Analysis and Characterization of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU)-resistant Euglena: II. Modifications Affecting Photosynthesis during Adaptation to Different Doses of DCMU

When grown in medium containing dl-lactate at 27 C in the light, Euglena gracilis Z populations underwent modifications of the pigment system in response to 0.05 to 250 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU).Chlorophyll content dropped dramatically, the only remaining form being Chl a(673). Light-driven O(2) evolution was no longer detectable for the two highest DCMU concentrations tested. The energy-capture cross-section of detectable photosystem II units remained unchanged, although intersystem energy transfer no longer occurred. Euglena at this stage had chloroplast membranes destacked and swollen. A recovery phase then occurred, marked by enhanced photosynthetic properties. The initial forms of chlorophyll which were accumulated were highly efficient for O(2) evolution. The newly formed photosystem II antennae were connected and of small size. Finally, the third phase involved the recovery of photosynthetic capacity similar to that of the controls as the thylakoids regained their normal structures.Since these modifications occurred in the entire population and DCMU resistance persisted through successive cell generations, these adapted Euglena were considered to be a variant of the Z strain, designated ZR.     

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.     

Titration with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) as Evidence for Several Photophosphorylation Sites in Scenedesmus

Photophosphorylation was measured in intact cells of Scenedesmus obtusiusculus, which were made phosphate starved before the start of the experiments. Photophosphorylation was titrated with narrow intervals of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) from 10^-7M upwards. Plots of the reciprocal of photophosphorylation against concentration of DCMU gives three essentially straight lines; one between 10^-7 and 10^-6M DCMU; one between 10^-6 and 6 · 10^-6M DCMU; and one for more than 6 · 10^-6M DCMU, the last-mentioned line being parallel to the abscissa. The stoichiometry between the three reactions is roughly 2: 1: 1. At least three sites for photophosphorylation are indicated, and the assumption that all sites work with approximately the same efficiency would make them four.     

Isolation and characterization of a DCMU (3–(3,4–dichlorophenyl)-1,1–dimethylurea) resistant strain of a marine alga, Dunaliella bioculata

A DCMU-resistant strain was isolated from cell cultures of a wall-less alga, Dunaliella bioculata (Butcher 19/4 CCAP). This variant strain is essentially characterized by its ability of grow autotrophically in the presence of 4 μM DCMU and by the stability of this property even after numerous subcultures in the absence of the herbicide.
Chloroplastic structures and photosynthetic activities of whole and fragmented cells of the wild and the variant strains were compared for their respective sensitivities towards DCMU. The DCMU-resistant strain was also insensitive to other photosynthetic inhibitors such as chloroxuron, but not to atrazine. The results suggest that the DCMU-resistance could be due to some modified plastid property.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) Inhibition of System II and Light-Induced Regulatory Changes in Energy Transfer Efficiency

Oxygen pulses produced in Chlorella by a xenon flash of 15 μsec half-width were measured by means of a rapid oxygen polarograph. Under appropriate conditions the height of the pulse caused by a saturating flash was a measure of the number of active reaction centers in system II. In pigment state II, caused by illumination during several minutes with light II, the number of active centers II was the same as in pigment state I. Oxygen pulses produced by about half-saturating flashes were diminished by about 7-10% in state II, showing that the fluorescence decrease in light II was at least partly caused by a decrease in energy transfer to reaction center II. After addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), only the first flash produced oxygen which gives additional support for the hypothesis that DCMU inhibits between Q and system I.     

Photodestruction of Pigments in Higher Plants by Herbicide Action: I. THE EFFECT OF DCMU (DIURON) ON ISOLATED CHLOROPLASTS

3-(3',4'-Dichlorophenyl)-1',1'-dimethyl urea (DCMU) inducedthe photobleaching of chlorophylls and carotenoids in isolatedchloroplasts of Hordeum vulgare. In chloroplasts illuminatedin both the absence and presence of DCMU (5.0 mmol m^–3),the destruction of carotenoid preceded that of the chlorophylls.The rate of photodestruction was accelerated by the presenceof DCMU. After only 2 h illumination the rates of loss of ß-caroteneand of the epoxyxanthophylls, neoxanthin and violaxanthin, weresimilar (approximately 40–50% loss in the presence of5–0 mmol m^–3 DCMU) but weremuch greater than thatof lutein (25% loss). Analysis of the individual pigment-proteincomplexes, isolated from chloroplasts following such treatment,showed that whilst pigment destruction had occurred in all complexes,the relative content of the LHCP2/CPa complexes (containingthe PSII core) had fallen to the greatest extent. Further illuminationof the chloroplasts, for up to 22 h, resulted in far greaterbleaching but showed a similar pattern of pigment loss, withDCMU again accelerating the rate at which this loss occurred.ß-Carotene-5,6-epoxide was identified as a productof such photo-oxidative conditions. Key words: DCMU, carotenoids, chlorophylls, photobleaching, ß-carotene-5,6-expoxide     

Growth dynamics and domoic acid production of Pseudo-nitzschia sp. in response to oil and dispersant exposure

The diatom genus Pseudo-nitzschia is a common component of phytoplankton communities in the Gulf of Mexico and is potentially toxic as some species produce the potent neurotoxin domoic acid. The impact of oil and chemical dispersants on Pseudo-nitzschia spp. and domoic acid production have not yet been studied; preliminary findings from a mesocosm experiment suggest this genus may be particularly resilient. A toxicological study was conducted using a colony of Pseudo-nitzschia sp. isolated from a station off the coast of Louisiana in the Gulf of Mexico. The cultures were exposed to a water accommodated fraction (WAF) of oil and a diluted chemically enhanced WAF (DCEWAF) which was a mix of oil and dispersant (20:1). Exposure to WAF induced a lag phase but did not inhibit growth rates once in exponential growth. Cultures grown in DCEWAF did not experience a lag phase but had significantly lower growth rates than the Control and WAF cultures. The cellular quota of domoic acid was higher in cultures treated with DCEWAF and WAF relative to their control values, and half of the domoic acid had leaked out of the cells into the surrounding seawater in the DCEWAF cultures while all the domoic acid remained inside the cells in WAF-treated cultures. These results suggest that the presence of oil could lead to toxic blooms, but that the application of dispersant could decrease bioaccumulation of domoic acid through the food web.     

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.     

Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP,ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts.During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation.The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts.The fluorescence rise during a second illumination is still biphasic.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts.Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination.Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.     

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.

     

Effects of various buffers, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and NaN3 on menadione mediated photophosphorylation in isolated chloroplasts

The effects of DCMU and NaN₃ were studied on menadione-mediated photophosphorylation in broken spinach chloroplasts kept in low oxygen tension in Tricine or HEPES buffers at either high or reduced irradiances. – (A) At high irradiance (131 W. m^?2) and absence of NaN₃ the ATP formation was inhibited by DCMU regardless of the type of buffer used. – (B) At high irradiance and presence of NaN₃ some concentrations of DCMU stimulated, whilst others inhibited the ATP formation in a HEPES buffer. The ATP formation was predominantly inhibited by DCMU in a Tricine buffer. – (C) At reduced irradiance (57 W. m^?2) the chloroplasts in a HEPES buffer were almost insensitive towards DCMU both in the presence and absence of NaN₃. – (D) Chloroplasts in a Tricine buffer were slightly stimulated in their ATP formation by DCMU at reduced irradiance either with or without the presence of NaN₃ in the experimental medium. When menadione acts as a terminal electron acceptor, oxygen is consumed on its reoxidation. The results indicate that this process may occur with oxygen released by the splitting of water as the main oxidant. – The data also demonstrate the importance of caution when selecting buffering substances as well as when choosing light intensities for experiments on photophosphorylation in chloroplasts.     

The Effect of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) on Photosynthesis, Glycolate Excretion ('Photorespiration') and Amino Acid Metabolism in the Blue-Green Alga Anacystis

When photosynthesis of the blue-green alga Anacystis nidulans was measured as ¹⁴CO₂-fixation, the inhibitory effect of DCMU at low concentrations was greatest when mainly Photosystem 1 (PS 1) (excitation at 446 or 687 nm) was operative. At concentrations above 10-⁶M the inhibition on ¹⁴CO₂-fixation was greatest when mainly Photosystem 2 (PS 2) was operative (excitation at 619). During excitation of PS 1, the excretion of glycolate was stimulated at low concentrations of DCMU (5 × 10^-8M and lower), while higher concentrations inhibited excretion. All concentrations of DCMU inhibited glycolate excretion when mainly PS 2 was excited. The curves showing the relative effect of DCMU on the two photosystems, measured as PS 1/PS 2, had opposite shapes for ¹⁴CO₂-fixation and glycolate excretion. An increase in ¹⁴CO₂-fixation coincided with a decrease in glycolate excretion and vice versa. It appears that the increased rate of photosynthesis when mainly PS 1 was operative relative to that when mainly PS 2 was excited, increases the consumption of glycolate in an oxidation process associated with the excitation of PS 1, resulting in less excretion of glycolate to the medium. The influence of DCMU inhibition on labelled amino acid pools connected to the glycolate pathway (glycine-serine) is quite similar to that for ¹⁴CO₂-fixation. At concentrations below 10^-6M DCMU, inhibition of ¹⁴CO₂- incorporation into the amino acids was greatest when PS 1 was excited, while at the higher concentrations tested, inhibition was greater when PS 2 was excited. We conclude that the metabolism of glycine and serine is closely connected to the rate of photosynthesis.     

DCMU INDUCED INHIBITION OF GROWTH,PHOTOSYNTHESIS AND MOTILITY IN EUDORINA ELEGANS CULTURES

Studies with Eudorina elegans L. were done to provide additional information on the effect of phenyl urea herbicides on phytoplankton. Colonies were grown in various concentrations of DCMU, 3(3,4-dichlorophenyl)-1,1-dimethylurea. DCMU at 10^-5 m induced an algicidic response. DCMU at 10^-7 M and 10^-9 M caused a significant reduction in the growth of colonies. Photosynthesis was significantly inhibited at all concentrations of DCMU. A rapidly growing population of algae treated with 10^-7 m and 10^-9 m DCMU showed a reduced motile/non-motile balance of colonies.     

The cellular control of growth in cultures of Tetrahymena 2 In fluence of Fe medium and aeration on culture growth and cellular exchange of Ca.

Cultures of Tetrahymena pyriformis were grown exponentially with and without the addition of Fe and with no aeration. During the prestationary growth phase of all the cultures, there was a decrease in the cellular Fe concentration in the water insoluble cell fraction (IS) containing membranes and mitochondria, simultaneous with an increase in the Ca concentration in the water soluble cell fraction (S) containing ribosomes. This has been correlated to an energy deficit in the cells at the transition to the prestationary growth phase. In spite of the ability of Fe-deficient cultures to concentrate Fe, cultures grown in media with low Fe levels soon showed the lowest cellular Fe content. The high Fe levels seen in cultures grown with no aeration may reflect cellular adaptation to a different gaseous tension in the medium. Determinations with 45Ca showed an initial, large and rapid increase in cell radioactivity which was not correlated to cellular metabolism. After this there was differentiated increase due to the metabolic status of the cells. The following sequence was seen in all the cultures: (1) an increase in the exchange of S-and (mostly) IS-Ca at the end of the exponential growth phase, (2) an accumulation of Ca in the S fraction without an increased exchange (the IS-Ca is less exchangeable), and (3) a renewed increase in the exchange of Ca when the concentration is further increased at the end of the prestationary growth phase.     

Stimulation of Photosystem I Electron Transport by High Concentration of 3-(3,4-Dichlorophenyl)-1,1-dimethyl Urea in Uncoupled Chloroplasts

The light saturated rate of photosystem I-dependent electron transport (ascorbate/dichlorophenol-indophenol → methyl vilogen in presence of 1 micromolar 3-[3,4-dichlorophenyl]-1,1-dimethyl urea [DCMU]) was increased by a high concentration of DCMU added to broken and uncoupled chloroplasts isolated from pea (Pisum sativum). At 50 micromolar DCMU, the increase was around 50%. No stimulation was observed under limiting intensity of illumination, indicating that the relative quantum yield of electron transport was not affected by high DCMU. The light-saturated rate in coupled (to proton gradient formation) chloroplasts was unchanged by 50 micromolar DCMU, suggesting that the rate-limitation imposed by energy coupling was not affected. Using N,N,N′,N′-tetramethyl-p-phenylene diamine as electron donor, essentially no DCMU stimulation of the rate was observed, indicating further that the electron donation at a site close to P700 was not affected by high DCMU. It is concluded that DCMU, in the range of 10 to 50 micromolar, affected the thylakoid membranes in such a way that the rate constant of electron donation by dichlorophenol-indophenol at the site prior to the site of energy coupling increased. Further observations that DCMU at 100 micromolar stimulated the rate in coupled chloroplasts indicated an additional DCMU action, presumably by uncoupling the chloroplasts from phosphorylation, as suggested by Izawa (Shibata et al., eds, Comprehensive Biochemistry and Biophysics of Photosynthesis, University Press, State College, Pennsylvania, pp 140-147, 1968). A scheme has been proposed for multiple sites of DCMU action on the electron transport system in chloroplasts.     

Non-Mendelian Inheritance of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-Resistant Thylakoid Membrane Properties in Chlamydomonas

A uniparentally inherited 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-resistant mutant of Chlamydomonas reinhardii, Dr2, which has a resistance mechanism of the type defined as `primary,' has been isolated. In vitro Hill reactions catalyzed by isolated thylakoid membranes reveal a reduced apparent affinity of the thylakoids for DCMU. These changes in membrane properties quantitatively account for the resistance of mutant Dr2 to herbicide inhibition of growth. The properties of this mutant show that all of the Hill reaction-inhibiting DCMU binding sites are under identical genetic control. Mutant Dr2 is a useful new uniparental genetic marker, since it has a novel phenotype and it may be possible to identify its altered gene product. The low cross-resistance of Dr2 to atrazine suggests that there may be considerable flexibility in exploiting induced herbicide resistance of crop plants for improving herbicide specificity.     

Cellular control of growth in cultures of Tetrahymena. 3. The influence of Fe-medium and aeration on cellular ultrastructure.

Cultures of Tetrahymena pyriformis were grown with and without the addition of Fe and with no aeration. The same cultures were used both for determinations of the cellular Fe and Ca concentrations and the exchange of Ca (reported earlier), and for the ultrastructural study. In all cultures there was an increase in rounded, tubuli-deficient mitochondria at the transition to the prestationary growth phase. In the non-aerated culture (high cellular Fe content) these changes were less marked. In the Fe-deficient culture, however, these mitochondrial changes were seen as early as the late exponential growth phase, and tubuli-degeneration then increased during the prestationary growth phase. In this culture an irregular infolding of the outer mitochondrial membranes occurred. These changes are discussed in correlation with cytochromes, Fe-dependent desaturation of fatty acids, and high Ca concentration (non-aerated cells). During the prestationary growth phase of the non-aerated culture there was a marked increase in the amount of mitochondrial tubuli. In the organelles identified as peroxisomes there was, in all the cultures, an increased granular density of the matrix at the transition to the prestationary growth phase (in the Fe-deficient cells this occurred in the late exponential growth phase). This was correlated with an increased peroxisomal activity. The Fe-deficient culture has cells with very irregularly formed peroxisomes. This organelle was in all the cell-material very sensitive to the method of fixation. In the Fe-deficient late exponential cells there are long, bifacial pieces of RER (one side rough and the other smooth) which later undergo degradation. Many lipid droplets were seen at the ends of RER. Structures which in the literature have been called 'ergoplasm-like stacks of flattened rough cisternae' were found in the non-aerated exponential cells. They were absent from prestationary cells. In all the cultures there was an increased aggregation of the ribosomes in the cytoplasm during the prestationary growth phase. This was correlated with the accumulation of Ca in this cell fraction. An explanation is suggested regarding the earlier reported variations in the exchange of Ca, found in all the types of cultures at the transition to the prestationary growth phase.     

Effects of calcium channel blockers and DCMU on motility and the photophobic response of Gyrodinium dorsum

The effects of the calcium channel blockers, verapamil, diltiazem and lanthanum ions and the Ca²⁺ dependency on motility as well as the photophobic response (stop-response) of Gyrodinium dorsum were studied. At Ca²⁺ concentrations below 10^-3 M, motility was inhibited. La³⁺ inhibits the stop-response, in contrast to verapamil and diltiazem. The only calcium channel blocker that increased the amount of non-motile cells was verapamil. The results indicate that motility are Ca²⁺ dependent and that the stop-responses of G. dorsum could be affected by extracellular Ca²⁺. Effects of the photosythesis inhibitor (DCMU) on the stop-response was also determined. With background light of different wavelength (614, 658 and 686 nm) the stop-response increased. DCMU inhibited this effect of background light. Negative results with the monoclonal antibody Pea-25 directed to phytochrome and the results with DCMU, indicate that the stop-response of G. dorsum is coupled to photosynthesis rather than to a phytochrome-like pigment. Oxygen evolution, but not cell movement, was completely inhibited by 10^-6 M DCMU.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-methylurea - DILT diltiazem - DMSO dimethylsulfoxide - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - VER verapamil     

Light-Induced Hg Volatilization and O2 Evolution in Chlorella and the Effect of DCMU and Methylamine

The rate of volatilization of Hg²⁺ as metallic Hg is accelerated by illumination of Chlorella cells. In the presence of the uncoupler methylamine the rate of volatilization in the light is greatly but transiently increased. DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl urea) prevented the light response. In the presence of Hg²⁺, O₂ evolution by the cells was not completely inhibited by DCMU. Hg²⁺ appears to prevent DCMU reaching its binding site. Light seems to increase the amount of or leakage from the cells of a metabolite capable of reducing Hg²⁺ to Hg°.     

Inhibition of O2 evolution by NADP in isolated potato tuber chloroplast and its identity with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibition site.

Chloroplast from greening potato tuber showed good photosynthetic capacity. The evolution of O₂ was dependent upon the intensity of light. A light intensity of 30 lux gave maximum O₂ evolution. At higher intensities inhibition was observed. The presence of bicarbonate in the reaction mixture was essential for O₂ evolution. NADP was found to be a potent inhibitor of O₂ evolution in this system. NADP and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibited the O₂ evolution completely at a 3 μm concentration level, which was reversed by oxidized 2,6-dichlorophenol-indophenol (DCIP). Cyanide (CN)-treated chloroplasts showed full O₂ evolution capacity, when a lipophilic electron acceptor like N-tetramethyl-p-phenylenediamine (TMPD) or DCIP was used along with ferricyanide. Ferricyanide alone showed only 20% reduction. NADP or DCMU could inhibit O₂ evolution only when TMPD was the acceptor but not with DCIP. Photosystem II (PS II) isolated from these chloroplasts also showed inhibition by NADP or DCMU and its reversal by DCIP. Here also the evolution of O₂ with only TMPD as acceptor was sensitive to NADP or DCMU. In the presence of added silicotungstate in PS II NADP or DCMU did not affect ferricyanide reduction or oxygen evolution. The chloroplasts were able to bind exogenously added NADP to the extent of 120 nmol/mg chlorophyll. It is concluded that the site of inhibition of NADP is the same as in DCMU, and it is between the DCIP and TMPD acceptor site in the electron transport from the quencher (Q) to plastoquinone (PQ).