Mechanism of KCN inhibition of photosystem I.

Experiments with chloroplasts and purified spinach plastocyanin suggest a mechanism for KCN inhibition of Photosystem I. KCN inhibition can be bypassed by a detergent or reversed by replacement of the inactive plastocyanin. KCN bleaches and inactivates purified plastocyanin. KCN releases copper from chloroplast membranes and from purified plastocyanin. Cyanide does not bind to the apoprotein produced when plastocyanin is treated with KCN, and KCN-produced apoplastocyanin has a N-ethylmaleimide-reactive sulfhydryl group not found in holoplastocyanin. Apoplastocyanin is not active in restoring Photosystem I activity to plastocyanin-depleted membranes. Holoplastocyanin restores Photosystem I activities to plastocyanin-depleted membranes prepared from either control or KCN-treated chloroplasts to about the same extent. KCN-treated chloroplast membranes are found to have higher amounts of apoplastocyanin than do control chloroplast membranes. These results offer evidence that KCN removes the copper from plastocyanin in the chloroplast membrane, leaving the inactive apoplastocyanin which is unable to transfer electrons to Photosystem I.     

A new protein factor functional in the ferredoxin-independent light activation of chloroplast fructose 1,6-bisphosphatase

A protein purified from chloroplasts (the “new protein factor”) activated Fru-P₂ase in a photochemical reaction that depended only on chloroplast membranes. The results suggest that chloroplasts utilize the newly found mechanism for the photoregulation of Fru-P₂ase in addition to the recently described ferredoxin/thioredoxin system.     

Effect of 4-Thiouridine on Photosystems of a Higher Plant

Effect of 4-thiouridine, which was proved to inhibit selectively and “light-reversibly” the synthesis of chloroplast ribosomal RNAs in radish cotyledons, on the photo-induced development of photosystem I, II and a complete electron transport chain was investigated with plastids obtained from 4-thiouridine treated dark-grown radish cotyledons after various times of development in the light. It was demonstrated that the 4-thioridine treated chloroplasts showed a higher activity of photoreduction than the control untreated chloroplasts in every system on a chlorophyll basis during the development after 24 hr illumination. This specific activity decreased in both chloroplasts, as the chloroplasts matured with the time of illumination. The activity per g of fresh cotyledons treated with 4-thiouridine, especially in the early stage of development, was lower than that of ones untreated with the drug because total chlorophyll content was poor, but the activity of the former was enhanced with the increase of total chlorophyll content upon illumination while the activity of the latter decreased on 24 hr illumination. Moreover, Hill reaction measurements showed that 4-thiouridine treated chloroplasts were saturated at lower light intensity than untreated ones inspite of the same content of chlorophyll in both the chloroplasts: photoreduction of NADP⁺ was saturated at 3000 lux for the former and at 5000 lux for the latter. Based upon these results, specific development of the chloroplast is discussed.     

Complex origins of chloroplast membranes with photosynthetic machineries: multiple transfers of genes from divergent organisms at different times or a single endosymbiotic event?

The paradigm “cyanobacterial origin of chloroplasts” is currently viewed as an established fact. However, we may have to re-consider the origin of chloroplast membranes, because membranes are not replicated by their own. It is the genes for lipid biosynthetic enzymes that are inherited. In the current understandings, these enzymes became encoded by the nuclear genome as a result of endosymbiotic gene transfer from the endosymbiont. However, we previously showed that many enzymes involved in the synthesis of chloroplast peptidoglycan and glycolipids did not originate from cyanobacteria. Here I present results of comprehensive phylogenetic analysis of chloroplast enzymes involved in fatty acid and lipid biosynthesis, as well as additional chloroplast components related to photosynthesis and gene expression. Four types of phylogenetic relationship between chloroplast enzymes (encoded by the chloroplast and nuclear genomes) and cyanobacterial counterparts were found: type 1, chloroplast enzymes diverged from inside of cyanobacterial clade; type 2, chloroplast and cyanobacterial enzymes are sister groups; type 3, chloroplast enzymes originated from homologs of bacteria other than cyanobacteria; type 4, chloroplast enzymes diverged from eukaryotic homologs. Estimation of evolutionary distances suggested that the acquisition times of chloroplast enzymes were diverse, indicating that multiple gene transfers accounted for the chloroplast enzymes analyzed. Based on the results, I try to relax the tight logic of the endosymbiotic origin of chloroplasts involving a single endosymbiotic event by proposing alternative hypotheses. The hypothesis of host-directed chloroplast formation proposes that glycolipid synthesis ability had been acquired by the eukaryotic host before the acquisition of chloroplast ribosomes. Chloroplast membrane system could have been provided by the host, whereas cyanobacteria contributed to the genes for the genetic and photosynthesis systems, at various times, either before or after the formation of chloroplast membranes. The origin(s) of chloroplasts seems to be more complicated than the single event of primary endosymbiosis.

     

Effects of cations upon chloroplast membrane subunit interactions and excitation energy distribution

When isolated chloroplasts from mature pea (Pisum sativum) leaves were treated with digitonin under “low salt” conditions, the membranes were extensively solubilized into small subunits (as evidenced by analysis with small pore ultrafilters). From this solubilized preparation, a photochemically inactive chlorophyll · protein complex (chlorophyll $\text{a}\text{b}$ ratio, 1.3) was isolated. We suggest that the detergent-derived membrane fragment from mature membranes is a structural complex within the membrane which contains the light-harvesting chlorophyll $\text{a}\text{b}$ protein and which acts as a light-harvesting antenna primarily for Photosystem II.Cations dramatically alter the structural interaction of the light-harvesting complex with the photochemically active system II complex. This interaction has been measured by determining the amount of protein-bound chlorophyll b and Photosystem II activity which can be released into dispersed subunits by digitonin treatment of chloroplast lamellae. When cations are present to cause interaction between the Photosystem II complex and the light-harvesting pigment · protein, the combined complexes pellet as a “heavy” membranous fraction during differential centrifugation of detergent treated lamellae. In the absence of cations, the two complexes dissociate and can be isolated in a “light” submembrane preparation from which the light-harvesting complex can be purified by sucrose gradient centrifugation.Cation effects on excitation energy distribution between Photosystems I and II have been monitored by following Photosystem II fluorescence changes under chloroplast incubation conditions identical to those used for detergent treatment (with the exception of chlorophyll concentration differences and omission of detergents). The cation dependency of the pigment · protein complex and Photosystem II reaction center interactions measured by detergent fractionation, and regulation of excitation energy distribution as measured by fluorescence changes, were identical. We conclude that changes in substructural organization of intact membranes, involving cation induced changes in the interaction of intramembranous subunits, are the primary factors regulating the distribution of excitation energy between Photosystems II and I.     

The effect of abscisic acid on cold tolerance and chloroplasts ultrastructure in wheat under optimal and cold stress conditions

The effect of abscisic acid (0.1 mM) on cold tolerance of leaf cells and ultrastructure of chloroplasts in wheat (Triticum aestivum L.) under optimal (22 °C) and cold stress conditions (4 °C) was studied. Results indicated that exogenous abscisic acid induces a rise in the cold tolerance of wheat along with a number of significant ultrastructural changes in chloroplasts both at 22 and at 4 °C. Some of them (increase in density of chloroplasts stroma, formation of “distorted” and “dilated” thylakoids, appearance of invaginations, changes in the shape of chloroplasts and increase of their dimension owing to the stroma area) were common to the two types of treatments. At the same time, the character of changes in the membrane system of plastids was temperature specific, i.e. if at 22 °C the hormone caused a considerable increase in the length of photosynthetic membranes in chloroplast owing the length of both appressed and non-appressed membranes of thylakoids, then in cold stress conditions observed an increase in the number of grana and the length of appressed membranes of thylakoids. These results suggested that the rise in the cold tolerance of abscisic acid-treated plants is associated with the ultrastructural reorganization of chloroplasts aimed to defense plant cells against chilling injury and to maintain the activity of the photosynthetic system.     

Photosynthetic activity and membrane polypeptide composition of supergranal chloroplasts from plant tissue cultures containing a viruslike particle

Tissue culture cells of Streptanthus tortuosus (Kell.) var. orbiculatus (Greene) Hall (Cruciferae), having a viruslike particle in their nucleoli, the STV cell line, contain “supergranal” chloroplasts. Freeze-fracture studies of chloroplasts of a control cell line, which lacks the viruslike particles, reveal two complementary faces similar to those observed in spinach chloroplasts. Replicas of freeze-fractured STV supergranal chloroplasts, however, show that one membrane face (B) contains widely spaced 80 Å particles and the other face (C) is essentially smooth. Isolated STV supergranal chloroplasts lack photosystem II activity as indicated by their inability to reduce dichlorophenolindophenol and are unable to reduce NADP with electrons from photosystem II or from ascorbate-reduced dichlorophenolindophenol. However, partial photosystem I activity is indicated by the reduction of methyl viologen with electrons from dichlorophenolindophenol-ascorbate. This supports the concept that there is not a direct correspondence between grana formation and photosystem II activity. Electrophoresis shows that all of the major polypeptide bands present in the STV supergranal chloroplasts are also present in the control chloroplast membranes. One band, molecular weight 33,000, is present in a greatly increased amount in the STV supergranal chloroplast membranes and may be associated with grana stacking.     

Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach

The effects of nano-TiO₂ (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO₂, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg²⁺-ATPase and chloroplast coupling factor I (CF₁)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO₂ might be related to activation of photochemical reaction of chloroplasts of spinach.     

The Ultrastructure of Chloroplasts,Content of Photosynthetic Pigments,and Photochemical Activity of Maize (Zea mays L.) as Influenced by Different Concentrations of the Herbicide Amitrole

The effect of three different concentrations of amitrole (AM), a bleaching herbicide affecting carotenogenesis, on chloroplast ultrastructure, photosynthetic pigment contents, and photochemical activity was studied in two maize genotypes differing in photosynthetic characteristics. The content of photosynthetic pigments in leaves of plants treated with low (20 M) AM concentration was similar to control plants and no damaging effect of the herbicide on the ultrastructure of either mesophyll (MC) or bundle-sheath (BSC) cell chloroplasts was observed. Higher (60 and 120 M) concentrations of AM caused a significant decrease in the content of carotenoids (especially xanthophylls), which was followed by photooxidative destruction of chlorophylls and some alterations of chloroplast ultrastructure. MC chloroplasts appeared more sensitive to the damaging effect of AM compared to BSC chloroplasts. A significant decrease in the amount of both granal and intergranal thylakoids in MC chloroplasts was observed with the increasing concentration of AM. As regards BSC chloroplasts, rapid decrease in the volume density of starch inclusions was found in plants treated with higher concentrations of AM. When 120 M AM was used, both MC and BSC chloroplasts contained just a few thylakoid membranes that were strongly altered. The changes in the ultrastructure of MC chloroplasts were accompanied by the changes in their photochemical activity. The formation of chloroplast protrusions after treatment of plants with AM as well as in control plants was also observed.     

Photosynthetic apparatus of chilling-sensitive plants. X. Relationship between superoxide dismutase activity and photoperoxidation of chloroplast lipids

(1) The rate of photoperoxidation of chloroplast lipids, as measured by malondialdehyde formation following the illumination of either leaves or chloroplast preparations, is found to be approx. 2-fold higher in chloroplasts from both cold- and dark-stored as well as stored and illuminated tomato leaves than in those from fresh leaves. (2) Enhanced lipid photoperoxidation can also be observed in chloroplasts from fresh leaves treated with cyanide as well as in superoxide dismutase-depleted chloroplasts following washing with Tris or Hepes. (3) Cyanide-sensitive superoxide dismutase activity is not detected in chloroplasts isolated from cold- and dark-stored leaves. Their illumination does not reactivate the enzyme activity. (4) On the basis of these observations, it is concluded that inactivation of chloroplast cyanide-sensitive superoxide dismutase due to cold and dark treatment of leaves, rather than diminished electron transport, is responsible for accelerated chloroplast lipid photoperoxidation.     

Chemical cross-linking and its effect on fatty acid synthetase activity in intact chloroplasts from Euglena gracilis

Intact chloroplasts were isolated from Euglena gracilis variety bacillaris, aliquots were exposed to several different chemical cross-linking reagents. The reagents penetrated the triple membrane of Euglena chloroplasts. This was shown by gradient acrylamide gel electrophoresis under denaturing conditions. The activity of the nonaggregated fatty acid synthetase of Euglena was located within the chloroplast stroma, and the effects of dimethylsuberimidate cross-linking on the activity of the enzyme system were examined. The acyl-carrier protein concentration in the chloroplast was measured at about 0.24 mM.     

RIBOSOMES BOUND TO CHLOROPLAST MEMBRANES IN CHLAMYDOMONAS REINHARDTII

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.     

Characterization of the stromal protease(s) degrading the cross-linked products of the D1 protein generated by photoinhibition of photosystem II

When photosystem (PS) II-enriched membranes are exposed to strong light, cross-linking of the intrinsic D1 protein with the surrounding polypeptides and degradation of the D1 protein take place. The cross-linking of the D1 protein with the alpha-subunit of cytochrome b(559) is suggested to be an early event of photoinduced damage to the D1 protein (Barbato et al., FEBS Lett. 309 (1992) 165-169). The relationship between the cross-linking and the degradation of the D1 protein, however, is not yet clear. In the present study, we show that the addition of stromal extract from chloroplasts degrades the 41 kDa cross-linked product of D1/cytochrome b(559) alpha-subunit and enhances the degradation of the D1 protein. Incubation of the preilluminated PS II-enriched membranes with the stromal extract at 25 degrees C causes the degradation of the cross-linked product by more than 70%. The activity of the stromal extract showed a pH optimum at 8.0, and was enhanced by the addition of ATP or GTP. Consistent with the nucleotide effect, this stromal activity was eliminated by the preincubation of the stromal extract with apyrase, which hydrolyzes nucleotides. Also, the stromal activity was nearly fully inhibited by a serine-type protease inhibitor, 3,4-dichloroisocoumarin, which suggests participation of a serine-type protease(s).     

Interactions of rotor subunits in the chloroplast ATP synthase modulated by nucleotides and by Mg2+

ATP synthases - rotary nano machines - consist of two major parts, F(O) and F(1), connected by two stalks: the central and the peripheral stalk. In spinach chloroplasts, the central stalk (subunits gamma, epsilon) forms with the cylinder of subunits III the rotor and transmits proton motive force from F(O) to F(1), inducing conformational changes of the catalytic centers in F(1). The epsilon subunit is an important regulator affecting adjacent subunits as well as the activity of the whole protein complex. Using a combination of chemical cross-linking and mass spectrometry, we monitored interactions of subunit epsilon in spinach chloroplast ATP synthase with III and gamma. Onto identification of interacting residues in subunits epsilon and III, one cross-link defined the distance between epsilon-Cys6 and III-Lys48 to be 9.4 A at minimum. epsilon-Cys6 was competitively cross-linked with subunit gamma. Altered cross-linking yields revealed the impact of nucleotides and Mg(2+) on cross-linking of subunit epsilon. The presence of nucleotides apparently induced a displacement of the N-terminus of subunit epsilon, which separated epsilon-Cys6 from both, III-Lys48 and subunit gamma, and thus decreasing the yield of the cross-linked subunits epsilon and gamma as well as epsilon and III. However, increasing concentrations of the cofactor Mg(2+) favoured cross-linking of epsilon-Cys6 with subunit gamma instead of III-Lys48 indicating an approximation of subunits gamma and epsilon and a separation from III-Lys48.     

Evidence for cyanide and mercury inactivation of endogenous plastocyanin

Cyanide and mercury treatment of chloroplast membranes inactivates plastocyanin as shown by the inability of the extracted plastocyanin to restore electron transport in a bioassay on chloroplasts depleted of their endogenous plastocyanin by digitonin treatment. The extraction procedure did remore the enzyme from cyanide and mercury treated chloroplasts as shown by sodium dodecyl sulfate polyacrylamide electrophoresis of the extracts. This procedure normally shows a plastocyanin band at 11,000 dalton molecular weight and the band was present in extracts from control and cyanide or mercury treated membranes.     

Regulation of chloroplast phosphoribulokinase by the ferredoxin/thioredoxin system

A newly found form of chloroplast phosphoribulokinase (designated the “regulatory form”) required reduced thioredoxin for activity. A second form of the enzyme (the “nonregulatory form”) was not appreciably affected by thioredoxin. The thioredoxin required for activation of the regulatory enzyme could be reduced (i) photochemically by chloroplast membranes that were supplemented with ferredoxin and ferredoxin-thioredoxin reductase or (ii) chemically in the dark with the sulfhydryl reagent dithiothreitol. Following activation by reduced thioredoxin, phosphoribulokinase was deactivated by the soluble chloroplast oxidants dehydroascorbate and oxidized glutathione. The results suggest that the regulatory form of phosphoribulokinase resembles fructose 1,6-bisphosphatase in its mode of regulation by the ferredoxin/thioredoxin system.     

Chloroplast biogenesis. Regulation of lipid transport to the thylakoid in chloroplasts isolated from expanding and fully expanded leaves of pea

To study the regulation of lipid transport from the chloroplast envelope to the thylakoid, intact chloroplasts, isolated from fully expanded or still-expanding pea (Pisum sativum) leaves, were incubated with radiolabeled lipid precursors and thylakoid membranes subsequently were isolated. Incubation with UDP[(3)H]Gal labeled monogalactosyldiacylglycerol in both envelope membranes and digalactosyldiacylglycerol in the outer chloroplast envelope. Galactolipid synthesis increased with incubation temperature. Transport to the thylakoid was slow below 12 degrees C, and exhibited a temperature dependency closely resembling that for the previously reported appearance and disappearance of vesicles in the stroma (D.J. Morré, G. Selldén, C. Sundqvist, A.S. Sandelius [1991] Plant Physiol 97: 1558-1564). In mature chloroplasts, monogalactosyldiacylglycerol transport to the thylakoid was up to three times higher than digalactosyldiacylglycerol transport, whereas the difference was markedly lower in developing chloroplasts. Incubation of chloroplasts with [(14)C]acyl-coenzyme A labeled phosphatidylcholine (PC) and free fatty acids in the inner envelope membrane and phosphatidylglycerol at the chloroplast surface. PC and phosphatidylglycerol were preferentially transported to the thylakoid. Analysis of lipid composition revealed that the thylakoid contained approximately 20% of the chloroplast PC. Our results demonstrate that lipids synthesized at the chloroplast surface as well as in the inner envelope membrane are transported to the thylakoid and that lipid sorting is involved in the process. Furthermore, the results also indicate that more than one pathway exists for galactolipid transfer from the chloroplast envelope to the thylakoid.     

Toc64, a new component of the protein translocon of chloroplasts

A subunit of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) of 64 kD is described, Toc64. Toc64 copurifies on sucrose density gradients with the isolated Toc complex. Furthermore, it can be cross-linked in intact chloroplasts to a high molecular weight complex containing both Toc and Tic subunits and a precursor protein. The 0 A cross-linker CuCl(2) yields the reversible formation of disulfide bridge(s) between Toc64 and the established Toc complex subunits in purified outer envelope membranes. Toc64 contains three tetratricopeptide repeat motifs that are exposed at the chloroplast cytosol interface. We propose that Toc64 functions early in preprotein translocation, maybe as a docking protein for cytosolic cofactors of the protein import into chloroplasts.     

Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. II. Activity of the purified and membrane-bound enzymes

Cyanobacterial (Spirulina platensis) photosynthetic membranes and isolated F1 ATPase were characterized with respect to ATP activity. The following results indicate that the regulation of expression of ATPase activity in Spirulina platensis is similar to that found in chloroplasts: the ATPase activity of Spirulina membranes and isolated F1 ATPase is mostly latent, a characteristic of chloroplast ATPase activity; treatments that elicit ATPase activity in higher plant chloroplast thylakoids and isolated chloroplast coupling factor (CF1) greatly stimulate the activity of Spirulina membranes and F1, and the cation specificity of chloroplast ATPase activity, e. g., light-induced membrane activity that is magnesium dependent and trypsin-activated CF1 activity that is calcium dependent, is also observed in Spirulina. Thus, an 8- to 15-fold increase in specific activity (to 13-15 mumol Pi min-1 mg chl-1) is obtained when Spirulina membranes are treated with trypsin (CaATPase) or with methanol (MgATPase): a light-induced, dithiothreitol-dependent MgATPase activity is also found in the membranes. Purified Spirulina F1 is a CaATPase when activated with trypsin (endogenous activity increases from 4 to 27-37 mumol Pi min-1 mg protein-1) or with dithiothreitol (5.6 mumol Pi min-1 mg-1), but a MgATPase when assayed with methanol (18-20 mumol Pi min-1 mg-1). The effects of varying calcium and ATP concentrations on the kinetics of trypsin-induced CaATPase activity of Spirulina F1 were examined. When the calcium concentration is varied at constant ATP concentration, the velocity plot shows a marked sigmoidicity. By varying Ca-ATP metal-nucleotide complex concentration at constant concentrations of free calcium or ATP, it is shown that the sigmoidicity is due to the effect of free ATP, which changes the Hill constant to 1.6 from 1.0 observed when the free calcium concentration is kept constant at 5 mM. Therefore not only is ATP an inhibitor but it is also an allosteric effector of Spirulina F1 ATPase activity. At 5 mM free calcium, the Km for teh Ca-ATP metal-nucleotide complex is 0.42 mM.