A zinc finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice

Abstract

Aluminum (Al) toxicity is the major limiting factor of crop production on acid soils, but some plant species have evolved ways of detoxifying Al. Here, we report a C2H2-type zinc finger transcription factor ART1 (for Al resistance transcription factor 1), which specifically regulates the expression of genes related to Al tolerance in rice (Oryza sativa). ART1 is constitutively expressed in the root, and the expression level is not affected by Al treatment. ART1 is localized in the nucleus of all root cells. A yeast one-hybrid assay showed that ART1 has a transcriptional activation potential and interacts with the promoter region of STAR1, an important factor in rice Al tolerance. Microarray analysis revealed 31 downstream transcripts regulated by ART1, including STAR1 and 2 and a couple of homologs of Al tolerance genes in other plants. Some of these genes were implicated in both internal and external detoxification of Al at different cellular levels. Our findings shed light on comprehensively understanding how plants detoxify aluminum to survive in an acidic environment.

Figure 1.

Phenotype of the art1 Mutant. (A) Response to Al. Five-day-old seedlings of both the wild-type rice and art1 were exposed to a 0.5 mM CaCl₂ solution containing 0, 10, 30, or 50 μM AlCl₃, pH 4.5, for 24 h. Data are means ± sd (n = 10). (B) Growth on acid soil. Germinated seeds were sowed on acidic soil, pH 4.5, or neutral soil, pH 6.5, and grown for 6 d. (C) Response to different pHs. Seedlings were exposed to a buffered solution at different pHs for 24 h. Data are means ± sd (n = 10). (D) Effect of toxic metals on root elongation. Five-day-old seedlings were exposed to a 0.5 mM CaCl₂ solution, pH 4.5, containing 0, 30 μM Al, 20 μM Cd, 5 μM La, 100 μM Zn, or 0.5 μM Cu in their chloride form for 24 h. Root elongation was measured before and after the treatment and relative root elongation, (root elongation with metals)/(root elongation without metals) × 100. Data are means ± sd (n = 8 to 10). The asterisk shows a significant difference between the wild type and art1 (P < 0.05 by Student's t test).

Figure 2.

Sequence and Complementation Test of ART1. (A) Amino acid sequence of ART1. Predicted C2H2 domain (red frame with underline), putative nuclear targeting signal (black frame), mutation site (reversed triangle), and defect region in art1 due to frame shift by 1-bp deletion (blue letters) are shown. (B) Complementation test. Relative root elongation of the wild type, art1, transgenic rice with empty vector (vector control), and three independent transgenic lines with ART1 genomic region (lines 1 to 3) were measured. Data are means ± sd (n = 13 to 15 biological replicates). Different letters indicate significant differences at P < 0.05 by Tukey's test. (C) Phylogenic tree of ART1-like C2H2 zinc finger proteins in rice (Os-) and Arabidopsis (At-). The sequence alignment used to generate the phylogeny is presented in Supplemental Data Set 1 online. Bootstrap values from 1000 trials are indicated. The 0.1 scale shows substitution distance. [See online article for color version of this figure.]

Figure 3.

Expression, Localization, and Transcriptional Activation Analysis of ART1. (A) Relative mRNA expression of ART1 in different tissues. Five-day-old seedlings (cv Koshihikari) were exposed to a 0.5 mM CaCl₂ solution containing 0 or 50 μM Al for 6 h, and then root tips (0 to 10 mm), basal roots (10 to 20 mm), and shoots were used for quantitative RT-PCR to determine relative expression of ART1. Histone H3 was used as an internal control. Expression relative to the root tip expression without Al treatment is shown. Data are means ± sd (n = 6; three biological replicates and two technical replicates). Different letters indicate significant differences at P < 0.05 by Tukey's test. (B) Transient expression of the ART1-GFP fusion and GFP alone as control in rice callus protoplasts. (C) and (D) Immunostaining of root longitudinal section with an anti-ART1 antibody at 1 mm (C) and 20 mm (D) from the tip treated with Al (50 μM, 6 h). Arrows indicate root epidermal cells with (C) or without (D) ART1 expression. (E) and (F) Magnified images for single root cells at 20 mm from the tip treated with (F) or without (E) Al (50 μM, 6 h). Bars = 100 μm in (C) to (F). (G) Transcriptional activation analysis in yeast. Yeast strain AH109 carrying fusion gene of GAL4 DNA binding domain and ART1 (DB_GAL4-ART1) or DB_GAL4 alone as control (NC) were cultured on SD medium with or without histidine (His) at 30°C for 3 d.

Figure 4.

Regulation of STAR1 and STAR2 by ART1. (A) and (B) Relative expression levels of STAR1 (A) and STAR2 (B) in root tip (0 to 10 mm) of the wild type and art1 mutant treated with 0 to 50 μM Al, pH 4.5, or without Al, pH 5.6, for 6 h, were determined by quantitative RT-PCR. Expression relative to the wild type expression without Al treatment, pH 4.5, is shown. Data are means ± sd (n = 3). (C) Yeast one-hybrid assay. A pair of plasmids, (1 to 4) pHIS-ProSTAR1 [HIS reporter gene with STAR1 promoter, containing a region between −939 (1), −629 (2), −436 (3), or −297 (4) to −172 from STAR1 start codon] and pGAD-ART1 (fusion of GAL4 activation domain and ART1), (NC1) pHIS2.1 (without STAR1 promoter) and pGAD-ART1, (NC2) pHIS-ProSTAR1 and pGADT7 (GAL4 activation domain only) were introduced into yeast strain Y187 and cultured on SD medium containing 0 or 20 mM 3-amino-1,2,4-triazole, a competitor of HIS3 at 30°C for 3 d in the absence of His. (D) Relative Al sensitivity of art1, star1, and the art1 star1 double mutant. Five-day-old seedlings were exposed to a 0.5 mM CaCl₂ solution containing 0 or 5 μM Al, pH 4.5, for 24 h. Relative root elongation (root elongation with Al/root elongation without Al × 100) is shown. Data are means ± sd (n = 10). Different letters indicate significant differences at P < 0.05 by Student's t test.

Figure 5.

Position of Candidate Genes Regulated by ART1 and QTLs for Al Tolerance. Values indicate position of genes on each chromosome (Mb), and QTLs found by different studies are indicated by colored lines. Genes located at a similar position to the QTL are indicated with red boxes. [See online article for color version of this figure.]