1. Volume 6, Issue 2, Pages 323-336 (March 2013)
Thioredoxin Reductase Type C (NTRC) Orchestrates Enhanced Thermotolerance to Arabidopsis by Its Redox-Dependent Holdase Chaperone Function 
Ho Byoung Chae, Jeong Chan Moon, Mi Rim Shin, Yong Hun Chi, Young Jun Jung, Sun Yong Lee, Ganesh M. Nawkar, Hyun Suk Jung, Jae Kyung Hyun, Woe Yeon Kim, Chang Ho Kang, Dae-Jin Yun, Kyun Oh Lee, Sang Yeol Lee 
Molecular Plant 
Volume 6, Issue 2, Pages (March 2013)
DOI: /mp/sss105
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Thermotolerance of WT, NTRC°E, ntrc1, and hot1 Arabidopsis. (A) Growth sections of WT, NTRC°E, ntrc1, and hot1 plants are indicated. (B) Expression level of NTRC was examined by immunoblot analysis using an anti-NTRC antibody. (C, D) Two sets of Arabidopsis seedlings grown under the same conditions at 22°C for 3 d were used in (C) and (D). (C) Three-day-old seedlings were further grown at 22°C for 7 d as a control. (D) Three-day-old seedlings were heat shocked as indicated, and thermotolerance of heat-treated plants after growth under optimal conditions for 7 d was compared. (E) Electrolyte leakage of 30-day-old plants were compared with and without heat treatment. The percentage of electrolyte leakage was calculated as outlined in the Methods section. Data represent the mean ± SD from the three independent experiments.
Molecular Plant 2013 6, DOI: ( /mp/sss105)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Structural Analyses of NTRC Proteins. (A) Purified recombinant NTRC was separated on a 12% reducing SDS–PAGE (left panel), 12% non-reducing SDS–PAGE (mid panel), and 10% native-PAGE (right panel) gels followed by silver staining. (B) Purified NTRC was subjected to SEC using a Superdex 200 HR column. Molecular weight standards were run under identical conditions as indicated in the Methods section. (C) Each fraction of SEC in (B) was separated on a native-PAGE (upper panel) or reducing SDS–PAGE (bottom panel) and subjected to silver staining (left panel) or Western blotting with an anti-NTRC antibody (right panel).
Molecular Plant 2013 6, DOI: ( /mp/sss105)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Electron Microscopic Analyses of the Different Forms of NTRC. Negatively stained fields of SEC-separated proteins are shown; HMW complexes (A), mid-sized molecular weight proteins (C), and LMW fractions (E). The inset image in (E) shows the enlarged image of the field taken from the area of concentrated LMW fraction. Black arrows in (A) and black and white arrowheads in panels (C) and (E) indicate individual complexes found in each fraction, respectively. In the right panels (B, D, F), representative averaged images of NTRC processed from the individual complexes of the left panels (A, C, E) are shown. Averaged images of monomeric and dimeric forms are marked with an asterisk and cross in panel (F). The class averages represent 10–20 (B) and 20–50 particles (D, F). Scale bar = 50 nm applied to all the averaged images.
Molecular Plant 2013 6, DOI: ( /mp/sss105)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Comparison of Disulfide Reductase Activity of NTRC Using Either Chemical (DTNB) and Protein (Insulin) Substrates. (A) Schematic diagram of NTRC protein structure. SP, signal peptide; TR, thioredoxin reductase-domain; Trx, thioredoxin-domain. (B, C) Disulfide reduction activity of NTRC. (B) NADPH-dependent disulfide reduction activity was measured using 5 mM DTNB in the presence of 150 µM NADPH. (C) Insulin reduction activity was measured in the presence of 0.5 mM DTT. Concentrations of NTRC used were 5 µM (●), 10 µM (■), 20 µM (▲), and 30 µM (◆). Reactions using 30 µM ovalbumin (□) instead of NTRC were used as a control. (D, E) the concentration-dependent relative activity of the disulfide reductase function was measured using DTNB (D) and insulin (E) as substrates, respectively.
