Photosynthetic Properties of ac-31, a Mutant Strain of Chlamydomonas reinhardi Devoid of Chloroplast Membrane Stacking

A pale-green mutant strain of Chlamydomonas reinhardi, ac-31, is characterized by the absence of any stacking of its chloroplast membranes. The capacity for photosynthetic electron transport, phosphorylation, and CO₂ fixation in ac-31 is substantial, and it is concluded that these photosynthetic activities occur within the single membrane. The photosynthetic capacities of wild type and ac-31 as a function of increasing light intensity are compared. Saturation is attained at higher light intensities in ac-31, and the kinetics of the 2 sets of curves are distinctly different. The possibility that energy transfer is enhanced by membrane stacking is suggested by these results. The repeatedly-observed correlation between reduced stacking and disfunctional Photosystem II activities is discussed in view of the observation that ac-31 has no stacking but retains a functional Photosystem II.     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi: VIII. The 520 nm Light-induced Absorbance Change in the Wild-type and Mutant Strains 1

The 520 nm light-induced absorbance change in wild-type and 4 mutant strains of Chlamydomonas reinhardi was investigated. In the wild-type strain the absorbance change is composed of at least 2 components, P520 I and P520 II, sensitized by Systems I and II respectively. Some of the properties of these components can be studied by using the appropriate photosynthetic mutant strain. A group of mutant strains modified in the photochemical complex of System II shows only the P520 I absorbance change, whereas a mutant strain deficient in active P700 exhibits only the P520 II absorbance change. The possible relationship between these absorbance changes and the photosynthetic electron transport pathway is discussed.     

Characterization of a Photosynthetic Mutant Strain of Chlamydomonas reinhardi Deficient in Phosphoribulokinase Activity

A mutant strain of the unicellular green alga, Chlamydomonas reinhardi, is unable to fix carbon dioxide by photosynthesis because it is deficient in phosphoribulokinase activity. The absence of light-dependent carbon dioxide fixation in cells of the mutant strain supports the operation of the Calvin-Benson scheme of photosynthetic carbon dioxide fixation in this organism. No deficiency other than low phosphoribulokinase activity was found which would account for the inability of cells of the mutant strain to fix carbon dioxide by photosynthesis. Activities comparable to those in the wild-type strain were found for eight other enzymes of the Calvin cycle and two enzymes associated with the C₄ dicarboxylic acid pathway. The normal rates of nicotinamide adenine dinucleotide phosphate photoreduction and of photosynthetic phosphorylation observed in chloroplast fragments prepared from cells of the mutant strain indicated that the photosynthetic electron transport chain in the mutant is intact.     

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.     

Fluorescence Properties of Wild-Type Chlamydomonas reinhardi and Three Mutant Strains Having Impaired Photosynthesis

The wild-type strain of Chlamydomonas reinhardi and 3 mutant strains ac-21, ac-141, and ac-115 have been compared for their fluorescence (and luminescence) properties. The different fluorescence levels, the rapid and slow photochemical responses affecting fluorescence, and the intensity of luminescence have been studied under various conditions: air, nitrogen, 3(p-chlorophenyl)-1,1-dimethylurea. The strain ac-21 exhibits fluorescence properties only quantitatively different from those of the wild-type strain, and it is believed to be affected in some component of the electron transport chain between the 2 light reactions. Both ac-141 and ac-115 have an abnormally high initial fluorescence level; ac-115 does not show the normal photochemical response associated with System II and has a very low luminescence. Mutant strains ac-141 and ac-115 both seem to be modified in the System II photochemical center. These conclusions are compared with a previous analysis based on absorbance changes of cytochrome 559.     

A uniparentally inherited mutation affecting photophosphorylation in Chlamydomonas reinhardi.

