Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. III. Light-Induced Absorbance Changes in Chloroplast Fragments of the Wild Type and Mutant Strains

Light-induced absorbance changes were investigated in chloroplast fragments of wild type Chlamydomonas reinhardi and 5 different mutant strains having impaired photosynthesis. Two absorbance changes were detected, 1 having a maximum at 553 nm and the other at 559 nm. The component exhibiting the 553 nm change is a cytochrome similar to cytochrome f from higher plant chloroplasts. The component exhibiting the 559 nm change has the properties of a cytochrome similar to cytochrome b(3). Two of the mutant strains (ac-115 and ac-141) were found to lack the 559 cytochrome and light induced only the oxidation of the 553 cytochrome. A third mutant strain (ac-206), previously shown to lack the 553 cytochrome, exhibited only the light-induced reduction of the 559 cytochrome. A fourth mutant strain (ac-208), shown to lack plastocyanin, exhibited absorbance changes attributable to both cytochromes. However, light was capable of inducing the reduction of the 559 cytochrome but not its oxidation. On the other hand, light induced the oxidation of the 553 cytochrome but not its reduction.These observations are discussed in terms of the series formulation for photosynthetic electron transport in which the 559 cytochrome is reduced by system II and transfers electrons via the component affected in ac-21 to the 553 cytochrome. Accordingly, system I sensitizes the oxidation of the 3 components of the electron transport chain.     

The photosynthetic electron transport chain of a mutant strain of Chlamydomonas reinhardi lacking P700 activity

Components and reactions of the photosynthetic electron transport chain were investigated in a mutant strain of the unicellular green alga Chlamydomonas reinhardi which is virtually devoid of the System I reaction center pigment, P700. The plastocyanin and ferredoxin isolated from this mutant strain are both qualitatively and quantitatively indistinguishable from that isolated from the wild-type strain. Cytochromes with absorption maxima at 553 and 559 nm cannot be oxidized by far-red light in the mutant strain, but they are reduced by red light. The Fe(CN)₆³⁻-Hill reaction in the mutant strain is about 50% of that of wild type at high light intensities; however, at low light levels, it is not significantly different from the rate of wild type. These results are interpreted to indicate that P700 is not so closely involved or complexed with adjacent electron carriers or with the reaction center of System II that destruction of P700 necessarily leads to alteration of these other components of the electron transport chain. It is suggested that the Hill reaction data can be explained by the existence of two separate sites for photoreduction of Fe(CN)₆³⁻ in wild type, whereas only one remains operative in the mutant strain.     

The 520 nm absorbance changes in Scenedesmus obliquus and its relation to Photosystem I

The kinetics (region of seconds) of the light-induced 520 nm absorbance change and its dark reversal have been studied in detail in the wild type and in some pigment and photosynthetic mutants of Scenedesmus obliquus. The following 5 lines of evidence led us to conclude that the signal is entirely due to the photosystem I reaction modified by electron flow from Photosystem II.Gradual blocking of the electron transport with 3(3,4-dichlorophenyl)-1,1-dimethylurea resulted in diminution and ultimate elimination of the biphasic nature of the signal without reducing the extent of the absorbance change or of the dark kinetics. On the contrary, blocking electron flow at the oxidizing side of plastoquinone with 2, $\text{5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isoprophyl}\text{-p-}\text{benzoquinone}$ or inactivating the plastocyanin with KCN, prolonged the dark reversal of the absorbance change apart from abolishing the biphasic nature of the signal.Action spectra clearly indicate that the main signal (I) is due to electron flow in Photosystem I and that its modification (Signal II) is due to the action of Photosystem II.Signal I is pH independent, whereas Signal II demonstrates a strong pH dependence, parallel to the O₂-evolving capacity of the cells.Chloroplast particles isolated from the wild type Scenedesmus cells demonstrated in the absence of any added artificial electron donor or acceptor and also under non-phosphorylation conditions the 520 nm absorbance change with approximately the same magnitude as whole cells. The dark kinetics of the particles were comparatively slower. Removal of plastocyanin and other electron carriers by washing with Triton X-100 slowed down the kinetics of the dark reversal reaction to a greater extent. A similar positive absorbance change at 520 nm and slow dark reversal was also observed in the Photosystem I particles prepared by the Triton method.Mutant C-6E, which contains neither carotenoids nor chlorophyll $\text{b}$ and lacks Photosystem II activity, demonstrates a normal signal I of the 520 nm absorbance change. This latter result contradicts the postulate that carotenoids are the possible cause of the 520 nm absorbance change.     

