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.     

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.     

Poly(A)+ RNA synthesis in germinating pollen ofNicotiana tabacum L

Poly(A)⁺RNA is synthesized during the first hours of pollen germination and is rapidly incorporated into polysomal structures. After a 2-h pulse with uracil-¹⁴C, 42% of the transcribed fraction of polysomal RNA is polyadenylated. Following 4 h of germination the amount of the newly-made poly(A)⁺RNA decreases steadily at the rate of about 14% per h, whereas that of rapidly-labelled poly(A)⁻RNA continues to grow. Beginning 1 h of cultivation the ratio of poly(A)⁻/poly(A)⁺RNA increases exponentially. Similarly as in non-polyadenylated mRNA the main portion of the synthesized polysomal poly(A)⁺RNA sediments at a rate of 4 to 14 S and its mean size decreases slightly with the time of labelling. RNA isolated from nuclei and cell wall containing pollen tube fraction differed from the polysomal one in higher apeoific radioactivity and the polyadenylated RNA exhibited higher size distribution. The comparison of the results with earlier observations suggests the involvement of poly(A)in mRNA translation in pollen tubes.     

Polyribosomes Conserved during Desiccation of the Moss Tortula ruralis Are Active

During desiccation of the moss Tortula ruralis (Hedw.) (Gaertn, Meyer and Scherb) polyribosomes are conserved. On rehydration, protein synthesis is rapidly resumed. In the presence of protein synthesis initiation inhibitors ribosome run-off from the conserved polyribosomes takes place, confirming that these retain their activity as intact structures during desiccation.     

Stability and Synthesis of Phospholipids during Desiccation and Rehydration of a Desiccation-Tolerant and a Desiccation-Intolerant Moss

The fatty acid composition of the phospholipids from the desiccation-tolerant moss Tortula ruralis (Hedw.) Gaertn, Meyer and Scherb and the desiccation-intolerant moss Cratoneuron filicinum has been determined. No changes in composition occur in either moss as a consequence of rapid drying, but, after slow drying, there is a decline in some unsaturated fatty acids. Upon rehydration of T. ruralis after slow drying, these acids decline further; however, within 105 minutes, they regain the same levels as those in undesiccated controls. A smaller and more transient decline occurs after rapid desiccation. Most phospholipid unsaturated fatty acids decrease during rehydration of C. filicinum, and their levels are not recovered. After both rapid and slow drying of T. ruralis, acetate and glycerol are incorporated into the phospholipid fraction, although de novo synthesis, alone, might not account for the increase in unsaturated fatty acids upon rehydration. Very little acetate or glycerol is incorporated during rehydration of C. filicinum. Loss of unsaturated fatty acids from the phospholipids of T. ruralis does not appear to be associated with increased lipoxygenase activity. Furthermore, there is little correlation between the extent of peroxidation of fatty acids due to desiccation and changes in the phospholipid fraction.     

Reprogramming of Protein Synthesis from a Developmental to a Germinative Mode Induced by Desiccation of the Axes of Phaseolus vulgaris

Immature seeds of Phaseolus vulgaris cv Taylor's Horticultural removed from the pod at 32 days of development do not germinate unless first subjected to desiccation. Our results show that premature drying not only redirects metabolism from a developmental to a germination program but it does so permanently, thus effecting an irreversible switch. This is shown by in vitro protein synthesis, and analysis of poly(A)⁺ mRNA with a cDNA probe specific for phaseolin message. For example, the pattern of proteins synthesized in vitro by the mRNA fraction from fresh and prematurely dried axes show strong similarities; on the other hand, the mRNA population from rehydrated axes code for a different set of proteins. Also, the message for phaseolin is preserved following the normal maturation process and premature desiccation of seeds. Following rehydration of immature seeds at the desiccation-tolerant stage, this message is no longer detectable in the axes.     

Membrane Organization of the Desiccation-Tolerant Moss Tortula ruralis in Dehydrated States

Membrane organization of the desiccation tolerant moss Tortula ruralis was studied in several intensely dehydrated states (75% relative humidity [RH], 90% RH, plasmolysis in molar salt, freezing to −20°C) by ³¹P nuclear magnetic resonance and ultrastructural analyses. Both methods revealed that even at 75% RH (−400 bars), the moss cellular membranes retained extended phospholipid bilayers. Ultrastructural analyses of the fully hydrated moss showed an extensive proliferation of membrane vesicles in the endoplasmic reticulum. During dehydration, these vesicles form layers of membrane under the plasmalemma and in some cases appear to fuse with the surface membrane. This suggests that these vesicles may serve as a reservoir of membranes to accommodate for membrane surface area changes during desiccation and subsequent rehydration.     

