A methyl viologen-resistant mutant of Arabidopsis, which is allelic to ozone-sensitive rcd1, is tolerant to supplemental ultraviolet-B irradiation

Abstract

To better understand the role of active oxygen species (AOS) in acquired resistance to increased levels of ultraviolet (UV)-B irradiation in plants, we isolated an Arabidopsis mutant that is resistant to methyl viologen, and its sensitivity to UV-B was investigated. A complementation test revealed that the obtained mutant was allelic to the ozone-sensitive radical-induced cell death1-1 (rcd1-1). Therefore, this mutant was named rcd1-2. rcd1-2 was recessive and nearly 4-fold more resistant to methyl viologen than wild type. It exhibited a higher tolerance to short-term UV-B supplementation treatments than the wild type: UV-B-induced formation of cyclobutane pyrimidine dimers was reduced by one-half after 24 h of exposure; the decrease in quantum yield of photosystem II was also diminished by 40% after 12 h of treatment. Furthermore, rcd1-2 was tolerant to freezing. Steady-state mRNA levels of plastidic Cu/Zn superoxide dismutase and stromal ascorbate peroxidase were higher in rcd1-2 than in wild type, and the mRNA level of the latter enzyme was enhanced by UV-B exposure more effectively in rcd1-2. UV-B-absorbing compounds were more accumulated in rcd1-2 than in wild type after UV-B exposure for 24 h. These findings suggest that rcd1-2 methyl viologen resistance is due to the enhanced activities of the AOS-scavenging enzymes in chloroplasts and that the acquired tolerance to the short-term UV-B exposure results from a higher accumulation of sunscreen pigments. rcd1 appears to be a mutant that constitutively shows stress responses, leading to accumulation of more pigments and AOS-scavenging enzymes without any stresses.

Figure 1.

Complementation test between rcd1-1 and rcd1-2 as determined by chlorophyll contents of crossed lines grown on agar medium containing 0 (white column), 0.4 (gray column), or 0.8 μm (black column) methyl viologen for 2 weeks. Values are means ± se of three independent experiments, in which 25 seedlings were used.

Figure 2.

Growth and morphology of Arabidopsis wild type (left in A, and top in B) and rcd1-2 mutant (right in A, and bottom in B). A, Six-week-old plants grown for 3 weeks on agar medium containing Murashige and Skoog salts and 1% (w/v) Suc followed by photoautotrophic growth on soil. Bar = 5 cm. B, Three-week-old plants grown on the agar medium. Bar = 5 mm.

Figure 3.

Effects of methyl viologen (paraquat) on root growth (A) and chlorophyll content (B). Wild type (white circle) and rcd1-2 (black circle) were grown for 2 weeks on agar medium containing methyl viologen. A, Growth of primary roots. Values are means of eight replicates ± sd. B, Chlorophyll was extracted with methanol from aerial parts of plants. Values are means ± se of three independent experiments, in which four plants were used.

Figure 4.

Effects of supplemental UV-B radiation (10.8 kJ m^-2 d^-1) on wild type and rcd1-2. A, F_v/F_m was measured with the third or fourth leaf with petiole, which was excised from 3-week-old wild type (white symbol) and rcd1-2 (black symbol). The leaf explant was planted into moistened precut floral foams and was then irradiated with white light with (circle) or without (triangle) UV-B before measurements. Values are means of six replicates ± sd. B, CPD was quantified by ELISA with monoclonal antibodies specific for CPD. DNA was isolated from aerial parts of plants irradiated with white light and UV-B for 24 h. Values are means of three independent experiments ± sd.

Figure 5.

Effects of freezing treatment on survival of leaf cell. Ion leakage from 2-week-old wild-type (white circle) and rcd1-2 (black circle) leaves were determined after freezing treatment. Values are means ± se of five independent experiments, in which three plants were used.

Figure 6.

Effects of supplemental UV-B irradiation on mRNA levels of cytosolic Cu/ZnSOD (CSD1), plastidic Cu/ZnSOD (CSD2), FeSOD (FSD), and MSD genes in wild type and rcd1-2. RNA gel-blot analysis (top row) was carried out with 20 μg of total RNA prepared from aerial parts of the 3-week-old plants irradiated with (+UV-B) or without (-UV-B) supplemental UV-B for 24 h. cDNAs of the genes were used as a probe. The ethidium bromide stain of rRNA (bottom row) is shown for each lane to allow assessment of equal loading.

Figure 7.

Effects of supplemental UV-B irradiation on mRNA levels of APX genes in wild type and rcd1-2. For more details, see the legend to Figure 6.

Figure 8.

Effects of supplemental UV-B irradiation on H₂O₂ accumulation in wild type (top panel) and rcd1-2 (bottom panel). Plants were irradiated with white light (-UV) or white light with UV-B (+UV) for 24 h. H₂O₂ was detected by DAB-HCl staining. A withered cotyledon was stained dark brown in the UV-B-irradiated rcd1-2. Bar = 5 mm.

Figure 9.

Effects of supplemental UV-B irradiation on contents of UV-B-absorbing compounds. Contents of UV-B-absorbing compounds were measured in leaf explants prepared from the third or fourth leaf of wild type (white symbol) and rcd1-2 (black symbol) that had been irradiated with white light (triangle) or white light with UV-B (circle) for 24 h, as described in the legend to Figure 4A. Values are means ± se of three independent experiments, in which four to six replicates were taken.

Figure 10.

Effects of supplemental UV-B irradiation on mRNA levels of PAL1, CHS, and CHI genes in wild type and rcd1-2. cDNAs of the genes were used as a probe. For more details, see the legend to Figure 6.