Manipulation of catalase levels produces altered photosynthesis in transgenic tobacco plants

Abstract

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalatic [corrected] form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO2 compensation point (gamma) at a leaf temperature of 38 degrees C. A significant linear increase was observed in gamma with decreasing catalase (at 50% lower catalase activity gamma increased 39%). There was a significant temperature-dependent linear decrease in gamma in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase gamma decreased 17%). At 29 degrees C, gamma also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO2 were significantly reduced with increasing catalase activities at 38 degrees C, indicating that the stoichiometry of photorespiratory CO2 formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.

Figure 1

Northern-blot analysis of RNA from WT and transgenic plants (9.8, 2.2A, 2.3B; Table I) with altered catalase specific activities. The blots were obtained on Nytran membranes using total RNA (15 μg) from the tip region of leaves that was hybridized to radiolabeled N. tabacum catalase DNA and SSu DNA. Plant 9.8, mean catalase activity, 2.04 times that of WT, plant 2.2A; mean catalase activity, 0.62 times that of WT; and plant 2.3B, mean catalase activity, 0.66 times that of WT. The catalase signals relative to WT were: plant 9.8, 0.3×; plant 2.2A, 0.5×; plant 2.3B, 0.2×. The SSu signals relative to WT were: plant 9.8, 1.7×; plant 2.2A, 2.1×; plant 2.3B, 2.9×.

Figure 2

Elution profile showing separation by chromatofocusing of catalase isozymes in stems and sepals of WT and transgenic tobacco in which catalase activity was overexpressed in leaves. The fractions encompassing the catalase isoform with enhanced peroxidatic activity are shown in the shaded areas. Recoveries of catalase activities of 79 to 90% were obtained from the chromatofocusing columns. A, An extract of stem tissue (18 g) of transformant plant 9.8 (Table I) with a leaf catalase specific activity 2.51 times that of WT yielded 3830 units of catalase activity and 30.5 mg of protein for chromatofocusing. B, An extract of WT stem tissue (31 g) yielded 6560 units of catalase activity and 44.8 mg of protein for chromatofocusing. C, An extract of sepal tissue (4.1 g) of transformant plant 9.11 (Table I) with a leaf catalase specific activity 2.50 times that of WT yielded 1800 units of catalase activity and 10.2 mg of protein for chromatofocusing. D, An extract of WT sepal tissue (5.0 g) yielded 4060 units of catalase activity and 16.5 mg of protein for chromatofocusing.

Figure 3

Comparison of NADH-hydroxypyruvate reductase and catalase specific activities in transgenic plants in the T₂ generation with decreased and increased catalase specific activities relative to WT. The ratio of transgenic-to-WT activities of NADH-hydroxypyruvate reductase and catalase were determined on the same leaf extracts. The solid line is a least-squares fit with a slope of 0.009 and r² = 0.001 (P = 0.97) over a range of catalase activities that varied from 0.2 to 2.0 times that of WT.

Figure 4

The effect of antisense catalase constructs in the T₂ generation on Γ at a leaf temperature of 38°C. The results are expressed as a ratio of transgenic-to-WT activities. Transgenic leaves were from the progeny of three different self-pollinated transformants containing the pBZ1 construct (Tables I and II). The mean Γ for WT (± se) was 133 ± 6.3 μL CO₂ L⁻¹ (n = 6). Each point represents experiments done on different leaves on different days. The solid line is a least-squares fit with a slope of −0.87 ± 0.41 and r² = 0.36 (P < 0.05).

Figure 5

The effect of enhanced catalase activity in transgenic plants of the T₂ generation on Γ at leaf temperatures of 38°C (•) and 29°C (○). The results are expressed as a ratio of transgenic-to-WT activities. Transgenic leaves were used from progeny of two different self-pollinated transformants containing the pBZ8 construct (Tables I and II). The mean Γ for WT (± se) at 38°C was 132 ± 4.7 μL CO₂ L⁻¹ (n = 10), and at 29°C was 74 ± 1.6 μL CO₂ L⁻¹ (n = 10). For each experiment the points at leaf temperatures of 38 and 29°C represent results with transgenic leaf samples taken at the same time from the same leaf and compared with WT samples also taken at the same time. The solid lines are least-square fits with slopes of −0.21 ± 0.053 and −0.13 ± 0.071 for 38 and 29°C, respectively. The r² = 0.67 (P < 0.01) at 38°C, and r² = 0.32 (P > 0.10) at 29°C.