The presence and synthesis of the NADPH-protochlorophyllide oxidoreductase in barley leaves with a high temperature-induced deficiency of plastid ribosomes

High-temperature-induced deficiency of plastid ribosomes in barley plants (Hordeum vulgare L.) was used as a system for studying the role of the cytoplasm in the synthesis of the NADPH-protochlorophyllide oxidoreductase. The enzyme is present in 33° C-grown plants. The failure of high-temperature-grown plants to accumulate chlorophyll during illumination is not caused by the absence of the protochlorophyllide-reducing enzyme. The synthesis of the NADPH-protochlorophyllide oxidoreductase was studied by feeding [³⁵S]methionine to the seedling and by following the incorporation of the radioactively labeled amino acid into plastid proteins. The NADPH-protochlorophyllide oxidoreductase was labeled in high-temperature-grown barley plants to the same extent as in control plants grown at 25° C. It is concluded that the 36,000-M_r polypeptide of the NADPH-protochlorophyllide oxidoreductase is synthesized outside the plastid on cytoplasmic 80S ribosomes.     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Capacity for RNA synthesis in 70S ribosome-deficient plastids of heat-bleached rye leaves

In the leaves of rye seedlings (Secale cereale L.) grown at an elevated temperature of 32°C the formation of plastidic 70S ribosomes is specifically prevented. The resulting plastid ribosome-deficient leaves, which are chlorotic in light, represent a system for the identification of translation products of the 80S ribosomes among the chloroplastic proteins. Searching for the primary heat-sensitive event causing the 70S ribosome-deficiency, the thermostability of the chloroplastic capacity for RNA synthesis was investigated. The RNA polymerase activity of isolated normal chloroplasts from 22°-grown rye leaves was not inactivated in vitro at temperatures between 30° and 40°C. The ribosome-deficient plastids purified from bleached 32°-grown leaf parts contained significant RNA polymerase activity which was, however, lower than in functional chloroplasts. After application of [³H]uridine to intact leaf tissues [³H]uridine incorporation was found in ribosome-deficient plastids of 32°C-grown leaves. The amount of incorporation was similar to that in the control chloroplasts from 22°C-grown leaves. According to these results, it is unlikely that the non-permissive temperature (32°C) causes a general inactivation of the chloroplastic RNA synthesis in rye leaves.     

Dark-induced chloroplast dedifferentiation in Euglena gracilis

Transfer of light-grown autotrophic Euglena gracilis cells to darkness and carbon (glucose) containing heterotrophic media causes structural and functional decomposition of the photosynthetic apparatus. The process can be ascribed to a strict diluting-out mechanism of stroma constituents among the progeny, as shown for ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39), and aminoacyl-tRNA synthetases (Aa-RS; especially Leu-RS, EC 6.1.1.4) activities. The diluting-out effect of thylakoid membranes and chlorophyll seems to be superimposed by additional degradations, beginning soon after the transfer of cells to darkness. Cultivation of cells in darkness in 0.03 M KCl or without utilizable organic carbon (resting media) preserves chloroplast structure and function over a long period, indicating negligible turnover in these cells. Thus, under both growing and resting conditions, darkness induces the arrest of synthesis of plastid constituents. Experiments with the inhibitors cycloheximide, chloramphenicol, and nalidixic acid demonstrate that chloroplast dedifferentiation does not require organelle gene expression, but it is more strictly dependent on biosynthetic events in the nucleo-cytoplasmic compartment than the reverse process, light-induced chloroplast formation. Since cycloheximide at low concentrations in growth medium causes a marked suppression of precursor uptake or re-utilization similar to that in cells of resting media, intracellular precursor deficiency is suggested to control the observed blockade in cytoplasmic synthesis of plastid proteins. On the other hand, darkness might signalize the stop of gene expression in the organelles.Abbreviations Aa aminoacid - CH cycloheximide - CM chloramphenicol - Leu-RS leucyl-tRNA synthetase - RuBP ribulose-1,5-bisphosphate - TCA trichloroacetic acid     

Heat sensitivity and protein synthesis during heat-shock in the tobacco hornworm,Manduca sexta

