Plant desiccation and protein synthesis: an in vitro system from dry and hydrated mosses using endogenous and synthetic messenger ribonucleic Acid

The conditions and requirements for an in vitro protein synthesizing system from the moss Tortula ruralis are outlined. Using this system the effects of desiccation, achieved quickly or slowly, were studied. Slowly dried moss retained fewer polyribosomes on desiccation but more active ribosomes than rapidly dried moss. Even in the completely desiccated moss the polyribosomes and/or free ribosomes present have retained their synthetic capacities. On rehydration, the slowly dried moss resumed protein synthesis more quickly than moss previously desiccated rapidly. Moss ribosomes are cycloheximide sensitive and chloramphenicol insensitive and thus the major protein synthesis occurs within the cytoplasm on rehydration. Extracted polyribosomes per se can withstand desiccation to a significant extent, suggesting that protection by the cytoplasm might not be necessary. The aquatic moss Hygrohypnum luridum can retain polyribosomal and ribosomal activity during desiccation, but this decreases greatly on rehydration.     

Plant Desiccation and Protein Synthesis : VI. Changes in Protein Synthesis Elicited by Desiccation of the Moss Tortula ruralis are Effected at the Translational Level

Upon rehydration of the moss Tortula ruralis following desiccation at a rapid or slow rate, there is increasing utilization of newly synthesized-poly(A)⁺ RNA for protein synthesis. Initially, poly(A)⁺ RNA conserved in the dry moss is associated with polysomes, but by 2 hours of rehydration there is an overwhelming recruitment of newly synthesized poly(A)⁺ RNA, at the expense of conserved messages. In rehydrated moss, there is a marked synthesis in vivo of new proteins, which are separable by two-dimensional electrophoresis, and identifiable by fluorography. These new proteins, termed rehydration proteins, are synthesized after both rapid and slow desiccation, but their synthesis persists longer after rapid desiccation. The protein patterns obtained following in vitro translation of bulk RNA from hydrated, desiccated, and rehydrated moss were qualitatively identical. Thus the differences in protein patterns observed in vivo must result from preferential selection of specific mRNAs from the same pool, which is indicative of control of protein synthesis at the translational level. The implications of these observations in relation to the response of the moss to drying in its natural environment are discussed.     

Inhibitory action of erythromycin on protein biosynthesis by isolated polyribosomes

Consistent with the previous work by Pestka (Antimicrob. Agents Chemother.5, 255, 1974) on the binding of erythromycin to polyribosomes, we found that erythromycin does not inhibit protein synthesis catalyzed by polyribosomes. This is due to the presence of nascent peptidyl tRNA on the naturally occurring polyribosomes. In a soluble extract from E. coli pretreated to remove the ribosome releasing factor, polyribosomes without nascent polypeptides remain intact and can catalyze protein synthesis in the absence of initiation. In this system erythromycin effectively inhibited protein synthesis. The inhibition by erythromycin was caused by premature release of oligopeptidyl tRNA from polyribosomes.     

Endosperms and embryos of mature dry castor bean seeds contain active ribosomes

Mature dry endosperms and embryos of castor bean contain ribosomes which are capable of catalysing protein synthesis in vitro. This observation contradicts previous reports that ribosomes are absent from mature dry endosperms. On hydration of the mature seeds the polyribosome content of both embryos and endosperms increases. These polyribosomes are lost on subsequent dehydration but, again, ribosomes are conserved. REFERENCES     

PERSISTENCE OF MESSENGER RNA THROUGH MITOSIS IN HELA CELLS

The decrease in protein synthesis which occurs in mammalian cells during cell division is associated with significant disaggregation of polyribosomes. For determining whether messenger RNA survives this disaggregation, the reformation of polyribosomes was investigated in synchronized HeLa cells as they progressed from metaphase into interphase in the presence of 2 µg/ml Actinomycin D. The persistence of messenger during cell division was evidenced by: (1) a progressive increase in the rate of protein synthesis in both treated and untreated cells for 45 min after metaphase; (2) reformation of polyribosomes, as determined by both sucrose gradients and electron microscopy, within 30 min after the addition of Actinomycin D to metaphase cells; (3) the persistence of approximately 50% of the rapidly labeled nonribosomal RNA which had associated with polyribosomes just before metaphase; (4) the resumption of synthesis, following cell division, of 6 selected peptides in Actinomycin-treated cells.     

Plant Desiccation and Protein Synthesis : V. Stability of Poly (A) and Poly (A) RNA during Desiccation and Their Synthesis upon Rehydration in the Desiccation-Tolerant Moss Tortula ruralis and the Intolerant Moss Cratoneuron filicinum

Upon desiccation of gametophytes of the desiccation-tolerant moss Tortula ruralis preexisting pools of poly(A)⁻ RNA (rRNA) remain inact, regardless of the speed at which desiccation is achieved. Preexisting poly(A)⁺ RNA pools (mRNA) are unaffected by slow desiccation but are substantially reduced during rapid desiccation. Poly(A)⁻ RNA involved in protein synthesis is also unaffected by desiccation, whereas the levels of polysomal poly(A)⁺ RNA in rapid- and slow-dried moss closely reflect the state of the protein synthetic complex in these dried samples.

Poly(A)⁻ RNA pools, both total and polysomal, are also stable during the rehydration of both rapid- and slow-dried moss. The total poly(A)⁺ RNA pool decreases upon rehydration, but this reduction is simply an expression of the normal turnover of poly(A)⁺ RNA in this moss. Analysis of polysomal fractions during rehydration reveals the continued use of conserved poly(A)⁺ RNA for protein synthesis. The rate of synthesis of poly(A)⁺ RNA upon rehydration appears to depend upon the speed at which prior desiccation is administered. Rapidly dried moss synthesizes poly(A)⁺ RNA at a faster rate, 60 to 120 minutes after the addition of water, than does rehydrated slowly dried moss. Recruitment of this RNA into the protein synthetic complex also follows this pattern. Comparative studies involving the aquatic moss Cratoneuron filicinum are used to gain an insight into the relevance of these findings with respect to the cellular mechanisms associated with desiccation tolerance.

     

Studies on Protein Synthesis in Tortula ruralis: POLYRIBOSOME REFORMATION FOLLOWING DESICCATION

Desiccation of Tortula ruralis was achieved rapidly by placingthe moss on the laboratory bench, or more slowly by placingit in desiccators with atmospheres of high relative humidities.Unlike the rapidly desiccated moss, the slower desiccated mossretained no polyribosomes in the dehydrated state, althoughpolyribosome reformation and protein synthesis resumed on reintroductionof the moss to water. Protein synthesis commenced on rehydrationof the slower desiccated moss at a greater rate than on rehydrationof the faster desiccated moss. A lack of correlation betweenendogenous ribonuclease activity and polyribosome levels extractedfrom the moss suggests that the observed reduction in polyribosomesduring desiccation was not due to their degradation but wasmore likely a consequence of stress-induced restriction on reinitiationof existing messenger RNA. The observed protein synthesis onrehydration of the moss was largely independent of any priorRNA synthesis.     

Elongation and termination reactions of protein synthesis on maize root tip polyribosomes studied in a homologous cell-free system

We show that the control of gene expression at the level of elongation and termination of protein synthesis can be observed in vitro. Free cytoplasmic polyribosomes were isolated from maize (Zea mays) root tips, and translated in root tip extracts that had been fractionated with ammonium sulfate to contain elongation factors, and be depleted in initiation factors. The root tip extract performs elongation and termination reactions as efficiently as wheat germ extracts. The translation products of the maize system are the same as made in vivo. The dependence of these in vitro elongation and termination reactions on pH was determined. Total protein synthesis in this system exhibits an optimum at pH ~7.5. However, the pH dependence of rates of synthesis of individual proteins is not at all uniform; many polyribosomes become stalled when translated at low pH. These data were compared with the elongation and termination capacity of polyribosomes isolated from oxygenated and hypoxic root tips (tissue having, respectively, high and low cytoplasmic pH values). We observed an inverse relationship between the relative abundance of many specific translatable mRNAs in polyribosomes of hypoxic root tips, and the relative rates of elongation and termination reactions on the different mRNAs at low pH in vitro. These results suggest that changes in intracellular pH in hypoxic root tips can be sensed directly by the translational machinery and thereby selectively modulate gene expression.     

Effects of Various Rates of Freezing on the Metabolism of a Drought-tolerant Plant, the Moss Tortula ruralis

The response of the drought-tolerant moss Tortula ruralis ([Hedw.] Gaertn., Meyer, Scherb.) to freezing and thawing at controlled rates has been studied. Slow freezing (at 3 C per hour to −30 C) of hydrated T. ruralis leads to only temporary, reversible changes in metabolism. These changes can be considered to result from desiccation due to extracellular ice formation. In contrast, rapid freezing in liquid N₂ and thawing in 20 C water leads to deterioration in all aspects of metabolism studied: ribosome, protein, and ATP levels decrease, and in vivo and in vitro protein synthetic activity is lost rapidly. Such changes probably result from intracellular ice formation. Following freezing and thawing at an intermediate rate (60 C per hour), only ATP levels and in vivo protein synthesis are reduced. The protein-synthesizing apparatus (the polyribosomes) remains intact and active in an in vitro protein-synthesizing system even 24 hours after one 60 C per hour freeze-thaw cycle. These metabolic responses are discussed in terms of the two-factor hypothesis of Mazur et al. (1972 Exp. Cell Res. 71: 345-355).     

