Inducible antisense suppression of glycolate oxidase reveals its strong regulation over photosynthesis in rice.
Tobacco (Nicotiana tabacum var Havana Seed) leaf discs were supplied tracer quantities of [2-(14)C]- and [3-(14)C]pyruvate for 60 minutes in steady state photosynthesis with 21% or 1% O(2), and the glycolate oxidase inhibitor alpha-hydroxy-2-pyridinemethanesulfonic acid was then added for 5 or 10 minutes to cause glycolate to accumulate. The [3-(14)C]pyruvate was converted directly to glycolate as shown by a 50% greater than equallabeled (14)C in C-2 of glycolate, and the fraction of (14)C in C-2 increased in 1% O(2) to 80% greater than equal-labeled. This suggests the pathway using pyruvate is less O(2)-dependent than the oxygenase reaction producing glycolate from the Calvin cycle. The formation of glycolate from pyruvate in the leaf discs was time-dependent and with [2-(14)C]- and [3-(14)C]pyruvate supplied leaf discs the C-2 of glyoxylate derived from C-2 of isocitrate was labeled asymmetrically in a manner similar to the asymmetrical labeling of C-2 of glycolate under a number of conditions. Thus glycolate was probably formed by the reduction of glyoxylate. Isocitric lyase activity of tobacco leaves was associated with leaf mitochondria, though most of the activity was in the supernatant fraction after differential centrifugation of leaf homogenates. The total enzyme activity was at least 35 micromoles per gram fresh weight per hour. The relative contribution of the pathway to the glycolate pool is unknown, but the results support the existence of a sequence of reactions leading to glycolate synthesis during photosynthesis with pyruvate, isocitrate, and glyoxylate as intermediates.