Summary Fifth instar larvae of the tobacco hornworm,Manduca sexta, tolerate 1-h exposures to temperatures as high as 42°C. Above 42°C, survival declines rapidly to 18% at 44°C and 0% at 48°C. As in other insects, the heat-shock response ofManduca sexta involves the induction of synthesis of heat-shock proteins very similar in size to theDrosophila heat-shock proteins (84, 73, 71, 27, 25, 23, and 22 kd). In the epidermis, heat-shock protein synthesis peaks at 42°C, correlating with the heat sensitivity of both the tissue itself and the intact larva. Some heat-shock proteins have different isoelectric forms depending on tissue. Also, the heat-shock proteins are synthesized over a wider range of temperatures in the imaginal discs and the fat body as compared to the epidermis. In contrast to dipteran insects,Manduca sexta does not exhibit a strong repression of non-heat-shock protein synthesis under tolerable conditions.Abbreviations TCA trichloroacetic acid - PAGE polyacrylamide gel electrophoresis - AZT arbitrary Zeitgeber time - kd kilodaltons     

The effects of temperature acclimation on the photoinhibitory responses of Ulva rotundata Blid.

The effect of acclimation to 25, 18, or 10° C on the relationship between photoprotection and photodamage was tested in low-light-grown (80 mol · m^–2 · s^–1) Ulva rotundata Blid. exposed to several higher irradiances at the acclimation temperature. Changes in chlorophyll fluorescence parameters (minimum fluorescence, F₀, and the ratio of variable to maximum fluorescence, F_v/F_m, measured after 5 min darkness) were monitored during 5 h transfers to 350, 850, and 1700 mol · m^–2 · s^–1, and during recovery after 1- or 5-h treatments. At all temperatures, rate of onset and final extent of photoinhibition, measured by a decrease in F_v/F_m, increased with increasing irradiance. At a given photoinhibitory irradiance, rate of onset was most rapid at 10 ° C, but the extent was temperature-independent. Recovery rates from mild light stress were similar at all temperatures, but recovery from the most extreme photoinhibitory treatment lagged 2 h at 10° C. De-epoxidation of xanthophyll-cycle components proceeded faster and to a lower epoxidation status at 25° C, but there was little difference in the pool size among the three growth conditions. Using chloramphenicol to inhibit chloroplast protein synthesis and dithiothreitol to inhibit violaxanthin de-epoxidation, it was shown that at the lowest light treatment given, the extent of photoinhibition could be attributed both to greater amounts of photodamage and to greater zeaxanthin-related photoprotection at 25 than at 10° C. While these two mechanisms for high-light-induced loss of photosynthetic efficiency were operating at 10° C, there was evidence for a relatively greater proportion of zeaxanthin-unrelated photoprotection at the low temperature. This photoprotective mechanism is related to a rapidly reversible increase in F₀ and is insentivite to both chloramphenicol and dithiothreitol.Abbreviations and Symbol CAP chloramphenicol - DTT dihiothreitol - F₀, F_m, F_v minimum, maximum, and variable fluorescence - quantum yield This research was conducted in partial fulfillment of the requirements for the Ph. D. degree in the Department of Botany, Duke University. The author wishes to thank E.-M. Aro, W.J. Henley, G. Levavasseur, C.B. Osmond, and J. Ramus for helpful discussions, and C. Lovelock for pigment standards. Funding was provided by Grants-in-Aid of Research from Sigma Xi and the Phycological Society of America, and a Lynde and Harry Bradley Foundation Fellowship to L.A.F., and National Science Foundation grant OCE-8812157 to C.B.O. and J.R.     

Photoinhibition of photosynthesis in intact bean leaves: role of light and temperature,and requirement for chloroplast-protein synthesis during recovery

Photoinhibition of photosynthesis was induced in intact leaves of Phaseolus vulgaris L. grown at a photon flux density (PFD; photon fluence rate) of 300 mol·m^-2·s^-1, by exposure to a PFD of 1400 mol·m^-2·s^-1. Subsequent recovery from photoinhibition was followed at temperatures ranging from 5 to 35°C and at a PFD of either 20 or 140 mol·m^-2·s^-1 or in complete darkness. Photoinhibition and recovery were monitored mainly by chlorophyll fluorescence emission at 77K but also by photosynthetic O₂ evolution. The effects of the protein-synthesis inhibitors, cycloheximide and chloramphenicol, on photoinhibition and recovery were also determined. The results demonstrate that recovery was temperature-dependent with rates slow below 15°C and optimal at 30°C. Light was required for maximum recovery but the process was light-saturated at a PFD of 20 mol·m^-2·s^-1. Chloramphenicol, but not cycloheximide, inactivated the repair process, indicating that recovery involved the synthesis of one or more chloroplast-encoded proteins. With chloramphenicol, it was shown that photoinhibition and recovery occurred concomitantly. The temperature-dependency of the photoinhibition process was, therefore, in part determined by the effect of temperature on the recovery process. Consequently, photoinhibition is the net difference between the rate of damage and the rate of repair. The susceptibility of chilling-sensitive plant species to photoinhibition at low temperatures is proposed to result from the low rates of recovery in this temperature range.Abbreviations and symbols Da Dalton - Fo, Fm, Fv instantaneous, maximum, variable fluorescence emission - PFD photon flux density - PSII photosystem II - photon yield C.I.W.-D.P.B. Publication No. 871     

