The Kok Effect in Chlamydomonas reinhardil

A Haxo-Blinks rate-measuring oxygen electrode together with a modulated light source gave an average current signal (change in net Oa exchange) and a modulated current signal (photosynthetic 02 evolution). Using this apparatus, net 02 exchange and photosynthetic 02 evolution at low intensities have been studied in the green alga, Chlamydomonas reinhardi. At both 645 nm and 695 nm, the curves of net 02 exchange as a function of light intensity were steeper at lowest intensities than about compensation, indicative of the Kok effect. The effect was greater at 695 nm than at 645 nm. The corresponding curves of photosynthetic 02 evolution, on the other hand, showed no Kok effect; here, the slope was lowest at lowest intensity. The absence of the Kok effect in 02 evolution, together with its sensitivity to monofluoroacetic acid, show that it is due to an interaction of photosynthesis and respiration. The effect was exaggerated by 3-(3,4-dichlorophenyl).1,1dimethylurea. In the presence of concentrations of this inhibitor sufficient to inhibit 02 evolution completely, a lightinduced change in net 02 exchange remained. This was interpreted as a system I dependent depression of respiratory 02 uptake. The Kok effect remained undiminished in concentrations of carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol which partially uncoupled either oxidative phosphorylation alone or both oxidative and photosynthetic phosphorylations. The above results can be explained within a model of the Kok effect in which 02 uptake is depressed by diversion of reductant away from respiratory electron transport and into photosystem I. The same photodepression of 02 uptake also appears to account for a transient in net 02 exchange seen in several algae upon turning off the light. Attention was first drawn to a nonlinearity in the curve of net 02 exchange as a function of light intensity by Kok (29), who studied it in Chlorella sp. Now known as the Kok effect, this phenomenon has also been observed in Haematococcus sp. (29), Osillatoria sp. and Symploca sp. (42), Anacystis nidulans and Anabaena variabilis (24, 27), Porphyridium sp. (15), Fragillaria sublinearis (5), Chlorella fusca (39), and under certain conditions in leaves of higher plants (12, 29). The effect is absent or very small in Chlorella pyrenoidosa (9, 35) and Phaeodactylum tricornutum (32). 1This study was supported by Grant GM11300 from the National Institutes of Health. 2 Present address: Fisheries Research Board of Canada, Freshwater Institute, 501 University Crescent, Winnipeg 19, Manitoba, Canada. Kok (29) suggested that the effect may be due to a lightdependent depression of respiration. A depression in the rate of uptake of isotopic 02 in the light has been seen in Anacystis nidulans (24) and Fragillaria sublinearis (5). The results of both Hoch et al. (24) and of Jones and Myers (27) show that preferential activation of photosystem I exaggerates the Kok effect. Activation of system I in excess of activation of system II could depress respiratory 02 uptake in at least two ways. Hoch et al. (24) proposed that cyclic phosphorylation driven by system I could raise the ratio of cellular ATP to ADP sufficiently to restrict respiratory electron transport. Alternately, preferential activation of system I could shift the flow of respiratory reductant away from respiratory electron transport and into system I of photosynthetic electron transport, as suggested by Jones and Myers (27). Recent observations on anaerobic, DCMU3-resistant "4CO2 fixation by Chlamydomonas reinhardi (47), and on photoevolution of H2 by Scenedesmus obliquus (45) and Chlamydomonas moewusii (18) show that the latter pathway may operate in anaerobic cells. The observation of a marked Kok effect in C. moewusii and C. reinhardi led to a study of the phenomenon in the latter alga in an attempt to gain a fuller understanding of its mechanism. MATERIALS AND METHODS Stock cultures of Chlamydomonas reinhardi Dangeard (Indiana culture collection No. 89, 44) were maintained in liquid media by weekly transfers. Cultures for experimental use were inoculated from week-old stock cultures and harvested during exponential growth about 40 hr later. Both stock and experimental cultures were grown in tubes containing 25 ml of FW-2 medium (18), aerated with 1% CO2 in air at a rate of about 1 liter per hour. The cultures were maintained at 25 C in a water bath with constant illumination by fluorescent tubes from two sides giving about 3000 lux from each side. The cells were harvested by centrifugation and resuspended in FW-2 medium lacking trace elements and buffered at pH 6.8 to 7.0 with 0.02 M phosphate (unless otherwise mentioned). Absolute measurements of 02 exchange were made in a 2.1-ml cuvette with 1-cm light path. A Beckman oxygen electrode, polarized at -0.65 v and placed in circuit with a Keithley Model 417 picoammeter, gave a sensitivity of about 1.6 /ul 02/ml full scale on a recorder. Relative measurements made on a Haxo-Blinks rate-measuring electrode formed the major part of the study. In conjunction with a modulated light beam, this apparatus gave an average current signal (change in net 02 exchange) and a modulated current signal (photosynthetic 02 evolution). Joliot (26) has presented evidence that the modulated signal is due to photosynthetic 02 alone. In 'Abbreviations: CCCP: carbonyl cyanide m-chlorophenylhydrazone; CMU: 3-(p-chlorophenyl)-1-, l-dimethylurea; DCMU: 3-(3 ,4-dichlorophenyl)-1, ldimethylurea; DNP: 2,4-dinitrophenol; MFA: monofluoroacetic acid.