1. Volume 7, Issue 7, Pages 1228-1247 (July 2014)
Identification of Target Genes and Transcription Factors Implicated in Translation- Dependent Retrograde Signaling in Arabidopsis 
Leister Dario , Romani Isidora , Mittermayr Lukas , Paieri Francesca , Fenino Elena , Kleine Tatjana  
Molecular Plant 
Volume 7, Issue 7, Pages (July 2014)
DOI: /mp/ssu066
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Phenotypic Characterization of 4-Week-Old Wild-Type (Col-0) and prors1-2 Mutant Plants, and Constitutive (35S:PRORS1 RNAi) and Inducible (iPRORS1 RNAi) PRORS1-Targeting RNAi Lines, Grown under Long-Day (16h Light/8h Dark) Conditions.
(A) Real-time PCR analysis of PRORS1 mRNA levels of Col-0, 35S:PRORS1 (line c3 and line c11) and prors1-2 mutant plants. Expression values are reported relative to PRORS1 transcript levels in Col-0.
(B) Phenotypes of Col-0, 35S:PRORS1 (lines c3 and c11) and prors1-2 mutant plants. The scale bar corresponds to 2 cm.
(C) Real-time PCR analysis of PRORS1 mRNA levels in iPRORS1 RNAi lines (lines i10, i14, and i35) treated for the indicated lengths of time with ethanol vapor. Expression values are reported relative to PRORS1 transcript levels at time 0h in the respective lines.
(D) Phenotypes of Col-0, iPRORS1 RNAi line i35 and prors1-2 mutant plants treated for the indicated lengths of time with ethanol vapor. The scale bar corresponds to 2 cm.
Real-time values were derived from three technical replicates on at least two biological replicates (each a pool of at least 10 different plants). The data are shown as mean values ± SD. Statistically significant differences (t-test; p < 0.05) between wild-type (time point 0h) and mutant or time series samples are indicated by an asterisk. All real-time results were normalized with respect to the expression level of AT4G36800.
Molecular Plant 2014 7, DOI: ( /mp/ssu066)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Basic Physiological Characterization of 4-Week-Old Wild-Type (Col-0), Col-0 Plants Harboring the Inducible GUS RNAi Construct (GUS), prors1-2 Plants, and the Inducible iPRORS1 RNAi Line i35, Grown under Long-Day (16h Light/8h Dark) Conditions and Exposed for the Indicated Lengths of Time to Ethanol Vapor.
(A) Analysis of plastid protein synthesis. An autoradiogram of thylakoid membrane and stromal proteins isolated from leaves, after pulse-labeling with [35S]Met for 1, 30, and 60min, and subsequent fractionation by SDS–PAGE is shown. The portion of the Coomassie (Coom.)-stained SDS-PA gel corresponding to the LHCII migration region served as a loading reference. Note that Col-0 and prors1-2 on one side and GUS and i35 samples on the other side were analyzed on the same gels, respectively. Levels of [35S]Met incorporation into RbcL was quantified in three replicates and is reported in the bar-plot. The values of prors1-2 and line i35 were normalized to the signal intensities obtained in Col-0 and GUS leaves, respectively (prors1-2/Col-0 and line i35/GUS).
(B) Acetone-extracted chlorophyll was measured photometrically, and chlorophyll concentrations were calculated as described (Lichtenthaler, 1987) and are reported in μg mg–1 fresh weight.
(C) Photosynthetic parameters were derived from measurements on at least three leaves from different plants. Actinic light intensity was 95 μmol photons m−2 s−1.
The data are shown as mean values ± SD. Statistically significant differences (t-test; p < 0.05) between wild-type (time point 0h) and mutant samples are indicated by an asterisk.
Fv/Fm, maximum quantum yield of PSII; ΦII, effective quantum yield of PSII; Chl, chlorophyll.
Molecular Plant 2014 7, DOI: ( /mp/ssu066)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 Identification of Putatively OGE-Dependent Target Genes.
