MVKFLFSVIILFFLLSAVGSSARNIEEDGVIRLPSEVKDFINGKNIDDDSVGGTRWAVLI 60 AGSSGYWNYRHQADVCHAYQVLKRGGVKDENIVVFMYDDIALNEENPRPGVIINHPKGED 120 VYAGVPKDYTGRDVTAHNFYSVLLGNKTAVKGGSGKVIDSGPNDHIFIYYSDHGGPGVLG 180 MPTYPYLYADDLVNVLKQKHALGAYKSLVFYLEACESGSIFEGILPKGLNIYATTASNAE 240 ESSWGTYCPGEFPSPPSEYETCLGDLYSVAWMEDSDVHNLRSETLKQQYHLVKERTQNAN 300 SAYGSHVMQYGDLEVSKEDLFLYMGTNPANDNNKFIEQNSLPSLSGSVNQREADLIHFWQ 360 KYRKAPEGSQRKADAQKQFVEVMAHRMHVDHSIKLIGKLLFGFEKGPQVLEAVRPAGQPL 420 VDDWDCLKTMVRTFEAQCGSLSQYGMKHMRSVANICNAGIKKEQMAEAASQACVTIPNGS 480 WSSTHQGFSA 490 Bosch et al. Supplemental Figure 1A. Predicted amino acid sequence of Papaver pollen VPE. Supplemental Fig 1a. Predicted amino acid sequence of Papaver pollen VPE. Arrow indicates predicted signal peptide cleavage site. The red box indicates the IRLPS predicted vacuolar targeting motif. The essential cysteine and histidine amino acid residues for VPE activity, based on Kuroyanagi et al., (2002), Plant and Cell Physiology 43, , are indicated by green boxes; a further conserved His residue required for catalytic activity and found in other legumains is indicated by a blue box. Amino acids essential for caspase-1 activity, based on Hara-Nishimura et al., (2005), Curr Opin Plant Biol. 8, 404-8, that are conserved in poppy VPE are indicated by stars. Arrowheads indicate putative processing sites of pro-peptides. The black arrowhead indicates the predicted N-terminal cleavage site at the C-terminal side of Asn-45 based on (Kuroyanagi et al., 2002), Plant and Cell Physiology 43, , while the green arrowhead indicates this cleavage site to be at Asp-49, based on Hiraiwa et al., (1999) FEBs Letters 447, The red arrowhead indicates the position of the C-terminal cleavage site based on Kuroyanagi et al., (2002), Plant and Cell Physiology 43, ), who deduced the cleavage site of the C- terminal peptide to be Asn in PEVLN·KVRS. Note however that the Asn residue is missing in the corresponding poppy VPE sequence (PQVLEAVRP). The blue arrowhead indicates the C- terminal cleavage site to be at Asp-423 based on Hiraiwa et al., (1999), FEBs Letters 447, The pro-peptides that were removed to produce the recombinant mVPE and pVPE forms for expression studies, are underlined.

Bosch et al. Supplemental Figure 1B. Amino acid alignment of poppy VPE with other known plant VPE sequences. Papaver VPE Mouse Legumain Human Legumain NtVPE3 AtγVPE AtαVPE AtδVPE Rice NP1 AtβVPE Maize See2a Castor bean VPE Malus hupehensis VPE Vitis vinifera VPE LeVPE1 Papaver VPE Mouse Legumain Human Legumain NtVPE3 AtγVPE AtαVPE AtδVPE Rice NP1 AtβVPE Maize See2a Castor bean VPE Malus hupehensis VPE Vitis vinifera VPE LeVPE1 Papaver VPE Mouse Legumain Human Legumain NtVPE3 AtγVPE AtαVPE AtδVPE Rice NP1 AtβVPE Maize See2a Castor bean VPE Malus hupehensis VPE Vitis vinifera VPE LeVPE1 Papaver VPE Mouse Legumain Human Legumain NtVPE3 AtγVPE AtαVPE AtδVPE Rice NP1 AtβVPE Maize See2a Castor bean VPE Malus hupehensis VPE Vitis vinifera VPE LeVPE1 Supplemental Fig 1b. Amino acid alignment of Papaver VPE with other known VPE/legumain sequences. Full-length sequences were aligned using the ClustalW algorithm (Thompson et al., 1994, Nucleic Acids Research 22, ). The same symbols are used to indicate the features as shown in Fig. S1a, except that a red line indicates the IRLPS predicted vacuolar targeting motif. The region containing the presumed cleavage sites for the N-terminal and C-terminal pro-regions are indicated by a green and blue line respectively. Black boxes indicate completely conserved residues; dark grey indicates identical residues and light grey, similar residues. The red boxes indicate the highly conserved tetrapeptide of hydrophobic residues 2 amino acids N-terminal to the catalytic residues. See Fig. S2 for GenBank accession numbers.

Vegetative type Seed-type Bosch et al. Supplemental Figure 2. Phylogenetic analysis of selected legumain/VPE proteins. (BAA09614) (BAA18924) (BAA76744) (BAA76745) (P49043) (O24325) (BAC54827) (BAC54828) (BAC54829) (BAC54830) (CAC43295) (ACB30368) (EEF45813) (ACR24644) (CAO43774) (CAB64544) (AAD04883) (AAL40390) (BAC76418) (AAD04882) (AAF89679) (CAB42651) (P49045) (AAF89646) (O24326) (P49046) (CAB16318) (P49042) (BAA09615) (CAB51545) (CAB42650) (CAE84598) (BAC65233) (EEE88287) (BAC41386) (CAA04439) (AAH03061) Supplemental Fig 2. Phylogenetic analysis of selected legumain/VPE proteins. Full-length sequences were aligned using the ClustalW algorithm (Thompson et al., 1994, Nucleic Acids Research 22, ), and a PHYLIP rooted phylogenetic tree was prepared using the Drawgram algorithm (Felsenstein, 1989, Cladistics 5, ). All sequence analyses were performed in the Biology Workbench web-based environment ( under default conditions. The GenBank accession numbers are indicated within brackets. The Papaver VPE falls between the two major classes of VPEs (seed and vegetative type), together with two maize VPEs that are also expressed in reproductive tissues.