Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses

Abstract

We studied the response of each ascorbate peroxidase (APX) isoenzyme in spinach leaves under stress conditions imposed by high light intensity, drought, salinity, and applications of methyl viologen and abscisic acid. The steady-state transcript level of cytosolic APX remarkably increased in response to high-light stress and methyl viologen treatment, but not in response to the other stress treatments. The transcript levels of the chloroplastic (stromal and thylakoid-bound) and microbody-bound APX isoenzymes were not changed in response to any of the stress treatments. To explore the responses of the APX isoenzymes to photooxidative stress, the levels of transcript and protein and activities of each isoenzyme were studied during high-light stress and following its recovery. The cytosolic APX activity increased in parallel with transcript abundance during high-light stress, while the protein level was not altered. The other isoenzymes showed no significant changes in transcript and protein levels and activities, except for the gradual decrease in chloroplastic isoenzyme activities.

Figure 1

Changes in the steady-state transcript levels of the APX isoenzymes in response to environmental and chemical stresses. Four-week-old plants were exposed to stresses of high light, salinity, drought, and treatments with ABA and MV. A, Northern-blot analysis of APX isoenzyme transcripts. Total RNA was isolated from spinach leaves, separated by electrophoresis (30 μg each), blotted on a membrane, and hybridized with cAPX, tAPX, mAPX, and SAP1 cDNA probes as described in “Materials and Methods.” B, Relative mRNA levels. The mRNA level of each sample was quantified with a phosphor imager and normalized to the respective 18S ribosomal RNA, and the values shown represent the mean value ± sd of three individual experiments. The value of the control plants was set equal to 1. Asterisks indicate that the mean values are significantly different compared with those of the control plants (P < 0.05).

Figure 2

Photosynthetic parameters measured in spinach leaves exposed to high-light stress. Four-week-old plants were exposed to high light intensity (1,600 μE m⁻² s⁻¹). After 5 h, stressed plants were re-exposed to moderate light at 300 μE m⁻² s⁻¹. The CO₂ fixation (A) and chlorophyll fluorescence (F_v/F_m) (B) were measured as described in “Materials and Methods.” The data are the mean value ± sd of three individual experiments. ●, Control plants; ○, stressed plants. Asterisks indicate that the mean values are significantly different compared with those of the control plants (P < 0.05).

Figure 3

(Continued from facing page.)The effect of high-light stress and its recovery on transcript and protein levels and activities of APX isoenzymes. A, Northern-blot analysis. Each APX isoenzyme transcript was detected as described in the legend of Figure 1. B, Relative mRNA levels. The mRNA levels of each sample were quantified with a phosphor imager and normalized to the respective 18S ribosomal RNA, and the values shown represent the mean value ± sd of three individual experiments. The value at time 0 was set to 1. C, Immunoblot analysis. The crude homogenates (50 μg each) of spinach leaves were subjected to SDS-PAGE and immunoblotting using mAb-I for sAPX and tAPX or EAP1 for cAPX and mAPX, as described in “Materials and Methods.” D, Relative protein levels. The protein levels of each sample were densitometrically quantified and represent the mean value ± sd of three individual experiments. The value at time 0 was set to 1. E, Activities. Detailed procedures are described in “Materials and Methods.” The data represented are the mean value ± sd of three individual experiments. ●, Control plants; ○, stressed plants. Asterisks indicate that the mean values are significantly different compared with those of the control plants (P < 0.05).

Figure 3

(Continued from facing page.)The effect of high-light stress and its recovery on transcript and protein levels and activities of APX isoenzymes. A, Northern-blot analysis. Each APX isoenzyme transcript was detected as described in the legend of Figure 1. B, Relative mRNA levels. The mRNA levels of each sample were quantified with a phosphor imager and normalized to the respective 18S ribosomal RNA, and the values shown represent the mean value ± sd of three individual experiments. The value at time 0 was set to 1. C, Immunoblot analysis. The crude homogenates (50 μg each) of spinach leaves were subjected to SDS-PAGE and immunoblotting using mAb-I for sAPX and tAPX or EAP1 for cAPX and mAPX, as described in “Materials and Methods.” D, Relative protein levels. The protein levels of each sample were densitometrically quantified and represent the mean value ± sd of three individual experiments. The value at time 0 was set to 1. E, Activities. Detailed procedures are described in “Materials and Methods.” The data represented are the mean value ± sd of three individual experiments. ●, Control plants; ○, stressed plants. Asterisks indicate that the mean values are significantly different compared with those of the control plants (P < 0.05).

Figure 4

The effect of high-light stress and its recovery on the H₂O₂ level, which was determined as described in “Materials and Methods.” The data represented are the mean value ± sd of three individual experiments. ●, Control plants; ○, stressed plants. Asterisks indicate that the mean values are significantly different compared with those of the control plants (P < 0.05).

Figure 5

The effect of high light intensity and its recovery on the levels of ascorbate and dehydroascorbate and the redox status of ascorbate. Experimental conditions are described under “Materials and Methods.” The redox status of ascorbate (ascorbate/[ascorbate + dehydroascorbate]) was calculated. The data represented are the mean value ± sd of three individual experiments. ●, Control plants; ○, stressed plants. Asterisks indicate that the mean values are significantly different compared with those of the control plants (P < 0.05).