Atrazine absorption and effects on photosynthesis in wheat leaf protoplasts

Protoplasts isolated from wheat (Triticum aestivum cv Anza) leaves were used to study atrazine (6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-dia mine) absorption and effect on protoplast photosynthetic oxygen evolution. There was a linear relationship between protoplast volume and chlorophyll content; 1 microliter usually contained about 5 micrograms of chlorophyll. Protoplast O2 evolution in saturating NaHCO3 and light (photosynthetic photon flux densities greater than 300 micromoles photons m-2sec-1) was about 100 micromoles O2 mg chl-1 hr-1; activity was stable for several hours following isolation. Leaves from which the protoplasts were isolated evolved 233 micromoles O2 mg chl-1 hr-1. Protoplast and leaf segment oxygen evolution in intermittent light (saturating 5 Hz flashes) were 6.8 and 12.9 micromoles O2 mg chl-1 hr-1, respectively. Protoplasts and leaf segments had, respectively, 735 and 390 chlorophyll molecules per active photosystem II reaction center as calculated from the oxygen pulse measurements in intermittent light. Variable chlorophyll a fluorescence decay following a single saturating flash was slower in leaf protoplasts than in leaf segments. The oxygen evolution and fluorescence measurements indicated that protoplast isolation affected thylakoid mediated reactions in leaf protoplasts. This effect was probably due to the osmotic stress caused by the hypertonic solutions used to isolate and stabilize the protoplasts. Atrazine concentrations greater than 0.05 micromolars inhibited protoplast O2 evolution; the I50 concentration was 0.2 micromolar atrazine. Atrazine absorption increased linearly as the atrazine concentration in the external solution was increased from 6.5 to 112 micromolars. The leaf protoplasts accumulated atrazine against a concentration gradient. The accumulation ratio (Ci/Co, concentration inside/ concentration outside) was 12.5 at an external atrazine concentration of 6.5 micromolars. At external atrazine concentrations of 14, 27, 57, and 112 micromolars, the accumulation ratio was 8.5. It was calculated that atrazine binding to photosystem II reaction centers could account for only a small portion of the atrazine accumulated by leaf protoplasts. Atrazine accumulation was attributed to a large extent to the partitioning of atrazine into leaf protoplast lipid membranes.