New insights into aluminum tolerance in rice: the ASR5 protein binds the STAR1 promoter and other aluminum-responsive genes

Abstract

Aluminum (Al) toxicity in plants is one of the primary constraints in crop production. Al³⁺, the most toxic form of Al, is released into soil under acidic conditions and causes extensive damage to plants, especially in the roots. In rice, Al tolerance requires the ASR5 gene, but the molecular function of ASR5 has remained unknown. Here, we perform genome-wide analyses to identify ASR5-dependent Al-responsive genes in rice. Based on ASR5_RNAi silencing in plants, a global transcriptome analysis identified a total of 961 genes that were responsive to Al treatment in wild-type rice roots. Of these genes, 909 did not respond to Al in the ASR5_RNAi plants, indicating a central role for ASR5 in Al-responsive gene expression. Under normal conditions, without Al treatment, the ASR5_RNAi plants expressed 1.756 genes differentially compared to the wild-type plants, and 446 of these genes responded to Al treatment in the wild-type plants. Chromatin immunoprecipitation followed by deep sequencing identified 104 putative target genes that were directly regulated by ASR5 binding to their promoters, including the STAR1 gene, which encodes an ABC transporter required for Al tolerance. Motif analysis of the binding peak sequences revealed the binding motif for ASR5, which was confirmed via in vitro DNA-binding assays using the STAR1 promoter. These results demonstrate that ASR5 acts as a key transcription factor that is essential for Al-responsive gene expression and Al tolerance in rice.

Keywords: ASR.; Aluminum; ChIP-Seq; RNA-Seq; rice.

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.

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.

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.

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.

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 1.204 genes that were affected by ASR5 silencing but were unresponsive to Al.

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.

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).