Volume 6, Issue 2, Pages 411-422 (March 2013) HsfA1d, a Protein Identified via FOX Hunting Using Thellungiella salsuginea cDNAs Improves Heat Tolerance by Regulating Heat-Stress-Responsive Gene Expression  Yukari Higashi, Naohiko Ohama, Tomoko Ishikawa, Taku Katori, Ayaka Shimura, Kazuya Kusakabe, Kazuko Yamaguchi-Shinozaki, Junko Ishida, Maho Tanaka, Motoaki Seki, Kazuo Shinozaki, Yoichi Sakata, Takahisa Hayashi, Teruaki Taji  Molecular Plant  Volume 6, Issue 2, Pages 411-422 (March 2013) DOI: 10.1093/mp/sst024 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Heat Tolerance of T. salsuginea and Arabidopsis. (A) Ten-day-old seedlings of T. salsuginea and Arabidopsis were exposed to 42°C for 90 min. The seedlings were then moved to normal growth conditions (22°C) for 7 d. (B) Three-week-old soil-grown plants of T. salsuginea and Arabidopsis were exposed to 37°C for 9 d. (C) Callus was generated from hypocotyls of T. salsuginea and Arabidopsis (ecotypes Columbia (Col-0) and Wassilewskija (Ws)) seedlings. The callus was exposed to 37°C for 7 d. Molecular Plant 2013 6, 411-422DOI: (10.1093/mp/sst024) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Heat Tolerance of Wild-Type and HeatFOX38 Plants. (A) Ten-day-old seedlings of wild-type (WT), HeatFOX38-1, and HeatFOX38-2 (upper panel) were exposed to 42°C for 70 min and then moved to normal growth conditions (22°C) for 16 d (lower panel). (B) The expression levels of the transgenes in lines HeatFOX38-1 and -2 under normal growth conditions were confirmed by Northern blot analysis. Molecular Plant 2013 6, 411-422DOI: (10.1093/mp/sst024) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Sequence Analyses of the HeatFOX38/TsHsfA1d Genes and Proteins. (A) Phylogenetic tree of 21 AtHsf proteins and TsHsfA1d. (B) Alignment of the predicted amino acid sequences encoded by HeatFOX38/TsHsfA1d and AtHsfA1d. Black boxes indicate identical amino acids in the two sequences; gray boxes indicate similar amino acids. Molecular Plant 2013 6, 411-422DOI: (10.1093/mp/sst024) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Expression Profiles of TsHsfA1d and AtHsfA1d during Heat Treatment of T. salsuginea and Arabidopsis Wild-Type Plants. Two-week-old wild-type plants of Arabidopsis and T. salsuginea were exposed to 37°C for 0, 1, 2, or 5 h. Total RNA was reversed-transcribed into cDNA and used as a template for quantitative RT–PCR. Relative transcript levels were normalized to Actin2 mRNA. Data are means ± SD for three individual experiments (n = 3). Molecular Plant 2013 6, 411-422DOI: (10.1093/mp/sst024) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Comparison of the AtHsfA1 and TsHsfA1d Regulons. Venn diagram shows genes up-regulated by TsHsfA1d overexpression (log2 ratio > 2) in transformants and genes down-regulated in hsfa1a/b/d and induced by heat stress in wild-type plants. Molecular Plant 2013 6, 411-422DOI: (10.1093/mp/sst024) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Expression of Genes Downstream of TsHsfA1d. Total RNAs were prepared from 2-week-old seedlings of HeatFOX38-1, HeatFOX38-2, and wild-type (WT) plants grown under normal growth conditions. The expression levels of TsHsfA1d and several genes that had expression log2 ratio > 2 in HeatFOX38 plants relative to WT plants in the microarray analysis (HSP70, HSP17, Rof2, MBF1c, and GolS1) were examined by Northern blot analysis. For each gene, the corresponding full-length cDNA was used as the probe. Molecular Plant 2013 6, 411-422DOI: (10.1093/mp/sst024) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Localization of TsHsfA1d Proteins and Their Interactions with HSP90. (A) Accumulation of sGFP–TsHsfA1d fusion protein in Arabidopsis T87 protoplasts. Confocal images of sGFP fluorescence and Nomarski images are shown. (B) Bimolecular fluorescence complementation visualization of interactions between TsHsfA1d and AtHSP90 proteins following transient expression in Arabidopsis T87 protoplasts. The constructs used in this experiment produced fusions of these proteins with either the N- or C-terminal fragment of yellow fluorescent protein (YFP N and YFP C, respectively). Labels to the left of each row indicate the YFP N fusion and the YFP C fusion, respectively. ‘Vector’ indicates that the construct encoded only the indicated YFP fragment. The appearance of YFP fluorescence in the co-transfected cells indicates that the two fusion proteins interacted within the cell. YFP fluorescence, cyan fluorescent protein (CFP) fluorescence, and merged images from the same field of transfected cells are shown for each transfection combination. A control plasmid (35S:CFP) was co-transfected to identify transformed cells before analysis of YFP fluorescence. Molecular Plant 2013 6, 411-422DOI: (10.1093/mp/sst024) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 8 Heat Tolerance and Expression Analysis of AtHsfA1d-Overexpressing Plants. (A) Ten-day-old seedlings of WT, 35S::AtHsfA1d-d, 35S::AtHsfA1d-e, and 35S::TsHsfA1d-2 were exposed to 42°C for 70 min and then moved to normal growth conditions at 22°C for 16 d. (B) Total RNAs were prepared from 2-week-old seedlings of WT, 35S::TsHsfA1d (lines -1 and -2), and 35S::AtHsfA1d (lines -b, -d, and -e) grown under normal growth conditions. The RNAs were then analyzed by Northern blot hybridization. The full-length cDNA was used as the probe for each gene. Molecular Plant 2013 6, 411-422DOI: (10.1093/mp/sst024) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions