Overexpression of iron superoxide dismutase in transformed poplar modifies the regulation of photosynthesis at low CO2 partial pressures or following exposure to the prooxidant herbicide methyl viologen

Abstract

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H2O2, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO2 assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O2) and at low (1%) O2. CO2-response curves for photosynthesis showed similar net CO2-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO2 (Ci) values below 200 microL L-1 but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O2 concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 micromol m-2 s-1) a progressive decrease in the ratio of variable (Fv) to maximal (Fm) fluorescence was observed with decreasing temperature. At 6(o)C the high-light-induced decrease in the Fv/Fm ratio was partially prevented by low O2 but values were comparable in all lines. Methyl viologen caused decreased Fv/Fm ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.

Figure 1

Polyacrylamide gels stained for SOD activity following IEF in the absence (A) or presence of the inhibitors CN (B) and H₂O₂ (C). The top gels show a comparison between SOD activity from A. thaliana leaves (At), WT, and three lines of FeSOD-transformed poplars (4SOD, 5SOD, and 6SOD). The bottom gels show FeSOD transformants 8SOD and 9SOD in addition to At, WT, and 4SOD. Extracts were loaded on a total soluble protein basis (5 μg per well for extracts from transformed poplar leaves, 50 μg per well for WT leaves, and 35 μg per well for A. thaliana leaves).

Figure 2

The relationship between the maximal extractable SOD activity of leaves and their position on the stem of WT (open bars) and transformed poplars overexpressing FeSOD (line 6 SOD [closed bars]). Position 1 contained the youngest green leaf on the stem, whereas leaf 16 was the oldest mature leaf. Leaf 3 was the youngest mature leaf on the stem when leaf 16 showed no sign of senescence, such as decreased total leaf-extractable soluble protein. U, Units; prot, protein.

Figure 3

Light (A)- and CO₂ (B)-response curves for chlorophyll a fluorescence quenching parameters and net CO₂ uptake by leaves from WT (○) and poplar overexpressing FeSOD (•). A, CO₂ concentration was 350 μL L⁻¹; B, PPFD was 590 μmol m⁻² s⁻¹. Values are means ± se of three (A) or four (B) individual plant measurements.

Figure 4

The relationship between Ci and q_P in leaves of WT (○) and transformed poplars overexpressing SOD (•) in air containing 1% O₂. The mean values ± se for three individual untransformed poplar leaves and three FeSOD transformants are given. PPFD was 590 μmol m⁻² s⁻¹.

Figure 5

The relationship between ΦPSII and ΦCO₂ in WT (○, ▵) and poplar overexpressing FeSOD (•, ▴) measured under nonphotorespiratory conditions, 1% O₂. Measurements were obtained with either varying irradiance (○, •) or varying CO₂ partial pressures (▵, ▴). Each point represents an individual measurement.

Figure 6

Net CO₂ uptake by WT (○) and transformed poplar (•) leaves after a 2-h high-light (1100 μmol m⁻² s⁻¹) treatment at different temperatures in 350 μL L⁻¹ CO₂, 21% O₂. Each point is an independent measurement on a different plant.

Figure 7

The effect of temperature during a high-light treatment on F_v/F_m ratios measured in leaves of WT (□ and white bars) and poplar overexpressing FeSOD (▪ and black bars). In A, the O₂ concentration of the surrounding air was maintained at 21%, whereas in B, the O₂ concentration was maintained at either 21 or 1% throughout the duration of the high-light treatment (2 h). Measurements of F_v/F_m were made on leaves maintained in the dark at 24°C for 1 h (○, •) or on leaves subjected to 2 h of high light and temperatures ranging from 6 to 24°C (squares or bars) and then allowed to recover in darkness for a further 2 h. Each point reflects a measurement on a different plant.

Figure 8

CO₂-dependent O₂ evolution by leaf discs from WT (white bars) and poplar overexpressing FeSOD (gray bars) incubated overnight in the presence of different concentrations of MV and then illuminated for 30 min in high light (1100 μmol m⁻² s⁻¹) and saturated CO₂ (5%). Values are the mean ± se of four separate experiments.

Figure 9

Dark-adapted F_v/F_m ratios measured on leaf discs from WT (white bars) and poplar overexpressing FeSOD (black bars) incubated overnight in the presence of different concentrations of MV and then illuminated for 2 h in high light (1100 μmol m⁻² s⁻¹) and saturating CO₂ (5%). Leaf discs were then allowed to recover in darkness for 2 h prior to F_v/F_m measurement. The F_v/F_m values measured at the end of the overnight incubation in water or MV (•) were compared with F_v/F_m values measured after 2 h of dark adaptation following exposure to high light (bars). Values are means ± se of six independent experiments.