Fig. S1

A622 1 MVVSEKSKILIIGGTGYIGKYLVETSAKSGHPTFALIRESTLKNPEKSKLIDTFKSYGVT 60 A622L 1 ..................................V.......V................. 60

A622 61 LLFGDISNQESLLKAIKQVDVVISTVGGQQFTDQVNIIKAIKEAGNIKRFLPSEFGFDVD 120A622L 61 ...............................A............................ 120

A622 121 HARAIEPAASLFALKVRIRRMIEAEGIPYTYVICNWFADFFLPNLGQLEAKTPPRDKVVI 180A622L 121 ..H.............K........................................... 180

A622 181 FGDGNPKAIYVKEEDIATYTIEAVDDPRTLNKTLHMRPPANILSFNEIVSLWEDKIGKTL 240A622L 181 ....................MK...............................E...... 240

A622 241 EKLYLSEEDILQIVQEGPLPLRTNLAICHSVFVNGDSANFEVQPPTGVEATELYPKVKYT 300A622L 241 ...........H......M...V..................I..S............... 300

A622 301 TVDEFYNKFV 311 A622L 301 ....Y..... 311 (Identity=95.5%, Similarity=98.4%)

Fig. S2

189 135 238 204 167

189 135 238 204 167

A622

A622L

(73.0%) (85.9%) (68.4%) (43.3%)

(98.4%) (98.5%) (97.1%) (96.6%) (93.4%)

231 220 103 135

236 211 136 245

(nt)

Fig. S3

Red: A622 Blue: ObEGS1+NADPHGreen: PtPCBER

Supplementary Figures

Figure S1. Alignment of the amino acid sequences of A622 and A622L. Dots indicate the A622L residues that are identical to those of the A622 sequence.Figure S2. Diagram of the A622 and A622L genes. Filled boxes indicate coding exons, whereas the lines show introns. The numbers indicate the lengths of the exons and introns in nucleotides. The sequence identity values between corresponding gene segments are indicated in parentheses. The A622L genomic sequence was deposited in GenBank under the accession number AB445396.Figure S3. Superposition of the polypeptide-chain backbones of A622, ObEGS1 (PDB entry no. 2R6J), and PtPCBER (PDB entry no. 1qyc). The color coding is shown in the inset. The NADPH cofactor in ObEGS1 is shown in the ball mode. The structural modeling of A622 protein and the comparison with other PIP-family proteins were carried out using MODELLER 8v2 (University of California San Francisco, San Francisco, CA) and PyMol version 0.99rc6 (DeLano Scientific LLC, Palo Alto, CA) software, respectively.

Supplementary Materials and Methods

Determination of the A622L genomic and cDNA sequencesSince the A622L genomic sequence in the N. tabacum genome database (http://www.tobaccogenome.org/) was incomplete, genomic PCR was carried out with the primers 5’-CCTCCACCTTAACCCGAAGC and 5’-TGCAGATTGATGTCGACAAC, using 10 ng of N. tabacum genomic DNA and TaKaRa Ex Taq DNA polymerase (TaKaRa Bio) under the following conditions: 30 cycles of 94 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 1.5 min. The PCR product was sequenced directly by using 5’-TCAGAGAAAGCACACTCGT and 5'-GCATATGGCCAAATTGACT. To determine the cDNA sequence of A622L, RT-PCR was carried out with 5’-CCTCCACCTTAACCCGAAGC and 5’-TGCAGATTGATGTCGACAAC, using 1 ng of N. tabacum root tissue cDNA and TaKaRa Ex Taq DNA polymerase (TaKaRa Bio) under the following conditions: 30 cycles of 94 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 1 min. The PCR product was cloned into pGEM-T easy vector (Promega, Madison, WI), and sequenced using 5’-TCAGAGAAAGCACACTCGT and 5'-GCATATGGCCAAATTGACT.