Volume 3, Issue 5, Pages 890-903 (September 2010) Complex Assembly and Metabolic Profiling of Arabidopsis thaliana Plants Overexpressing Vitamin B6 Biosynthesis Proteins  Leuendorf Jan Erik , Osorio Sonia , Szewczyk Agnieszka , Fernie Alisdair R. , Hellmann Hanjo   Molecular Plant  Volume 3, Issue 5, Pages 890-903 (September 2010) DOI: 10.1093/mp/ssq041 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Arabidopsis Plants Overexpressing myc:PDX Fusion Proteins. (A) Phenotype of Col0 wild-type (left plant) and P35S:myc:PDX1.3stunted plants (right plant, marked with an arrow) under long and (B) under short-day conditions. (C) Overview of Col0 and the different P35S:myc:PDX overexpressor lines under short-day conditions. Bars in (A) represent the size of 2 cm. Molecular Plant 2010 3, 890-903DOI: (10.1093/mp/ssq041) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 mRNA and Protein Expression Levels of the PDX Family in Col0 and Transgenic Plants. (A–E) qRT–PCR reflects total expression levels of endogenous gene plus the respective transgene. Expression is shown in relation to Col0 wild-type (= 1) (A) Col0 and P35S:myc:PDX1.1, (B) Col0 and P35S:myc:PDX1.2, (C) Col0 and P35S:myc:PDX1.3stunted, (D) Col0 and P35S:myc:PDX1.3normal, (E) Col0 and P35S:myc:PDX2. (F, G) Expression analysis of myc:PDX1.3 transgene (F) and endogenous PDX1.3 (G) in Col0, P35S:myc:PDX1.3stunted, and P35S:myc:PDX1.3normal plants in relation to P35S:myc:PDX1.3normal (= 1) (F) and Col0 wild-type (= 1) (G). Error bars are standard error (SE). (H) Western-blot analysis showing expression of myc:PDX proteins in the different transgenic lines. Molecular Plant 2010 3, 890-903DOI: (10.1093/mp/ssq041) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Complementation of pdx1.3G54S/rsr4-1 by myc:PDX1.1 and myc:PDX1.3. (A) myc:PDX1.1 can interact with all PDX proteins. GST fusion for the different PDX proteins and GST alone were expressed in E. coli and affinity purified before used in pull-down assays. Upper part: all GST-fusion proteins, except for sole GST, were able to precipitate in planta-expressed myc:PDX1.1. Input represents 30 μg of total protein extract. Lower part: Coomassie-stained gel showing all GST proteins and their respective sizes. Asterisks indicate correct size of each GST:PDX protein and GST alone. (B) Expression analysis by Western blot shows correlation of normalized plant growth with myc:PDX1.1 and myc:PDX1.3 levels. Ectopic expression of myc:PDX1.2 did not result in normalized pdx1.3G54S/rsr4-1 mutant plant growth. Ponceau staining is shown as a control for protein loading. (C) Phenotype of C24 wild-type, pdx1.3G54S/rsr4-1 mutant, and representative primary transformants of pdx1.3G54S/rsr4-1 plants expressing either myc:PDX1.1, myc:PDX1.2, or myc:PDX1.3 under the control of the 35S promoter and in the case of PDX1.3 also under the control of a 1.1-kb natural PDX1.3 promoter, respectively. Molecular Plant 2010 3, 890-903DOI: (10.1093/mp/ssq041) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 VitB6 Measurements (A) of Pyridoxamine (PM), Pyridoxal (PL), and Pyridoxamine (PN) and Metabolite Profiling (B–D) of Col0 and P35S:myc:PDX Overexpressor Plants Grown under Short-Day (SD) Conditions. n = 5 independent samples; error bars represent SE; circles (o) indicate t-test results p < 0.05, while asterisks (*) indicate p < 0.01. Molecular Plant 2010 3, 890-903DOI: (10.1093/mp/ssq041) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Split-YFP Approach Shows Homo- and Heteromeric Assemblies of PDX1 Proteins. (A) Transient expression assays in tobacco leaves were followed to investigate assembly of PDX1 proteins with pE-SPYNE-GW (NYFP) and pE-SPYCE-GW (CYFP) vectors. Yellow represents emitted fluorescence of re-assembled YFP-halves based PDX interactions. BPM4 served as a negative control. (B) Western-blot analysis on samples show in (A). Fusion proteins derived from NYFP vectors were detected with α-myc antibodies, while for CYFP-products, a specific α-HA antibody was used. 30 μg total protein extract was loaded in (B). Molecular Plant 2010 3, 890-903DOI: (10.1093/mp/ssq041) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 FPLC Analysis, Size-Exclusion Chromatography. (A) Chromatogram of standard proteins used for standardization of a HiLoadTM 16/60 Superdex 200 column. FPLC was performed at a flow rate of 1 ml min−1. (B) Representative FPLC purification-chromatogram of extracted 6–8 mg total protein from P35S:myc:PDX plants. (C) Western Blot analysis with α-myc antibody of different fractions from size-exclusion chromatography of P35S:myc:PDX plants. Corresponding retention volume is shown above. Black asterisks mark retention volumes of specific interest (for details, see main text). Smaller, cross-reacting bands that do not represent PDX proteins are marked with black arrows. Results shown are representative for three independent experiments. Molecular Plant 2010 3, 890-903DOI: (10.1093/mp/ssq041) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions