Respiration and SlGalLDH activity in isolated mitochondria from leaves
Respiration was measured in intact mitochondria prepared from young developing leaves of P35S:SlgalldhRNAi line 8, line 5, and control plants as described in the “Materials and Methods.” The reaction was conducted with 500 μg of mitochondria protein in 1 mL of respiration buffer in the presence of substrates at concentration as indicated. Prior to measuring the respiration in the presence of l-GalL, the mitochondria were incubated with 12.5 μm antimycine A. SlGalLDH activity was measured with 30 μg of mitochondrial protein as described in the “Materials and Methods.” Data represent mean ± sd of seven or eight individual mitochondrial isolations. Superscript a (a) indicates values that were determined by the t test to be significantly different (P < 0.01) from control.
| Respiration | Control | Line 5 | Line 8 | ||
|---|---|---|---|---|---|
| Electron Donor | |||||
| nmol O2 min−1 mg−1 protein | |||||
| 2 mm NADH | 48.7 ± 5.5 | 46 ± 4.5 | 73.6 ± 3.7a | ||
| + 2.5 mm ADP | 86.9 ± 8.4 | 75.8 ± 7.1 | 139.9 ± 4.1a | ||
| + 2 mm KCN | 10.4 ± 1.8 | 10.9 ± 3.8 | 20.6 ± 3.2a | ||
| + 1 mm SHAM | 8.4 ± 2.5 | 9.6 ± 1.9 | 12.5 ± 2.1a | ||
| Respiratory control | 1.78 | 1.65 | 1.90 | ||
| 5 mm succinate | 39.7 ± 7.8 | 48.4 ± 7.3 | 42.6 ± 10.9 | ||
| +2.5 mm ADP | 86.9 ± 13 | 97.1 ± 12.8 | 95.2 ± 20.7 | ||
| Respiratory control | 2.19 | 2.00 | 2.23 | ||
| 5 mm Pyr/malate | 29.5 ± 4.5 | 34 ± 4.4 | 30.6 ± 3.4 | ||
| +2.5 mm ADP | 42.8 ± 8.3 | 47.6 ± 8.8 | 46.8 ± 4.6 | ||
| Respiratory control | 1.45 | 1.40 | 1.53 | ||
| 5 mm AsA | 39.9 ± 6.4 | 42.1 ± 5.1 | 39.7 ± 3.9 | ||
| 2.5 mm TMPD | 409.5 ± 32.5 | 393.7 ± 44.3 | 380.1 ± 45.4 | ||
| 4.2 mm l-GalL | 9.7 ± 0.9 | 5 ± 0.9a | 5.4 ± 0.6a | ||
| +10 μm cytochrome c | 13.4 ± 1.6 | 6.9 ± 0.7a | 6.5 ± 0.8a | ||
| SlGalLDH Activity | Control | Line 5 | Line 8 | ||
| nmol cytochrome c min−1 mg−1 protein | |||||
| 96 ± 15.1 | 49.6 ± 10.5a | 20 ± 5a | |||