Volume 7, Issue 10, Pages 1586-1590 (October 2014) Ferredoxin:Thioredoxin Reductase Is Required for Proper Chloroplast Development and Is Involved in the Regulation of Plastid Gene Expression in Arabidopsis thaliana  Peng Wang, Jun Liu, Bing Liu, Qingen Da, Dongru Feng, Jianbin Su, Yang Zhang, Jinfa Wang, Hong-Bin Wang  Molecular Plant  Volume 7, Issue 10, Pages 1586-1590 (October 2014) DOI: 10.1093/mp/ssu069 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 FTR Is Required for PEP-Dependent Plastid Gene Expression at the Early Stages of Chloroplast Development. (A) Identification and characterization of VIGS–FTRc Arabidopsis plants. Phenotypes of VIGS–GFP (a negative control for the effect of the virus infection) and VIGS–FTRc plants. Plants were observed 3 weeks after infiltration. Three biological replicates were performed, and similar results were obtained. (B) Representative leaves from VIGS plants. (C) Immunodetection of FTRc in VIGS–FTRc plants. Aliquots of 50 μg (one-half VIGS–GFP), 100 μg (VIGS–GFP), and 100 μg (VIGS–FTRc) of total leaf proteins were loaded on the gels. A CBB (Coomassie brilliant blue)-stained gel of the samples is shown below to provide an estimate of gel loading. Three biological replicates were performed, and similar results were obtained. (D–G) Photosynthetic electron transfer properties of VIGS–FTRc Arabidopsis plants. Chlorophyll fluorescence parameters were examined in VIGS–FTRc plants under different actinic light intensities, including the quantum yield of PSI [D, Y(I)], quantum yield of PSII [E, Y(I)], excitation pressure of PSII (F, 1-qP), and non-photochemical quenching (G, NPQ). The data represent means ± SD of three biological replicates. (H) Transmission electron microscope images of chloroplast ultrastructures in VIGS–GFP (upper panel), VIGS–FTRc-G (middle panel), and VIGS–FTRc (lower panel); leaves were taken 3 weeks after infiltration. The images in the left panels show light micrographs of whole chloroplasts, which were observed further by electron microscopy in the magnified images in the right panels. Scale bars are indicated. Two biological replicates were performed, and similar results were obtained. (I) BN–PAGE analyses of thylakoid chlorophyll–protein complexes. Equal amounts of thylakoid membranes (7 μg chlorophyll) from VIGS–FTRc and VIGS–GFP plants were solubilized with 1% DM (w/v) and separated by BN–PAGE. Assignments of the thylakoid membrane macromolecular protein complexes indicated at right were identified. (J–L) 2D BN/SDS–PAGE fractionation of thylakoid protein complexes. Individual lanes from the BN–PAGE gels in (I) were subjected to second-dimension SDS–urea–PAGE followed by Coomassie brilliant blue staining. Identities of the relevant proteins are indicated by arrows. Three biological replicates were performed, and similar results were obtained. (M) Co-localization of FTRc:YFP fusion proteins (yellow) and PEND:CFP (blue) with chloroplast nucleoids. PEND (plastid envelop DNA binding) is a well characterized plastid DNA binding protein. Bars = 5 μm. Three biological replicates were performed, and similar results were obtained. (N) Changes in transcript abundance of plastid-encoded genes in VIGS–FTRc plants. The log2 (VIGS–FTRc/VIGS–GFP) value is given, where 3.32 corresponds to a 10-fold up-regulation, and –3.32 corresponds to a 10-fold down-regulation, in two different sectors in VIGS–FTRc plants relative to VIGS–GFP plants. I, class I genes; II, class II genes; III, class III genes; gray bars, VIGS–FTRc-G; dark gray bars, VIGS–FTRc-Y. The UBQ4 mRNA level was used as a reference. The data represent means ± SD of three biological replicates. Significant differences between VIGS–FTRc-G or VIGS–FTRc-Y leaves and VIGS–GFP leaves were calculated using Student's t-test and are indicated by * P ≤ 0.05 and ** P ≤ 0.01. (O) Immunoblot analysis of thylakoid membrane proteins in the green and yellow sectors of VIGS–FTRc leaves. Aliquots of 2.5 μg (one-quarter VIGS–GFP), 5 μg (one-half VIGS–GFP), 10 μg (VIGS–GFP), and 10 μg (VIGS–FTRc) of total thylakoid proteins were loaded on the gels. Designations of thylakoid membrane protein complexes and their diagnostic components are labeled on the left. Three biological replicates were performed, and similar results were obtained. Molecular Plant 2014 7, 1586-1590DOI: (10.1093/mp/ssu069) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions