- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MKL RL - - AL VL R - - - - AAWI AG I L P I L VAS I PSSRL SS - - - F HGA I L GF AKRG - - K I MQ - - - F SS - - - - - - - - - - - - - - NKF T VPQKF F L HF YVVAV I WT T VL L L MT WF YAC - RMVPL GSESL H - YST VASYL T GG - AH I F SM- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL G I WT VWL VRAAWI - - - - AT I L L MV I GS I P I SKL RL - - - F HEL ML I F AGRG - - K I L Q - - - PSSS - - - - - - - - - - - - - QKWT VPQKYF AHF YVVGVVWT T L L F AMT WMYAF - KMAP - - - - - - - - - - - - - - - L T GG - SH - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL G I - - VWL VR - - - - VAWI AA I L PML I AS I PNSKL T S - - - F HQL VL GF ARRG - - K I L Q - - - PSSSS - - - - - - - - - - - - QKWT L PQKHF AHF YVL GVAWT T L L L AAT WMYAF - KMT PL T SDEF H - L S - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MVKME I - - ERL L R - - - - VT WVAA I L P I L I ASF PSSYL NS - - - F HHT L L VF AGRG - - K I L Q - - - SSSRF - - - - - - - - - - - - SKL T VPQRF F SHF YVL AVVWT SL L L L T T WAYAH - K I GT L VSEST S - HSSVASYL T GG - SH I L S I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MESWL - - AGL L R - - - - AAWVAG I L PL I I ASL PSSRL NS - - - F HGAL L GF AKRG - - K I MQ - - - SSS - - - - - - - - - - - - - - HKF T VPQRF F CHF YVVAVVWT T L L L F T L WAYAY - KMAPSVSEPSL - YST I ASHL T GG - SHMF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MESWL - - AGL L R - - - - AAWVAG I L PL I I ASL PSSRL NS - - - F HGAL L GF AKRG - - K I MQ - - - SSS - - - - - - - - - - - - - - HKF T VPQRF F CHF YVVAVVWT T L L L F T L WAYAY - KMAPSVSEPSL - YST I ASHL T GG - SHMF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL DL - - VGL L R - - - - AAWVAG I L P I L I ASL PF SWT AS - - - F HGL VL GF AKRG - - K I MQ - - - ASS - K - - - - - - - - - - - - K - F T L PQKF F GHF YVVAVVWT T F L L VT T WL YAY - RT VPMVL KPF H - YT T I ANQL AGG - SH I F SW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MHCF PEKKKAL AL EDPVEPVL SF GRET SL F QL F KL CS I WVF AKMEEGL - - VGL L R - - - - T AWI AA I L PL L I ASVPSSRL SS - - - F HGAVL EF AKRG - - K I MK - - - SSS - Q - - - - - - - - - - - - K - F T VPQKF F CHF YL VAVVWT T L L L VT T GMYAY - KT VPL V - - - - - - YPT F PSQL T GG - SH I F SW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEVGL - - VGL L R - - - - T AWI AA I L PL L I ASVPSSRL SS - - - F HGAVL EF AKRG - - K I MK - - - SSS - Q - - - - - - - - - - - - K - F T VPQKF F CHF YL VAVVWT AL L L VT T GMYAY - KT VPL V - - - - - - YPT F PSQL T GG - SH I F SW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL G I - - VWL L R - - - - AAWVAG I L P I I VAF L PF SKL NW- - - F RG I L L RF AKRG - - K I L Q - - - SSS - Q - - - - - - - - - - - - K - F T VPQKF F CHF YVL AV I WT T F L L GT T WI YAY - VST PE I SEPF N - F PA I T SQL T GG - SSL F PW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MK - - - SSSH - - - - - - - - - - - - - HKF T VPQRF F SHF YVVAVVWT T L L L I AT WI YAH - RMAP I VSEPF F - YSDL GSYL AGR - SNT F SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL DL - - VAL L R - - - - AAWI AGT L P I L I AS I PSSRL HS - - - F HGL VL GF ARRG - - KT MP - - - SSS - - - - - - - - - - - - - - YKF T VPQRF F CHF YVVAVVWT T L L VL AT L L YAY - RMAPL VSEPL L - YST VASQL T GG - SH I F SL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MQ - - - SSSH - - - - - - - - - - - - - RKF T VPQRF F T HF YVVAVAWT T L L L L GT S I YAY - RMT P I VSEPF F - YSDL GSYL AGR - SN I F SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MQ - - - SSSH - - - - - - - - - - - - - HKF T VPQRF F SHF YVVAVAWT T L L L L GT S I YAY - RMT P I L SEPF F - YSDL GSYL AGR - SN I F S I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MK - - - SSSH - - - - - - - - - - - - - HKF T VPQRF F SHF YVVAVVWT T L L L I AT WI YAR - RMAP I VSEPF F - YSDL VSYL AGR - SNT F SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEVGV - - VGL L R - - - - T AWI AAT L P I L I AAL PF SWL SS - - - F RGAVL GF AKRG - - KT MQ - - - SSS - Q - - - - - - - - - - - - K - F T VPQKF F CHF YL VAVAWT T VL L VAT L MYAY - KAVPL VSDSL L - HPSL PNPYT GG - SH I F SW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEVGV - - VGF L R - - - - T AWI AAT L P I L I AAL PF SWL SS - - - F RGAVL GF AKRG - - KT MQ - - - SSS - Q - - - - - - - - - - - - K - F T VPQKF F CHF YL VAVAWT T VL L VAT L MYAY - KAVPL VSDSL L - HPSL PNPYT GG - SH I F SW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VT L L R - - - - AAWI AVT L P I V I AP I PSSKL SS - - - F HQL VSKF AKRG - - KT MQ - - - PCS - - - - - - - - - - - - - - CKF T VPHRF F SHVYMVAVVWT T I L L L T T WL YAY - RMAPL T SESF H - I L HL ASHL T GG - SH I L SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MAVGV - - VGL L R - - - - T AWVAAT MP I L VAAVPCSWL T S - - - F HGAVL GF AKRG - - K I MQ - - - SSS - Q - - - - - - - - - - - - K - F T VPQKL F CHF YVVAVAWT T L L L VAT SMYAY - KT VPL VSESL L - YPS I ASHF T GG - SH I F SW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MNL L GVL MEAL VSQS I NT I KT T L - - I WL L R - - - - T T WI VGT L P I L I ASL PSPCL NS - - - F HAL L L SF AKRG - - K I MP - - - SSS - - - - - - - - - - - - - - HRF T VPQSF F L HF YL L AV I WT T T L L MGT WYF AY - KVT PL ST ESL S - YL AAANHL T GG - SL VF SL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEVVGVL VEAF SL RSMDT I KT CF - - I WL L R - - - - AGWVAGT L P I L I ASL PSSRL GS - - - F HT L L L GF AKRG - - K I MP - - - SSS - - - - - - - - - - - - - - HKYT VPQSF F L HF YL L AVVWT T VL L MG I WWF AY - EVAPL SVESL S - YSAAASHL T GG - F HVF SL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MGMEVGV - - VEL VR - - - - T GWI AGT L P I L I ASL PL SCL NS - - - F HD I VL GF AKRG - - K I L Q - - - SSS - S - - - - - - - - - - - - KRF T VPQKF F WHF YVL AVVWT T L L L AT T WT YAY - KMVPL NADPF L - YST F ANR I T GG - SH I L SW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL G - - - - - - L T AL I RL AWVAA I AP I L L ASL PVR - SL G - - F L HRML T GF AARG - - KT RS - - - SSS - - - - - - - - - - - - - - - KF T VPQRYF L HF YL VAVML T T CL VVST WF YAYRKML PL SSESL H - YST I ASHL T GG - SHVF S I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VT L L R - - - - AAWVAA I L PVV I AL I PSSK I SF - - - F QQF VL GF AKRG - - K I MQ - - - SSS - - - - - - - - - - - - - - NKF AVPQKL F I HF Y I L ASVWT T L L L AAT WL YAY - ST VPK I SEPYL - F SS I ASHL T GG - SR I F SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL G - - - - - - L T AL L RL AWVAAT AP I L L ASL PVR - SL G - - F L HQML T GF AARG - - KT RS - - - SSS - - - - - - - - - - - - - - - KF T VPQRYF L HF YL VAL VL T T CL VL CT WF YAYGKML PL SSESL Q - YST I ASHL T GG - SHVF S I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ML L HDQPMEEVL - - VQ I L R - - - - AAWL AAT L P I V I ASL P I PNL SF - - - L RRT L I GF AGRG - - K I MQ - - - SSS - - - - - - - - - - - - - - QKF T I PQRL F L HF YAVAF L WT T L L L VAT WAYAY - SMVPL VAEPF S - YST I T SF L T GG - - - - - ST- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M I SV - - VSL L R - - - - AAWI AGT L P I V I AS I PSPKL NA - - - F HEL I I GF ARRG - - K I MQ - - - SSS - - - - - - - - - - - - - - KKF SL PQRF F CHF Y I L AVVWT T F L L VAT SL YAH - RT APT VSEPVL - YSS I ASHL T GV - SHEL SL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VT L L R - - - - AAWI AAT L P I V I AL I PSSKL RS - - - F QQF L L GF AKRG - - K I MQ - - - SSS - - - - - - - - - - - - - - NKL SVPQKL F I HF YVL AF VWT T L L L AAT WL YAY - ST T PK I SEPL L - F SS I T SHL T GG - SR I F SL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MQ - - - SSSH - - - - - - - - - - - - - HKF T VPQRF F SHF YVVAVAWT T L L L L GT S I YAY - RMT P I L SEPF F - YSDL GSYL AGR - SN I F S I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ML PGKKMEL GL - - VT L L R - - - - AAWVAA I L PVV I AL I PSSK I SF - - - F QKF VL GF AKRG - - K I MQ - - - SSS - - - - - - - - - - - - - - NKL SVPQKL F I HF Y I L AF VWT T L L L VAT WL YDY - CT MPK I T EPL L - F SS I ASHL T GG - SR I F SS- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL G I - - VWL L R - - - - AAWF AG I L P I I I AF L PF SRL NW- - - F RA I L L GF AKRG - - K I L Q - - - SSS - Q - - - - - - - - - - - - K - F T VPQKF F CHF YVL AV I WT T L L L GT T WT YAY - VST PE I SEPF N - F PG I T SQL T GG - SSL F PW- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEVVL - - VQML R - - - - L AWVAAT L P I I VAS I PT PKL NC - - - L RGT L L GF ARRG - - K I MH - - - SSS - - - - - - - - - - - - - - QKVT I SQRF F L HF YVVAF I WT T L L L VGT WAYAY - SMVPL VVEPF S - YST I T SF L T GG - - - - - ST- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MVF L F HNQPMEVVL - - VQML R - - - - L AWVAAT L P I I VAS I PT PKL NC - - - L RGT L L GF ARRG - - K I MH - - - SSS - - - - - - - - - - - - - - QKVT I SQRF F L HF YVVAF I WT T L L L VGT WAYAY - SMVPL VVEPF S - YST I T SF L T GG - - - - - ST- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VT PL R - - - - AAWI AAT L P I V I AL I PSST L RS - - - F QQF L L GF AKRG - - K I MQ - - - SSS - - - - - - - - - - - - - - NKL SVPQKL F I HF YVL AF VWT T L L L AAT WL YAY - ST T PK I SEPL L - F SS I ASHL T GG - SR I F SL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ML F HNQPMEL VL - - L Q I L R - - - - L AWL AAT L P I I VAS I PT PKL SC - - - L RAT L L AF ARRG - - K I MH - - - SS - - - - - - - - - - - - - - - QKF T I SQRF F L HF Y I VAF I WT T L L L L GT WAYAY - NMVPL VVEPF S - YST I T SF L T GG - - - - - ST- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MD I RL - - DWL L R - - - - AAWI SAT L P I L I ASF PSSRL NS - - - F HQL L L GF SRRG - - KT MQ - - - PSS - - - - - - - - - - - - - - - HF T VPQRF F L HF YL VGVAL T T F L L I I I WGYAY - KL ASL DSGSL D - YST AASQL VGG - SQ I YSL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MK - - - SSSH - - - - - - - - - - - - - HKF T VPQRF F SHF YVVAVVWT T L L L I AT WI YAR - RMAP I VSEPF F - YSDL VSYL AGR - SNT F SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MQ - - - SSSH - - - - - - - - - - - - - HKF T VPQRF F SHF YVVAVAWT T L L L L GT S I YAY - RMT P I L SEPF F - YSDL GSYL AGR - SN I F S I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ML GL G - - - - - - F T AL L RL AWL AAT VP I L VVSL P I R - PVG - - F L HRM I MGF AARG - - KT MM - - - NSST Q - - - - - - - - - - - - - KF T VPQRYF L HF YWWSL AL T T SL AL ST WF YAYRKMVPL ASESL S - YST VASHL I GG - SH I F SSML AVL GMSE I PGVVQVDSVPVQPGRGAGVVAF GVGRSPKSAL AVVL L PPT I AVL QVT AL L DVAGVPSAAVK I CT DVVPGVSPPPPL YPQ I HVL T F RR I I REDPHPSHHT SL SDENE I RF L T SSF T L PST T PR I I SSH I T MNL SF L SNDQHQ I MKT L L - - VSL L R - - - - L SWI AAT L P I I I VS I P I PNL NF - - - L RQ I L L GF AKRG - - KT S - - - - SSS - - - - - - - - - - - - - - QKF T VPQRF F L HF YVVSS I WT T F L L VAT WI YAY - RMAPL VADPL S - YST L T GF L T GG - - - - - L T- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MDL HG I L MEAF T SL SVNT I KT GL - - I WL L R - - - - VAWI AGT L P I L I ASL PF P I L NS - - - F RAL L L SF AKRG - - K I MP - - - SSS - - - - - - - - - - - - - - HRF T VPHSF F L HF YL L AVVWT T SL L T CT WYF AY - KVAPL GT ESL S - YPF AASHL T GG - SHVF S I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MET EG - - WPAL QPL L CL AWI AT T L P I I VAAL P I PAAAGGHL L RRL L SAF SSRG - - KT VRPSPASSSGSSSSK - - - - - - - AKF T VPQKYF MHF YVVGVL AT T I L L L A I WF YAYMKMT PL L PESSS - YST I ASHL VG - - SNSF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MNL L GVL MEAL VSQS I NT I KT T L - - I WL L R - - - - T T WI VGT L P I L I ASL PSPCL NS - - - F HAL L L SF AKRG - - K I MP - - - SSS - - - - - - - - - - - - - - HRF T VPQSF F L HF YL L AV I WT T T L L MGT WYF AY - KVT PL ST ESL S - YL AAANHL T GG - SL VF SL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MAL RGS I QMDT GS - - GPAL QYL L CL AWVAAT L P I AAAAL P I PAAAGGRF I HGL L CAF SSRG - - KT VR - - PSSSSSSKA - - - - - - - - - - RF T VSQKCF L HF YVVGAVVT T SL L L A I WF YAYMKMT PL MPEQSS - YST I ASHL VGG - SNSF HL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEKGD - - GPAL QPL L CL AWL AAT L P I AAAAL P I PAAAGGRF L HSL L AAF SSRG - - KT VR - - PSSSSK - - - - - - - - - - - - AKF T VPQKYF L HF YVVGVAVT T SL L L A I CF YAYMKMT PL L AEPSS - YST I ASHL I G - - SNSF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MK - - - SSSH - - - - - - - - - - - - - HKF T VPQRF F SHF YVVAVVWT T L L L I AT WI YAR - RMAP I VSEPF F - YSDL VSYL AGR - SNT F SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MAPMASEPF L - YSS I ASHL T GG - SHPF T L- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEMES - - WPAL QPML CL AWL AAT L P I I T AAL P I PAAAGGHL L HRL L SAF SSRG - - KT VR - - - ASSASSST SK - - - - - - - AKF T VPQKYF MHF YVVGVVAT T T L L L A I WF YAYMKMT PL VPESSN - YST I ASHL VG - - SNSF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL GL - - VEL L R - - - - AAWI AGT L P I L I ASL PCSWL GS - - - F HGL VL GF ARRG - - K I MQ - - - SSSH - - - - - - - - - - - - - HKF T VPQRF F SHF YVVAVAWT T L L L L GT S I YAY - RMT P I L SEPF F - YSDL GSYL AGR - SN I F S I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ME I HL - - DWL L R - - - - L AWI AGT L P I L L ASL PSSSL NS - - - F HQL L L GF AKRG - - K I MQ - - - L SSSSSSF - - - - - - - - - HKF T VPQRF F L HF YL L GVVWT T F L L I VT WHYAY - KMT L L GSGSL I - YSVASAHMAGG - SRML PFML AVL GMSE I PGVVQVDSVPVQPGRGAGVVAF GVGRSPKSAL AVVL L PPT I AVL QVT AL L DVAGVPSAAVK I CT DVVPGVSPPPPL YPQ I HVL T F RR I I REDPHPSHHT SL SDENE I RF L T SSF T L PST T PR I I SSH I T MNL SF L SNDQHQ I MKT L L - - VSL L R - - - - L SWI AAT L P I I I VS I P I PNL NF - - - L RQ I L L GF AKRG - - KT S - - - - SSS - - - - - - - - - - - - - - QKF T VPQRF F L HF YVVSS I WT T F L L VAT WI YAY - RMAPL VADPL S - YST L T GF L T GG - - - - - L T- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 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T T MQHMEF DWL EF VSSL L RL RWL QWT GAV I F F WGWI HQRNCHA I L G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - SL REHSG - - - QNDEYV I PHGDWF EVVSSPHYL AE I V I YT GL VVASGGT DPT I WL L F - - - - - - - - - GF VVSNL VF AAAET HRWY - - - - - L K - - KF KNYP - - - - - - - - - - - - - - - - - - -QKSH - SSWREHGF L VWKAVF L I L L MEVQVL RRL YET I YVF NYSPSARMH I F GYL T GL F F YT AAPL SL CSNC I PEVYHF AASGL AQF I VQGR - RHMPDVE I SPL DAV I PL SNL GWCQWI GAT L F F WGWI HQHRCHQ I L G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - SL RVRRE - - - QSEEYR I PHGDWF EVVSSPHYL AE I V I YGGL VVASGGKDL T I WL L F - - - - - - - - - GF VVVNL AF VAAET HRWY - - - - - F R - - KF DNYP - - - - - - - - - - - - - - - - - - -HRSQ - L I NSENKF RVWL SVF L L L L MEVQVL RRL F ET L YVF KYSPSARMH I F GYL T GL F F YT AMPL T L CCT CAPNVF KF GT SEVT EF I VKDR - SSMQA I EF DWWDF VNPF SKL GWCQWI GAA I F L WGWI HQHRCHA I L G - - - - - - - - - - - - - - - - - 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NQMPA I E I DWCGSVNPL MKL GWSQWI GSA I F L WGWI YQCRCHASL G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - SVQAHAE - - - QVDDYV I PT GDWF E I VSSPHYL AE I V I YAGMVVASGGGDL T VWL L F - - - - - - - - - VF VVANL GF AAAET HKWY - - - - - L C - - KF ENYP - - - - - - - - - - - - - - - - - - -DKVH - SVPVSYRYS I WRSVF L L L L MEVQVL RRL F ET I YVF NF SSSARMH I F GYL T GL F F YT AAPL SL CCNNVL EVYKF S I SGVAEF I VKGK - SSMRQVEF DWL QF VNSL L KL GWL RWI GAA I F F WGWI HQRRCHA I L G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - SL RDHSE - - - PNDEYV I PHGDWF E I VSSPHYL AE I V I YAGL VVASGGT DL T I WL L F - - - - - - - - - GF VVSNL VL SAAET HRWY - - - - - L Q - - KF ESYP - - - - - - - - - - - - - - - - - - -HKSR - F SSVDDRF NVWKAVF L L L L ME I HVL RRL YET F QVF NYSPSARMH I F GYL T G I YF YT AAPL SL CT F CVL EAF NF AADQVAEF KVEGQ - EMVS I T DF DL WGYMKPL T RL GWCQWAGAV I F AWGWF HQL RCHA I L G - - - - - - - - - - - - - - - - - - - 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- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - SRRKAL F PWL Y - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - KHRKAF L PF L F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - VAPYA I I PF I I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - 4 5 + 3 7 9 8 + 9 7 6 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - P - - - - - - - - P - - - - G - - RNR+ A I I PF VYT AMS - - - - - - - - - - - - - - - - - - - L - - G - - G - S - - - - I - - - G - - - - - - - - - - - K - F - - - - - - - - - - - - - - - - - - - - G I - - - - - - - - - - - -

Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

2

Supplemental Figure 2. Evolutionary Tree of Proteins with Steroid 5α-Reductase Domain. Amino acid sequences of proteins from 60 plant species, yeast (Dfg10p) and human (hSRD5A3) were aligned as above and the tree was constructed using the neighbor-joining method. Related to Supplemental Figure 1.

AtPPRD2 Arabidopsis thaliana

AlPPRD Arabidopsis lyrata

CsPPRD2 Camelina sativa

AtPPRD1 Arabidopsis thaliana

CrPPRD Capsella rubella

BnaPPRD2 Brassica napus

ArPPRD Arabis alpina

EsPPRD1 Eutrema salsugineum

EsPPRD2 Eutrema salsugineum

BrPPRD2 Brassica rapa

BnPPRD1 Brassica napus

ThPPRD2 Tarenaya hassleriana

BrPPRD2-likeBrassica rapa

SiPPRD2 Sesamum indicum

StPPRD2-like Solanum tuberosum

SlPPRD2 Solanum lycopersicum

NsPPRD2 Nicotiana sylvestris

NtPPRD2 Nicotiana tomentosiformis

FvPPRD2-like Fragaria vesca

CsPPRD2-like Citrus sinensis

CcPPRD Citrus clementina

GaPPRD Gossypium arboreum

GaPPRD2 Gossypium arboreum

TcPPRD2 Theobroma cacao

GaPPRD1 Gossypium arboreum

JcPPRD Jatropha curcas

RcDFG10 Ricinus communis

BvPPRD2 Beta vulgaris

PdPPRD1 Phoenix dactylifera

EgPPRD1 Elaeis guineensis

MaPPRD1 Musa acuminata

OsPPRD2 Oryza sativa Japonica Group

OsPPRD2 Oryza sativa Indica Group

TuPPRD1 Triticum urartu

HvPPRD Hordeum vulgare

BdPPRD1 Brachypodium distachyon

OsPPRD1 Oryza sativa Japonica Group

ObPPRD1-like Oryza brachyantha

SiPPRD1-like Setaria italica

ZmPPRD1 Zea mays

ZmPPRD2 Zea mays

PmPPRD2-like Prunus mume

PpPPRD Prunus persica

MdPPRD2 Malus domestica

MdPPRD2-like Malus domestica

PbPPRD2-likePyrus x bretschneideri

VvPPRD2 Vitis vinifera

MnPPRD2 Morus notabilis

CmPPRD 2 Cucumis melo

CsPPRD2-like Cucumis sativus

TcPPRD1 Theobroma cacao

PePPRD1-like isoform X2 Populus euphratica

PePPRD1-like isoform X3 Populus euphratica

PePPRD1-like isoform X1 Populus euphratica

PtPPRD1 Populus trichocarpa

PtPPRD2 Populus trichocarpa

PePPRD2-like isoform X4 Populus euphratica

PePPRD2-like isoform X2 Populus euphratica

PePPRD2-like isoform X3 Populus euphratica

PePPRD2-like isoform X1 Populus euphratica

NnPPRD2 Nelumbo nucifera

EgPPRD2-like Eucalyptus grandis

EgPPRD Eucalyptus grandis

CaPPRD2-like Cicer arietinum

MtPPRD2 Medicago truncatula

MtPPRD1Medicago truncatula

GsPPRD1 Glycine soja

GsPPRD2 Glycine soja

GmPPRD2-like Glycine max

PvPPRD Phaseolus vulgaris

ApPPRD2 Auxenochlorella protothecoides

SRD5A3 Homo sapiens

Dfg10p Saccharomyces cerevisiae S288c9 8

9 7

9 8

6 6

5 4

4 6

2 3

9 9

3 8

3 5

3 7

4 8

3 6

9 0

2 8

1 4

2 1

4 1

8 9

9 9

8 2

9 4

2 5

8 2

7 7

8 1

3 4

9 8

3 0

2 5

9 4

7 5 9 8

3 8

3 8

5 7

8 4

8 8

9 8

8 6

6 8

2 4

2 1

3 5

2 8

9 0

4 0

6 0

4 4

1 4

2 4

1 4

3 0

1 4

1 5

2 7

1 8

3 2

1 0

2

6

1

2

0

0

1

0.1

Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

3

Supplemental Figure 3. Models of PPRDs, Related to Supplemental Figure 1.

d transmembrane domains in PPRD1, PPRD2 and PPR(A) Predicte D3. Please note that numbering of X-axis used for PPRD3 does not align to the amino acid numbers in this protein. (B) Topology model of PPRD2; five predicted transmembrane domains (in red) and putative steroid reductase domain (in green) are indicated. Amino acid residues of particular interest are indicated, namely H321 and H336 subjected to site-directional mutagenesis (this report) and natural polymorphisms observed for PPRD2 isolated from Arabidopsis thaliana hairy roots (T78S, G193R, G274R and T301S).

0

0,2

0,4

0,6

0,8

1

1,2

0 50 100 150 200 250 300

prob

abili

ty

TMHMM posterior probabilities for PPRD3

transmembrane inside outside

0

0,2

0,4

0,6

0,8

1

1,2

0 50 100 150 200 250 300

prob

abili

ty

TMHMM posterior probabilities for PPRD1

transmembrane inside outside

A

B

Supplemental Figure 3. Models of PPRDs, Related to SupplementalFigure 1.(A) Predicted transmembrane domains in PPRD1, PPRD2 andPPRD3 .Please note, that numbering of X-axis used for PPRD3 does notalign to the amino acid numbers in this protein.(B) Topology model of PPRD2; five predicted transmembrane domains (inred) and putative steroid reductase domain (in green) are indicated. Aminoacid residues of particular interest are indicated, namely H321 and H336subjected to site-directional mutagenesis (this report) and naturalpolymorphisms observed for PPRD2 isolated from Arabidopsis thaliana hairy roots (T78S, G193R, G274R and T301S).

0

0,2

0,4

0,6

0,8

1

1,2

0 50 100 150 200 250 300

prob

abili

ty

TMHMM posterior probabilities for PPRD2

transmembrane inside outside

I II III IV V

NH2

COOH

Cytosol

ER lumen

steroid 5-α-reductase domain

7

29 66

88 166

188 221

243 285

307

T78S

G274R

G193R

T301S

H321L

H336L

Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

4

Supplemental Figure 4. Schematic Representation and Expression of PPRDs, Related to Figure 2. (A) Structure of coding sequences of PPRD1, PPRD1-INT3, PPRD1-INT4 and PPRD2. Premature STOP codons are present in PPRD1-INT3 and PPRD1-INT4. The two latter DNA fragments were obtained during cloning of PPRD1 cDNA. (B) PPRD1 transcripts detected by (RT-PCR) in the leaves of 21- and 38-day-old WT plants. Bands corresponding to PPRD1 and PPRD1-INT3/ PPRD1-INT4 are indicated.

Supplemental Figure 4. Schematic Representation and Expression ofPPRDs, Related to Figure 2.(A) Structure of coding sequences of PPRD1, PPRD1-INT3, PPRD1-INT4and PPRD2. Premature STOP codons are present in PPRD1-INT3 and PPRD1-INT4. The two latter DNA fragments were obtained during cloning ofPPRD1 cDNA. (B) PPRD1 transcripts detected by (RT-PCR) in the leaves of 21- and 38-day old WT plants. Bands corresponding to PPRD1 and PPRD1-INT3/ PPRD1-INT4 are indicated.

I II III IV V VI

4 x STOP

I II III IV V VI

STOP

Exon Intron

AtPPR1_int3

AtPPR1_int4

I II III IV V VI 6xHis

I II III IV V VI GST

I II III IV V VI G3GFP

AtPPR2_6xHis

AtPPR2_GST

AtPPR2_G3GFP

I II III IV V VI

I II III IV V VI AtPPR1

AtPPR2

PPRD1

PPRD1-INT3/INT4

Col-0 leaves 38 days

Col-0 leaves 21 days

PPRD1

PPRD1-INT3

PPRD1-INT4

PPRD2

STOP

4 x STOP

A

B

Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

5

Supplemental Figure 5. Subcellular Localization of PPRD1 and PPRD2. Related to Figure 2. Co-localization of GFP-tagged PPRD2 or PPRD1 (green) with organelle markers – ER:mCherry and AtPIP2A:mCherry (red). Scale bar = 10 µm.

PPRD1:GFP AtPIP2A:mCherry Merge

PPRD2:GFP ER:mCherry Merge

PPRD2

PPRD1

Supplemental Figure 5. Subcellular Localization of PPRD1 and PPRD2. Relatedto Figure 2. Co-localization of GFP-tagged PPRD2 or PPRD1 (green) with organellemarkers – ER:mCherry and AtPIP2A:mCherry (red). Scale bar = 10 µm.

Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

6

Supplemental Figure 6. Phenotypes of WT, pprd2-1+/-

, PPRD1-OE, pprd2 PPRD1-OE andpprd2 PPRD1-OE

+/- Plants. Related to Figure 4.

(A) Leaves, (B) seed number and silique length and (C) expression of BiP2 in WT and studied mutants. White arrows indicate undulated leaves. Statistically significant differences from WT are indicated; *** - P<0.001.

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Supplemental Figure 6. Phenotypes of WT, pprd2-1+/-, PPRD1-OE, pprd2 PPRD1-OE and pprd2 PPRD1-OE+/- Plants. Related to Figure 4.(A) Leaves, (B) seed number and silique length and (C) expression of BiP2in WT and studied mutants. White arrows indicate undulated leaves.Statistically significant differences from WT are indicated; *** - P<0.001.

***

***

Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

7

Supplemental Figure 7. Analysis of Glycosylated Proteins in WT, pprd2-1+/-

, pprd2 PPRD1-OE and pprd2 PPRD1-OE

+/- Plants. Protein extracts from leaves and flowers were separated

by SDS-PAGE and gel was stained with Glycoprotein Gel Stain (upper image) or Coomassie Brilliant Blue (lower image). Marker lines contain, respectively, Glycoprotein Molecular Weight Standard or PageRuler

TM Prestained Protein Marker. Asterisks mark bands present in WT

extracts but absent in pprd2 lines.

Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

8

Supplemental Figure 8. (A) Alexander Staining of Mature Anthers of WT and pprd2-1+/-

Plants and (B) Aniline Blue Staining of Pollen Tube in the Pistil. For the latter analysis pollen grains from wild-type, pprd2

+/- or pprd2 PPRD1-OE mutants were

used to pollinate wild-type stigma. Pollen tubes were visualized by aniline blue staining. White frame indicates region of highest accumulation of inhibited pollen tubes of mutant origin. Scale bar = 100 µm. Related to Figure 5 and 6.

Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

9

Supplemental Figure 9. Effect of PPRD1 Overexpression on Plant Tolerance to Stress. Effect of sodium chloride and sorbitol (A, B) and tunicamycin (C) on WT and PPRD1-OE seed germination. Percentage of seedlings with roots formed (A, B, C); cotyledons (A’, B’) and root growth (C’). Fifty seeds of each genotype were germinated per experiment; values are means ± SD of three independent experiments. (D) Expression of PPRD1 (D) and PPRD2 (D’) in Arabidopsis seedlings treated with sorbitol or tunicamycin normalized to control conditions. Bars represent mean value ±SD of four independent experiments. Statistically significant differences are indicated; * - P<0.05 and ** - P<0.01.

Since dolichol is postulated to participate in cell response to environmental stress PPRD1-OE plants (Figure 3B) containing higher Dol level (Figure 3E) were challenged with various stimuli. Therefore the plant germination test was performed in the presence of sorbitol or NaCl. Overexpression of PPRD1 improved the formation of cotyledons upon NaCl and especially sorbitol treatments, while it did not affect the appearance of roots comparing to WT plants (Panels A/A’ and Panels B/B’). Additionally, effect of tunicamycin, an inhibitor of protein glycosylation on seed germination was tested too. Overexpression of PPRD1 enabled development of roots for all the seedlings in contrast to WT plants (only 50% of germinating seeds). Moreover the roots of the PPRD1-OE seedling were substantially longer than WT (Panels C/C’).Thus, overexpression of PPRD1 increases plant tolerance to sorbitol and salinity and prevents tunicamycin toxicity. To study further the relations between the PPRDs and stress we determined their expression profiles in WT plants treated with sorbitol and tunicamycin (Panels D/D’). PPRD2 expression was induced 3-4-fold after four days of treatment with either compound and increased slightly with time. In contrast, the PPRD1 expression was induced 3-fold by sorbitol after fourteen days and somewhat inhibited by tunicamycin during ten days of treatment. Taken together, PPRD2 undergoes induction in response to various adverse environmental factors.

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WT root PPRD1-OE root

WT root PPRD1-OE root

WT cotyledon PPRD1-OE cotyledon

WT cotyledon PPRD1-OE cotyledon

C WT root PPRD1-OE root

WT root PPRD1-OE root

D D’

C’

* * *

*

*

**

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Supplemental Figure 9. Effect of PPRD1 Overexpression on Plant Tolerance to Stress.Effect of sodium chloride and sorbitol (A, B) and tunicamycin (C) on WT and PPRD1-OE seed germination. Percentage of seedlings with roots formed (A, B, C);cotyledons (A’, B’) and root growth (C’). Fifty seeds of each genotype were germinated per experiment; values are means ± SD of three independent experiments.(D) Expression of PPRD1 (D) and PPRD2 (D’) in Arabidopsis seedlings treated with sorbitol or tunicamycin normalized to control conditions.Bars represent mean value ±SD of four independent experiments.Statistically significant differences are indicated; * - P<0.05 and ** - P<0.01.

Since dolichol is postulated to participate in cell response to environmental stress PPRD1-OE plants (Figure 3B) containing higher Dol level (Figure 3E) were challengedwith various stimuli. Therefore the plant germination test was performed in the presence of sorbitol or NaCl. Overexpression of PPRD1 improved the formation of cotyledons upon NaCl and especially sorbitol treatments, while it did not affect the appearance of roots comparingto WT plants (Panels A/A’ and Panels B/B’).Additionally, effect of tunicamycin, an inhibitor of protein glycosylation on seed germination was tested too. Overexpression of PPRD1 enabled development of roots for all the seedlings in contrast to WT plants (only 50% of germinating seeds). Moreover the roots of the PPRD1-OE seedling were substantially longer than WT (Panels C/C’).Thus, overexpression of PPRD1 increases plant tolerance to sorbitol and salinity and prevents tunicamycin toxicity.To study further the relations between the PPRDs and stress we determined their expression profiles in WT plants treated with sorbitol and tunicamycin (Panels D/D’).PPRD2 expression was induced 3-4-fold after four days of treatment with either compound and increased slightly with time. In contrast, the PPRD1 expression was induced 3-fold by sorbitol after fourteen days and somewhat inhibited by tunicamycin during ten days of treatment.Taken together, PPRD2 undergoes induction in response to various adverse environmental factors.

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Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463

10

Supplemental Figure 10. Dolichol Cycle in Plant Cells, Related to Discussion. Genes crucial for the pathway are indicated, lethal phenotypes are marked. Two insets present alternative, yet not documented steps of Prenol to Dolichol conversion. Abbreviations used: MVA - mevalonate, Ac-CoA - acetyl-CoA, GA-3P - D-glyceraldehyde-3-phosphate, HMGS - 3-hydroxy-3-methylglutaryl-CoA synthase, HMG-CoA - 3-hydroxy-3-methylglutaryl-coenzyme A, HMGR - 3-hydroxy-3-methylglutaryl-CoA reductase, IPP - isopentenyl diphosphate, DMAPP - dimethylallyl diphosphate, DXS - 1-deoxy-D-xylulose-5-phosphate synthase, DXR - 1-deoxy-D-xylulose 5-phosphate reductoisomerase, DXP - 1-deoxy-D-xylulose 5-phosphate, MEP - 2-C-methylerythritol 4-phosphate, IDI - isopentenyl diphosphate isomerase, FPPS - farnesyl diphosphate synthase, CPT - cis-prenyltransferase, PPRD – polyprenol reductase, DOK – dolichol kinase, LEW - leaf wilting, DPMS - DolPMan synthase, ALG5 - putative dolichyl-phosphate beta-glucosyltransferase, ALG10 - alpha-1,2-glucosyltransferase, OST - dolichyl-diphosphooligosaccharide-protein glycosyltransferase, STT - dolichyl-diphosphooligosaccharide-protein glycosyltransferase, GCS - alpha-glucosidase 2, Phos – phosphatase.

P P

P

P OH

OH

P P

P

fps1 fps2

idi1 idi2

cpt6 lew1

CPT1-9 LEW1

FPPS1 FPPS2

IDI1 IDI2

DOK

dok dpms1

DPMS1 PPRD1 PPRD2

pprd2

P

P

LEW3

lew3

MEP pathway

MVA pathway Ac-CoA HMG-CoA Mevalonate HMGS

fkp

HMGR1 HMGR2

hmgr1 hmgr2

GA-3P + Pyruvate DXP MEP DXS

cla1

DXR

dxr

OST dgl1

STT stt3a stt3b

protein

GCS1 gcs1

Endoplasmic Reticulum Lumen

P P

P

Key:

phosphate

diphosphate

GlcNAc

Man

Glc

lethal mutation

Flippase

IPP DMAPP

FPP

Pren-PP Pren-OH Dol-OH Dol-P

Dol-P-Man Cytoplasm

P P

P

P

P

P

ALG5

P P

alg10

ALG10

P P

Dol-P-Glc

P

P P

P

P

P P

DOK

dok

PPRD1 PPRD2

pprd2

Pren-PP Pren-P Dol-P Dol-PP

P P OH

P

Phos (?)

Phos (?)

Kinase (?)

Dol-OH

P

P OH

OH

P

DOK

dok

PPRD1 PPRD2

pprd2

Pren-PP Pren-OH Dol-OH Dol-P

OH

IPOH

P P P P

IPP

IPP DMAPP

Supplemental Figure 10. Dolichol Cycle in Plant Cells, Related to Discussion. Genes crucial for the pathway are indicated, lethal phenotypes are marked. Two insets present alternative, yet not documented steps of Prenol to Dolichol conversion. Abbreviations used: MVA - mevalonate, Ac-CoA - acetyl-CoA, GA-3P - D-glyceraldehyde-3-phosphate, HMGS - 3-hydroxy-3-methylglutaryl-CoA synthase, HMG-CoA - 3-hydroxy-3-methylglutaryl-coenzyme A, HMGR - 3-hydroxy-3-methylglutaryl-CoA reductase, IPP - isopentenyl diphosphate, DMAPP - dimethylallyl diphosphate, DXS - 1-deoxy-D-xylulose-5-phosphate synthase, DXR - 1-deoxy-D-xylulose 5-phosphate reductoisomerase, DXP - 1-deoxy-D-xylulose 5-phosphate, MEP - 2-C-methylerythritol 4-phosphate, IDI - isopentenyl diphosphate isomerase, FPPS - farnesyl diphosphate synthase, CPT - cis-prenyltransferase, PPRD – polyprenol reductase, DOK – dolichol kinase, LEW - leaf wilting, DPMS - DolPMan synthase, ALG5 - putative dolichyl-phosphate beta-glucosyltransferase, ALG10 - alpha-1,2-glucosyltransferase, OST - dolichyl-diphosphooligosaccharide-protein glycosyltransferase, STT - dolichyl-diphosphooligosaccharide-protein glycosyltransferase, GCS - alpha-glucosidase 2, Phos – phosphatase.

n

n

n

n

n

n

n n n nn n n n n n n n n n n n n n n n

nn nn nn nn nn

n n n nn n n n

n

n

n n n nn n n n n n

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Supplemental Table 1. Comparison of Amino Acid Sequences of Putative PPRDs with Human and Yeast Polyprenol Reductases Based on the Alignment, Related to Supplemental Figure 1. Protein Gene locus Comparison to hSRD5A3 Comparison to Dfg10p

Identity [%]

Similarity [%]

BLASTP E-value

Identity [%]

Similarity [%]

BLASTP E-value

PPRD1 At1G72590 29 50 8E-36 26 39 2E-15 PPRD2 At2G16530 28 47 3E-38 27 42 2E-13 PPRD3 At3G43840 30 54 0.031 27 47 1E-04

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Supplemental Table 2. Localisation of Predicted 3-oxo-5-α-steroid 4-dehydrogenase Domain in Arabidopsis Polyprenol Reductases. Related to Supplemental Figure 3. Protein Protein length [a.a.] Predicted 3-oxo-5-α-steroid

4-dehydrogenase domain [a.a.] E-value

PPRD1 320 199-320 7.2E-24 PPRD2 343 223-343 1.1E-22 PPRD3 84 14-84 6.7E-10

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Supplemental Table 3. Seed Germination of pprd2-1+/- and pprd2-2+/- Plants on Solid Medium, Related to Figure 4.

Seeds pprd2-1+/- pprd2-2+/- WT Sown 203 (100%) 210 (100%) 200 (100%)

Germinated 197 (97%) 206 (98%) 198 (99%)

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Supplemental Table 4. Mass Spectrometry-based Differential Proteomics of WT and pprd2 PPRD1-OE+/- Plants. Proteins extracted from the flowers of 10-week-old plants were subjected to MS analysis. Listed are the proteins present in WT but totally absent from pprd2 PPRD1-OE+/-. Predicted number of N-glycosylation sites, function (described / predicted) and site of expression of particular proteins according to NetNGlyc 1.0 Server and TAIR database, respectively.

No. Protein Predicted glycosylation sites

Gene locus Function Expressed in (organs with highest expression are in bold)

1 ASN2, ASPARAGINE SYNTHETASE 2

2 AT5G65010 L-asparagine biosynthetic process, ammonium ion metabolic process, asparagine biosynthetic process, cysteine biosynthetic process, glutamine metabolic process, hyperosmotic salinity response

carpel, cauline leaf, collective leaf structure, cotyledon, flower, guard cell, hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, plant sperm cell, pollen tube cell, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

2 GLYCOSYL HYDROLASE FAMILY PROTEIN

4 AT5G20950 carbohydrate metabolic process, glucan catabolic process, response to endoplasmic reticulum stress, systemic acquired resistance

adult vascular leaf, carpel, cauline leaf, collective leaf structure, cotyledon, flower, fruit, guard cell, hypocotyl, inflorescence meristem, juvenile vascular leaf, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

3 ABNORMAL INFLORESCENCE MERISTEM, AIM1

3 AT4G29010 fatty acid beta-oxidation, flower development, jasmonic acid biosynthetic process, multicellular organismal development, seed germination

carpel, cauline leaf, collective leaf structure, cotyledon, flower, fruit, guard cell, hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

4 PECTIN LYASE-LIKE SUPERFAMILY PROTEIN

7 AT4G23500 carbohydrate metabolic process, cell wall organization

guard cell, inflorescence meristem

5 SKS11, SKU5 SIMILAR 11

6 AT3G13390 oxidation-reduction process carpel, collective leaf structure, flower, pedicel, petal, plant embryo, pollen, pollen tube cell, sepal, stamen, stem

6 A. THALIANA 1 AT3G10160 one-carbon metabolic process, carpel, cauline leaf, collective leaf structure,

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DHFS-FPGS HOMOLOG C, DFC

photorespiration, seedling development, tetrahydrofolylpolyglutamate biosynthetic process

cotyledon, flower, guard cell, hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

7 Transducin/WD40 repeat-like superfamily protein

2 AT2G20330 N-terminal protein myristoylation, biological_process, photoperiodism, flowering, protein targeting to mitochondrion

carpel, cauline leaf, collective leaf structure, cotyledon, cultured plant cell, flower, guard cell, hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, plant sperm cell, pollen, pollen tube cell, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

8 HOP1 2 AT1G12270 cullin deneddylation, nucleotide biosynthetic process, photomorphogenesis

carpel, cauline leaf, collective leaf structure, cotyledon, cultured plant cell, flower, guard cell, hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

9 O-Glycosyl hydrolases family 17 protein

8 AT1G11820 carbohydrate metabolic process, defense response

guard cell, inflorescence meristem

10 HDA5, HISTONE DEACETYLASE 5

1 AT5G61060 fatty acid beta-oxidation, histone deacetylation, protein N-linked glycosylation, protein import into peroxisome matrix, regulation of transcription, DNA-templated, transcription, DNA-templated

carpel, cauline leaf, collective leaf structure, cotyledon, flower, guard cell, hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

11 PCK1, PEPCK, PHOSPHOENOLPYRUVATE CARBOXYKINASE

2 AT4G37870 cellular response to phosphate starvation, defense response to fungus, incompatible interaction, gluconeogenesis, response to cadmium ion

carpel, cauline leaf, collective leaf structure, cotyledon, cultured plant cell, flower, guard cell, hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, plant sperm cell, pollen, root, rosette leaf, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

12 1-DEOXY-D-XYLULOSE 5-

5 AT4G15560 1-deoxy-D-xylulose 5-phosphate biosynthetic process, aromatic amino

carpel, cauline leaf, cotyledon, epidermis, flower, fruit, guard cell, hypocotyl, inflorescence

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PHOSPHATE SYNTHASE, CLA, DXS,

acid family biosynthetic process, aromatic amino acid family metabolic process, carotenoid biosynthetic process, cell differentiation, cellular amino acid biosynthetic process, chlorophyll biosynthetic process, chlorophyll metabolic process, coenzyme biosynthetic process, cysteine biosynthetic process, glycine catabolic process, isopentenyl diphosphate biosynthetic process, methylerythritol 4-phosphate pathway, jasmonic acid biosynthetic process, leaf morphogenesis, lipoate metabolic process, nucleotide metabolic process, oxidoreduction coenzyme metabolic process, oxylipin biosynthetic process, phosphatidylglycerol biosynthetic process, positive regulation of transcription, DNA-templated, protein autophosphorylation, regulation of lipid metabolic process, regulation of protein localization, regulation of proton transport, response to light stimulus, secondary metabolic process, sulfur amino acid metabolic process, sulfur compound biosynthetic process, thiamine biosynthetic process, vitamin metabolic process

meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, stigma, trichome, vascular leaf

13 PAL4, PHENYLALANINE AMMONIA-LYASE 4

3 AT3G10340 L-phenylalanine catabolic process, cinnamic acid biosynthetic process, glucuronoxylan metabolic process, xylan biosynthetic process

carpel, collective leaf structure, flower, guard cell, hypocotyl, inflorescence meristem, petal, petiole, plant embryo, pollen, root, sepal, shoot apex, shoot system, stamen, vascular leaf

14 ABCF3 2 AT1G64550 cytoskeleton organization, defense response to bacterium,

carpel, cauline leaf, collective leaf structure, cotyledon, cultured plant cell, flower, guard cell,

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gluconeogenesis, glycolytic process, mRNA export from nucleus, proteasomal protein catabolic process, protein import into nucleus, response to cadmium ion, response to salt stress

hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

15 GH3.17

1 AT1G28130 auxin homeostasis, brassinosteroid biosynthetic process, response to auxin

carpel, collective leaf structure, flower, hypocotyl, petal, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

16 AN, ANGUSTIFOLIA

2 AT1G01510 actin nucleation, cell adhesion, chromatin silencing, floral organ morphogenesis, fruit morphogenesis, histone H3-K9 methylation, histone phosphorylation, inductive cell-cell signaling, leaf morphogenesis, microtubule cytoskeleton organization, monopolar cell growth, oxidation-reduction process, regulation of cell shape, regulation of epidermal cell differentiation, regulation of epidermal cell division, regulation of trichome morphogenesis, trichome branching, trichome morphogenesis

carpel, cauline leaf, collective leaf structure, cotyledon, flower, guard cell, hypocotyl, inflorescence meristem, leaf apex, leaf lamina base, pedicel, petal, petiole, plant embryo, pollen, root, seed, sepal, shoot apex, shoot system, stamen, stem, vascular leaf

Methodology: Peptide mixtures were analyzed by liquid chromatography coupled to an LTQ FT ICR mass spectrometer (Hybrid-2D-Linear Quadrupole Ion Trap – Fourier Transform Ion Cyclotron Resonance Mass Spectrometer, Thermo Electron Corp., Bremen, Germany). Gel slices were subjected to a standard digestion procedure, during which proteins were reduced with 100 mM DTT for 30 min at 56°C, alkylated with iodoacetamide for 45 min in a darkroom at 22°C and digested overnight with Trypsin. The resulting peptides were eluted from the gel with 0.1% TFA and 2% ACN. The peptide mixture was applied to an RP-18 pre-column (LC Packings) using water that contained 0.1% TFA as the mobile phase and than transferred to a nano-HPLC RP-18 column (nanoACQUITY UPLC BEHC18, Waters) using an acetonitrile gradient (0–60% ACN in 30 min) in the presence of 0.05% formic acid with a flow rate of 150 nL/min The column outlet was directly coupled to an ion source of LTQ-FT-MS that worked in the regime of data dependent MS to MS/MS switch. A blank run that ensured a lack of cross-contamination from previous samples preceded each analysis. Acquired raw data were processed by Mascot Distiller (version 2.1.1, Matrix Science, London, UK) followed by Mascot Search database search engine (v.2.1, Matrix Science).

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Supplemental Table 5. Effect of Polyprenol, Dolichol and Tunicamycin on Pollen Germination on Solid Medium, Related to Figure 6. Statistically significant differences are indicated: * - P<0.05; *** - P<0.001

Regular medium Medium supplemented with Prenol-16 Dolichol - MIX Tunicamucin, WT pollen

0 ng/ml 10 ng/ml 50 ng/ml Pollen grains

WT, n=1208 pprd2-1+/-, n=1424

WT, n=482 pprd2-1+/-, n=438

WT, n=522 pprd2-1+/-, n=508

n=164 n=203 n=241

normal 1160 (96%) 812 (57%) 458 (95%) 228 (52%) 506 (97%) 488 (96%) 161 (98%) 171 (84%) 94 (39%)

deformed 48 (4%) 612 (43%)*** 24 (5%) 210 (48%)*** 16 (3%) 20 (4%) 3 (2%) 32 (16%)* 147 (61%)*

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Supplemental Table 6. Phenotypes of Arabidopsis Mutants in Genes Encoding Elements of Isoprenoid Synthesis or Protein Glycosylation Pathways, Related to Discussion. Gene Function Phenotype Reference MVA pathway encoding genes HMGS 3-hydroxy-3-

methylglutaryl-CoA synthase

fkp (flaky pollen) null mutant is lethal due to male-sterility; fkp1-4 pollen, carrying null allele of the gene, has normal 3 nuclei as visualized by DAPI stain, but does not germinate normally and is 100% sterile; weak fkp1-1 allele does not affect the vegetative growth, but pollen grains formed on heterozygote are infertile due to abnormal development of tapetum-specific organelles - elaioplasts and tapetosomes - leading to reduced pollen coat deposition and, as a consequence, adhesion and hydratation on stigma

Ishiguro et al., 2010

HMGR1 HMGR2

3-hydroxy-3-methylglutaryl-CoA reductase responsible for formation of mevalonate

hmgr1 hmgr2 double mutant is lethal due to male-sterility; hmgr1+/- hmgr2 heterozygous plants - half of pollen grains are malformed, shrunken and have underdeveloped ER structures, pollen lethality is due to both gametophytic and maternal sporophytic effects; hmgr1 single mutant - plants are dwarfed and homozygotes are male sterile, short empty siliques are formed, male sterility comes from sporophytic, maternal defect of tapetum and can be reversed by external application of squalene, but not brassinosteroids on inflorescence

Suzuki et al., 2009 Suzuki et al., 2004

IDI1 IDI2

isopentenyl diphosphate isomerase responsible for formation of dimethylallyl diphosphate

idi1 idi2 double mutant is lethal due to male-sterility

Phillips et al., 2008

MEP pathway encoding genes DXS 1-deoxy-D-

xylulose 5-phosphate synthase

cla1 mutant is albino lethal; chloroplast biogenesis is affected

Mandel et al., 1996 Budziszewski et al, 2001

DXR 1-deoxy-D-xylulose 5-phosphate reducto-

dxr mutant is albino lethal; chloroplast biogenesis is affected

Budziszewski et al., 2001 Xing et al.,

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isomerase 2010 Isoprenoid chain formation FPPS1 FPPS2

farnesyl diphosphate synthase responsible for synthesis of FPP

fps1 fps2 double mutant is lethal due to block on embryo development at pre-globular stage, transmission through male gametophyte is severely reduced (12% of expected frequency), pollen grains develop 3 nuclei, pollen germination is not impaired, but growth of pollen tube is severely affected; fps1 and fps2 single mutants are viable and show minor biochemical defects;

