supplemental data. jozwiak et al. (2015). plant cell 10 ... · pdf filesupplemental data....
TRANSCRIPT
Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463
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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
1111111111111111111111111111111111111111111111111111111111111111111111111
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KMAPL SSEEF Q - L SD I ASRL AGG - SHVF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MQL E I - - VWL VRF AWI T VWI VS I L PL L I AS I PT SKL DS - - - F REL VL SL AGRG - - K I L H - - - PSS - - - - - - - - - - - - - - QKF T I PQKCF AHF YV I GVVWT T L L L AAT WL YAC - EMAPL SSEEF Q - L SD I ASHL T GG - SHVF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MDL E I - - VWF VRAAWI T VWI VS I L PL L I AS I PT SKL DS - - - F RDL VL SF AGRG - - K I L H - - - PSS - - - - - - - - - - - - - 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- K I L H - - - PSS - - - - - - - - - - - - - - QKF T VPQKF F GHF YVVGVVWT T L L L AAT WMYAC - KMA - - - - - - - - - - - - - - - - - - GG - SHVF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MV I AS I VMEVG I - - VWL VR - - - - AAWI AS I L P I L I AS I PSSKL T S - - - F HQL VL GF AGRG - - K I L H - - - L SS - - - - - - - - - - - - - - QKWT VPQKYF AHF YVVGAAWT T L L L AT T WMYAC - KT APL SSEEF H - L SD I ASHL T GG - SHVF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MV I AS I VMEL G I - - VWL ERAAWI AAWI AA I L PML I AS I PSSKL T S - - - F HQL VL GF AGRG - - K I L H - - - L SS - - - - - - - - - - - - - - QKWT VPQKYF AHF YVVGAAWT T L L L AT T WMYAC - KT APSSSEEF H - L SD I ACHL T GG - SHVF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEL G I - - VWL VR - - - - AAWI AA I L PMV I AF I P I SKL T S - - - F HQL L L GF ARRG - - K I I Q - - - PSS - - - - - - - - - - - - - - QKWT VPQKF F AHF YVL GVAWT T F L L AT T WMYAC - KT APL F SEEF Q - F SD I ASSL T GGSSHVF SF- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MEF WF - - 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.
***
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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’
* * *
*
*
**
*
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
Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463
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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
Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463
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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).
Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463
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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.,
Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463
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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
Supplemental Data. Jozwiak et al. (2015). Plant Cell 10.1105/tpc.15.00463
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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’