A Survey of Plants for Leaf Peroxisomes

Abstract

Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose.

In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 μmoles × min⁻¹ × g⁻¹ wet weight or a specific activity of 0.02 to 0.05 μmole × min⁻¹ × mg⁻¹ protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 μmoles × min⁻¹ × g⁻¹ wet weight or 90 to 300 μmoles × min⁻¹ × mg⁻¹ protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial.

Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 μmole × min⁻¹ × mg⁻¹ protein; for catalase. 8000 μmoles × min⁻¹ × mg⁻¹ protein, and for malate dehydrogenase, 40 μmoles × min⁻¹ × mg⁻¹ protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 μmole of glycolate oxidase × min⁻¹ × g⁻¹ tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane.

Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration.

Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation.

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