Water Stress and Protein Synthesis: V. Protein Synthesis, Protein Stability, and Membrane Permeability in a Drought-sensitive and a Drought-tolerant Moss

The effects have been studied of water stress and desiccation on protein synthesis in the drought-tolerant moss Tortula ruralis and the drought-sensitive moss Hygrohypnum luridum. At any particular level of steady state water stress, the inhibition of protein synthesis was greater in H. luridum than in T. ruralis. Water stress-induced changes in the pattern of protein synthesis, as determined by the double label ratio technique, were minor in T. ruralis, but major in H. luridum. Proteins of both mosses were found to be stable during desiccation and subsequent rehydration. Changes in membrane permeability, as indicated by the leakage of amino acid, were observed during rehydration of desiccated moss and were dependent on the rate of desiccation. The leakage was small and reversible in T. ruralis but large and irreversible in H. luridum. Although H. luridum failed to recover from complete desiccation (80% loss in fresh weight), it was able to recover fully from steady state stress under conditions where a maximum loss of 55% in fresh weight was recorded.     

THE IN VIVO PROTEIN SYNTHETIC ACTIVITIES OF FREE VERSUS MEMBRANE-BOUND RIBONUCLEOPROTEIN IN A PLASMA-CELL TUMOR OF THE MOUSE

Cytoplasmic extracts of the transplantable RPC-20 plasma-cell tumor were fractionated by sucrose density gradient centrifugation. Four major fractions were distinguished: (a) microsomes and mitochondria; (b) membrane-free polyribosomes; (c) free monomeric ribosomes; and (d) soluble fraction. The fractions were analyzed for RNA and lipid phosphorus, and their particulate components were characterized by electron microscopy. Particular attention was paid to the problem of membrane contamination of the free polyribosome fraction. It was shown that this contamination was small in relation with the total content of ribosomes in the fraction, and that it consisted primarily of smooth-surfaced membranes which were not physically associated with the polyribosomes themselves. In vivo incorporation studies were carried out by injecting tumor-bearing animals intravenously with leucine-C¹⁴, removing the tumors at various times thereafter, and determining the distribution of protein radioactivity among the gradient-separated cytoplasmic fractions. The free polyribosome and the microsome-mitochondria fractions constituted active centers for protein synthesis. It was shown that nascent protein of the free polyribosome fractions was not associated significantly with the contaminating membranes. The kinetics of labeling during incorporation times up to 11 min suggested that protein synthesized on the free polyribosomes was rapidly transferred in vivo to the soluble fraction of the cell, while protein synthesized by the microsomes and mitochondria remained localized within these elements. It was estimated that the free polyribosome fraction and the microsome-mitochondria fraction accounted for approximately equal proportions of the total cytoplasmic protein synthesis in vivo.     

PRODUCTION OF MEMBRANE WHORLS IN RAT LIVER BY SOME INHIBITORS OF PROTEIN SYNTHESIS

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [³H]leucine and of [³H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [³H]leucine incorporation into cytoplasmic membranes was inhibited, while [³H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.     

Three-Dimensional Organization of Polyribosomes–A Modern Approach

Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed tworow forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes. At the same time, modern cell-free translation systems have opened the possibility of investigating polyribosomes on mRNA of a given structure to elucidate the mechanism of polyribosome structure formation, especially of circular polyribosomes. There is an increasing amount of data supporting the idea of interdependence between polyribosome structure and their translational activity. Moreover, participation of polyribosomes in mRNA transport and localization of protein synthesis in the cell has been shown. Improvement of the resolution and the development of the cryo electron tomography technique for the analysis of polyribosomes in situ will enable further progress in understanding the process of protein synthesis in cells.     

Some effects of antibiotics on bacterial polyribosomes as studied by gel electrophoresis

Bacterial polyribosomes possess characteristic electrophoretic mobilities in agarose-acrylamide composite gels. In cells whose normal protein synthesis is inhibited by certain antibiotics, the resolution of the gel electrophoresis technique has permitted the detection of specific increases in the mobility of the polyribosomes. Antibiotics producing these changes in polyribosome mobility include inhibitors of the 30 S as well as the 50 S subunit.The in vivo action of streptomycin has been studied in some detail. Streptomycin alters the polyribosomes of sensitive strains, haploid as well as heterodiploid, but does not alter polyribosomes of strains resistant to or dependent upon streptomycin. Streptomycin-altered polyribosomes are stable in vivo for more than one hour and exhibit a considerably prolonged run-off time following rifampicin treatment. They are also significantly more resistant to the in vitro RNase degradation than control ribosomes. The subunit composition ($\text{50}\text{S}\text{30}\text{S}$) of the altered polyribosomes remains unchanged from the control (1:1).Since the electrophoretic mobility of monosomes remains unchanged during the antibiotic treatment, the evidence presented suggests that the alteration of polyribosome mobility involves a stacking of the ribosomes on mRNA.     

Protein synthesis,polyribosomes, and peptide elongation in early development of Strongylocentrotus purpuratus

The absolute rate of protein synthesis in developing embryos of Strongylocentrotus purpuratus has been measured by lysine incorporation. Protein synthesis rises to about 240 pg hr^?1 embryo^?1 from the two- to eight-cell stage, and then gradually increases to a maximum of over 500 pg hr^?1 embryo^?1 in the blastula. The changes in protein synthesis are accompanied by similar increase in the polyribosomes in the embryo, so that 60–65% of the ribosomes are in polyribosomes by the blastula stage. The data are used to calculate an average peptide elongation rate of 1.8 amino acids ribosome^?1 sec^?1.     

Preferential synthesis of a membrane-associated protein by free polyribosomes (Short Communication)

Isolated free polyribosomes from rat liver appear to synthesize NADPH–cytochome c reductase in vitro four times as efficiently as do membrane-bound polyribosomes. The observed synthesis by the latter could result from contamination with free polyribosomes, but an alternative explanation is also suggested.     

Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

Naturalized soil Escherichia coli populations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soil E. coli strains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day⁻¹) than that of MG1655 (0.85 day⁻¹). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among the E. coli strains. All E. coli strains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman''s ρ = −1.0; P = 0.02). De novo trehalose synthesis was further determined for 15 E. coli strains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. Most E. coli strains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soil E. coli strains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).     

Plant Desiccation and Protein Synthesis: III. Stability of Cytoplasmic RNA during Dehydration, and Its Synthesis on Rehydration of the Moss Tortula ruralis

RNA species from the haploid gametophyte generation of the moss Tortula ruralis exhibit typical eukaryotic characteristics. The major ribosomal and soluble RNA species are stable during drying and rehydration. RNA synthesis occurs rapidly on reintroduction of the moss to water and incorporation into high molecular weight RNA fractions was detected after 20 to 30 minutes of rehydration and into low molecular weight fractions after 30-60 minutes. Newly synthesized ribosomal RNA was detected in ribosomes within 2 hours of rehydration, but not in polysomes. It is apparent that the ribosomal and transfer RNA conserved during desiccation is involved in the re-establishment of early protein synthesis during subsequent rehydration and that, initially, there is no requirement for newly synthesized material.     

Influence of Protoplasmic Water Loss on the Control of Protein Synthesis in the Desiccation-Tolerant Moss Tortula ruralis: Ramifications for a Repair-Based Mechanism of Desiccation Tolerance

Desiccation tolerance of the moss Tortula ruralis is characterized by a desiccation-induced change in gene expression that becomes evident upon rehydration. As reported earlier, this change in gene expression is apparently brought about by a change in the control of translation and does not include a major shift in mRNA abundance. A full qualitative and quantitative analysis of the alteration in gene expression, which is characterized by the loss of (or greater than fivefold decrease in) the synthesis of 25 hydration (h) proteins and initiation (or greater than fivefold increase) of the synthesis of 74 rehydration (r) proteins, is given in this report. Exposure to a desiccating atmosphere, for times that result in varying levels of water loss, enabled the determination that the control of synthesis of r proteins is different from the control of synthesis of h proteins. The r and h protein synthesis responses are internally coordinate, however. Similarly, the return to normal levels of h protein synthesis differs from that of the r proteins. The return to normal synthetic levels for all h proteins is synchronous, but the rate of loss of r protein synthesis varies with each individual r protein. Run-off translation of polysomes isolated from gametophytes during the drying phase demonstrates that there are no novel mRNAs recruited and no particular mRNA is favored for translation during desiccation. These findings add credence to the argument that translational control is the major component of the desiccation-induced alteration in gene expression in this plant, as discussed. Aspects of the response of protein synthesis to desiccation are consistent with the hypothesis that T. ruralis exhibits a repair-based mechanism of desiccation tolerance.