Mechanism of gene expression of Arabidopsis glutathione S-transferase, AtGST1, and AtGST11 in response to aluminum stress

Abstract

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5'-upstream region of each gene was fused to a beta-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.

Figure 1.

Gene expression level of AtGST1 and AtGST11 after various Al treatments. According to Richards et al. (1998), total RNA was extracted from the whole plant of 8-d-old seedlings that were completely immersed in the one-sixth MS medium containing Al at 0, 10, 30, or 50 μm for 4 h. White square, AtGST1; black square, AtGST11. The cDNA molecules were synthesized from the extracted total RNA and then used as templates in semiquantitative RT-PCR. RT-PCR experiments were performed for three independent RNA extractions and error bars = se values.

Figure 2.

GUS enzyme activity determined by a fluorescent method after Al stresses. A, Three transgenic plants (p35S::GUS line 17-2, black symbols in the left graph; pAtGST1::GUS line 14-1, white symbols in the left graph; pAtGST11::GUS line 15-1, white symbols in the right graph), and Ler (black symbols in the right graph) were exposed to various Al stresses: 0 (○ or •), 50 (▵ or ▴), 100 (□ or ▪), and 200 μm (▿ or ▾) for 24 h. Each point represents the mean value for two independent experiments. B, Increase of GUS activity by 100 μm Al stress in the constructed transgenic lines. Two pAtGST1::GUS lines, 7-2 and 14-1, and three pAtGST11::GUS lines, 3-3, 5-2, and 15-1, were exposed to 0 or 100 μm Al stress for 0, 2, or 8 h: white square, 0 h; dotted square, without Al for 2 h; square with diagonal lines, with 100 μm Al for 2 h; gray square, without Al for 8 h; black square, with 100 μm Al for 8 h. Error bars = se values (n = 3). In both A and B, soluble proteins extracted from the whole plant were used for detection of GUS enzyme activity. The activity was shown in arbitrary units per 1 μg of extracted protein.

Figure 3.

GUS staining for plants exposed to Al and other stresses. Roots of three transgenic lines [the p35S::GUS line (17-2, positive control), the pAtGST1::GUS line (14-1), or the pAtGST11::GUS line (15-1)] and a nontransgenic plant (Ler; negative control) were exposed to Al stress and then the whole plant was stained to detect GUS activity. A, Gene expression patterns in young seedlings (8–10 d old) exposed to 0 or 100 μm Al. Since similar staining patterns were obtained in Ler and p35S::GUS line (17-2) independent of Al stress, their staining patterns at 0 h were shown. −Al, 0 μm Al; +Al, 100 μm Al. B, Gene expression patterns for various stresses in the pAtGST1::GUS and the pAtGST11::GUS transgenic lines. Young seedlings (8–10 d old) of each line were exposed to various stresses as described in “Materials and Methods” for 24 h and then stained.

Figure 4.

GUS enzyme activity for other stresses determined by a fluorescent method. The pAtGST1::GUS line (14-1, white squares) and the pAtGST11::GUS line (15-1, black squares) were exposed to various stresses for 24 h. Soluble proteins extracted from the whole plant were used for determination of enzyme activity and the activity was shown in arbitrary units per 1 μg of extracted protein. Error bars were calculated from the three independent experiments.

Figure 5.

Structural comparison of the pAtGST1 and pAtGST11. DNA sequences of approximately 1-kb fragments of each gene were compared and homologous regions were shown here. Each initiation ATG exists in 483 to 485 bp and in 417 to 419 bp, respectively. Identical nucleotides were marked with asterisks. Several gaps (-) were inserted in both sequences to obtain maximum identities. Two big gaps seen in pAtGST11 were named ΔGap1 and ΔGap2. Repeated sequences (32 bp), T-rich region, and A-rich region existing in pAtGST1 were shown with thin, bold, and broken upper lines, respectively. The bottom picture shows an image of structural differences between the two sequences.

Figure 6.

Characterization of a proposed signaling system for Al stress. A, Time-course experiments using adult plants (25 d old) of three lines [Ler, p35S::GUS line (17-2), and pAtGST11::GUS line (15-1)]. Only roots were exposed to 100 μm Al. −Al, 0 μm Al; +Al, 100 μm Al. B, Microscopic observation of leaves and roots of the Al-treated seedlings. The p35S::GUS line (17-2) and pAtGST11::GUS line (15-1; 8–10 d old) were exposed to 100 μm Al for 8 h and then stained. Three leaves stained in different degrees were shown for 15-1 line. Blue spots observed in vein were shown by arrows.

Figure 7.

Al-dependent expression of pAtGST11::GUS gene in shoot region under with or without Ca condition. A, Effect of depletion of Ca ion on the gene expression of the AtGST11::GUS in shoot region of 15-1 line. The seedlings (8–10 d old) were kept in four media (see “Materials and Methods”) for 28 h (pretreatment for 16 h and treatment with or without 100 μm Al for another 12 h). Control medium (+Ca medium), ○ or •; −Ca medium, ▵ or ▴; +La medium, □ or ▪; +EGTA medium, ▿ or ▾. White and black symbols indicate conditions with and without Al, respectively. After cutting off the roots, the shoots were used for extraction of soluble proteins. GUS enzyme activity is shown in arbitrary units per 1 μg of extracted protein. Error bars = se values (n = 3–5). B, Effect of Ca ion on the gene expression in three pAtGST11::GUS lines. Young seedlings of three pAtGST11::GUS lines (3-3, 5-2, and 15-1) were treated with two media (+Ca medium or −Ca medium) for 24 h (pretreatment for 16 h and treatment with or without 100 μm Al for another 8 h). White square, pretreatment with +Ca medium for 16 h; dotted square and gray square, pretreatment with +Ca medium for 16 h and then continuous treatment with +Ca medium for another 8 h without and with 100 μm Al, respectively; white square with lines, pretreatment with −Ca medium for 16 h; black square with lines and black square, pretreatment with −Ca medium for 16 h and then continuous treatment with −Ca medium for another 8 h without and with 100 μm Al, respectively. Two independent experiments were performed for 3-3 line. Error bars for 5-2 and 15-1 lines = se values, which were calculated from three independent experiments. Statistical analysis was performed for the latter two lines and significant differences were seen in 5-2 line (P < 0.01; double asterisk) and in 15-1 line (P < 0.05, single asterisk) between +Al−Ca and +Al+Ca conditions.