Analysis and Characterization of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU)-resistant Euglena: I. Growth, Metabolic and Ultrastructural Modifications during Adaptation to Different Doses of DCMU

Cultures of Euglena gracilis Klebs strain Z Pringsheim were grown photoorganotrophically in the presence of different concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in the range of 0.05 to 250 micromolar. Cultures were serially transferred and various metabolic parameters were followed for 10 weeks. A process of adaptation occurred which was divided operationally into three phases. A phase of ultrastructural disorganization occurred, succeeded by a recovery phase; their intensity and duration were functions of the dose of DCMU. A stable adaptation phase then ensued. This phase was observed in all cultures except that exposed to the highest DCMU concentration. Adapted cells from all of the DCMU cultures contained twice the protein and half the paramylon of the control cells and thus utilized the carbon source to accumulate cellular reserves with only half the efficiency of controls. DCMU affected cellular metabolism as well as photosynthesis.     

Analysis and characterization of DCMU-resistant Euglena gracilis

Dark-grown, DCMU-adapted Euglena gracilis Z (ZR) are able to undergo light-induced chloroplast development in the presence or absence of DCMU. The differentiated chloroplasts are photosynthetically active and are resistant not only to DCMU, but also to an analog, o-phenanthrolene. When DCMU overdoses are added to ZR cells or to chloroplasts isolated from these cells, photosynthesis is partially inhibited. A brief period of darkness removes this inhibition. This recovery phenomenon is related to DCMU resistance, since it is not exhibited by non-resistant control cells. The chloroplast protein synthesis apparatus is not involved in DCMU resistance. Rather, this phenomenon is apparently related to new characteristics of thylakoids. It is shown that photosynthetic recovery by ZR cells depends on the accessibility and fluid properties of membranes. The analysis of fluorescence induction kinetics shows that changes in the environmental conformation of photosystem II units occur during recovery.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ZR DCMU-adapted Euglena gracilis Z I and II=Calvayrac et al., in press (a, b)     

Studies on the Component Responsible for the Oxidation of the Primary Electron Acceptor of Photosystem II in the Presence of 3-(3',4'-Dichlorophenyl)-1,1-Dimethyl Urea: Reactivity to External Reductants

The dark reoxidation of the photochemically reduced primaryelectron acceptor of photosystem II, Q., in the presence of3-(3',4'-dichlorophenyl)-l,l-dimethyl urea (DCMU) by the redoxcounterpart (here designated Z) of Q, was studied by monitoringthe dark recovery of the induction of chlorophyll fluorescence. In normal chloroplasts, the dark reoxidation of reduced Q inthe presence of DCMU was not affected by the externally addedhydrophilic reductants; ascorbate, hydroquinone, hydrogen peroxide,manganous chloride, potassium iodide and potassium ferrocyanide.In chloroplasts whose oxidizing side of photosystem II had beeninactivated by heat- or Tris-treatments, reoxidation was inhibitedpartially. This inhibition increased on the addition of hydrophilicreductants, but was relieved by increasing the redox potentialof the suspension medium with the chloroplasts. We concluded that the redox counterpart, Z, of Q, in the presenceof DCMU is located in a hydrophobic environment which can bedenatured by heat- or Tris-treatments to allow the access ofnormally extruded hydrophilic electron donors. (Received January 10, 1981; Accepted March 12, 1981)     

Response of multiple herbicide resistant strain of diazotrophic cyanobacterium, Anabaena variabilis, exposed to atrazine and DCMU

Effect of two photosynthetic inhibitor herbicides, atrazine (both purified and formulated) and [3-(3,4-dichlorophenyl)-1,1-dimethyl urea] (DCMU), on the growth, macromolecular contents, heterocyst frequency, photosynthetic O2 evolution and dark O2 uptake of wild type and multiple herbicide resistant (MHR) strain of diazotrophic cyanobacterium A. variabilis was studied. Cyanobacterial strains showed gradual inhibition in growth with increasing dosage of herbicides. Both wild type and MHR strain tolerated < 6.0 mg L(-1) of atrazine (purified), < 2.0 mg L(-1) of atrazine (formulated) and < 0.4 mg L(-1) of DCMU indicating similar level of herbicide tolerance. Atrazine (pure) (8.0 mg L(-1)) and 4.0 mg L(-1) of atrazine (formulated) were growth inhibitory concentrations (lethal) for both wild type and MHR strain indicating formulated atrazine was more toxic than the purified form. Comparatively lower concentrations of DCMU were found to be lethal for wild type and MHR strain, respectively. Thus, between the two herbicides tested DCMU was more growth toxic than atrazine. At sublethal dosages of herbicides, photosynthetic O2 evolution showed highest inhibition followed by chlorophyll a, phycobhiliproteins and heterocyst differentiation as compared to carotenoid, protein and respiratory O2 uptake.     

Identification of the pheophytin-QA-Fe domain of the reducing side of the photosystem II as the Cu(II)-inhibitory binding site.

Oxygen evolution by photosystem II membranes was inhibited by Cu(II) when 2,6-dichlorobenzoquinone or ferricyanide, but not silicomolybdate, was used as electron acceptor. This indicated that Cu(II) affected the reducing side of the photosystem II. The inhibition curves of Cu(II), o-phenanthroline and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), were compared; the inhibitory patterns of Cu(II) and o-phenanthroline were very similar and different in turn from that of DCMU. Cu(II) did not eliminate or modify the electron paramagnetic resonance signal at g = 8.1 ascribed to the non-heme iron of the photosystem II reaction center, indicating that the inhibition by Cu(II) was not the result of the replacement of the iron by Cu(II). Controlled trypsin digestion of thylakoid membranes inhibited oxygen evolution using 2,6-dichlorobenzoquinone, but had no effect when using ferricyanide or silicomolybdate. Using ferricyanide, oxygen evolution of trypsin-treated thylakoids was insensitive to DCMU but became even more sensitive to Cu(II) and o-phenanthroline than nontreated thylakoids; however, trypsinized thylakoids were insensitive to inhibitors in the presence of silicomolybdate. We conclude that Cu(II) impaired the photosystem II electron transfer before the QB niche, most probably at the pheophytin-QA-Fe domain.     

Chlorophyll Formation and Photosynthetic Competence in Euglena During Light-Induced Chloroplast Development in the Presence of 3, (3,4-dichlorophenyl) 1,1-Dimethyl Urea (DCMU)

The possibility that photosynthetic competence is gratuitous for light-induced chloroplast development in Euglena gracilis var. bacillaris was examined by incubating dark-grown resting cells in the light with DCMU, an inhibitor of photosynthesis. Under these conditions photosynthetic carbon dioxide fixation was inhibited essentially completely at all times during chloroplast development, but about 70% of the chlorophyll was formed with essentially the same pattern of accumulation found for cells incubated in the absence of the inhibitor. Electron microscopy of cells incubated with DCMU in the light revealed the formation of morphologically recognizable chloroplasts having comparable overall dimensions and structural elements to those found in normally developed chloroplasts, but frequently lacking a readily detectable pyrenoid with paramylum sheaths, and often containing increased numbers of discs per lamella. Such abnormalities are considered minor since upon removal of DCMU by centrifugation, the cells usually regained almost full photosynthetic competence on a chlorophyll basis.

It is concluded that photosynthetic competence is not necessary for chloroplast development in Euglena and supports the hypothesis, already suggested from other evidence, that light induction results in activation of synthetic machinery external to the developing chloroplast.

     

Biogenesis of Thylakoid Membranes in Chlamydomonas reinhardtii y1 (A Kinetic Study of Initial Greening)

Initiation of thylakoid membrane assembly was examined in degreened cells of Chlamydomonas reinhardtii y1 cells depleted of thylakoid membranes and photosynthetic activity by growth in the dark for 3 to 4 d. Photoreductive activities of photosystem II (PSII) and photosystem I (PSI) increased with no apparent lag when degreened cells were exposed to light at 38[deg]C. However, fluorescence transients induced by actinic light, which reflect the functional state of PSII, changed only slightly during the first 2 h of greening. When these cells were treated with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or saturating light, fluorescence increased commensurate with the cellular content of chlorophyll. In similar experiments with greening cells of C. reinhardtii CC-2341 (ac-u-g-2.3), a PSI-minus strain, fluorescence increased with chlorophyll without treatment with DCMU. These data suggested that fluorescence of initial PSII centers in greening y1 cells was quenched by activity of PSI. Continuous monitoring of fluorescence in the presence or absence of DCMU showed that assembly of quenched PSII centers occurred within seconds after exposure of y1 cells to light. These results are consistent with initial assembly of PSI and PSII within localized domains, where their proximity allows efficient energy coupling.     

Photobleaching of photosynthetic pigments in spinach thylakoid membranes. Effect of temperature, oxygen and DCMU

The time dependence of photobleaching of photosynthetic pigments under high light illumination of isolated spinach thylakoid membranes at 22 and 4 degrees C was investigated. At 22 degrees C, the bleaching at 678, 472 and 436 nm was prominent but lowering the temperature up to 4 degrees C during illumination prevented the pigments from bleaching almost completely. The accelerating effect on pigment photobleaching by the presence of 3-(3,4 dichlorophenyl)-1,1-dimethyl-urea)-(DCMU), a well-known inhibitor of the electron transport and known to prevent photosystem I (PSI) and photosystem II (PSII) against photoinhibitory damage, was also suppressed at low temperature. At 22 degrees C in the presence and absence of DCMU, the decrease of the absorption at 678 and 472 nm was accompanied by a shift to the shorter wavelengths. To check the involvement of reactive oxygen species in the process, pigment photobleaching was followed in anaerobiosis. The effects of the three different environmental factors--light, temperature and DCMU--on the dynamics of photobleaching are discussed in terms of different susceptibility of the main pigment-protein complexes to photoinhibition.     

Studies on thermoluminescence,delayed light emission and oxygen evolution from photosynthetic materials: UV effects

The effect of ultraviolet light on thermoluminescence, oxygen evolution and the slow component of delayed light has been investigated in chloroplasts and Pothos leaves. All peaks including peak V (48°C) were inhibited by UV. However, the peak at 48°C which was induced by DCMU was enhanced following UV irradiation of chloroplasts at ambient temperature (23°C) whereas peak II (-12°C) and peak III (10°C) which were also induced by DCMU were inhibited. Chloroplasts treated with DCMU and dark incubated for several minutes at ambient temperature prior to recording of glow curves have also shown enhancement of peak at 48°C. A slow component of delayed light and photosystem II activity of chloroplasts were inhibited by UV whereas photosystem I activity was marginally affected. These results corroborate involvement of photosystem II in generating thermoluminescence and slow components of delayed light in photosynthetic materials.Abbreviations DCIP Dichlorophenol Indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCQ 2,6 Dichloro-p-benzoquinone - DLE delayed light emission - MOPS Morpholino propane sulfonic acid - PSI Photosystem I - PS II Photosystem II - TL thermoluminescence     

Putative Second Binding Site of DCMU on the Oxidizing Side of Photosystem II in Photosystem II Membranes Depleted of Functional Mn

The effects of DCMU on the oxidizing side of PS II were studiedwith Triton-solubilized PS II membranes depleted of functionalMn. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) non-competitivelyinhibited the diphenylcarbazide-supported (DPC-supported) photoreductionof silicomolybdate (SiMo) at concentrations more than ten timeshigher than that required for inhibition of the DPC-supportedphotoreduction of 2,6-dichlorophenolindophenol (DCIP). The maximumfluorescence intensity was also reduced by DCMU at a similarconcentration to that required for the inhibition of the SiMophotoreduction. These findings suggest two inhibitory sitesof action of DCMU in PS II: one on the reducing side and oneon the oxidizing side of PS II. The inhibition constant forDCMU in the DPC-supported SiMo-photoreduction was 10 µMin every examination. The extent of inhibition was attenuatedby modifications of the PS II oxidizing side by the presenceof functional Mn, by photoinhibition and by chemical modificationsof histidine residues and acidic amino acid residues. Our resultssuggest that DCMU binds to the PS II oxidizing side near Z,D and the high-affinity Mn-binding sites. ¹ Present Address and address for all communications: NoriakiTamura (Dr.), Plant Physiology Laboratory Fukuoka Women's University,Kasumigaoka 1-1, Higashi-ku, Fukuoka, 813 Japan. FAX 092-661-2415.     

On the specific site of action of 3-)3,4-dichlorophenyl)-1,1-dimethylurea in chloroplasts: inhibiion of a dark acid-induced decrease in midpoint potential of cytochrome b-559.

Acidification of chloroplasts in the dark causes a decrease in the ability of ferrocyanide to reduce the oxidized cytochrome, which is reversible upon raising the pH. This effect is inhibited if 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) is added to the medium before acidification. DCMU inhibition of the loss of ferrocyanide reduction at pH 5.0 occurs at low DCMU concentrations, half-inhibition requiring 1 DCMU:400 chlorophyll molecules under conditions where half-inhibition of oxygen evolution requires the addition of 1 DCMU: 100 chlorophylls. Potentiometric titration shows that the midpoint potential of “high potential” cytochrome b-559 is +395 mV at pH 7.8, +335 mV at pH 5.0, and in the presence of DCMU +380 mV at pH 5.O. The ability of low concentrations of DCMU to exert a specific effect on a property associated with “high potential” cytochrome b-559 implies that this cytochrome, which is known to be in close structural proximity to the reduction center of photosystem II, is a principle site of action of DCMU.     

Temperature and Light Dependence of Photosynthetic Activities in Wheat Seedlings Grown in the Presence of DCMU [3-(3,4-Dichlorophenyl)-1,1-Dimethylurea]

Photosynthetic electron transfer was studied in thylakoids isolated from control and DCMU-grown wheat (Triticum aestivum L.) seedlings. When exposed to high temperature (HT) and high iradiance (HI), thylakoids showed large variations in the photosynthetic electron transport activities and thylakoid membrane proteins. A drastic reduction in the rate of whole electron transport chain (H₂O MV) was envisaged in control thylakoids when exposed to HT and HI. Such reduction was mainly due to the loss of photosystem 2, PS2 (H₂O DCBQ) activity. The thylakoids isolated from seedlings grown in the presence of DCMU showed greater resistance to HT and HI treatment. The artificial exogenous electron donors MnCl₂, DPC, and NH₂OH failed to restore the HI induced loss of PS2 activity in both control and DCMU thylakoids. In contrast, addition of DPC and NH₂OH significantly restored the HT induced loss of PS2 activity in control thylakoids and partially in DCMU thylakoids. Similar results were obtained when F_v/F_m was evaluated by chlorophyll fluorescence measurements. The marked loss of PS2 activity in control thylakoids was evidently due to the loss of 33, 23, and 17 kDa extrinsic polypeptides and 28-25 kDa LHCP polypeptides.     

Effects of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea on the Cell Cycle in Euglena gracilis

The cell cycle of the photosynthetic unicellular alga Euglena gracilis growing in phototrophic medium is regulated by light. To investigate the relationship of this cell cycle response to light stimulated photosynthesis, we have tested the effect of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on Euglena cell cycle transit. While DCMU does not block light stimulated cells from entering the S phase of the cell cycle, it does inhibit the transit through G₂/M. The specificity of this response and its relationship to photosynthesis was studied by looking at the effect of DCMU on dark grown wild-type cells, and on two bleached variants of Euglena (W₃BUL and W₁₀BSmL) that lack chloroplasts. The drug does block G₂/M in these cells, but not entrance into the cell cycle. Our studies show that entrance of cells into the cell cycle from a quiescent state does not require active photosynthesis, and that DCMU has effects on G₂/M transit that are independent of the photosynthetic capacity of the cells.     

Photosynthesis of Prochlorothrix hollandica under Sulfide-Rich Anoxic Conditions

The photosynthetic activity and photosystem II fluorescence of Prochlorothrix hollandica were studied under anoxic, sulfide-rich conditions. Oxygenic photosynthetic activity with water as the electron donor was highly resistant to inhibition by sulfide. Cells still retained 50% of their oxygenic photosynthetic activity at >1 mM sulfide. In the presence of DCMU [N-(3,4-dichlorophenyl)-N(prm1)-dimethylurea], an inhibitor of photosystem II activity, P. hollandica cells exhibited a low but significant anoxygenic photosynthetic activity when sulfide was present. This activity increased with higher sulfide concentrations and reached maximal rates at concentrations exceeding 1 mM sulfide. The effects of hydroxylamine on both oxygen evolution and fluorescence induction kinetics were similar to those observed for sulfide. It was concluded that the oxidizing site of photosystem II was the site of sulfide action leading to reduced or even fully inhibited electron donation to photosystem II. These observations bear similarity to the situation in some cyanobacteria in which both hydroxylamine and sulfide inhibit electron donation from H(inf2)O to P(inf680). The high resistance of photosystem II to sulfide is related to the hydrophobic nature of the manganese-stabilizing protein in P. hollandica (T. S. Mor, A. F. Post, and I. Ohad, Biochim. Biophys. Acta 1141:206-212, 1993). The observed sulfide tolerance of P. hollandica may confer a competitive advantage in its natural environment, where it forms a dominant fraction of phytoplankton in waters in which sulfide presence is a recurring phenomenon.     

Desiccation-time limits of photosynthetic recovery in Equisetum hyemale (Equisetaceae) spores

The chlorophyllous spores of Equisetum survive desiccation, yet cannot tolerate this quiescent state for more than ~2 wk. The hypothesis that spore viability of Equisetum hyemale L. is limited by inhibition of photosynthetic recovery was tested using chlorophyll a fluorescence and oxygen-exchange analyses. Experimental spores were desiccated at 2% relative humidity and 25C for time periods of 24 h, 1 wk, and 2 wk, and then rehydrated at 200 mmol photons/m2s (PAR) and 25C for up to 24 h. Spores desiccated for 24 h recovered photosynthetic competence very rapidly during rehydration, reaching the O2 compensation point in 6.3 ~ 0.3 (mean +/- SE) min. Recovery of photosynthetic performance of spores desiccated for 1 wk was slower, as judged by significantly slower increases of (1) photochemical efficiency of photosystem (PS) II, (2) PS II quinoneB-reducing center concentration, (3) quinoneB concentration, (4) water-oxidation activity, (5) rate of light-induced O2 evolution, and (6) apparent quantum yield of net O2 exchange. Photosystem-II and whole-spore photosynthetic competence of 2-wk desiccated spores was increasingly impaired, and did not recover during rehydration. Origin fluorescence yield and dark respiration were not affected by desiccation time following rehydration. The results suggest that the extremely short viability of disseminated spores of Equisetum hyemale is due to the inability to recover losses of water oxidation and photosystem II-core function following 2 wk of desiccation.     

Water oxidation chemistry of photosystem II

The O(2)-evolving complex of photosystem II catalyses the light-driven four-electron oxidation of water to dioxygen in photosynthesis. In this article, the steps leading to photosynthetic O(2) evolution are discussed. Emphasis is given to the proton-coupled electron-transfer steps involved in oxidation of the manganese cluster by oxidized tyrosine Z (Y(*)(Z)), the function of Ca(2+) and the mechanism by which water is activated for formation of an O-O bond. Based on a consideration of the biophysical studies of photosystem II and inorganic manganese model chemistry, a mechanism for photosynthetic O(2) evolution is presented in which the O-O bond-forming step occurs via nucleophilic attack on an electron-deficient Mn(V)=O species by a calcium-bound water molecule. The proposed mechanism includes specific roles for the tetranuclear manganese cluster, calcium, chloride, Y(Z) and His190 of the D1 polypeptide. Recent studies of the ion selectivity of the calcium site in the O(2)-evolving complex and of a functional inorganic manganese model system that test key aspects of this mechanism are also discussed.     

Evolution of enzymes involved in carbon metabolism (phosphoenolpyruvate and ribulose-bisphosphate carboxylases,phosphoenolpyruvate carboxykinase) during the light-induced greening of Euglena gracilis strains Z and ZR

Phosphoenolpyruvate carboxykinase activity decreases when Euglena gracilis Z and ZR undergo light-induced chloroplast development in batch resting medium lacking utilizable organic carbon and CO₂. This enzyme is present in heterotrophically grown cells (Briand et al. 1981) and assures gluconeogenesis. It was consistently more active in strain ZR. Decreased carboxykinase activities were accompanied by parallel increases in the activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase. The rates of O₂ evolution in light were much lower than those of CO₂ fixed simultaneously. The incorporation of ¹⁴CO₂ into early C-4 dicarboxylic acids was higher in green cells than in etiolated cells, and it was even higher in green cells assayed in light in the presence of (DCMU). A hypothesis has been proposed, according to which there is a possible cooperation of phosphoenolpyruvate carboxylase in photosynthetic CO₂ fixation, especially under conditions of limiting CO₂.High temperatures (34° C) depress carboxylation enzyme activities to a greater extent than that of the carboxykinase without a great effect on cellular chlorophyll content. In the presence of 25 m DCMU, however, chlorophyll accumulation is reduced without any detectable changes in enzyme activities in the Z strain. The ZR strain displayed its characteristic resistance to DCMU.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose bisphosphate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea To whom all correspondence and reprint request should be addressed     

Herbicide-induced changes in charge recombination and redox potential of Q(A) in the T4 mutant of Blastochloris viridis

To gain new insights into the function of photosystem II (PSII) herbicides DCMU (a urea herbicide) and bromoxynil (a phenolic herbicide), we have studied their effects in a better understood system, the bacterial photosynthetic reaction center of the terbutryn-resistant mutant T4 of Blastochloris (Bl.) viridis. This mutant is uniquely sensitive to these herbicides. We have used redox potentiometry and time-resolved absorption spectroscopy in the nanosecond and microsecond time scale. At room temperature the P(+)(*)Q(A)(-)(*) charge recombination in the presence of bromoxynil was faster than in the presence of DCMU. Two phases of P(+)(*)Q(A)(-)(*) recombination were observed. In accordance with the literature, the two phases were attributed to two different populations of reaction centers. Although the herbicides did induce small differences in the activation barriers of the charge recombination reactions, these did not explain the large herbicide-induced differences in the kinetics at ambient temperature. Instead, these were attributed to a change in the relative amplitude of the phases, with the fast:slow ratio being approximately 3:1 with bromoxynil and approximately 1:2 with DCMU at 300 K. Redox titrations of Q(A) were performed with and without herbicides at pH 6.5. The E(m) was shifted by approximately -75 mV by bromoxynil and by approximately +55 mV by DCMU. As the titrations were done over a time range that is assumed to be much longer than that for the transition between the two different populations, the potentials measured are considered to be a weighted average of two potentials for Q(A). The influence of the herbicides can thus be considered to be on the equilibrium of the two reaction center forms. This may also be the case in photosystem II.     

Titration of Photophosphorylation in Spinach Chloroplasts with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)

The kinetics of the inhibition of photophosphorylation in chloroplasts from spinach (Spinacia oleracea) was investigated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in small concentration intervals, starting at 10^-7M. Plots of the reciprocal of photophosphorylation against concentration of DCMU gave essentially the same straight line with 2 mM nicotinamide adenine dinucleotide phosphate (NADP) together with saturating amounts of ferredoxin or with 4 mM K₃Fe(CN)₆ as the final acceptors for electrons. Practically complete inhibition was obtained at 3 x 10^-6M DCMU. With 0.1 mM flavin mononucleotide (FMN) and ferredoxin, the inhibition between 10^-7M and 10^-6M DCMU was a little slower than in the other two cases. At 10^-6M DCMU a break occurred to a new straight line in the plots, indicating that another reaction was inhibited. Total photophosphorylation without DCMU was about 77 μmol ATP per mg chlorophyll and hour. At the breaking point 20% remained, and inhibition was not complete even at 8 x 10^-6M DCMU. The inhibitor constant for the high-DCMU reaction was in the order of 2 x 10^-5M; for the low-DCMU reaction some complication made the “constant” appear negative. With phenazine methosulfate (PMS) added, DCMU was without effect on photophosphorylation. – As earlier shown by us, titration curves for intact cells of the microalga Scenedesmus show the break at 10^-6M DCMU; and above 6 x 10^-6M photophosphorylation in the algae is not further decreased by DCMU. The data are compared and their possible significance is discussed.     

Photoassimilation of Glycolate, Glycine and Serine by Euglena gracilis

SYNOPSIS. Glycolate was readily utilized for growth by Euglena gracilis , strain Z, in the light at pH 3.8 under a variety of atmospheric conditions, including CO₂-free air and nitrogen. Glycolate did not support growth in the dark as sole carbon source; no significant uptake of glycolate was observed under these conditions. However, cells grown in the light with glycolate as sole carbon source were still capable of glycolate uptake for up to 3 hr after transfer to darkness, and glycolate was taken up by cells utilizing glucose in the dark. The energy requirement for glycolate utilization could thus be met either by light, or by the aerobic metabolism of glucose in the dark. DCMU, an inhibitor of photosystem II, inhibited photoassimilation of glycolate. In the light, but again not in the dark, glycine and serine also served as sole source of carbon under CO₂-free air, but not under nitrogen. Net release of ammonia to the medium accompanied the photoassimilation of glycine and serine. Of the several metabolicallyrelated compounds tested, only glycolate was utilized as sole carbon source in the light under "anaerobic" conditions. A lag in net chlorophyll synthesis occurred during the photoassimilation of glycolate glycine or serine. Determination of rates of photosynthetic ¹⁴CO₂ fixation confirmed that some inhibition of photosynthetic capacity had occurred in response to utilization of glycolate and related compounds.