Studies on the functional mechanism of system II herbicides in isolated chloroplasts

The effect of specific proteolytic enzymes on variable fluorescence, p-benzoquinone-m ediated oxygen evolution, PS II herbicide (atrazine and brom oxynil) binding, and protein degradation has been analyzed in isolated class II pea chloroplasts. It was found that: 1. Trypsin and a lysine-specific protease effectively reduce the m axim um chlorophyll-a fluorescence yield, whereas the initial fluorescence remains alm ost constant. At the sam e number of enzymatic activity units both proteases have practically the sam e effect. 2 Trypsin and a lysine-specific protease inhibit the/»-benzoquinone-m ediated flash-induced oxygen evolution with trypsin being markedly more effective at the same number of activity units of both enzymes. Unstacked thylakoids exhibit a higher sensitivity to proteolytic degradation by both enzymes. 3. Trypsin and a lysine-specific protease reduce the binding capacity of [¹⁴C]atrazine, but enhance that of [¹⁴C]bromoxynil (at long incubation tim es trypsin treatment also impairs bromoxynil binding). At the same specific activity a markedly longer treatment is required for the lysine-specific protease in order to achieve the same degree of m odification as with trypsin. 4. Trypsin was found to attack the rapidly-turned-over 32 kD a-protein severely, whereas the lysine-specific protease does not modify this polypeptide. On the other hand, the lysine-specific protease attacks the light harvesting com plex II. 5. Under our experimental conditions an arginine-specific protease did not affect chlorophyll-a fluorescence yield, p-benzoquinone-mediated oxygen evolution, herbicide binding and the polypeptide pattern. Based on these results a mechanism is proposed in which an as yet unidentified polypeptide with exposable lysine residues, as well as the lysine-free “Q_B-protein” regulate the electrontransfer from Q_A⁻ to Q_B and are involved in herbicide binding.