1. Volume 7, Issue 4, Pages 709-721 (April 2014)
New Insights into Aluminum Tolerance in Rice: The ASR5 Protein Binds the STAR1 Promoter and Other Aluminum-Responsive Genes 
Arenhart Rafael Augusto , Bai Yang , Valter de Oliveira Luiz Felipe , Bucker Neto Lauro , Schunemann Mariana , Maraschin Felipe dos Santos , Mariath Jorge , Silverio Adriano , Sachetto-Martins Gilberto , Margis Rogerio , Wang Zhi-Yong , Margis-Pinheiro Marcia  
Molecular Plant 
Volume 7, Issue 4, Pages (April 2014)
DOI: /mp/sst160
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Expression Pattern of ASR5 prom:GUS in Rice Roots.
(A) View of the root elongation zone.
(B) Longitudinal section of the root elongation zone.
(C) Macroscopic view of the root cap.
(D) Longitudinal section of the root cap.
(E) Root cap of the lateral root and (F) mechanical damage in the cortical cells.
(G) Root cap with unstructured cells (root border cells).
(H) Transverse section of the root elongation zone showing a GUS-positive reaction in the exodermal cells (arrow), cortex, pericycle, parenchymatic cells of the xylem, and companion cells of the phloem. The bars in A = 150 μm, B = 50 μm, C = 50 μm, D = 50 μm, E = 100 μm, F = 100 μm, and G = 100 μm.
Molecular Plant 2014 7, DOI: ( /mp/sst160)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Al-Responsive Genes in Non-Transformed (NT) and ASR5_RNAi Plants.
(A) Venn diagram showing the overlap of the Al-responsive up- and down-regulated genes between the NT and ASR5_RNAi plants.
(B) Number of genes affected by ASR5 silencing in the ASR5_RNAi plants.
Molecular Plant 2014 7, DOI: ( /mp/sst160)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Quantitative Real-Time RT–PCR of Four Selected Genes from the RNA-Seq Analysis.
Total RNA was extracted from the roots and used to synthesize cDNA. The relative expression was plotted using the expression levels of the FDH and Actin 2 genes as a reference. The roots of the Nipponbare cultivar were collected after 8h of treatment with AlCl3 (450 μM). The bars with different letters are significantly different (ANOVA, P < 0.05). Cnt, non-transformed plants under control conditions; Al, non-transformed plants under aluminum treatment; RNAi_Cnt, ASR5_RNAi plants under control conditions; RNAi_Al, ASR5_RNAi plants under aluminum treatment; NQ, not quantified.
Molecular Plant 2014 7, DOI: ( /mp/sst160)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
ChIP-Seq Analysis of ASR5 Target Genes in Al-Treated Rice Plants.
(A) Western blot showing increased ASR5 protein levels in rice plants in response to Al. ASR5 was detected with anti-ASR5. (Cnt) indicates the control untreated plants, whereas (Al) indicates the plants that were treated with 450 μM AlCl3 for 8h.
(B) Number and percentage of loci found in each binding region.
(C) Distribution of the binding sites. The x-axis displays the relative distance; the promoter region group is indicated in the top yellow bar; the coding region group is indicated by top blue and black bars; and the downstream region group is indicated by the top red bar. The y-axis displays the number of binding sites located in the different groups.
Molecular Plant 2014 7, DOI: ( /mp/sst160)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Overlap between the Genes Affected by ASR5_RNAi or Al Treatment and the Genes that Bound to ASR5 (ChIP-Seq Loci).
(A) Venn diagram showing the overlap of the Al-responsive genes between the non-transformed (NT) and ASR5_RNAi plants and the genes affected by ASR silencing.
(B) Venn diagram showing the overlap between the 469 Al-responsive genes found in the NT plants and ChIP-Seq loci. Venn diagram showing the overlap between the 367 Al-responsive genes found in the NT plants and those affected in the ASR5_RNAi plants due to ASR silencing with ChIP-Seq loci.
(C) Venn diagram showing the overlap between the genes that were affected by ASR5 silencing but were unresponsive to Al.
Molecular Plant 2014 7, DOI: ( /mp/sst160)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

7. Figure 6 Discriminative Motif Discovery in the ASR5 ChIP-Seq Data Set.
(A) The most significant motif identified using only the promoter regions of the binding peaks from the ChIP-Seq data identified by DREME.
(B) Enrichment of possible motifs identified by DREME compared to the reference rice genome.
Molecular Plant 2014 7, DOI: ( /mp/sst160)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

8. Figure 7 ASR5 Binds to the STAR1 Promoter In Vitro.
(A) ChIP qPCR results showing enrichment of the STAR1 promoter region using the α-ASR5 antibody.
(B) Scheme showing the amplification sites for the STAR1 promoter.
(C) SAPA pull-down system showing that ASR5–GST binds to an F1 biotinylated DNA fragment (F1*). Fragments F1, F2, F3, and Di-Dc were used as competitors. GST alone was used as a negative control.
(D) Transient gene expression assays demonstrating the regulation of STAR1 by ASR5 using GUS/luciferase assays.
(E) A proposed model for the ASR5–STAR1 promoter interaction. ART1 does not respond to Al in rice but maintains a housekeeping expression level of STAR1 under control conditions. In response to Al, ASR5 binds to the STAR1 promoter to enhance its expression.
Adapted from Delhaize et al. (2012).
Molecular Plant 2014 7, DOI: ( /mp/sst160)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions