Ali A.M. Ahmed, Nancy Roosens, Eric Dewaele, Michel Jacobs and Geert Angenon

Overexpression of a novel feedback-desensitized Δ1-pyrroline-5-carboxylate synthetase increases proline accumulation and confers salt tolerance in transgenic Nicotiana plumbaginifolia.

Supplementary figures

LB Eco72I BglII RB

3’ 35S hpt p35S p35Se mP5CS 3’ nos

Supplementary Fig. 1 Structure of T-DNA in the plasmid pCAM-mP5CS. LB, left T-DNA border; 3’35S, cauliflower mosaic virus 35S 3’ processing and polyadenylation signal; hpt, hygromycin phosphotransferase gene; P35S, cauliflower mosaic virus 35S promoter; P35Se, enhanced cauliflower mosaic virus 35S promoter; mP5CS, mutated Δ1-pyrroline-5-carboxylate synthetase; 3’nos, nopaline synthase 3’ processing and polyadenylation signal; RB, right T-DNA border.

1 CLUSTAL W (1.83) multiple sequence alignment

SaP5CS ------MATADRTRTFMKDVKRVIIKVGTAVV 26 SoP5CS ------MATVDRTRTFMKDVKRVIIKVGTAVV 26 SbP5CS2 ------MATADRTRSFMRDVKRVIIKVGTAVV 26 ZmP5CS ------MATADRTRTFMKDVKRVIIKVGTAVV 26 OsP5CS ------MASVDPSRSFVRDVKRVIIKVGTAVV 26 TaP5CS ------MAGADPNRSFMKDVKRIIIKVGTAVI 26 SbP5CS1 ------MGRGGIGGAVGMAMENADSARAFVKDVKRIIIKVGTAVV 39 AcP5CS ------MDR-VDSTRAFVKGVKLIVVKVGTAVV 26 AdP5CS ------MDA-VDSTRAFVKGVKRVIIKVGTAVV 26 AvP5CS ------MDSNVNATRAFVKDVKRVIVKVGTAVV 27 SlP5CS2 ------MET-VDSTRAFVKNVKRLIVKVGTAVV 26 PeP5CS ------MS-DLDRSRAFFNDVKRLIIKVGTAVV 26 RcP5CS ------MNGGMDRSRAFVKDVKRLVIKVGTAVV 27 EjP5CS ------MDPSRAFVKNVKRIIIKVGTAVV 23 VvP5CS ------MHMDAMDPTRAFVKDVKRLVIKFGTAVV 28 GmP5CS ------MEN-TDPCRHLLKDVKRIIIKVGTAVV 26 PvP5CS2 ------MEN-TDPCRHFLKDVKRIVIKVGTAVV 26 MSP5CS ------MAN-ADPCRDFVKDVKRIIIKVGTAVV 26 MtP5CS ------MAN-ADPCRDFVKDVKRIIIKVGTAVV 26 PvP5CS1 ------MEGAVDPSRSFMKDVKRVIIKVGTAVV 27 VaP5CS ------MESAVDPSRGFMKDVKRVIIKVGTAVV 27 Mutated AtP5CS1 ------MEELDRSRAFARDVKRIVVKVGTAVV 26 AtP5CS1 ------MEELDRSRAFARDVKRIVVKVGTAVV 26 CbP5CS ------MEELDRSRAFGKDVKRIVVKVGTAVV 26 AtP5CS2 ------MTEIDRSRAFAKDVKRIVVKVGTAVV 26 BnP5CS ------MEDEIDRSRAFAKDVKRIVVKVGTAVV 27 McP5CS ------MDATRAFVKDVKRVVVKVGTAVV 23 VuP5CS MELLQNGTKAKPSEIPLVNGTALTHLNSLFQTQFSGNIDPSRAFVSKVKRLIVKVGTAVV 60 SlP5CS1 ------MSEVVDQAPIAKQWRRVVVKVGTSSI 26 ScP5CS ------MKDANESKSYT-----IVIKLGSSSL 21 :::*.*:: :

SaP5CS TR-HDGRLALGRLGALCEQVKELNALGYEVIIVTSGAVGVGKQRLKYRKLVNSSFADLQK 85 SoP5CS TT-HDGRLALGRLGALCEQVKELNALGYEVIIVTSGAVGVGKQRLKYRKLVDSSFADLQK 85 SbP5CS2 TR-HDGRLALGRLGALCEQVKELNALGYEVIIVTSGAVGVGKQRLKYRKLVNSSFADLQK 85 ZmP5CS TRGDDGRLAVGRLGCLCEQVKELNVLGYEVILVTSGAVGVGKQRLKYRKLVNSSFADLQK 86 OsP5CS SR-QDGRLALGRVGALCEQVKELNSLGYEVILVTSGAVGVGRQRLRYRKLVNSSFADLQK 85 TaP5CS TR-NDGRLALGRIGALCEQVKDLNAQGYEVIMVTSGAVGVGRQRLRYRKLVNSSFADLQK 85 SbP5CS1 TG-QNGRLAMGGLGSLCEQVKQLNFQGYEVILVTSGAVGVGRQRLQYRKLIHSSFADLQN 98 AcP5CS TR-SDGRLALGRLGALCEQIKELITLGHEVILVASGAVSVGRQRLRYRRLINSSFADLQK 85 AdP5CS TR-ADGRLALGRLGALCEQIHELNSQGFEVILVTSGAVGVGRQRLRYRKLVNSSFADLQK 85 AvP5CS TR-ADGRLALGRLGALCEQIHELNSQGYEVILVTSGAVGVGRQRLKYRRLVNSSFADLQK 86 SlP5CS2 TR-ADGRLALGRLGALCEQLQELNSQGYEVILVTSGAVGVGRQRLRYRKLLNSSFLDLQK 85 PeP5CS TR-ADGRLALGRLGALCEQIKDLSSLGYEVIVVTSGAVGLGRQRLKYRRFVNSSFSDLQK 85 RcP5CS TR-GDGRLALGRLGAFCEQVKELNSQGYEVIVVSSGAVGLGRQRLRYRKLVNSSFADLQK 86 EjP5CS TR-SDGRLALGRLGSLCEQVKELNSQGYEVIVVTSGAVGLGRQRLRYRRLINSSLADLQK 82 VvP5CS TR-SDGRLALGRLGALCEQIKELNSQGYQVIVVTSGAVGLGRQRLRYRSLLNSSFADLQK 87 GmP5CS TR-QDGRLAVGELGALCEQIKELNSLGYEIILVSSGAVGLGRQRLRYRKLINSSFADLQK 85 PvP5CS2 TR-QDGRVAVGKLGALCEQIKELNSLGYEIILVSSGAVGLGRQRLRYRKLINSSFADLQK 85 MSP5CS TR-QDGRLAVGKLGALCEQIKELNILGYEVILVSSGAVGLGRQRLRYRKLIQSSFADLQK 85 MtP5CS TR-QDGRLAVGKLGALCEQIKELNILGYEVILVSSGAVGLGRQRLRYRKLIQSSFADLQK 85 PvP5CS1 TR-EEGRLAVGRLGALCEQIKQLNSLEYDIILVSSGAVGIGRQRLRFRKLIHSSFADLQK 86 VaP5CS TR-EEGRLAVGRLGALCEQIKQLNSLGYDIILVSSGPVGIGRQRLRFRKLINSSFADLQK 86 Mutated AtP5CS1 TG-KGGRLALGRLGALFEQLAELNSDGFKVILVSSGAVGLGRQRLRYRQLVNSSFADLQK 85 AtP5CS1 TG-KGGRLALGRLGALCEQLAELNSDGFEVILVSSGAVGLGRQRLRYRQLVNSSFADLQK 85 CbP5CS TG-KGGRLALGRLGALCEQLAELNSDGFEVILVSSGAVGLGRQRLRYRQLVNSSFADLQK 85 AtP5CS2 TG-KGGRLALGRLGAICEQLAELNSDGFEVILVSSGAVGLGRQRLRYRQLVNSSFADLQK 85 BnP5CS TG-KGGRLALGRLGAICEQLAELNSDGFEVILVSSGAVGLGRQRLRYRQLVNSSFADLQK 86 McP5CS TR-SDGRLALGRLGSLCEQLKELNSDGYEVILVTSGAVSAGRQRLRFRKLVNSSFADLQK 82 VuP5CS TR-SDGRLALGRIGALCEQLKELSSQGFEVILVTSGAVGLGRQRLRYRKLANSSFSDLQK 119 SlP5CS1 VD-AAGEIKYPVINRLAQTLTQLEQTGYEVILVTSGAIGVALKQMHQT------KR 75 ScP5CS VDEKTKEPKLAIMSLIVETVVKLRRMGHKVIIVSSGGIAVGLRTMRMNK------R 71 . :. : : : .* ..:*:*:** :. . : :: .

SaP5CS PQMELDGKACAAVGQSGLMALYDMLFTQLDVSSSQLLVTDSDFENPNFRERLRETVESLL 145 SoP5CS PQMELDGKACAAVGQSGLMALYDMLFTQLDVSSSQLLVTDSDFENPNFRERLRETVESLL 145 SbP5CS2 PQMELDGKACAAVGQSGLMALYDMLFTQLDVSSSQLLVTDSDFENPNFRERLRETVESLL 145 ZmP5CS PQMELDGKACAAVGQSGLMALYDMLFTQLDVSSSQLLVTDSDFENPNFRERLCETVESLL 146 OsP5CS PQMELDGKACAAVGQSGLMALYDMLFNQLDVSSSQLLVTDSDFENPKFREQLTETVESLL 145 TaP5CS PQMELDGKACAAVGQSGLMALYDMLFTQLDVSSSQLLVTDSDFDNSNFRERLRETVESLL 145 SbP5CS1 PQMNFDGKACAAVGQSGLMAICDTLFSQLDVTSSQLLVTDRDFRDPSFGDQLRETVFALL 158 AcP5CS PQVELDGKACAAVGQNGLMALYDTLFSQLDVTSSQLLVTDNDFRDPEFRKQLSGTVKTLL 145 AdP5CS PQIELDGKACAAVGQNGLLALYDTLFSQLDVTSAQLLVTDNDFRDPEFRKQLTETVESLL 145 AvP5CS LQNELDGKACAAVGQNGLMALYDSLFTQLDVTSAQLLVTDNDFRDPDFRRQLNEAVNSLL 146

2 SlP5CS2 PQTELDGKACAAVGQNGLMALYDSLFSQLDVTSAQLLVTDNDFRDPDFRRQLNDTVNSLL 145 PeP5CS PQVDLDGKACAAVGQNSLMALYDTLFSQLDVTSAQLLVTDRDFKNKDFRKQLDETVQSLL 145 RcP5CS PQVEIDGKASAAVGQSSLMALYDTMFSQLDVTSAQLLVTDNDFRDKDFRKQLNETVESLL 146 EjP5CS PQVDLDGKACAAVGQNSLMALYDTLFSQLDITSAQLLVTDSDFRDKDFRKQLDETVISLL 142 VvP5CS PQAELDGKACAAVGQNNLMALYDTLFSQLDVTSAQLLVTDNDFRDEAFRNQLTQTVDSLL 147 GmP5CS PQVELDGKACAAVGQNSLMALYDVLFSQLDVTSAQLLVTDNDFRDKDFRMQLSETMKSLL 145 PvP5CS2 PQVELDGKACAAVGQSSLMALYDALFSQLDVTSAQLLVTDNDFRDKDFRKQLSETVKSLL 145 MSP5CS PQVELDGKACAAVGQSSLMATYDDLFSQLDVTSAQLLVTDNDFRDQDFRKQLSETVKSLL 145 MtP5CS PQVELDGKACAAVGQSSLMATYDDLFSQLDVTSAQLLVTDNDFRDQDFRNQLSETVKSLL 145 PvP5CS1 PQLELDGKACAAVGQNSLMALYDTLFTQLDVTSAQLLVTDNDFRDKDFRKQLTETVESLL 146 VaP5CS PQLELDGKACAAVGQNSLMALYDTLFTQLDVTSAQLLVTDNDFRDKDFRKQLTETVKSLL 146 Mutated AtP5CS1 PQTELDGKACAGVGQSSLMAYYETMFDQLDVTAAQLLVNDSSFRDKDFRKQLNETVKSML 145 AtP5CS1 PQTELDGKACAGVGQSSLMAYYETMFDQLDVTAAQLLVNDSSFRDKDFRKQLNETVKSML 145 CbP5CS PQTELDGKACAGVGQSSLMAYYETMFDQLDVTAAQLLVNDSSFRDKEFRQQLNETVKPML 145 AtP5CS2 PQMELDGKACAGVGQSSLMAYYETMFDQLDVTVAQMLVTDSSFRDKDFRKQLSETVKAML 145 BnP5CS PQTELDGKACAGVGQSSLMAYYETMFDQMDVTVAQMLVTDSSFRDKDFRKQLSETVKAML 146 McP5CS PQVELDGKACAAVGQNGLMALYDTLFSQLDLTAAQLLVTDNDFRDPSFRTQLTETVYQLL 142 VuP5CS PQGELDGKACAAVGQSSLMALYDTMFSQLDVTSSQLLVNDGFFRDAGFRKQLSDTVNALL 179 SlP5CS1 PTEIPAQQALAAIGQSYLMAIYNQSFAFYQQHVGQVLLTYDVFHNKQMLRHTIDAIAAML 135 ScP5CS PKHLAEVQAIAAIGQGRLIGRWDLLFSQFDQRIAQILLTRNDILDWTQYKNAQNTINELL 131 :* *.:**. *:. : * : .*:*:. : : . :: :*

SaP5CS DLKVVPIFNENDAISTRKAPYEDSSGIFWDNDSLAGLLAIELKADLLVLLSDVDGLY-NG 204 SoP5CS DLKVVPIFNENDAISTRKAPYEDSSGISWDNDSLAGLLAIELKADLLVLLSDVDGLY-NG 204 SbP5CS2 DLKVVPIFNENDAISTRKAPYEDSSGIFWDNDSLAGLLAIELKADLLVLLSDVDGLY-SG 204 ZmP5CS DLKVVPIFNENDAISTRKAPYEDSSGIFWDNDSLAGLLAIELKADLLVLLSDVDGLY-SG 205 OsP5CS DLKVIPIFNENDAISTRKAPYEDSSGISWDNDSLAGLLALELKADLLILLSDVDGLY-SG 204 TaP5CS ELRVIPIFNENDAISTRKAPYEDSSGIFWDNDSLAGLLALELKADLLVLLSDVDGLY-SG 204 SbP5CS1 NLKVIPLFNENDAISTRRQSDEDSSGVFWDNDSLAALLAAELNADLLIMLSDVEGLY-SG 217 AcP5CS SLKVIPIFNENDAVSTRRAPYEDSSGIFWDNDSLAALLALELKADLLILLSDVEGLF-SG 204 AdP5CS NLKVIPIFNENDAVSTRKAPYEDASGIFWDNDSLAALLALELKADLLVLLSDVEGLY-SG 204 AvP5CS ALKVVPVFNENDAVSTRRAPYEDSSGIFWDNDSLAALLALELKADLLVLLSDVEGLY-SG 205 SlP5CS2 SLKVIPIFNENDAISTRRAPYEDSSGIFWDNDSLAALLALELKADLLVLLSDVDGLY-SG 204 PeP5CS ALRVIPIFNENDAVSTRKAPYEDSSGIFWDNDSLAALLALELKADLLVLLSDVEGLY-SG 204 RcP5CS SLRVIPIFNENDAVSTRKAPYEDASGIFWDNDSLSALLALELKADLLVLLSDVDGLY-SG 205 EjP5CS SLKVIPILNENDAISTRKAPYEDSSGIFWDNDSLAALLALGLKADLLILLSDVEGLY-SG 201 VvP5CS ALRVIPIFNENDAEVTRKAPYEDSSGIFWDNDSLAGLLALQLKADLLVLLSDVDGLY-SG 206 GmP5CS ALKVIPIFNENDAVSTRKAPYEDSSGIFWDDDSLSALLALELKADLLILLSDVEGLY-SG 204 PvP5CS2 GLKVIPIFNENDAVSTRKAPYEDSSGIFWDNDSLSALLALELKADLLILLSDVEGLY-SG 204 MSP5CS ALKVIPIFNENDAVSTRKAPYEDSSGIFWDNDSLSALLALELKADLLILLSDVDGLY-SG 204 MtP5CS ALKVIPIFNENDAVSTRKAPYEDSSGIFWDNDSLSALLALELKADLLILLSDVDGLY-NG 204 PvP5CS1 ALKVIPVFNENDAVSTRKAPYEDSSGIFWDNDSLSALLALELKADLLVLLSDVEGLY-SG 205 VaP5CS ALKVIPVFNENDAVSTRKAPYEDSSGIFWDNDSLSALLALELKADLLVLLSDVEGLY-SG 205 Mutated AtP5CS1 DLRVIPIFNGNDAISTRRAPYQDSSGIFWDNDSLAALLALELKADLLILLSDVEGLY-TG 204 AtP5CS1 DLRVIPIFNENDAISTRRAPYQDSSGIFWDNDSLAALLALELKADLLILLSDVEGLY-TG 204 CbP5CS DLRVIPIFNENDAISTRRAPYQDSSGIFWDNDSLAALLALELKADLLILLSDVEGLY-TG 204 AtP5CS2 RMRVIPVFNENDAISTRRAPYKDSTGIFWDNDSLAALLSLELKADLLILLSDVEGLY-TG 204 BnP5CS KMRVIPVFNENDAISTRKAPYKDSTGIFWDNDSLAALLALELKADLLILLSDVEGLY-TG 205 McP5CS DLKVVPVLNENDAVSTRKAPYEDSSGIFWDNDSLAALLALELKADLLILLSDVDGLY-NG 201 VuP5CS DLRVIPIFNENDAVSTRKAPYEDSSGIFWDNDSLAGLLALELKADLLVLLSDVEGLY-SG 238 SlP5CS1 QQRVIPIINENDVIAVDELDHQHS---FGDNDRLAAMVTHETNADALIVLSDIDALYTAN 192 ScP5CS NMGVIPIVNENDTLSVREIK------FGDNDTLSAITSALIHADYLFLLTDVDCLYTDN 184 *:*:.* **. . . *:* *:.: : :** *.:*:*:: *: .

Supplementary Fig. 2 Multiple sequence alignment of the N-terminal part of P5CS amino acid sequences (The substitution introduced in mP5CS1 is indicated in red) SaP5CS, Saccharum arundinaceum (GenBank accession no. EU113257); SoP5CS, Saccharum officinarum (GenBank accession no. EU005373); SbP5CS2, Sorghum bicolour (GenBank accession no. GQ377720); ZmP5CS, Zea mays (GenBank accession no. EU953366); OsP5CS, Oryza sativa (GenBank accession no. AY574031); TaP5CS, Triticum aestivum (GenBank accession no. AY888045); SbP5CS1, Sorghum bicolor (GenBank accession no. GQ377719); AcP5CS, Aegiceras corniculatum (GenBank accession no. DQ431113); AdP5CS, Actinidia deliciosa (GenBank accession no. U92286); AvP5CS, Apocynum venetum (GenBank accession no. EF160132); SlP5CS2, Solanum lycopersicum (GenBank accession no. SLU60267); PeP5CS, Populus euphratica (GenBank accession no. EF412967); RcP5CS, Ricinus communis (Gen Bank accession no. XM_002524184); EjP5CS, Euonymus japonicus (GenBank accession no. EU652695); VvP5CS, Vitis vinifera (GenBank accession no. AJ005686); GmP5CS, Glycine max (GenBank accession no. FM999730); PvP5CS2, Phaseolus vulgaris (GenBank accession no. EU340347); MsP5CS, Medicago sativa (GenBank accession no. GU180149); MtP5CS, Medicago truncatula (GenBank accession no. AJ278818); PvP5CS1, Phaseolus vulgaris (GenBank accession no.

3 EU340347); VaP5CS, Vigna aconitifolia (GenBank accession no. P32296); AtP5CS1, A. thaliana (GenBank accession no. AY150430); CbP5CS, Chorispora bungeana (GenBank accession no. AY804246); AtP5CS2, A. thaliana (GenBank accession no. NM_115419); (BnP5CS) Brassica napus (GenBank accession no. AF314812); McP5CS, Mesembryanthemum crystallinum (GenBank accession no. AF067967); VuP5CS, Vigna unguiculata (GenBank accession no. AB518058); SlP5CS1, Solanum lycopersicum (GenBank accession no.U27454); ScP5CS, Saccharomyces cerevisiae (GenBank accession no. M85293). Identical amino acids (*) and similarities (.) and (:) are indicated below the sequence comparison.

4 1 MEELDRSRAF ARDVKRIVVK VGTAVVTGKG GRLALGRLGA LCEQLAELNS 50 51 DGFEVILVSS GAVGLGRQRL RYRQLVNSSF ADLQKPQTEL DGKACAGVGQ 100 101 SSLMAYYETM FDQLDVTAAQ LLVNDSSFRD KDFRKQLNET VKSMLDLRVI 150 151 PIFNENDAIS TRRAPYQDSS GIFWDNDSLA ALLALELKAD LLILLSDVEG 200 201 LYTGPPSDPN SKLIHTFVKE KHQDEITFGD KSRLGRGGMT AKVKAAVNAA 250 251 YAGIPVIITS GYSAENIDKV LRGLRVGTLF HQDARLWAPI TDSNARDMAV 300 301 AARESSRKLQ ALSSEDRKKI LLDIADALEA NVTTIKAENE LDVASAQEAG 350 351 LEESMVARLV MTPGKISSLA ASVRKLADME DPIGRVLKKT EVADGLVLEK 400 401 TSSPLGVLLI VFESRPDALV QIASLAIRSG NGLLLKGGKE ARRSNAILHK 450 451 VITDAIPETV GGKLIGLVTS REEIPDLLKL DDVIDLVIPR GSNKLVTQIK 500 501 NTTKIPVLGH ADGICHVYVD KACDTDMAKR IVSDAKLDYP AACNAMETLL 550 551 VHKDLEQNAV LNELIFALQS NGVTLYGGPR ASKILNIPEA RSFNHEYCAK 600 601 ACTVEVVEDV YGAIDHIHRH GSAHTDCIVT EDHEVAELFL RQVDSAAVFH 650 651 NASTRFSDGF RFGLGAEVGV STGRIHARGP VGVEGLLTTR WIMRGKGQVV 700 701 DGDNGIVYTH QDIPIQA.

Supplementary Fig. 3 Amino acid sequence of AtP5CS1 with indication of the residues that when modified lead to proline feedback insensitivity (shown in red). Modified residues are reported for a series of species: 125 and 128 for Vigna aconitifolia (Zhang et al. 1995); 157,159,163,172,175 for tomato (Fujita et al. 2003); 156,163,164,168,182 for yeast (Sekine et al. 2007); 163 for E. coli (Rushlow et al. 1985); and 137,160,162 for Listeria (Sleator et al. 2001). Position 155 (shown in green): amino acid substituted in this work.

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