Volume 2, Issue 2, Pages 357-367 (March 2009) Redox-Dependent Regulation of the Stress-Induced Zinc-Finger Protein SAP12 in Arabidopsis thaliana  Elke Ströher, Xin-Jia Wang, Nils Roloff, Peter Klein, Arne Husemann, Karl-Josef Dietz  Molecular Plant  Volume 2, Issue 2, Pages 357-367 (March 2009) DOI: 10.1093/mp/ssn084 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Bioinformatic Analysis for Arabidopsis thaliana SAP Gene Family. (A) Phylogenetic tree. The alignment was generated using the ClustalW program, and the tree was drawn using the neighbor-joining method of MEGA. Scale bar represents 0.2 amino acid substitutions per site. (B) Transcript analysis for developmental series using the Meta-Profile Analysis tool from Genevestigator Version 3 (www.genevestigator.ethz.ch). Data for SAP1, SAP8, SAP11, and SAP14 are not available in the database. Indicated are the stage group symbols and the corresponding age in days. (C) Transcript analysis for the indicated abiotic stress treatments using the Meta-Profile Analysis tool from Genevestigator (NascArrayRefNo. 138). Red color marks up-regulation, and green color down-regulation of transcript abundance. Molecular Plant 2009 2, 357-367DOI: (10.1093/mp/ssn084) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Transcript Regulation in Wild-Type Arabidopsis thaliana. Gene-specific RT–PCR was used to assess the transcript abundances of selected genes under different stress conditions (150 and 300 mM NaCl, 15% (w/v) PEG-6000 and 4°C) at time points 6 (A), 24 (B), and 48 h (C). In (A), the first lane gives the transcript at t = 0 h as an additional reference. The second lane in (A) and the first lanes in (B) and (C) represent the transcript amount at the given time point and is employed as the reference for the calculation of the relative signal intensity. The cDNA samples were standardized on actin transcript amount. The numbers give the means of the proportion of signal intensity relative to the control from three independent experiments. (D) qRT–PCR for the transcript abundance of SAP12 during a 4°C cold treatment at different time points as indicated. Molecular Plant 2009 2, 357-367DOI: (10.1093/mp/ssn084) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Amino Acid Sequence Alignment of SAP11 to SAP14. The alignment was performed using the ClustalW and visualized using Jalview as described in the method section. Grayscale level indicates for degree of amino acid conservation. Grey and black circles indicate Cys- or His-residues involved in Zn–S-cluster formation, while grey stars mark other conserved Cys residues. Positions of the first and the second AN1 domain are marked. Molecular Plant 2009 2, 357-367DOI: (10.1093/mp/ssn084) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Quaternary Structure Regulation of SAP12 in Dependence on a Changing Redox Milieu. Protein separation was performed on 5–12% gradient SDS–PAGE and detected via specific SAP12 antibody. (A) Purified recombinant SAP12 was incubated in the presence of different concentrations of H2O2 and DTT, respectively. (B) Re-reduction of H2O2-oxidized SAP12 was achieved by incubation with 50 mM DTT. The different disulfide-linked forms are indicated. Molecular Plant 2009 2, 357-367DOI: (10.1093/mp/ssn084) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Redox Titration of SAP12 Protein. The quaternary structure of recombinant SAP12 was analyzed relative to the redox poise of the medium by 5–12% gradient SDS–PAGE separation, Western blotting and detection with anti-6xHis antibody. Molecular Plant 2009 2, 357-367DOI: (10.1093/mp/ssn084) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Reductive Regeneration of Oxidized SAP12 Protein. Separation was performed on 12% SDS–PAGE and detected via specific SAP12 antibody. (A) In the presence of different DTT concentrations, the recombinant SAP12 was incubated either without (top) or with (bottom) recombinant E. coli thioredoxin A (TrxA). (B) In the presence of the complete Trx regeneration system (recombinant E. coli thioredoxin reductase (TR), TrxA), SAP12 was incubated with different concentrations of NADPH. Molecular Plant 2009 2, 357-367DOI: (10.1093/mp/ssn084) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 7 Quantification of SAP12 Protein in Arabidopsis thaliana Leaf Extracts. Protein extracts were separated with 12% SDS–PAGE and analyzed with specific SAP12 antibody. (A) Detection sensitivity and cross-reaction of the raised SAP12 antibody. 2, 5, and 10 ng purified recombinant SAP2 and SAP12 proteins (with its 6xhis tag about 24.7 kDa) were employed to analyze the specificity of the raised antibody. (B) Quantification of the SAP12 protein in leaf protein extract (native SAP12 with 20.7 kDa). Purified recombinant SAP12 protein and protein extract isolated from leaves of 4–5-week-old A. thaliana wild-type plants were loaded at increasing concentrations and signal intensities were compared using Gelscan software package. (C) SAP12 protein amount was analyzed in protein extract isolated from A. thaliana wild-type plants grown on soil under control conditions or after treatment at 4°C or with 300 mM NaCl. Molecular Plant 2009 2, 357-367DOI: (10.1093/mp/ssn084) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 8 Bioinformatic Analysis of Co-Expressed Transcripts with SAP12. (A) Extraction of co-expressed interesting transcripts (for full list, see Supplemental Table 2). Analysis was performed using the tool ACT (Arabidopsis co-expression data mining tool; www.arabidopsis.leeds.ac.uk/act/index.php; Manfield et al., 2006). (B) Transcript abundance analysis using the Meta-Profile Analysis tool from Genevestigator (NascArrayRefNo. 138) for SAP12 and cytosolic thioredoxins from A. thaliana. Red color marks up-regulation, and green color down-regulation of transcript abundance. Molecular Plant 2009 2, 357-367DOI: (10.1093/mp/ssn084) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions