Planta (1985)164:201-206 P l a n t a 9 Springer-Verlag 1985 Enhancement of chloroplast energization in Chlorella by treatments inactivating the nitrate-reducing system Yuri K. Chemeris, Antoaneta V. Popova and Pavel S. Venediktov* Biophysics Department, Faculty of Biology, M.V. Lomonosov State University, Lenin Hills, Moscow W-234, USSR 119899 Abstract. Inactivation of the nitrate-reducing sys- tem in whole cells of Chlorella vulgaris Bejerinck by darkening, nitrogen starvation, ammonium, or cycloheximide brings cells into a state with a high yield of the millisecond-delayed fluorescence of chlorophyll. Activation of this system by illumina- tion, by adding glucose to dark-adapted cells or nitrate to nitrogen-starved cells brings the cells into a low-yield state. The transitions between the low- and high-yield state induced by alternating light and dark periods are suppressed by tungstate and restored by subsequent molybdate addition. The drop in the delayed-fluorescence yield upon activa- tion of the nitrate-reducing system is associated with the decrease of the amplitude of the electro- chemical proton gradient across the thylakoid membrane of the chloroplast, as evidenced by the kinetics of the light-induced adsorption changes at 520 rim. The decrease of the proton gradient may be caused by the electron flow diverting from the cyclic path in photosystem I as a result of the activation of the electron transfer from ferredoxin to nitrite. Key words: Chlorella - Chloroplast energization - Fluorescence (delayed) - Nitrate-reducing system. cence has earlier been observed by us upon illumi- nation of dark-adapted cells (Venediktov et al. 1981). The light-induced decrease in yield is revers- ible, and the high yield is recovered in the dark. The changes in the emission yield may possibly be related to changes in the activity of the nitrate- reducing system, because nitrate and nitrite reduc- tases are inducible in Chlorella by light and sub- strates (Hodler et al. 1972; Johnson 1979; Tischner and Lorenzen 1979). If this is true, one may expect that nitrate starvation or an addition of ammoni- um (Johnson 1979; Losada et al. 1970), cyclohexi- mide (Tischner 1976), or tungstate (Paneque et al. 1972; Johnson 1979; Tischner and Lorenzen 1979) - treatments which inactivate nitrate reductase - will induce an increase in the yield of delayed fluo- rescence. On the other hand, activation of the ni- trate-reducing system by adding glucose in the dark (Paneque et al. 1972) should diminish the de- layed-fluorescence yield, as light does. In the present work, we report that such chan- ges in the delayed fluorescence do take place. Re- sults are presented indicating that activation of the nitrate-reducing system causes a decrease in the amplitude of the proton gradient across the thylak- oid membrane of the chloroplast, probably as a consequence of reduced cyclic electron flow in pho- tosystem I. Introduction We have noticed recently that the addition of ni- trate to nitrogen-starved cells of Chlorella, causes a twofold decrease in the yield of the millisecond- delayed fluorescence of chlorophyll (Chemeris et al. 1983). Also, a decrease in delayed fluores- * To whom correspondence should be addressed Abbreviation: DCMU = 3-(3",4'-dichlorophenyl)-l,l-dimethyl- urea Material and methods Cells of the thermophylic strain S-39/64688 of Chlorella vulgaris Bejerink (Algal Collection, Inst. of Biology, Leningrad State Universtiy, USSR) were grown in a cultivation vessel, aerated with sterile air at 37 ~ C under continuous illumination (energy fluence rate 10 W m -z) in a Tamiya medium (Tamiya et al. 1953), containing KNO 3 as a nitrogen source. For starvation experiments, exponentially growing cells were centrifuged, washed twice in the Tamiya medium free of KNO 3, diluted with the same medium to a concentration of about 7" 106 cells ml-1 and transferred again into the cultivation vessel. The design of the cultivator and the apparatus for measur-