(A) Validation of microarray results. Real-time PCR analysis of TRY (AT5G53200), OVA2 (AT5G49040), ELIP2 (AT4G14690), and LHCB2.1 (AT2G05100) mRNA levels was carried out (three technical replicates) on 4-week-old leaves from the pooled (three biological replicates) iPRORS1 RNAi lines i10, i14, and i35 that had been exposed to ethanol vapor for the indicated time periods. Expression values are reported relative to the transcript levels at time point 0h. The results were normalized with respect to the expression level of AT4G Bars indicate standard deviations. Statistically significant differences (t-test; p < 0.05) between time point 0h and the time series samples (48h and 56h) are indicated by an asterisk. Numbers represent differential expression data derived from microarray analyses. Note that more validation data are shown in Supplemental Table 1.
(B) Genes whose transcript levels were found to be differentially regulated at least two-fold at all time points (48h, 51h, 56h) were first filtered for control sets of genes (Col-0 48h versus Col-0 0h and RNAi 0h versus Col-0 0h), then for genes known to show circadian regulation. Seven diurnally regulated genes were subsequently included in the list, because they were regulated at all time points. The final ‘working list’ comprised 1020 genes.
(C) Venn diagrams depicting the degree of overlap between the set of genes whose expression levels were altered at least two-fold (up or down) in the prors1-2 mutant and the collection on the ‘working list’.
Molecular Plant 2014 7, DOI: ( /mp/ssu066)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Identification of Co-Regulated Gene Sets and Gene Ontology (GO) Analysis.
(A, B) Venn diagrams depicting the overlap between sets of genes whose expression were levels were down- (A) or up-regulated (B) at least two-fold or at least 1.5-fold (numbers in brackets).
(C, D) GO analysis of genes down- (C) and up-regulated (D) at the different time points compared with the permanent state in the prors1-2 mutant. The bar lengths indicate the frequency of assignment (expressed as fold change compared to the whole genome) to the GO category ‘cellular component’. GO annotations were extracted from The Arabidopsis Information Resource (TAIR; and include endoplasmic reticulum (ER), mitochondria (mt), chloroplasts (cp), and nucleus (nc). Note that TAIR does not calculate statistical significances.
Molecular Plant 2014 7, DOI: ( /mp/ssu066)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

6. Figure 5 Relationships between the Sequence Motifs Identified.
A similarity tree for all 47 sequence motifs identified in the mtdown (mt down 1–5), cpdown (cp down 1–9), ncdown (nc down 1–10), mtup (mt up 1–5), cpup (cp up 1–10), or ncup (nc up 1–8) gene sets was generated by Phylip implemented in STAMP, and illustrated with MEGA. Motifs were considered distinct if the length of the branch between the two nodes concerned was ≥ When this cut-off was applied, 19 motif groups were obtained (indicated by a light-gray rectangle). The sequence motifs which were already identified by PScan associated with bZIP910 and bZIP911 are marked. Furthermore, sequence logos of three top similarity motifs with an E-value < 1.0E-09 are displayed. The six identified transres elements are shown in bold.
Molecular Plant 2014 7, DOI: ( /mp/ssu066)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Identified transres cis-Elements in the Co-Regulated Gene Sets.
(A) Sequence logos of the most enriched cis-regulons in down- and up-regulated sets of genes whose products are localized to mitochondria (mt), chloroplasts (cp), or the nucleus (nc).
(B) Dendrogram of the most highly enriched cis-regulons identified in the down- and up-regulated sets of genes. nc_down_transres, mt_up_transres, and nc_up_transres are related and share a family binding profile (FBP) which has weak similarity to the plant SORLIP1 or the AtERF_twoSite motifs, but higher similarity to the mouse B-cell nuclear factor NF-muE1.
(C) Sequence logos of motifs resembling the FBP described in (B).
Fold enrichment and E-values of the identified motif sequences were calculated based on comparison of the 750-bp upstream sequences of the different gene sets with the whole-genome 750-bp upstream sequences of A. thaliana. E-values for the best hits were calculated in STAMP.
Molecular Plant 2014 7, DOI: ( /mp/ssu066)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

8. Figure 7 Regulated Transcription Factors (TFs).
(A) Venn diagram depicting the overlap of genes from the ‘working list’ with genes encoding TFs identified in the Database for Arabidopsis Transcription Factors (DATF; and TAIR.
(B) The differential expression pattern of selected TFs is shown. An asterisk marks those genes that contain the nc_down_transres or nc_up_transres element in their promoters.
Molecular Plant 2014 7, DOI: ( /mp/ssu066)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions