Protoplasts from Oat Primary Leaves as Tools for Experiments on the Compartmentation in Lipid and Flavonoid Metabolism

Abstract

Abstract Mesophyll protoplasts were isolated from defined stages of developing primary leaves of Avena sativa by a simple procedure in high and reproducible yield of about 50%. Rates of photosynthesis were measured by different techniques and turned out to be lower by a factor of 2 - 5 with protoplasts as compared to parent tissues, whereas dark respiration rates were about the same. Stability of protoplasts during storage in suspension medium at room temperature was checked by various methods. In contrast to protoplast numbers and accessibility of soluble enzymes, which suggested reasonable stability, photosynthetic oxygen evolution seems to be a more sensitive criterion. This rate dropped to about 50% of its original value during storage for 3 h. The protoplasts from differ­ent developmental stages did not show significant differences with respect to these various param­eters. Mechanical rupture or chemical destruction of the plasma membrane by filipin resulted in release of chloroplasts, which according to activity profiles of soluble marker enzymes after gra­dient centrifugation were intact. Centrifugation in a vertical rotor separated mitochondria from chloroplasts. Irrespective of the developmental stage of the parent tissue each protoplast contained about 200 chloroplasts. In conjunction with chlorophyll determinations it was calculated that about 2 × 109 chloroplasts account for 1 mg of chlorophyll. Lipid mixtures from leaves, protoplasts and chloroplasts were compared. They did not contain compounds which usually indicate enzy­matic alteration due to destruction of cellular compartmentation. Flavonoid profiles obtained from mesophyll protoplasts deviate significantly from epidermal patterns and prove the occurrence of flavonoids in photosynthetically active m esophyll cells.