Plastid and nuclear DNA synthesis are not coupled in suspension cells ofNicotiana tabacum

The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following³H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA. Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of exposure to the cells. The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels of ethidium bromide has no effect on nuclear DNA synthesis. These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the biosynthesis of nuclear and plastid DNA.     

Lipid Formation by Aqueous Fischer-Tropsch-Type Synthesis over a Temperature Range of 100 to 400 °C

The formation of lipid compounds during anaqueous Fischer-Tropsch-type reaction was studied withsolutions of oxalic acid as the carbon and hydrogensource. The reactions were conducted in stainlesssteel vessels by heating the oxalic acid solution atdiscrete temperatures from 100 to 400 °C, atintervals of 50 °C for two days each. Themaximum lipid yield, especially for oxygenatedcompounds, is in the window of 150–250 °C. At atemperature of 100 °C only a trace amount oflipids was detected. At temperatures above150 °C the lipid components ranged from C₁₂to >C₃₃ and included n-alkanols, n-alkanoic acids, n-alkyl formates, n-alkanals, n-alkanones, n-alkanes, andn-alkenes, all with essentially no carbon numberpreference. The n-alkanes increased inconcentration over the oxygenated compounds attemperatures of 200 °C and above, with a slightreduction in their carbon number ranges due tocracking. It was also noted that the n-alkanoicacids increased while n-alkanols decreased withincreasing temperature above 200 °C. Attemperatures above 300 °C synthesis competeswith cracking and reforming reactions. At 400 °Csignificant cracking was observed and polynucleararomatic hydrocarbons and their alkylated homologswere detected. The results of this work suggest thatthe formation of lipid compounds by aqueous FTTreactions proceeds by insertion of a CO group at theterminal end of a carboxylic acid functionality toform n-oxoalkanoic acids, followed by reductionto n-alkanoic acids, to n-alkanals, thento n-alkanols. The n-alkenes areintermediate homologs for n-alkan-2-ones andn-alkanes. This proposed mechanism for aqueousFTT synthesis differs from the surface-catalyzedstepwise FT process (i.e., gaseous) of polymerization of methylene reported in the literature.     

Heat shock protein induction and the acquisition of thermotolerance in the psychrotrophic yeastTrichosporon pullulans

Two-dimensional gel electrophoretic analysis of the heat shock response in the psychrotrophic yeastTrichosporon pullulans revealed the induction of 11 heat shock proteins (hsps) after a 5° to 21°C heat shock, 12 hsps after a 5° to 26°C heat shock, and 12 hsps after a 5° to 29°C heat shock. Heat shock from 5° to 26° or 29°C resulted in a statistically significant increase in thermotolerance to a lethal heat challenge at 45°C for 5 min. When the protein synthesis inhibitor, cycloheximide, was added prior to the heat shock, no statistically significant thermotolerance was acquired. To confirm the correlation between the synthesis of hsps and the acquisition of thermotolerance, protein extracts of cells that had been heat shocked in the presence or absence of cycloheximide were electrophoretically analyzed. Addition of the same concentration of cycloheximide that prevented the acquisition of thermotolerance also inhibited the synthesis of any hsps.     

Growth of dimorphic human pathogenicfungi on media containing cycloheximide and chloramphenicol

Summary Studies of 24 strains ofBlastomyces dermatitidis confirmed previously published results that the yeast-phase of this fungus is more sensitive than the mycelial-phase to cycloheximide and chloramphenicol.Studies of 5 strains each ofHistoplasma capsulatum, Paracoccidioides brasiliensis andSporotrichum schenckii show that that these species also have a similar yeast-phase mycelial -phase sensitivity differential in regard to these antibiotics.A cycloheximide resistant strain ofB. dermatitidis was developed from a sensitive strain.The experimental results support the general practice of using 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol in media for the isolation of the four fungi at 25° C. The results indicate, however, that some strains would not be recovered at 37° C with similar concentrations of these antibiotics.It is recommended that a concentration of not more than 0.2 mg/ml chloramphenicol should be used to preserve sputum which is subsequently to be cultured forB. dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis orS. schenckii.     

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K _m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C.The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.     

Polypeptide synthesis on ribosomes of an extreme thermophilic hydrogen bacterium Calderobacterium hydrogenophilum

Ribosomes of the extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum interact with a broad spectrum of polyamines. In the absence of polyamines and at 70°C close to the growth optimum (75°C), high salt washed ribosomes lost their activity in the poly(UG)-directed polypeptide synthesis. At 70°C the in vitro system synthesized the polypeptide in the presence of spermidine, spermine or natural polyamines (tri-pentaamines) isolated from ribosomal extracts but not in the presence of putrescine. The activity of ribosomes was affected by a number of antibiotics interfering with functions of typical eubacterial 70S, such as tetracyclines, lincomycin, chloramphenicol and erythromycin. However, the ribosomes were relatively resistant to streptomycin and insensitive to 80S inhibitors, such as ricin and cycloheximide. The 30S and 50S subunits have structural features typical of eubacterial ribosomes.The authors dedicated this paper to Professor Ivan Málek on the occasion of his 80th birthdy to remember his contribution to the Czechoslovak microbiology     

The effects of temperature and host density on the development and survival of Copidosoma koehleri

Developmental rates for Copidosoma koehleri Blanchard (Hymenoptera: Encyrtidae) and its host, Phthorimaea operculella (Zeller), were determined at 10, 15, 23, 27, 29, 32, 34 and 35°C from host egg to adult. The developmental rates determined for both species showed good fit to mathematical models of insect development. At 15°C mean emergence of adult C. koehleri was 15 days after P. operculella adults emerged. At 29°C mean emergence of C. koehleri was only 5 days after that of P. operculella. P. operculella developed at 35°C, but parasitized larvae, and therefore C. koehleri, did not tolerate temperatures above 32°C. In the presence of C. koehleri, host survival was low (3%) at low host egg densities, but greater (20%) at higher host densities. Parasitized larvae of P. operculella were less able to compete for food resources, as measured by adult emergence.     

Effect of Calcium and Zinc on the Activity and Thermostability of Superoxide Dismutase

The effect of calcium and zinc ions on superoxide dismutase (SOD) from four plant species (Taxus baccata, Pinus sylvestris, Medicago rigidula, and Zea mays) was followed at three temperatures: optimal (20 °C), increased (50 °C), and high, inhibiting temperature (70 – 80 °C). At 20 and 50 °C in vitro added calcium increases SOD activity, but the degree was different for the plants investigated. The effect of zinc ions at the same temperatures varied in the investigated plants from activation to inhibition. An inhibiting effect of high temperature on SOD activity was diminished in the presence of calcium or zinc ions. It was shown that calcium and zinc ions can increase activity and thermostabilize different SOD isoforms.     

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus

The production of an extracellular collagenase and an alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37°C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5°C Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30°C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.     

Rapidly induced ethylene formation after wounding is controlled by the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis

Bean leaves from Phaseolus vulgaris L. var. Pinto 111 react to mechanical wounding with the formation of ethylene. The substrate for wound ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC). It is not set free by decompartmentation but is newly synthesized. ACC synthesis starts 8 to 10 min after wounding at 28°C, and 15 to 20 min after wounding at 20°C. Aminoethoxyvinylglycine (AVG), a potent inhibitor of ethylene formation from methionine via ACC, inhibits wound ethylene synthesis by about 95% when applied directly after wounding (incubations at 20°C). AVG also inhibits the accumulation of ACC in wounded tissue. AVG does not inhibit conversion of ACC to ethylene. Wound ethylene production is also inhibited by cycloheximide, n-propyl gallate, and ethylenediaminetetraacetic acid.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine - EDTA ethylenediaminetetraacetic acid     

Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli

Summary The temperature-sensitive dnaA46 mutation in Escherichia coli can be phenotypically suppressed at 42° C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 30° C. We found that the phenotype of dnaA46 showing this cold sensitivity was dominant over the phenotype of dnaA ⁺, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA46 strain with a GroELS-overproducing plasmid was shifted from 42° to 30° C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 4 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 42° C in the suppressed dnaA46 strain, or at either temperature in GroELS-oversupplied dnaA ⁺ cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding.A preliminary account of this work was presented at the Annual Meeting of the Molecular Biology Society of Japan in 1989.