Site-directed mutagenesis of the CPa-1 protein of photosystem II: alteration of the basic residue pair 384,385R to 384,385G leads to a defect associated with the oxygen-evolving complex.

The psbB gene encodes the intrinsic chlorophyll-a binding protein CPa-1 (CP-47), a component of photosystem II in higher plants, algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into a segment of the psbB gene encoding the large extrinsic loop region of CPa-1 in the cyanobacterium Synechocystis sp. PCC 6803. Altered psbB genes were introduced into a mutant recipient strain (DEL-1) of Synechocystis in which the genomic psbB gene had been partially deleted. Initial target sites for mutagenesis were absolutely conserved basic residue pairs occurring within the large extrinsic loop. One mutation, RR384385GG, produced a strain with impaired photosystem II activity. This strain exhibited growth characteristics comparable to controls. However, at saturating light intensities this mutant strain evolved oxygen at only 50% of the rate of the control strains. Quantum yield measurements at low light intensities indicated that the mutant had 30% fewer fully functional photosystem II centers than do control strains of Synechocystis. Immunological analysis of a number of photosystem II protein components indicated that the mutant accumulates normal quantities of photosystem II proteins and that the ratio of photosystem II to photosystem I proteins is comparable to that found in control strains. Upon exposure to high light intensities the mutant cells exhibited a markedly increased susceptibility to photoinactivation. However, Tris-treated thylakoid membranes from both the mutant and wild-type exhibited comparable rates of photoinactivation. Thylakoid membranes isolated from RR384385GG exhibited only 15% of the H2O to 2,6-dichlorophenolindophenol electron transport rate observed in wild-type strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photosynthetic generation of O2 and H2 by photosystem I-deficient chlamydomonas mutants

A comparative study of aerobic generation of O2 and anaerobic photoproduction of H2 in whole cells of a wild-type strain of Chlamydomonas reinhardtii and its photosystem I-deficient mutants B4 and F8 found no contribution of photosystem II to ferredoxin photoreduction, which is not consistent with data of recent studies by Greenbaum et al. (Nature, 1995, 376, 438-441; and Science, 1996, 273, 364-367) who reported that they had discovered such a capacity in these mutant strains. In the wild-type and mutant strains, action spectra showed that O2 was evolved by photosystem II, whereas photoinhibition of chlororespiration and evolution of H2 depended on the activity of photosystem I. Single-turnover flash measurements of H2 evolution showed that the contents of photosystem I in mutant strains amounted to 3-35% of that in the wild-type strain. This fraction of photosystem I in "leaky" mutants displayed abnormal kinetic features and was highly sensitive to photoinhibition.     

A nuclear mutant of Chlamydomonas reinhardtii defective in photosynthetic photophosphorylation. Characterization of the algal coupling factor ATPase

Use of the Flagyl selection procedure [Schmidt et al. (1977) Proc. Natl Acad. Sci. USA, 74, 610-614] led to the isolation of a nuclear mutant of Chlamydomonas reinhardtii designated thm-24. This mutant displays normal electron transport rates in vitro, possesses high latent ATPase activity bound to the thylakoid membrane, but is incapable of photophosphorylation. Decay of the transmembrane potential, as indicated by the kinetics of the 520-nm absorption change after illumination, is unusually slow and markedly biphasic. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of purified thylakoid membranes shows mutant thm-24 to be lacking a number of polypeptides including those previously designated 4.1, 4.2 and 8.1. Treatment of purified thylakoid membranes of wild-type and mutant algae, using the chloroform-release procedure of Beechey et al. [(1975) Biochem. J. 148, 533-537] resulted in the removal of ATPase activity from each strain. In wild-type cells, the ATPase activity was of heterogeneous enzymatic origin; fractionation of the chloroform-release extracts by non-denaturing polyacrylamide gel electrophoresis yielded three distinct bands displaying ATPase activity, designated ATPases I, II and III. In contrast, extracts from membranes of mutant thm-24 yielded only one ATPase-containing fraction, co-migrating with ATPase I from wild-type. Use of electrophoretic, immunological and enzymatic methods established a correspondence of the polypeptide subunits of ATPases II and III and those of spinach coupling factor, CF1. ATPase I from either algal strain was shown to be structurally distinct from high plant CF1 and to C. reinhardtii ATPases II and III.     

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

A chloroplast membrane lacking photosystem II. Thylakoid stacking in the absence of the photosystem II particle.

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

Water Flow in Wheat Seedlings after Small Water Deficits

Cultures of Scenedesmus obliquus when grown heterotrophically for 10 or 30 days without addition of fresh medium showed 85 and 98% loss of their photosynthetic capacity respectively. This loss in photosynthetic capacity was accompanied by an increase in quantum requirement. No major changes in the pigment amounts or types were detected which would explain the decay in photosynthetic capacity. Partial reactions mediated by photosystem II or I showed a more or less constant decay over a period of 30 days. Photosystem II reactions appeared less stable than those of photosystem I, decaying by 95% as compared with 70%, over this time period. The results of comparative studies on aged cells for their potential of cytochrome f photooxidation, fluorescence kinetics, 520 nm absorbance change and the variable influence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone on the photosynthetic capacity of such cells, suggest that it is the inherent ability of the cells to photooxidize plastohydroquinone which is affected primarily. In addition, secondary changes were noted in the activity of reactions on the water-splitting side of photosystem II and in the P700 — plastocyanin — cytochrome f complex.     

Acclimation of the Photosynthetic Apparatus to Growth Irradiance in a Mutant Strain of Synechococcus Lacking Iron Superoxide Dismutase

The acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus sp. PCC 7942 lacking detectable iron superoxide dismutase activity was studied. The growth of the mutant was inhibited at concentrations of methyl viologen 4 orders of magnitude smaller than those required to inhibit the growth of the wild-type strain. An increased sensitivity of photosynthetic electron transport near photosystem I (PSI) toward photooxidative stress was also observed in the mutant strain. In the absence of methyl viologen, the mutant exhibited similar growth rates compared with those of the wild type, even at high growth irradiance (350 [mu]E m-2 s-1) where chronic inhibition of photosystem II (PSII) was observed in both strains. Under high growth irradiance, the ratios of PSII to PSI and of [alpha]-phycocyanin to chlorophyll a were less than one-third of the values for the wild type. In both strains, cellular contents of chlorophyll a, [alpha]-phycocyanin, and [beta]-carotene, as well as the length of the phycobilisome rods, declined with increasing growth irradiance. Only the cellular content of the carotenoid zeaxanthin seemed to be independent of growth irradiance. These results suggest an altered acclimation to growth irradiance in the sodB mutant in which the stoichiometry between PSI and PSII is adjusted to compensate for the loss of PSI efficiency occurring under high growth irradiance. Similar shortening of the phycobilisome rods in the sodB mutant and wild-type strain suggest that phycobilisome rod length is regulated independently of photosystem stoichiometry.     

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.     

The development of structure and function in chloroplasts of greening mutants of Scenedesmus II. Development of the photosynthetic apparatus

Mutant C-2A' of Scenedesmus obliquus formed only traces of chlorophylland showed no detectable photosynthesis when grown heterotrophically.When transferred to light this mutant developed chlorophylland its photosynthetic capacity was established. Following ashort initial lag phase, both photosynthetic capacity and totallight absorption of the cells reached saturation more rapidlythan did the rate of chlorophyll synthesis. Consequently, thequantum requirement of photosynthesis showed a rapid declineduring the initial 6 hr of greening, to a best value of 8. Subsequently,a slow increase occurred as additional chlorophyll was synthesized.Behavior parallel to that of the quantum requirement was alsonoted for the relative fluorescence yield and for the onsetof the 520 nm light-induced absorbance change. Of the two photosystems,PS-I seemed to develop more rapidly than did PS-II. The appearanceof PS-II activity appeared to accompany linkage of the two photosystems,as revealed by analysis of the variable-yield fluorescence andthe kinetics of the 520 nm light-induced absorbance change.During the phase of greening at which photosynthetic capacitydeveloped its maximum quantum efficiency, no significant changesin type or content of the various chloroplast cytochromes weredetected. Analysis of the ratio of chlorophyll/plastoquinone,however, showed that changes in this value followed more closelythe observed increase in the quantum efficiency of photosynthesisand the other parameters of photosynthesis examined. (Received May 19, 1972; )     

Observations on Photosystem II mutants of Scenedesmus: Pigments and proteinaceous components of the chloroplasts

Nine mutants of the green alga, Scenedesmus obliquus, which are blocked in the Photosystem II portion of photosynthesis were analyzed for possible deletion or alteration of (1) various components of the photosynthetic electron transport system, (2) of chloroplast lipids, (3) of total chlorophyll or of the chlorophyll a/chlorophyllb ratio, and (4) of their content of carotenes and carotenoids. No changes in content or activity of ferredoxin, ferredoxin-NADP⁺ reductase, plastocyanin, cytochrome c-552, and the membrane-bound b-type or c-type cytochromes were observed. The most consistent differences noted between the mutant strains and the wild-type strain were in the molar ratio of chlorophyll/plastoquinone A, the total chlorophyll content, and a decreased content of - and β-carotene with a concomitant increase of carotenoids. The loss of Photosystem II activity in these mutant strains, as observed either with whole cells or with isolated chloroplast fragments, may be accounted for by their decreased content of plastoquinone A. Their decreased chlorophyll content and altered carotene/xanthophyll ratio also suggests possible alteration of chloroplast membrances resulting in increased internal oxidation of the photosynthetic pigments.     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. VII. Photosynthetic Phosphorylation by a Mutant Strain of Chlamydomonas reinhardi Deficient in Active P700

Electron transport activity and absorbance changes associated with P700 were investigated in a mutant strain of Chlamydomonas reinhardi with impaired photosynthesis. This mutant strain, ac-8oa, cannot reduce NADP with electrons from either water or dye and ascorbate, but it has considerable Hill activity. The mutant strain shows none of the absorbance changes characteristic of P700. Although unable to carry out cyclic photosynthetic phosphorylation, ac-8oa is able to synthesize ATP when ferricyanide is provided as an electron acceptor.

These observations lead to the conclusion that a site for the coupling of photosynthetic phosphorylation with electron transport must exist between the 2 photochemical systems.

     

E. coli RNAase P has a required RNA component

RNAase P has been partially purified from three thermosensitive strains of E. coli and the thermal inactivation characteristics of each preparation have been determined. The RNAase P preparations from two of these mutant strains, ts241 and ts709, and the wild-type strain have been separated into RNA and protein components. Various mixtures of the reconstituted components have been checked in vitro for complementation of their thermal sensitivity properties. The protein component of RNAase P from ts241 and the RNA component of RNAase P from ts709, respectively, account for the thermal sensitivity of the rnaase P from the two strains. The amount of the RNA component of RNAase P is lower in ts709 than in ts241 or the wild-type parent, 4273. RNAase P partially purified from a revertant of the third mutant strain, A49, which maps at or near the ts241 mutation, has an altered charge when compared to the RNAase P from the parent strain, BF265. We conclude that mutations which affect either the protein or RNA component of RNAase P can confer thermal sensitivity on the enzyme both in vivo and in vitro.     

The phosphorylation status of the chloroplast protein kinase STN7 of Arabidopsis affects its turnover

The chloroplast serine-threonine protein kinase STN7 of Arabidopsis (Arabidopsis thaliana) is required for the phosphorylation of the light-harvesting system of photosystem II and for state transitions, a process that allows the photosynthetic machinery to balance the light excitation energy between photosystem II and photosystem I and thereby to optimize the photosynthetic yield. Because the STN7 protein kinase of Arabidopsis is known to be phosphorylated at four serine-threonine residues, we have changed these residues by site-directed mutagenesis to alanine (STN7-4A) or aspartic acid (STN7-4D) to assess the role of these phosphorylation events. The corresponding mutants were still able to phosphorylate the light-harvesting system of photosystem II and to perform state transitions. Moreover, we noticed a marked decrease in the level of the STN7 kinase in the wild-type strain under prolonged state 1 conditions that no longer occurs in the STN7-4D mutant. The results suggest a possible role of phosphorylation of the STN7 kinase in regulating its turnover.     

The respiratory chain complex thresholds in mitochondria of a Drosophila subobscura mutant strain

Analysis of a mutant strain of Drosophila subobscura revealed that most (80%) mitochondrial genomes have undergone a large scale deletion (5 kb) in the coding region. Compared with the wild-type strain, complex I and III activities are, respectively, reduced by 50% and 30% in the mutant. However, the ATP synthesis capacities remain unchanged. In order to elucidate how the ATP synthesis is maintained at a normal level, despite a significant decrease in complex I and III activities, we progressively inhibited respiratory chain complex activities, respiration rate and ATP synthesis. Complex I, III and IV activities were inhibited by rotenone, antimycin and KCN, respectively. Threshold curves were thus determined for each complex. Our results demonstrated that in the mutant strain, both mitochondrial respiration and ATP synthesis had decreased when complex I activity was inhibited by more than 20%, whereas 70% inhibition is required to induce similar changes in the wild-type. The complex I inhibition pattern of the wild-type was restored by a backcross (mutant female/wild-type male). The complex III activity threshold is below 20% in both strains, and we observed some difference in antimycin sensitivity, suggesting a modification of the complex enzymatic properties in the mutant. In contrast, threshold values of 70% were measured for complex IV inhibition. Our data suggest that the difference in the complex I threshold curves between the wild-type and mutant strains could partially account for the absence of pathological phenotype in the mutant.     

An Investigation of Electron Spin Resonance in Wild Type Chlamydomonas reinhardi and Mutant Strains Having Impaired Photosynthesis

The electron spin resonance signals of wild type Chlamydomonas reinhardi and three mutant strains having impaired photosynthesis have been investigated. The wild type strain generates two different electron spin resonance signals. Signal I is obtained without illumination (i.e., dark signal) whereas signal II is generated preferentially only by red light. Signal I is missing from wild type cells that have been cultured in the dark, but it returns after these dark-grown cells have been illuminated. Chloroplast fragments obtained from the three mutant strains cannot photoreduce TPN. Two of the strains lack the dark signal I while the third strain has both signal I and signal II. Other studies have revealed that the two mutant strains which lack signal I give no Hill reaction but that they can photoreduce TPN if supplied with an artificial reductant. The mutant strain which has both electron spin resonance signals can carry out the Hill reaction, yet it too will not photoreduce TPN unless reductant is supplied. The electron spin resonance signals generated by the wild type and mutant strains are discussed in terms of the pathway of TPN photoreduction, and it is suggested that signal I is associated with one of the two light-dependent phases of this pathway.