A photosynthetically incompetent mutant strain of $\text{Chlamydomonas}\text{reinhardi}$ shows a uniparental mode of inheritance, a substantial level of photosynthetic electron transport activities, in whole cells and in a cell free system, but a negligible level of photosynthetic phosphorylation activity in a cell free system.     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi VI. Electron Transport in Mutant Strains Lacking Either Cytochrome 553 or Plastocyanin

A mutant strain of Chlamydomonas reinhardi, ac-206, lacks cytochrome 553, at least in an active and detectable form. Chloroplast fragments of this mutant strain are inactive in the photoreduction of NADP when the source of electrons is water, but they are active when the electron source is 2,6-dichlorophenolindophenol and ascorbate. The addition of either cytochrome 553 or plastocyanin, obtained from the wild-type strain, has no effect upon the photosynthetic activities of the mutant strain. Cells of the mutant strain lack both the soluble and insoluble forms of cytochrome 553, but they possess the mitochondrial type cytochrome c. Thus, the loss of cytochrome 553 appears to be specific.     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. IV. Purification and Properties of Plastocyanin

The copper protein plastocyanin has been found to be an essential component of the photosynthetic electron transport chain of Chlamydomonas reinhardi, and in this paper we describe a method for its isolation and purification from the wild-type strain. In addition, we describe some of its properties and compare them with those reported for spinach plastocyanin.     

An Electron Spin Resonance Study of Manganese in Wild-Type and Mutant Strains of Chlamydomonas reinhardi

Changes in the intensity of the electron spin resonance signal of divalent manganese were found to occur in suspensions of wild-type Chlamydomonas reinhardi. The observed manganese signal decreased in the light and increased in the dark. Through the use of a continuous-flow system it was possible to determine that the manganous ions responsible for the observed signal were localized solely in the medium. Changes in the signal intensity associated with wild-type cells were independent of the ability of fragments prepared from these cells to perform the Hill reaction with 2,6-dichlorophenol-indophenol (DPIP) as the oxidant.

The manganese signal changes were still evident, though smaller, in cell suspensions of wild-type cells treated with 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, and in mutant strains unable to carry out the Hill reaction, ac-115 and ac-141.

From these data it is concluded that the changes in intensity of the manganese resonance are not related to the function of manganese in photosynthesis but may reflect the capacity of cells for ion uptake in the light.

     

Chloroplast Ultrastructure in Mutant Strains of Chlamydomonas reinhardi Lacking Components of the Photosynthetic Apparatus

The fine structure of the chloroplast of wild-type and 9 photosynthetic mutant strains of Chlamydomonas reinhardi is described. The chloroplast phenotypes of the mutant strains are clearly distinct from the wild type in all but 2 cases. Moreover, strains with similar photosynthetic disabilities have structurally similar chloroplasts. These differences are apparently not the result of altered chlorophyll content, nor of photosynthetic inactivity. It is therefore proposed that the structural alterations are in some way related to the mutant strains' inability to synthesize active components of the photosynthetic electron transport chain.     

Energy and Electron Transfer Systems of Chlamydomonas reinhardi. I. Photosynthetic and Respiratory Cytochrome Systems of the Pale Green Mutant

The role of cytochromes in photosynthetic electron transfer system has been studied using the pale green mutant of Chlamydomonas reinhardi (ATCC 18302). The existence of cytochromes b₅₆₃ and f is confirmed, while no significant amount of ascorbate-reducible cytochrome b₅₅₉ is detected in this mutant. The presence of cytochrome c and a small amount of a-type cytochrome is determined in these cells.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : II. Photosynthetic Electron Transport

Photosynthetic electron transport is markedly affected in mixotrophic cells of ac-20 because they lack the capacity to form the wild-type level of cytochrome 559, as well as Q, the quencher of fluorescence of photochemical system II. The other components of the electron-transport chain, as well as reactions dependent upon photochemical system I, are unaffected in the mutant strain. These observations are discussed in terms of the previously reported effects of the ac-20 mutation on CO₂ fixation and ribulose-1,5-diphosphate carboxylase activity.     

The Kok Effect in Chlamydomonas reinhardi

A Haxo-Blinks rate-measuring oxygen electrode together with a modulated light source gave an average current signal (change in net O₂ exchange) and a modulated current signal (photosynthetic O₂ evolution). Using this apparatus, net O₂ exchange and photosynthetic O₂ evolution at low intensities have been studied in the green alga, Chlamydomonas reinhardi. At both 645 nm and 695 nm, the curves of net O₂ 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 O₂ evolution, on the other hand, showed no Kok effect; here, the slope was lowest at lowest intensity. The absence of the Kok effect in O₂ 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)-1,1-dimethylurea. In the presence of concentrations of this inhibitor sufficient to inhibit O₂ evolution completely, a light-induced change in net O₂ exchange remained. This was interpreted as a system I dependent depression of respiratory O₂ 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 O₂ uptake is depressed by diversion of reductant away from respiratory electron transport and into photosystem I. The same photodepression of O₂ uptake also appears to account for a transient in net O₂ exchange seen in several algae upon turning off the light.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : II. Plastid Differentiation during Greening of a Dark-Grown Algal Mutant (Chlamydomonas reinhardi)

Dark-grown cells of the y-1 mutant of Chlamydomonas reinhardi contain a partially differentiated plastid lacking the photosynthetic lamellar system. When exposed to the light, a rapid synthesis of photosynthetic membranes occurs accompanied by synthesis of chlorophyll, lipids, and protein and extensive degradation of the starch reserve. The process is continuously dependent on illumination and is completed within 6–8 hr in the absence of cell division. Photosynthetic activity (O₂ evolution, Hill reaction, NADP photo-reduction, and cytochrome f photooxidation) parallels the synthesis of pigment and membrane formation. During the greening process, only slight changes occur in the levels of soluble enzymes associated with the photosynthetic process (RuDP-carboxylase, NADP-linked G-3-P dehydrogenase, alkaline FDPase (pH 8)) as compared with the dark control. Also cytochrome f concentration remains almost constant during the greening process. The kinetics of the synthesis of chlorophyll, formation of photosynthetic membranes, and the restoration of photosynthetic activity suggest that the membranes are assembled from their constituents in a single-step process.     

Morphological and Photosynthetic Properties of Digitonin-treated Chloroplast Membranes from the Wild-type and ac-5 Strains of Chlamydomonas reinhardi

Chloroplast membranes of wild-type Chlamydomonas reinhardi, treated with digitonin, yield photosystem II-rich and photosystem I-rich fractions; this fractionation is accompanied by a separation of stacked (grana) lamella from unstacked (stroma) lamellae. Poor fractionation of the photosystems occurs when the treated chloroplast membranes derive from the ac-5 strain grown mixotrophically, whereas good fractionation occurs with ac-5 cells grown phototrophically; the mixotrophic cells possess only unstacked membranes, whereas the phototrophic cells possess stacked membranes. We concluded that digitonin fractionation is dependent on the stacked membrane configuration.     

Photochemical activities of two photosystems in Bratonia orchid protocorms cryopreserved by vitrification method

Functional activities of two photosystems in orchid-specific embryos (protocorms) of a tropical hybrid orchid Bratonia were investigated before and after their cryopreservation by vitrification method. The kinetics of light-induced absorbance changes at 830 nm was analyzed as indicator of P700 redox conversions; changes in the variable chlorophyll fluorescence served to indicate the oxidation-reduction changes of the primary acceptor Q_A. Untreated protocorms exhibited low photochemical activity of photosystem II (PSII). In freeze-treated Bratonia protocorms, examined immediately after thawing, photosynthetic electron transport was strongly inhibited. Nevertheless, the cells retained activities of noncyclic electron flow and of alternative electron transport pathways related solely to PSI. However, Bratonia protocorms subjected to deep-freezing lost the capability of P700 photooxidation during the first day of reculturing. Deep freezing of protocorms had virtually no effect on the kinetics of dark relaxation of chlorophyll variable fluorescence, when measurements were made immediately after thawing. Unlike chlorophyll fluorescence, the kinetics of dark reduction of P700⁺ in protocorms exposed to freezing-thawing was substantially modified compared to untreated protocorms. Two exponential components with half-decay times of 27 and 310 ms were distinguished in the kinetics of P700⁺ reduction in treated samples, whereas the absorbance relaxation attributed to P700⁺ reduction in untreated samples followed an exponential decay with a half-decay time of 24 ms. Despite the appearance of additional slow component in the kinetics of P700⁺ reduction, the dark relaxation of variable fluorescence remained unaltered after deep freezing of protocorms. This observation indicates that the freezing-thawing procedure caused partial disorders in linear electron transport between PSII and PSI. Apparently, the functional interactions among carriers in the electron-transport chain were disturbed between the plastoquinone pool and the PSI reaction center. It is concluded that the vitrification method applied to protocorm cryopreservation did not cause their immediate death, but the protocorms died later, on the first day after reculturing.     

Polypeptide Composition of Photosynthetic Membranes from Chlamydomonas reinhardi and Anabaena variabilis

Anabaena variabilis, a blue-green alga lacking chlorophyll b, shows an absence of the major 22 and 24 kilodalton polypeptides which are present in the photosynthetic membranes of Chlamydomonas reinhardi and higher plants. These data are consistent with other investigations which have shown that these polypeptides are associated with chlorophyll b in the chloroplasts of higher plants, and indicate the presence of a light harvesting chlorophyll-protein complex in higher plants which contains the chlorophyll b of the photosynthetic membrane.     

Photosynthetic electron transport and specific photoprotective responses in wheat leaves under drought stress

The photosynthetic responses of wheat (Triticum aestivum L.) leaves to different levels of drought stress were analyzed in potted plants cultivated in growth chamber under moderate light. Low-to-medium drought stress was induced by limiting irrigation, maintaining 20 % of soil water holding capacity for 14 days followed by 3 days without water supply to induce severe stress. Measurements of CO₂ exchange and photosystem II (PSII) yield (by chlorophyll fluorescence) were followed by simultaneous measurements of yield of PSI (by P700 absorbance changes) and that of PSII. Drought stress gradually decreased PSII electron transport, but the capacity for nonphotochemical quenching increased more slowly until there was a large decrease in leaf relative water content (where the photosynthetic rate had decreased by half or more). We identified a substantial part of PSII electron transport, which was not used by carbon assimilation or by photorespiration, which clearly indicates activities of alternative electron sinks. Decreasing the fraction of light absorbed by PSII and increasing the fraction absorbed by PSI with increasing drought stress (rather than assuming equal absorption by the two photosystems) support a proposed function of PSI cyclic electron flow to generate a proton-motive force to activate nonphotochemical dissipation of energy, and it is consistent with the observed accumulation of oxidized P700 which causes a decrease in PSI electron acceptors. Our results support the roles of alternative electron sinks (either from PSII or PSI) and cyclic electron flow in photoprotection of PSII and PSI in drought stress conditions. In future studies on plant stress, analyses of the partitioning of absorbed energy between photosystems are needed for interpreting flux through linear electron flow, PSI cyclic electron flow, along with alternative electron sinks.     

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b₆/f complex, plastocyanin, and cytochrome c₆, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700⁺) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700⁺ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700⁺ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

     

Evidence for a Block between Plastoquinone and Cytochrome f in a Photosynthetic Mutant of Lemna with Abnormal Flowering Behavior

Mutant strain 1073 of Lemna perpusilla is concluded to be blocked between plastoquinone and cytochrome f in the photosynthetic electron transport system. The location of the block is based on the following observations of activities in chloroplasts isolated from the mutant and wild-type plants. (a) Relative to wild type, electron flow rates from water to ferricyanide, 2,6-dichlorophenol indophenol or NADP were very low in the mutant, but rates of photosystem I-dependent electron flow and cyclic phosphorylation were high. (b) Chlorophyll a fluorescence induction curves for mutant and wild type were similar. (c) Silicomolybdate and lipophilic acceptors in the mutant were photoreduced at rates comparable to wild type. (d) Cytochrome f of the mutant chloroplasts was not reduced by red light, but was oxidized by red or far red light. (e) Reduction of the primary electron acceptor of photosystem II (Q) by ATP-driven reverse electron flow was not observed in the mutant.