Molecular Plant 2013 6, DOI: ( /mp/sss105)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Multiple Functions of NTRC Associated with Protein Structure Differences.(A, B) Holdase chaperone activity was measured with the use of 1.5 µM MDH (A) or 80 µM insulin (B) as substrates. (A) 1.5 µM MDH in 50 mM HEPES (pH 7.0) was incubated in a spectrophotometer cell at 45°C with various concentrations of NTRC.(B) Effect of NTRC concentration on the 10 mM DTT-induced chemical aggregation of insulin at 25°C, in the absence (●) or presence of NTRC. Molar ratios of NTRC to MDH (A) or insulin (B) were 0:1 (●), 0.25:1 (■), 0.5:1 (▲), and 1:1 (○). Ovalbumin (◆) with a 1:1 molar ratio of substrate to NTRC was included as a negative control in (A) and (B).(C) Foldase chaperone activity was measured using the Cys-free form of G6PDH (10 µM). G6PDH was denatured in 6 M urea and renatured by diluting the G6PDH in renaturation buffer containing 100 mM phosphate (pH 7.5), and without (●) or with 5 µM of NTRC (▲), 5 µM E. coli Trx (□), or 5 µM ovalbumin (◆). Activity of native G6PDH was set to 100%.(D) NTRC protein fractions (F-I∼F-II) separated by SEC were re-chromatographed by SEC.(E) The chaperone and disulfide reductase activities were measured using equal concentrations of proteins obtained from the fractions in SEC. The activities in each fraction were compared with those in total protein, whose activities were set to 100%.
Molecular Plant 2013 6, DOI: ( /mp/sss105)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Effect of Heat Shock on Structural and Functional Transition of NTRC.(A) Heat shock was applied to NTRC at various temperatures for 20 min and the resulting proteins were separated by native-PAGE (upper) (and SDS–PAGE gels (bottom).(B) Heat shock-mediated hydrophobicity changes of NTRC as measured by bis-ANS binding.(C) Relative activities of the holdase chaperone (■), foldase chaperone (■), and disulfide reductase (□) functions of heat-treated NTRC were measured and compared to those of NTRC incubated at 25°C set as 100%.
Molecular Plant 2013 6, DOI: ( /mp/sss105)
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8. Figure 7
Effect of NADPH on Heat Shock-Dependent Structural and Functional hanges of NTRC.(A) Heat shock (45°C)-dependent structural regulation of NTRC was monitored by native-PAGE (upper panel) and SDS–PAGE (lower panel) gels with or without 1 mM NADPH.(B) Structural change of mutant was compared with the native form of NTRC in native-PAGE gels in the presence of 1 mM NADPH under heat shock (45°C). Mutant represents the protein of C217/454S-NTRC.(C) Incubation time-dependent holdase chaperone activity of NTRC at 45°C was compared with or without 1 mM NADPH.
Molecular Plant 2013 6, DOI: ( /mp/sss105)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

9. Figure 8
Effect of Active Site Cys Residues in NTRC on the Thermotolerance of Various Lines of Transgenic Arabidopsis.(A) Holdase chaperone (■), foldase chaperone (■), and disulfide reductase (□) activities of C217/454S-NTRC were compared with those of NTRC, whose activities were set to 100%.(B) Growth of WT, ntrc1, hot1, NTRC°E, and C217/454S-NTRC°E plants is indicated (upper panel). Expression of NTRC in Arabidopsis was analyzed by Western blotting of SDS–PAGE gels (lower panel).(C) Five-day-old seedlings were grown at 22°C for 10 d served as controls. (D, E) Heat shock was applied as indicated, and thermotolerance is shown after a 10-d recovery period from heat shock.
Molecular Plant 2013 6, DOI: ( /mp/sss105)
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10. Figure 9
A Model of the Structural and Functional Switching of NTRC against Heat Shock in the Light. In plants exposed to high temperature, LMW forms of NTRC in chloroplasts polymerize into HMW protein complexes, parallel to a functional switch from a disulfide reductase and foldase chaperone to a holdase chaperone. LMW forms of NTRC predominantly function as disulfide reductases and foldase chaperones. The HMW complex forms of NTRC act as a holdase chaperone protecting intracellular substrates from denaturation by heat shock.
Molecular Plant 2013 6, DOI: ( /mp/sss105)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions