Desensitization of GSTF8 induction by a prior chemical treatment is long lasting and operates in a tissue-dependent manner

Abstract

The Arabidopsis (Arabidopsis thaliana) GSTF8 gene is a member of the glutathione S-transferase (GST) family whose expression is induced by defense signals, certain chemical stresses, and some pathogens. Here, we have used transgenic plants and an in vivo imaging system to demonstrate that GSTF8 expression is subject to a distinct desensitization phenomenon because prior chemical treatment significantly reduces reactivation of the GSTF8 promoter by hydrogen peroxide, auxin, and salicylic acid. A GSTF8 null line had similar desensitization properties to wild type, demonstrating that GSTF8 protein levels are not responsible for desensitization. The resulting refractory period is unusually long lasting, with full recovery taking 4 d. Expression of the GSTF8 promoter following a second treatment occurred predominantly in newly formed tissue at the root tip, suggesting that desensitization is lost upon cell division. Expression of the endogenous GSTF8 gene and another GST gene, GSTF6, is also desensitized following treatment with hydrogen peroxide. The desensitization phenomenon can be activated by a very low concentration of inducer that is not sufficient to activate the GSTF8 promoter. These results demonstrate that activation of the GSTF8 promoter is not essential for eliciting desensitization. A key promoter sequence within the GSTF8 gene, the ocs element, is also affected by desensitization. Treatment with a phosphatase inhibitor prevents desensitization of GSTF8 expression and ocs element activity, suggesting that dephosphorylation of one or more proteins is required for desensitization to occur.

Figure 1.

Response of the GSTF8 promoter to SA and H₂O₂ treatments. A, Four-day-old GSTF8∷LUC seedlings were treated with 1 mm H₂O₂ or 1 mm SA for 40 min and the bioluminescence over a 19-h time course was measured using a CCD camera. The average light units per seedling for each treatment for each hour, with ses, are presented. B, Four-day-old GSTF8∷LUC seedlings were treated at time 0 and then again at 13 h with 1 mm H₂O₂ for 40 min and then decanted. The average relative light units per GSTF8∷LUC seedling with ses is presented for each hour over a 19-h time course. C, In vivo imaging of a representative, 4-d-old GSTF8∷LUC seedling treated with 1 mm H₂O₂ at time 0 and 13 h and visualized using a CCD camera. The white signal comes from the cotyledons and represents the transient chlorophyll fluorescence, which was imaged prior to measuring the bioluminescence. Bioluminescence was monitored after 10 min in the dark and is imaged in blue. Bioluminescence images were captured at 0, 3, 6, and 13 h after the first treatment and 3 and 6 h after the second treatment. The fluorescence and bioluminescence images were superimposed in the figure. The left- and right-most sections are black and white photographs of the same seedling taken before the first and second H₂O₂ treatment, respectively.

Figure 2.

Expression of the endogenous GSTF8 gene is also regulated by desensitization. Eight-day-old GSTF8∷LUC seedlings were treated with 1 mm H₂O₂ and RNA was extracted at 0, 3, and 6 h after the first H₂O₂ treatment and 0, 3, and 6 h after the second H₂O₂ treatment. RT-PCR was performed using GSTF6, GSTF8, and β-tubulin-specific primers and the results for GSTF6 and GSTF8 were standardized to the β-tubulin control. The average and se of two biological repeats are graphed.

Figure 3.

GSTF8 protein does not regulate desensitization. A, AtGSTF8 mRNA is undetectable in ΔGSTF8 plants. Eight-day-old seedlings were mock treated with water or 10 mm H₂O₂ for 30 min. Whole seedlings were harvested 7 h after commencing the treatments and gene expression was measured using real-time PCR. Relative AtGSTF8 mRNA expression levels were normalized to cyclophilin. The average of three biological replicates is shown along with ses. B, AtGSTF8 protein is undetectable in ΔGSTF8 plants. Ten-day-old seedlings were mock treated with water or 1 mm SA for 30 min. Whole soluble cell extracts were prepared from whole seedlings harvested 24 h after commencing the treatments. GSTs were purified by glutathione affinity chromatography and resolved by isoelectric focusing/SDS-PAGE over a pI range of 3 to 10. The resulting two-dimensional gels were silver stained and GSTs were identified based on their relative mobilities according to our previous mass spectrometry analysis of Arabidopsis GSTs (Sappl et al., 2004). C, Plates containing 4-d-old wild-type (columns 1 and 2) and ΔGSTF8 (columns 3 and 4) seedlings in the GSTF8∷LUC background were pretreated with water (columns 1 and 3) or 1 mm H₂O₂ (columns 2 and 4). The seedlings were treated again with 1 mm H₂O₂ 18 h later. For each treatment, 3 mL were added to a 55-mm Murashige and Skoog plate containing 50 μm luciferin and decanted after 40 min. Bioluminescence was measured for each seedling (approximately 20 seedlings/treatment) 6 h after the second treatment using a CCD camera and the average light units per seedling with ses are presented.

Figure 4.

Desensitization of GSTF8 expression is regulated temporally. Four-day-old GSTF8∷LUC seedlings were pretreated with either water (white bars) or 1 mm H₂O₂ (black bars) on day 0. Bioluminescence was measured 4 h after a second 1 mm H₂O₂ treatment delivered either on days 1, 2, 3, or 4 after the first treatment. Separate seedlings were used for each treatment and discarded after each day's measurements. The average bioluminescence values of seedlings are presented with ses.

Figure 5.

Low concentrations of auxin can activate the desensitization phenomenon. A, Four-day-old plants were treated with 1 or 300 μm auxin and plant extracts were assayed biochemically for LUC activity and graphed as LUC activity per milligrams protein with sds. Samples were tested with technical and biological repeats. The averages of the biological means and sd are graphed. B, Four-day-old GSTF8∷LUC seedlings were pretreated with water or 1 μm auxin and 13 h later all seedlings were treated with 1 mm H₂O₂. Bioluminescence was measured for each seedling (approximately 20 seedlings/treatment) using a CCD camera and the average light units per seedling with ses are presented.

Figure 6.

Ocs element activity is also desensitized following treatment with 1 mm H₂O₂ or 1 μm auxin. Four-day-old 4 × ocs∷LUC seedlings were pretreated with water (white columns), 1 mm H₂O₂ (black columns in A), or 1 μm auxin (black columns in B) and 14 h later the seedlings were treated with 1 mm H₂O₂. Bioluminescence was measured for each seedling (approximately 20 seedlings/treatment) using a CCD camera. The relative bioluminescence for each seedling was recorded at 6 and 12 h after the second treatment. The average light units per seedling with ses are presented.

Figure 7.

Okadaic acid releases desensitization of the GSTF8 promoter and ocs element activity. A, Plates containing 4-d-old GSTF8∷LUC seedlings were pretreated with water (column 1), 1 μm auxin (column 2), 1 μm okadaic acid (column 3), or both 1 μm auxin and 1 μm okadaic acid (column 4). The seedlings were treated with 1 mm H₂O₂ 17 h later. For each treatment, 1 mL was added to a 55-mm Murashige and Skoog plate containing 50 μm luciferin and decanted after 40 min. Bioluminescence was measured for each seedling (approximately 20 seedlings/treatment) 6 h after the second treatment and the average light units per seedling with ses are presented. B, Plates containing 4-d-old 4 × ocs∷LUC seedlings were pretreated as in Figure 7A. The seedlings were treated with 1 mm H₂O₂ 24 h later. Bioluminescence was measured for each seedling (approximately 20 seedlings/treatment) 12 h after the second treatment and the average light units per seedling with ses are presented.