1. Volume 6, Issue 3, Pages 817-829 (May 2013)
In Vivo Function of Tic22, a Protein Import Component of the Intermembrane Space of Chloroplasts 
Mareike Rudolf, Anu B. Machettira, Lucia E. Groß, Katrin L. Weber, Kathrin Bolte, Tihana Bionda, Maik S. Sommer, Uwe G. Maier, Andreas P.M. Weber, Enrico Schleiff, Joanna Tripp 
Molecular Plant 
Volume 6, Issue 3, Pages (May 2013)
DOI: /mp/sss114
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Gene Expression and Protein Localization of atTIC22-III and atTIC22-IV.
(A) Expression levels were analyzed by quantitative RT–PCR. RNA for preparation of cDNA was isolated from 30 mg of seeds, 25–50 individual plants grown on MS-Medium (day 3, day 12, day 25), or of the respective tissues harvested from a soil-grown plant (roots, rosettes, stem, hypsophyll, siliques, flowers day 48 and day 66). Plants were grown under long-day conditions. PCR reactions were performed in triplicate, whereby cDNA from three independent RNA preparations was used. The data were normalized to the expression of UBIQUITIN, and presented relative to the expression in sample flowers day 48, which was set to 1.
(B) Chloroplasts (lane 1) were isolated from the double knockout line tic22-III/tic22-IV complemented with Tic22-III-Strep (panel 1–3) or Tic22-IV-Strep (panel 4–6) and incubated with thermolysin (Th, lane 3) or trypsin (Try, lane 5) as described in the Methods section. As control, chloroplasts were treated with the buffer for digestion in the absence of the protease (lanes 2, 4). The according fractions were immunodecorated with the indicated antibodies. Please note, all samples of the Tic40 analysis were subjected to the same gel and processed identically.
Molecular Plant 2013 6, DOI: ( /mp/sss114)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Genotypic and Expression Analysis of tic22-III and tic22-IV Knockout Lines.
(A) Schematic representation of the structure of the TIC22-III and -IV genes and position of T-DNA insertions. Exons are depicted as boxes, introns as lines. Primer binding sites for the PCR in (B) and (C) are indicated. LB, left border of the T-DNA; positions are presented in Table 1.
(B) Analysis of T-DNA insertions by PCR. PCR reactions were performed on genomic DNA isolated from wild-type and two independent single knockout lines tic22-III (III-1 and III-2) and -IV (IV-1 and IV-2), respectively. PCR was performed using a primer binding in the left border of the T-DNA insertion (LB) and gene-specific forward (F) or reverse primers (R) in the combinations indicated.
(C) RT–PCR analysis of the expression of TIC22-III and -IV in wild-type and T-DNA insertion lines. Actin was used for normalization. Primer binding sites are shown in (A).
(D) Western blot with total leaf protein extracts from wild-type and mutant plants. Antibodies used are indicated on the left. An antibody against cytoplasmic Hsp70 was used as a loading control.
(E) Analysis of T-DNA insertions by PCR as in (B) using genomic DNA isolated from WT and tic22-III/IV. PCR was performed using a primer binding in the left border of the T-DNA insertion (LB) and gene-specific forward (F) or reverse primers (R) in the combinations indicated.
(F) RT–PCR analysis of the expression of TIC22-III and -IV in WT and T-DNA insertion lines as in (C).
Molecular Plant 2013 6, DOI: ( /mp/sss114)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Phenotypic Analysis of Knockout Plants under Different Light Conditions.
(A) Phenotype of 4-day-old plants grown under long-day conditions with light intensities of 20, 140, and 400 µM m–2 s–1, respectively.
(B) Phenotype of plants grown for 17 d under conditions as in (A). The scale bar in (A) and (B) shows 5 mm and all figures of one panel are scaled to the same size.
(C) Stages of leaf development and inflorescence emergence under different light conditions (LL, NL, HL). Each stage of leaf development (left panel) indicates the formation of a new leaf pair >1 mm in length. The diagram on the right indicates the period from the inflorescence emergence to the opening of flowers. Five plants of each line were analyzed.
Molecular Plant 2013 6, DOI: ( /mp/sss114)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Photosynthetic Performance and Chloroplast Ultrastructure of the Double Mutant.
(A, B) The time (A) or light-intensity (B) dependence of the effective PSII quantum yield (Ф(II)) determined for WT, tic22 double knockout, and ppi1. Measurements were made on cotyledons of 7-day-old plants. Values shown are means from eight measurements (± SD). Lines represent the analysis of the data by equation 1 (A) and 2 (B). Values are given in Table 2.
(C) Cotyledons of 5-day-old plants grown on MS medium were analyzed by electron microscopy. Representative images of each line are shown.
Molecular Plant 2013 6, DOI: ( /mp/sss114)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Isolated Chloroplasts of tic22-III/IV Double-Knockout Plants Are Impaired in Protein Import Compared to Wild-Type.
(A) The 35S radiolabeled precursor of the small RubisCO subunit (pSSU) was incubated with isolated chloroplasts of Arabidopsis thaliana wild-type, tic22-III/IV double-knockout (III/IV), and Tic22-IV complementation (III/IV / IV-Strep) plants for the duration of 2, 5, and 10 min (wild-type: lanes 1–3; III/IV: lanes 4–6; III/IV/IV-Strep: lanes 7–9). After chloroplast re-isolation, the samples were subjected to SDS–PAGE and the radioactivity was visualized by autoradiography. For import quantifications, 1% of the translation product was analyzed (TP). Bands corresponding to the different processing states of the small subunit of rubisco (SSU) are marked by arrowheads (white: precursor; black: mature protein).
(B) Quantification of at least four independent experiments from (A). Error bars show the standard deviation (wild-type: black circles; tic22-III/IV double-knockout: white rectangles; and Tic22-IV complemented double-knockout: gray diamonds (III/IV)).
(C) The 35S-radiolabeled outer membrane proteins Oep24 (left) or Toc34 (right) were incubated for 2 min with isolated Arabidopsis thaliana wild-type (lanes 1, 3) and tic22-III/IV double-knockout (III/IV) chloroplasts (lanes 2, 4). 10% of the translation product is shown as input (TP). After carbonate extraction, samples were subjected to SDS–PAGE and radioactivity was visualized by autoradiography.
(D) Imported Oep24 and Toc34 was quantified as described for (B) and the import after 2 min was presented normalized to wild-type. Values for pSSU are taken from (B) and shown for comparison.
Molecular Plant 2013 6, DOI: ( /mp/sss114)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6 Affected Metabolites in the tic22 Double-Knockout Lines.
(A) Metabolites, for which an alteration of abundance was observed are shown and the change for the two genotypes with respect to the wild-type from 7-day or 7-week-old plants is indicated.
(B) The metabolic relation of the different metabolites identified to be altered in the tic22 double mutant is shown. The reactions in chloroplasts (green), peroxisomes (blue), or mitochondria (yellow) are framed. The observed change in the double mutant is shown as in (A) under each metabolite. For clarity, not all reaction intermediates are shown. Ala, alanine; E4P, erythrose-4P; F6P, fructose-6P; G6P, glucose-6P; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; R5P, ribulose-5P; Val, valine; X5P, xylulose-5P.
Molecular Plant 2013 6, DOI: ( /mp/sss114)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions