Inhibitor Of Cytoplasmic Protein Synthesis Research Articles

Post-Translational Regulation of a Bidomain Glycerol-3-Phosphate Dehydrogenase Catalyzing Glycerol Synthesis under Salinity Stress in Chlamydomonas reinhardtii

Core chlorophytes possess glycerol-3-phosphate dehydrogenases (GPDs) with an unusual bidomain structure, consisting of a glycerol-3-phosphate phosphatase (GPP) domain fused to canonical GPD domains. These plastid-localized enzymes have been implicated in stress responses, being required for the synthesis of glycerol under high salinity and triacylglycerols under nutrient deprivation. However, their regulation under varying environmental conditions is poorly understood. C. reinhardtii transgenic strains expressing constitutively bidomain GPD2 did not accumulate glycerol or triacylglycerols in the absence of any environmental stress. Although the glycerol contents of both wild type and transgenic strains increased significantly upon exposure to high salinity, cycloheximide, an inhibitor of cytoplasmic protein synthesis, abolished this response in the wild type. In contrast, GPD2 transgenic strains were still capable of glycerol accumulation when cultured in medium containing cycloheximide and NaCl. Thus, the pre-existing GPD2 protein appears to become activated for glycerol synthesis upon salt stress. Interestingly, staurosporine, a non-specific inhibitor of protein kinases, prevented this post-translational GPD2 protein activation. Structural modeling analyses suggested that substantial conformational rearrangements, possibly triggered by high salinity, may characterize an active GPD2 GPP domain. Understanding this mechanism(s) may provide insights into the rapid acclimation responses of microalgae to osmotic/salinity stress.

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells.

Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin’s main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.

A novel system to monitor mitochondrial translation in yeast.

The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.

Impact of some phytohormones, metabolic inhibitor and agrochemicals on senescing paddy leaves and its relevance to productivity

The influence of hormones viz. Indole acetic acid (IAA), Gibberellic acid (GA3), Cytokinins viz. Benzyladenine (BA), Benzimidazole (BZI), biocides viz. 2,4-Dichlorophenoxy acetic acid (2,4-D), Diathane M-45, Dimecron and metabolic inhibitors viz. Cycloheximide (CHI) and Actinomycin-D (Act-D) on senescence in detached paddy (Oryza sativa L.) leaves was studied under dark and illuminated conditions. Cytokinins were found to be more effective senescence retardants in light than in dark while auxins were effective in light. Biocides proved to be milder senescence retardants. Effective concentration, a concentration away from the ineffective and toxic range, chosen from IAA, GA3, BA, BZI, 2,4-D, Diathane M-45 and Dimecron were 25,100, 5, 100, 25, 1500 and 0.50 ppm respectively. Application of Act-D (inhibitor of mRNA synthesis) with in 15 min after detachment and cycloheximide (inhibitor of cytoplasmic protein synthesis) from the start of the incubation resulted in suppression of peroxidase activity both under light and dark. Catalase activity exhibited an inverse relation with peroxidase activity under both the experimental conditions. It is recommended that attempts must be made in a more sophisticated way to study the changes in activities of different enzymes suspected to be linked with leaf senescence at a deeper level using all modern techniques available so that the exact nature of enzymes, the isoenzymes pattern and their relation with yield can be understood. Such studies may also help in future in manipulating the projects for the improvement of this economically important cereal crop.

High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae.

Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase (PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and the K(m)'s for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNA(Phe) were determined to be 48, 200, and 1.2 µM, respectively, while the K(m)'s for S. pneumoniae PheRS with respect to phenylalanine, ATP and tRNA(Phe) were 21, 225 and 0.94 µM, respectively. P. aeruginosa and S. pneumoniae PheRS were used to screen a natural compound library and a single compound was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa and S. pneumoniae PheRS with IC50's of 2.3 and 4.9 µM, respectively. The compound had a K(I) of 0.83 and 0.98 µM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 µg/ml, respectively. The mechanism of action of the compound was determined to be competitive with the amino acid, phenylalanine, and uncompetitive with ATP. There was no inhibition of cytoplasmic protein synthesis, however, partial inhibition of the human mitochondrial PheRS was observed.

Microalgae—novel highly efficient starch producers

The freshwater alga Chlorella, a highly productive source of starch, might substitute for starch-rich terrestrial plants in bioethanol production. The cultivation conditions necessary for maximizing starch content in Chlorella biomass, generated in outdoor scale-up solar photobioreactors, are described. The most important factor that can affect the rate of starch synthesis, and its accumulation, is mean illumination resulting from a combination of biomass concentration and incident light intensity. While 8.5% DW of starch was attained at a mean light intensity of 215 µmol/(m2 s1), 40% of DW was synthesized at a mean light intensity 330 µmol/(m2 s1). Another important factor is the phase of the cell cycle. The content of starch was highest (45% of DW) prior to cell division, but during the course of division, its cellular level rapidly decreased to about 13% of DW in cells grown in light, or to about 4% in those kept in the dark during the division phase. To produce biomass with high starch content, it is necessary to suppress cell division events, but not to disturb synthesis of starch in the chloroplast. The addition of cycloheximide (1 mg/L), a specific inhibitor of cytoplasmic protein synthesis, and the effect of element limitation (nitrogen, sulfur, phosphorus) were tested. The majority of the experiments were carried out in laboratory-scale photobioreactors, where culture treatments increased starch content to up to about 60% of DW in the case of cycloheximide inhibition or sulfur limitation. When the cells were limited by phosphorus or nitrogen supply, the cellular starch content increased to 55% or 38% of DW, respectively, however, after about 20 h, growth of the cultures stopped producing starch, and the content of starch again decreased. Sulfur limited and cycloheximide-treated cells maintained a high content of starch (60% of DW) for up to 2 days. Sulfur limitation, the most appropriate treatment for scaled-up culture of starch-enriched biomass, was carried out in an outdoor pilot-scale experiment. After 120 h of growth in complete mineral medium, during which time the starch content reached around 18% of DW, sulfur limitation increased the starch content to 50% of DW.

Programmed cell death in plants: effect of protein synthesis inhibitors and structural changes in pea guard cells.

Pea leaf epidermis incubated with cyanide displayed ultrastructural changes in guard cells that are typical of apoptosis. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, and lincomycin, an inhibitor of protein synthesis in chloroplasts and mitochondria, produced different effects on the dynamics of programmed death of guard cells. According to light microscopy data, cycloheximide reinforced and lincomycin suppressed the CN(-)-induced destruction of cell nuclei. Lincomycin lowered the effect of cycloheximide in the light and prevented it in the dark. According to electron microscopy data, the most pronounced effects of cycloheximide in the presence of cyanide were autophagy and a lack of apoptotic condensation of nuclear chromatin, the prevention of chloroplast envelope rupturing and its invagination inside the stroma, and the appearance of particular compartments with granular inclusions in mitochondria. Lincomycin inhibited the CN(-)-induced ultrastructural changes in guard cell nuclei. The data show that programmed death of guard cells may have a combined scenario involving both apoptosis and autophagy and may depend on the action of both cytoplasm synthesized and chloroplast and mitochondrion synthesized proteins.

Mg-protoporphyrin IX and heme control HEMA, the gene encoding the first specific step of tetrapyrrole biosynthesis, in Chlamydomonas reinhardtii.

HEMA encodes glutamyl-tRNA reductase (GluTR), which catalyzes the first step specific for tetrapyrrole biosynthesis in plants, archaea, and most eubacteria. In higher plants, GluTR is feedback inhibited by heme and intermediates of chlorophyll biosynthesis. It plays a key role in controlling flux through the tetrapyrrole biosynthetic pathway. This enzyme, which in Chlamydomonas reinhardtii is encoded by a single gene (HEMA), exhibits homology to GluTRs of higher plants and cyanobacteria. HEMA mRNA accumulation was inducible not only by light but also by treatment of dark-adapted cells with Mg-protoporphyrin IX (MgProto) or hemin. The specificity of these tetrapyrroles as inducers was demonstrated by the absence of induction observed upon the feeding of protoporphyrin IX, the precursor of both heme and MgProto, or chlorophyllide. The HEMA mRNA accumulation following treatment of cells with light and hemin was accompanied by increased amounts of GluTR. However, the feeding of MgProto did not suggest a role for Mg-tetrapyrroles in posttranscriptional regulation. The induction by light but not that by the tetrapyrroles was prevented by inhibition of cytoplasmic protein synthesis. Since MgProto is synthesized exclusively in plastids and heme is synthesized in plastids and mitochondria, the data suggest a role of these compounds as organellar signals that control expression of the nuclear HEMA gene.

Analyses of CIA5, the master regulator of the carbon-concentrating mechanism in Chlamydomonas reinhardtii, and its control of gene expression

In numerous studies, the CIA5 gene of Chlamydomonas reinhardtii Dangeard has been shown to control the expression of several "CO2-responsive genes" when cells are shifted to higher or lower levels of CO2. Using DNA microarray analyses with arrays containing 2764 unique cDNA sequences, we have demonstrated that several additional genes are controlled by the CIA5 gene, some increasing in expression when CO2 levels are lowered and others decreasing. Not all genes that respond to changes in CO2 concentrations are controlled by CIA5. For example, the RH1 gene, is markedly induced when both wild-type and cia5 mutant cells are shifted to high levels of CO2. We demonstrate that cycloheximide (an inhibitor of cytoplasmic protein synthesis) has no apparent effect on the initial induction of CO2-responsive genes, suggesting constitutive presence of all the molecular machinery needed by the cell to immediately respond to changes in CO2 levels. This observation is consistent with our earlier suggestions that CIA5 or another key component(s) of the carbon-concentrating mechanism must be rapidly "activated" (or "inactivated") as part of the response of C. reinhardtii to changes in external CO2 levels. We present new, direct evidence that CIA5 is localized to the nucleus in both low- and high-CO2 conditions.Key words: CIA5, CCM, carbon-concentrating mechanism, photosynthesis, gene regulation, Chlamydomonas reinhardtii, CO2.

Effect of inhibitors of protein synthesis and DNA replication on the induction of proteolytic activities, caspase-like activities and cell death in the unicellular chlorophyte Dunaliella tertiolecta

When the chlorophyte alga Dunaliella tertiolecta is placed in darkness, a form of programmed cell death with many similarities to apoptosis (including the induction of caspase-like proteases) is induced. Many uncertainties about this process remain, two of which are whether it requires protein synthesis and whether there is potential viral involvement. In order to examine the relationship between the induction and/or activation of proteolytic activities and the cell death event, we used inhibitors of cytoplasmic protein synthesis (cycloheximide, CHX), organellar protein synthesis (chloramphenicol, CAP) and DNA synthesis (mitomycin C, MMC). Use of MMC also allowed us to examine whether temperate viruses were present in the D. tertiolecta isolate, since MMC treatment has been shown to induce lytic cycles. Addition of protein synthesis inhibitors (100 μM CHX or 1500 μM CAP applied singly or both inhibitors added together) did not prevent cell death from occurring when cultures were placed in the dark. There were no differences in caseinolytic activities visualized using zymograms, or in caspase 1, 3, 8 and 9 -like activities. Surprisingly, 100 μM MMC prevented the cell death event. Caseinolytic activities that appeared in darkness in controls did not appear in MMC-treated cultures, and caspase-like activities remained the same as in controls maintained in the light. The lack of effect of CHX and CAP suggests that the cell death programme we observe does not depend on protein synthesis, but rather on post-translational modification of pre-existing proteins. Results for MMC discount the involvement of temperate virus, but are difficult to interpret. MMC affects DNA synthesis and presumably transcription, but since inhibitors of translation did not prevent cell death, it is not clear why inhibiting transcription would. MMC affects cell cycle progression and cell division cycles, thus these processes may play as yet unexplained roles in mediating the cell death process observed.

Nitrate reductase from winter wheat leaves is activated at low temperature via protein dephosphorylation.

The effect of short-term low temperature treatment on nitrate reductase (NR, EC 1.6.6.1) activity, NR protein and NR transcript levels in excised leaves of winter wheat (Triticum aestivum L. cv. Sadovo-1) was investigated. NR activity, measured in the presence of Mg2+ (NRact), doubled within 2 h at 4 degrees C, whereas NR activity, measured in the presence of EDTA (NRmax), did not respond to the cold treatment. Such an activation of NR occurred only if leaves were exposed to low temperature in the light but not in the dark. It was not affected by feeding cytoplasmic protein synthesis inhibitor, cycloheximide, or protein kinase inhibitor, staurosporin, but was completely prevented by okadaic acid, an inhibitor of protein phosphatases of the type 1 and 2 A. This inhibitory effect decreased gradually when okadaic acid-concentration in the nutrient solution was lowered below 1 &mgr;M and tended to disappear when leaves were fed with 10 nM okadaic acid. It was demonstrated that the cold-induced NR activation was dependent neither on cold-triggered calcium influx nor on high endogenous abscisic acid levels. The increased NRact in cold-exposed leaves was found to correlate with a higher level of NR transcript but not with an increased NR protein level. Feeding okadaic acid to these leaves prevented the cold-induced accumulation of NR mRNA. These data point to protein phosphatases of the type 2 A being involved in NR protein dephosphorylation and NR transcript accumulation as targets of activation by low temperature treatment.

Modulator of heme biosynthesis induces apoptosis in leukemia cells.

The purpose of this research is the investigation of the possible cause(s) of the dark-cell death phenomenon induced by 1,10-phenanthroline (Oph), a porphyrin biosynthesis modulator. We have previously shown that porphyrin biosynthesis modulators, such as Oph, which is also an iron-chelating agent, enhance protoporphyrin IX (Proto) accumulation in mammalian neoplastic cells treated with delta-aminolevulinic acid (ALA). As a result of the enhanced Proto accumulation, a significant increase in photodynamic damage was observed under illumination. Also tetrapyrrole and heme-biosynthesis modulators have been shown to cause death in treated insect larvae in darkness, a phenomenon referred to as dark-cell death. Dark-cell death was also observed in Oph + ALA-treated transformed mammalian cells. Neoplastic cells were treated with ALA, Oph, and ALA + Oph, and the following cell properties were investigated: growth arrest, membrane permeability, cell survival, nucleosomal cleavage, and cell cycle alterations. It was observed that Oph but not ALA induced growth arrest, in a T-cell leukemia line (MLA 144) as assessed by reduction in DNA synthesis. Exogenous Proto and isomers of Oph lacking the iron-chelating property of Oph also caused a dose-dependent inhibition of proliferation in MLA 144 cells. Although the plasma membrane of Oph-treated cells remained intact following 3 h of dark-incubation, the cells exhibited DNA internucleosomal cleavage, characteristic of cells undergoing apoptosis. Cell cycle analysis using the DNA intercalating dye propidium iodide (PI) coupled to flow cytometry, indicated that 81 +/- 5.6% of Oph-treated MLA 144 cells were apoptotic, with the majority of the cells arrested in the early S phase. On the other hand, treatment with either ALA or Proto did not alter the cell cycle. Also, using a double-labeling protocol with Hoechst 33342, and PI, and analysis by flow cytometry, Oph-treated cells were found to be 82% apoptotic after 3 h of dark-incubation. Apoptosis was reduced by 75% (p < 0.05) by the cytoplasmic protein synthesis inhibitor cycloheximide. These results indicate that in addition to enhancing Proto accumulation, the heme biosynthesis modulator Oph also induces growth arrest and apoptosis in transformed cells in darkness.

Differential induction of three pathogenesis-related genes, PR10, PR1b and PR5 by the ethylene generator ethephon under light and dark in rice ( Oryza sativa L.) seedlings

Ethylene has been shown to be involved in triggering pathogenesis-related (PR) gene expression mainly in dicotyledonous species; however, less attention has been devoted identifying and characterizing PR genes in rice (Oryza sativa L.), a monocot and a model of cereal crop genera. Here, we demonstrate that ethylene induces at least three important rice PR genes, the PR10, PR1 basic (PR1b), and PR5 genes in rice (cv. Nipponbare) seedling leaf, upon treatment with the ethylene generator, ethephon (ET), in a light-, time- and dose-dependent manner. Induction of these PR genes was partially blocked by cycloheximide (CHX), a eukaryotic cytoplasmic protein synthesis inhibitor, which indicates an involvement of cytoplasmic de novo protein synthesis in their induction. These results clearly indicate a dynamic role for ethylene in PR genes induction in rice.

Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus.

Chlorophyll precursors Mg-protoporphyrin IX and its monomethylester are candidates for plastid-derived molecules involved in light signalling from the chloroplast to the nucleus. The pool sizes of these two Mg2+-containing porphyrins and of protoporphyrin IX transiently increased upon a shift of Chlamydomonas cultures from dark to light. This increase coincided with the accumulation of mRNAs encoded by the nuclear genes HSP70A and HSP70B. Analysis of a mutant (brs-1), previously shown to be defective in the light induction of these genes, revealed high levels of protoporphyrin IX but no light-induced increase in the levels of Mg2+-containing porphyrins. Inhibitors of cytoplasmic protein synthesis prevented both the light-induced rise in pool levels and induction of the HSP70 genes. Similarly, pre-gametes, intermediates of sexual differentiation, lacked both responses to light. The block in light induction of the HSP70 genes in inhibitor-treated cells and in pre-gametes could be circumvented by the exogenous addition of Mg-protoporphyrin IX in the dark. This suggests an essential role for light-induced Mg-protoporphyrin IX accumulation in this chloroplast-to-nucleus signalling pathway. However, accumulation of this porphyrin in the dark - presumably in the chloroplast - did not result in induction. A second crucial role for light in this signalling pathway is postulated which makes this plastidic compound accessible to the cytoplasm/nucleus where the downstream signalling pathway may be activated.

Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus

SummaryChlorophyll precursors Mg‐protoporphyrin IX and its monomethylester are candidates for plastid‐derived molecules involved in light signalling from the chloroplast to the nucleus. The pool sizes of these two Mg2+‐containing porphyrins and of protoporphyrin IX transiently increased upon a shift of Chlamydomonas cultures from dark to light. This increase coincided with the accumulation of mRNAs encoded by the nuclear genes HSP70A and HSP70B. Analysis of a mutant (brs‐1), previously shown to be defective in the light induction of these genes, revealed high levels of protoporphyrin IX but no light‐induced increase in the levels of Mg2+‐containing porphyrins. Inhibitors of cytoplasmic protein synthesis prevented both the light‐induced rise in pool levels and induction of the HSP70 genes. Similarly, pre‐gametes, intermediates of sexual differentiation, lacked both responses to light. The block in light induction of the HSP70 genes in inhibitor‐treated cells and in pre‐gametes could be circumvented by the exogenous addition of Mg‐protoporphyrin IX in the dark. This suggests an essential role for light‐induced Mg‐protoporphyrin IX accumulation in this chloroplast‐to‐nucleus signalling pathway. However, accumulation of this porphyrin in the dark – presumably in the chloroplast – did not result in induction. A second crucial role for light in this signalling pathway is postulated which makes this plastidic compound accessible to the cytoplasm/nucleus where the downstream signalling pathway may be activated.

Phytotoxicity of selected trichothecenes using Chlamydomonas reinhardtii as a model systemt.

Trichothecenes are potent inhibitors of cytoplasmic protein synthesis which can affect the severity of plant diseases such as wheat head scab. While many trichothecene-producing fungi share the initial biosynthetic intermediates, Fusarium sp. are unique in the production of trichothecenes containing an oxygen function at C-3. Although the initial trichothecene and the final products have a C-3 hydroxyl group, the intermediate steps are acetylated at C-3. By using Chlamydomonas reinhardtii, a unicellular plant with a well-defined genetic system, we were able to test the proposal that trichothecenes with a C-3 hydroxyl are more toxic to plants, as well as demonstrate that C. reinhardtii is a promising plant trichothecene bioassay system. Seven pairs of trichothecenes with either a C-3 hydroxyl or C-3 acetyl group were assayed. Our results confirm that trichothecenes acetylated at C-3 were far less toxic to Chlamydomonas than those with a C-3 hydroxyl group.

Degreening of Citrus unshiu Fruits Via Ethylene-Induced Soluble Chlorophyllase.

The mechanism of ethylene action on degreening in satsuma mandarin (Citrus unshiu Marc. cv. Nichinan No.1) fruits was investigated using inhibitors of protein synthesis and soluble chlorophyllase (chlorophyll-chlorophyllide hydrolase, Chlase ; E.C. 3.1.1.14). This enzyme which catalyzes the dephytylation of chlorophyll (Chl) to chlorophyllide (Chlide), was partially purified. Chlase catalyzes the first step in the Chl breakdown process. Ethylene treatments promoted the degreening and increased soluble Chlase activity of C. unshiu fruit peels held in the dark. Cycloheximide (CH), an inhibitor of cytoplasmic protein synthesis, inhibited the effects of ethylene, whereas chloramphenicol (CP), an inhibitor of plastid protein synthesis, also weakly inhibited the Chlase activity. These results suggest that ethylene appears to enhance the degreening of the peels through de novo synthesis of Chlase. Moreover, de novo cytoplasmic protein synthesis and chloroplast-dependent enzyme synthesis may regulate Chlase activity in the peel of ethylene-treated C. unshiu fruits. Soluble Chlase was precipitated with acetone from C. unshiu fruit peels and purified by the following method : after ammonium sulfate fractionation, gel-filtration through Sephadex G-25 and Phenyl Sepharose CL-4B (hydrophobic chromatography) were used. The soluble Chlase was purified ca. 203.5-fold with a yield of 45.5%.

Mutation of residue threonine-2 of the D2 polypeptide and its effect on photosystem II function in Chlamydomonas reinhardtii.

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by 32P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [14C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.

Chlorophyll breakdown in senescing barley leaves as correlated with phaeophorbidea oxygenase activity

Activities of phaeophorbide a oxygenase (PaO), a key enzyme of the catabolic pathway of chlorophyll (Chi) were measured in membrane preparations from barley primary leaves at different stages of senescence. Under conditions of natural senescence, degreening began about 3 weeks after sowing; it took about 2 weeks and coincided with appearance and level of PaO activity. Premature, accelerated Chl breakdown in detached leaves induced to senesce in darkness was also well correlated with PaO activity. In detached and darkened leaves treatment with cytokinin delayed the loss of Chl as well as the appearance of PaO activity. Treatment of detached leaves with abscisic acid caused the acceleration of degreening in conjunction with increased PaO activity, but only when incubations were performed under photoperiod. Jasmonic acid methylester caused a most rapid breakdown of Chl in detached leaves kept under photoperiod. In this case the real activities of PaO could not be determined because the membrane preparations contained an inhibitory factor. Heat shock accelerated breakdown of Chl and also caused the appearance of increased activities of PaO. Effects of inhibitors of cytoplasmic protein synthesis suggest that the PaO protein is newly synthesized upon the induction of senescence. They also suggest that PaO is not the sole pace-making protein of the catabolic pathway.

Ethylene-Enhanced Chlorophyll Catabolism in Radish (Raphanus sativus L.) Cotyledons.

The mechanism of ethylene action on degreening of intact radish (Raphanus sativus) cotyledons was studied using inhibitors of protein and nucleic acid synthesis. Ethylene treatments enhanced the degreening and increased Chl-oxidative enzyme activity of cotyledons held in the dark. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, and actinomycin D, an inhibitor of nuclear RNA synthesis, completely inhibited the effects of ethylene, but chloramphenicol, an inhibitor of plastid RNA synthesis, did not. These results suggest that ethylene appears to enhance the degreening of the radish cotyledons through de novo synthesis of Chl-oxidative enzyme(s). Furthermore, an inverse correlation between the contents of total Chls or Chl a, of radish cotyledons and Chl-oxidative enzyme activity was obtained, indicating that Chl-oxidative enzyme(s) is the key enzyme in the degreening process.