1. The Arabidopsis Cysteine-Rich GASA5 Is a Redox-Active Metalloprotein that Suppresses Gibberellin Responses 
Lior Rubinovich, Sharon Ruthstein, David Weiss 
Molecular Plant 
Volume 7, Issue 1, Pages (January 2014)
DOI: /mp/sst141
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Functional and Biochemical Analysis of GASA5.
(A) qRT–PCR analysis of GASA5 expression in 14-day-old seedlings of wild-type (WT) and three transgenic (GASA5OX, T-4,5,8) lines.
(B) qRT–PCR analysis of GA20OX gene expression in WT and transgenic plants (GASA5OX, T-4,5,8). Values are means of three biological replicates ± SE.
(C) Number of rosette leaves in flowering WT and GASA5-overexpressing (GASA5OX, T-5) plants grown under short days (8-h photoperiod). Values are means of 12 plants ± SE.
(D) WT and transgenic seeds (GASA5OX, T-4) were placed in Petri dishes on MS medium containing different concentrations of paclobutrazol. Germination was scored after 4 d. Values are means ± SE of four replicates (four dishes) each containing 40 seeds. The experiment was repeated four times, with similar results.
(E) WT and transgenic seeds (GASA5OX, T-8) were placed in Petri dishes on MS medium. 10-day-old seedlings were treated with water, 200mM NaCl, or 1 μM MV for 3 h. After 48 h, the seedlings were immersed in Tris–HCl solutions containing 50 μM of the H2O2-sensitive dye H2DCF-DA for 20 min and analyzed by fluorescence binocular microscopy. The experiment was repeated twice, with similar results. Representative seedlings are presented.
(F) GST- and GST–GASA5-expressing bacteria were incubated with different concentrations of H2O2 and bacterial growth was measured by spectrophotometry. Values are means ± SE of three replicates. The experiment was repeated 10 times, with similar results.
(G) Total proteins were extracted from E. coli expressing GST, GST–GASA4, or GST–GASA5 and added to Tris–HCl solutions containing 100 μM of the H2O2-sensitive dye H2DCF-DA. H2O2 (10mM) was added to the H2DCF-DA-containing solution and fluorescence (excitation 485nm and emission 530nm) was measured for 30 min by fluorometry (expressed in arbitrary units). Values are means ± SE of three replicates. The experiment was repeated five times, with similar results.
(H) GST and GST–GASA5 total protein pellets extracted from recombinant E. coli.
(I) ICP–AES analysis of iron in the purified GST and GST–GASA5 proteins. Values are means ± SE of three replicates. The analysis was repeated three times, with similar results.
(J) EPR spectrum of purified GST (lower line), GST–GASA5 (upper line), and GST–GASA5 with the addition of 1mM DTT (middle line). Continuous wave X-band (9.42 GHz) EPR spectra were obtained using Bruker E500 Elexsys, and an Oxford helium continuous flow cryostat (ESR900) system at 10.0 K, with modulation amplitude of 1.0 G, 10.0 mW power, and 164 ms time constant. The analysis was repeated three times, with similar results.
Molecular Plant 2014 7, DOI: ( /mp/sst141)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions