Foliar flavonoid distribution during Spinacia chloroplast isolation

The flavonoid glycoside accumulation level was compared in class A and in class C spinach chloroplast suspensions. Class A chloroplasts (up to 97% intact) contained about 0.4% of the total flavonoid glycosides present in leaves. Further purification of 97%-intact chloroplast suspensions, through a Percoll gradient, reduced the glycoside level to less than 0.15%. On the other hand, class C chloroplasts (100% broken plastids) contained between 10 to 30 times more flavonoids than intact Percoll purified chloroplasts. These results indicate that chloroplasts could bind vacuolar glycosides during their period of isolation. This hypothesis was confirmed by controlled contamination experiments using a cell-free supernatant as a source of vacuolar glycosides. Furthermore, the level of flavonoids in chloroplasts could be decreased to a level close to that obtained in intact Percoll purified chloroplasts by washing with soluble polyvinylpyrolidone. The results presented in this paper demonstrate the importance of maintaining the physiological integrity of plastids during the course of organelle isolation when investigating flavonic compartmentation in leaves.