A bacterial transgene for catalase protects translation of d1 protein during exposure of salt-stressed tobacco leaves to strong light

Abstract

During photoinhibition of photosystem II (PSII) in cyanobacteria, salt stress inhibits the repair of photodamaged PSII and, in particular, the synthesis of the D1 protein (D1). We investigated the effects of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum 'Xanthi') and in transformed plants that harbored the katE gene for catalase from Escherichia coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both wild-type and katE-transformed plants, but the effect of salt stress was less significant in the transformed plants than in wild-type plants. In the presence of lincomycin, which inhibits protein synthesis in chloroplasts, the activity of PSII decreased rapidly and at similar rates in both types of leaf disc during photoinhibition, and the observation suggests that repair of PSII was protected by the transgene-coded enzyme. Incorporation of [(35)S]methionine into D1 during photoinhibition was inhibited by salt stress, and the transformation mitigated this inhibitory effect. Northern blotting revealed that the level of psbA transcripts was not significantly affected by salt stress or by the transformation. Our results suggest that salt stress enhanced photoinhibition by inhibiting repair of PSII and that the katE transgene increased the resistance of the chloroplast's translational machinery to salt stress by scavenging hydrogen peroxide.

Figure 1.

Molecular characterization of katE-transgenic tobacco plants by means of PCR amplification of nptII and katE genes in the genomic DNA isolated from leaves of wild-type and katE-transformed tobacco plants. Conditions for PCR and probes for nptII and katE are described in the text. M, A 100-bp ladder of molecular markers; +C, positive control (pBI101-katE); T, katE-transgenic tobacco plants; W, wild-type plants.

Figure 2.

Effects of salt stress in the absence and presence of lincomycin on photoinhibition of PSII in leaf discs from wild-type and katE-transformed tobacco plants. Leaf discs were illuminated at 2,000 μE m⁻² s⁻¹ in the presence of NaCl at various concentrations in the presence or absence of lincomycin. At indicated times, PSII activity was examined by monitoring the ratio F_v/F_m after incubation in darkness for 30 min, as described in the text. The control value of F_v/F_m was 0.84 ± 0.01 and 0.82 ± 0.01 in the absence and presence of lincomycin, respectively. A and B, Leaf discs from wild-type and katE-transformed plants, respectively, that had been incubated in the absence of lincomycin; C and D, leaf discs from wild-type and katE-transformed plants, respectively, that had been incubated in 0.6 mm lincomycin for 2 h in darkness as described in the text. □, 50 mm NaCl, light; ▵, 0.5 m NaCl, light; ○, 1.0 m NaCl, light; •, 1.0 m NaCl, dark. Each point and bar represent the average ± se of results from three independent experiments. The absence of bar indicates that se fell within the symbol in this and other figures.

Figure 3.

Changes in levels of D1 during photoinhibition of PSII in leaf discs from wild-type and katE-transformed tobacco plants. A, Results of western-blotting analysis for wild-type plants; B, results of western-blotting analysis for katE-transformed plants; C and D, quantification of the hybridization signals shown in A and B, respectively. Leaf discs were illuminated at 2,000 μE m⁻² s⁻¹ in the presence of 50 mm NaCl (▪), 0.5 m NaCl (▴), and 1.0 m NaCl (•). Controls (C) were incubated in 300 μE m⁻² s⁻¹ for 16 h light. At indicated times, leaf discs were frozen in liquid N₂, ground, and homogenized in isolation buffer (see text for details), and then thylakoid membranes were isolated. Proteins were analyzed by SDS-PAGE as described in the text. The light intensity (μE m⁻² s⁻¹), the concentration of NaCl (NaCl), and the duration of illumination (h) are indicated above the lanes. Each point and bar represent the average ± se of results from three independent experiments.

Figure 4.

Effects of salt stress on the synthesis of membrane-bound proteins during photoinhibitory treatment of leaf discs from wild-type and katE-transformed tobacco plants. Leaf discs were incubated with [³⁵S]Met at 2,000 μE m⁻² s⁻¹ in the presence of 50 mm, 0.5 m, and 1.0 m NaCl, as indicated. At indicated times, leaf discs were frozen in liquid N₂, ground, and homogenized in isolation buffer (see text for details), and then thylakoid membranes were isolated. The thylakoid membranes were solubilized and proteins were separated by SDS-PAGE as described in the text. Proteins from thylakoid membranes corresponding to 5 μg of Chl were applied to each lane. A, Autoradiography of radioactively labeled proteins from wild-type plants after SDS-PAGE; B, autoradiography of radioactively labeled proteins from katE-transformed plants after SDS-PAGE; C and D, quantification of the hybridization signals shown in A and B, respectively. ▪, 50 mm NaCl; ▴, 0.5 m NaCl; •, 1.0 m NaCl. The light intensity (μE m⁻² s⁻¹), application of [³⁵S]Met, the concentration of NaCl (NaCl), and the duration of illumination (h) are indicated above the lanes. Each point and bar represent the average ± se of results from three independent experiments.

Figure 5.

Effects of salt stress on levels of the psbA transcript during photoinhibitory treatment of leaf discs from wild-type and katE-transformed tobacco plants. Leaf discs were illuminated at 2,000 μE m⁻² s⁻¹ in the presence of 50 mm, 0.5 m, and 1.0 m NaCl. Controls (C) were incubated in 300 μE m⁻² s⁻¹ for 16 h light. At indicated times, leaf discs were frozen and ground in liquid N₂, and then total RNA was extracted and subjected to northern-blotting analysis as described in the text. The psbA transcript was probed with a fragment of the psbA gene (see text for details). A, Wild-type plants; B, katE-transformed plants. The levels of the psbA transcript were normalized by reference to levels of 25S rRNA and results are shown quantitatively in the bottom panels. C and D, Quantification of the hybridization signals shown in A and B, respectively. ▪, 50 mm NaCl; ▴, 0.5 m NaCl; •, 1.0 m NaCl. The light intensity (μE m⁻² s⁻¹), the concentration of NaCl (NaCl), and the duration of illumination (h) are indicated above the lanes. Each point and bar represent the average ± se of results from three independent experiments.