Closa et al., 2010

CPT1-9 2 out of 9 putative genes characterized

cis-prenyltransferase responsible for formation of polyprenyl diphosphate (syn. dehydrodolichyl diphosphate)

cpt6 single mutant does not show phenotypic aberrations

Surmacz et al., 2014

LEW1 described as atypical AtCPT recently recognized as AtCPT-like accessory protein (ortholog of hNogoBR)

lew1 null mutant is lethal Zhang et al., 2008 Qu et al., 2015

PPRD1 PPRD2

polyprenol reductase responsible for conversion of polyprenol to dolichol

pprd2 null mutation is lethal due to nonfunctional pollen, transmission through male gametophyte is abolished; pprd2 point mutations - no defect observed; pprd2+/- heterozygous plant – sporophyte does not show phenotypic changes, approx. 50% pollen grains are wrinkled and pollen tubes are malformed

this report

DOK dolichol kinase responsible for formation of dolichyl phosphate

dok (evn) null mutation is lethal, both mutated pollen and female gametophytes are nonfunctional; dok+/- heterozygous plant pollen is infertile, suggesting that intrinsic rather than maternal effect contributes to the lethality, mutated pollen grains wrinkled and hollow, some ovules have aberrant morphology and probably are unable to be fertilized

Kanehara et al., 2015 Lindner et al., 2015

Protein glycosylation pathway DPMS1 catalytic subunit

of DolPMan dpms1 mutant is viable, possesses wrinkled seed coat and is hypersensitive to

Jadid et al., 2011

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synthase responsible for mannosylation of DolP

ammonium (chlorosis and strong reduction of root growth)

ALG1 beta-1,4-mannosyltransferase responsible for transfer of mannose to DolPP-GlcNAc2

tun null mutant is lethal due to defects in pollen tube reception.

Lindner et al., 2015

LEW3 alfa-1,2-mannosyltransferase responsible for mannosylation of DolPP-GlcNAc2Man3

lew3 null mutant is lethal; lew3 point mutant shows reduced fertility, short siliques, impaired cellulose synthesis, abnormal primary and secondary cell wall biosynthesis

Zhang et al., 2009

ALG10 alfa-1,2-glucosyltransferase responsible for the terminal glucosylation of DolPP-GlcNAc2Man9Glc2 precursor

alg10-1 mutant is viable, displays severe protein underglycosylation, reduced leaf size, reduced tolerance to salt stress

Farid et al., 2011

multigene OST complex

oligosaccharide transferase complex responsible for transfer of tetradecasaccharide structure from DolP-GlcNAc2Man9Glc3 to protein (Asn-X-Ser/Thr)

dgl1-2 null mutant in one of OST complex subunits is embryo-lethal; dgl1-1 point mutant shows developmental defects in early seedling development, including reduced cell elongation and collapse of cells of central cylinder due to changes in accumulation of callose; stt3a stt3b double mutant in OST complex subunit is gametophytic lethal

Lerouxel et al., 2005; Koiwa et al., 2003

GCS1 glycan trimming after oligosaccharide transfer onto Asn in a protein molecule

gcs1 mutant is embryolethal (heart stage of development) and shows abnormal cell enlargement and occasional cell wall disruptions, abnormal accumulation of seed storage proteins, cytokinesis and cellulose biosynthesis

Boisson et al., 2001

N- glycan processing of N-glycosylated

subsequent downstream steps of N- glycan

homozygous null mutants show abnormalities in root growth, aberrant cellulose deposition pattern

Liebminger et al., 2009; Schaewen et al., 1993;

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proteins processing Strasser et al., 2004; Strasser et al., 2004b; Strasser et al., 2006; Strasser et al., 2007

SETH1 SETH2

first step of GPI biosynthetic pathway

seth1 and seth2 mutations block male transmission and pollen function due to reduced pollen germination and tube growth, abnormal callose deposition

Lalanne et al., 2004

APTG1 mannosyltransferase involved in GPI-anchor biosynthesis

aptg1 (abnormal pollen tube guidance) mutant pollen tubes shows compromised micropylar guidance, embryo lethality

Dai et al., 2014

PNT1 mannosyltransferase involved in GPI-anchor biosynthesis

pnt1 mutants are embryo-lethal, at early stages show large reduction in crystalline cellulose and an increase in pectin content, ectopic deposition of xyloglucan, pectin, and callose, poor transmission through pollen, with no significant effect on egg transmission

Gillmor et al., 2005

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Supplemental Table 7. Primers Used for Genotyping, Cloning, Mutagenesis and Expression Studies, Related to Materials and Methods. Genotyping primers

Gene Allele Primer sequence

PPRD1 PPRD1-OE

gFP1 F: 5’-CATATTTATAGTGTGGGGTGGC-3’ gRP1 R: 5’-GAAAGAGAAAACGTGGGAACC-3’

PPRD2 pprd2-1 gFP2 F: 5’-GTCTGTGAATTTGCCCATCTG-3’

gRP2 R: 5’-TCCACTCGCAATCAGTAAACC-3’

pprd2-2 gFP2 F: 5’-GTCTGTGAATTTGCCCATCTG-3’ gRP2 R: 5’-TCCACTCGCAATCAGTAAACC-3’

LBb1.3 - 5’-ATTTTGCCGATTTCGGAAC-3’

qPCR primers

Gene Locus Primer sequence

PPRD1 At1g72590 qFP1 F: 5’- GTGGCTGCTAATCTGACATAC-3’ qRP1 R: 5’-GTATAAACTTGGCCAAAGTTGTG-3’

PPRD2 At2g16530 qFP2 F: 5’- GTGGCTGCTAATCTGACATAC-3’ qRP2 R: 5’- CAGCATTTACAGAAGACAACCA-3’

BiP2 At5g42020 F: 5’- GGTATCGAGACTGTAGGAGG-3’ R: 5’- GGTACGTTGTGAAAACCTGA-3’

ACTIN-2 At3g18780 F: 5’-GACCAGCTCTTCCATCGAGAA-3’ R: 5’-CAAACGAGGGCTGGAACAAG-3’

Cloning primers

Gene Tag type Primer sequence

PPRD1 No tag F: 5’-CACCATGGAGGTAGAGATTGTGTGG-3’

R: 5’-TTAGTAGACATGAGGGAAAATAGCATGC-3’

GST F: 5’-CACCATGGAGGTAGAGATTGTGTGG-3’ R: 5’-GTAGACATGAGGGAAAATAGCATGC-3’

PPRD2 No tag F: 5’- CACCATGGTGGAATTGGAGATTGTG-3’

R: 5’- TTAGTAGACATAAGGGAAAATGGCATGC-3’

GST F: 5’- CACCATGGTGGAATTGGAGATTGTG-3’ R: 5’- GTAGACATAAGGGAAAATGGCATGCCC-3’

PPRD1Pro nd F: 5’- CACCTTCTTATATAAAATAAGGTAAAGGAAGAATAT -3’ R: 5’- CTCCATACTTTTTTTTTTTGTGTGTGTGTG-3’

PPRD2Pro nd F: 5’- CACCAAGGCATAACTTAATAACAAGCTCCACTA-3’ R: 5’- ACTTTTTTGTTTAGTGTTTCTTCTTCTCTGAAATC-3’

Mutagenesis primers

Construct Primer sequence

PPRD2 H321L F: 5’- CACCATGGTGGAATTGGAGATTGTG-3’ R: 5’- CGTTTCTCCAGCCGCGTATGTCAGATTAGC-3’ F: 5’- TCTGACATACGCGGCTGGAGAAACGCTCCG-3’ R: 5’- TTAGTAGACATAAGGGAAAATGGCATGC-3’

PPRD2 H336L F: 5’- CACCATGGTGGAATTGGAGATTGTG-3’ R: 5’- TTAGTAGACATAAGGGAAAATGGCAAGCC-3’