Protein Synthesis during Rehydration of Rapidly Dried Tortula ruralis: Evidence for Oxidation Injury

Rapidly dried Tortula ruralis, a drought-tolerant moss, is known to synthesize proteins on rehydration at a much lower rate than the slowly dried moss. The reasons for this low rate of protein synthesis are unclear. We have found that during rehydration of rapidly dried moss, there is a negative correlation between the rate of protein synthesis and the tissue levels of oxidized glutathione (GSSG) and lipid peroxidation. When rapidly dried moss, which is known to show extensive solute leakage, is rehydrated in the presence of 100 millimolar K⁺, 5 millimolar Mg²⁺, 1 millimolar ATP, and 1 millimolar GTP, either separately or together, there is no stimulation of protein synthesis. When it is hydrated in the presence of either 5 millimolar glucose-6-phosphate or 0.1 millimolar NADPH, protein synthesis is stimulated but the stimulation is transitory. A second addition of either of these two chemicals causes a second transient stimulation of protein synthesis. A transitory decrease in the rate of GSSG accumulation is observed during rehydration in the presence of glucose-6-phosphate or NADPH. Both glucose-6-phosphate and NADPH are known to reverse GSSG-induced inhibition of protein synthesis in rabbit reticulocyte lysate. Results of the present study suggest that the rate of protein synthesis during rehydration of rapidly dried moss is not limited by the availability of ions or energy sources. Since exogenously applied GSSG has been shown to inhibit in vivo and in vitro protein synthesis and since it is known to accumulate during rehydration of rapidly dried, but not slowly dried, moss, it is suggested that the low rate of protein synthesis during rehydration of the rapidly dried moss is, at least in part, due to endogenous GSSG.     

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.     

Respiration in Relation to Adenosine Triphosphate Content during Desiccation and Rehydration of a Desiccation-tolerant and a Desiccation-intolerant Moss

O₂ consumption by the desiccation-tolerant moss Tortula ruralis and the desiccation-intolerant Cratoneuron filicinum increased markedly during the latter stages of desiccation. ATP content of the mosses during desiccation was not correlated with O₂ consumption, but was influenced by the rate at which the mosses lost water. The more rapid the water loss, the more ATP that was present in the dry mosses. The pattern of O₂ consumption on rehydration also was influenced by the previous rate of desiccation. After rapid desiccation of T. ruralis O₂ consumption upon rehydration was considerably elevated, and for up to 24 hours. After very slow desiccation the elevation was small and brief. Normal O₂ consumption did not occur in C. filicinum after rapid desiccation, but did so within a few hours of rehydration after slower speeds of drying. ATP levels in T. ruralis returned to normal within 5 to 10 minutes of rehydration. In C. filicinum, increases in ATP were closely correlated with O₂ consumption. These observations are considered to be related to differential damage caused to mitochondria and to cellular integrity by different speeds of water loss. The desiccation-tolerant moss appears to be able to repair the severe damage imposed by rapid desiccation whereas the desiccation-intolerant moss cannot.     

Poly(A+)mRNA sequences in free mRNP and polysomes of mouse ascites cells

The poly(A⁺)RNA of the free mRNP of mouse Taper ascites cell contains a very reduced number of different mRNA sequences compared to the polysome poly(A⁺)RNA. By the technique of mRNA:cDNA hybridization we have determined that the free mRNP contains approximately 400 different mRNA sequences while the polysomes contain about 9000 different mRNAs. The free mRNP poly(A⁺)RNA sequences are present in two abundance classes, the abundant free mRNP class containing 15 different mRNA sequences and the less abundant free mRNP class containing 400 different mRNAs. The polysome poly(A⁺)RNA consists of three abundance classes of 25, 500 and 8500 different mRNA sequences.Despite its intracellular location in RNP structures not directly involved in protein synthesis the poly(A⁺)RNA purified from the free RNP of these cells was a very effective template for protein synthesis in cell-free systems. Cell-free translation products of free mRNP and polysome poly(A⁺)RNAs were analyzed by two-dimensional gel electrophoresis. This analysis confirmed the hybridization result that the free mRNP poly(A⁺)RNA contained fewer sequences than polysomal poly(A⁺)RNA. The abundant free RNP-mRNA directed protein products were a subset of the polysome mRNA-directed protein products. The numbers of more abundant products of cell-free protein synthesis directed by the free RNP-mRNA and polysomal mRNA were in general agreement with the hybridization estimates of the number of sequences in the abundant classes of these two mRNA populations.     

Plant Desiccation and Protein Synthesis: II. On the Relationship between Endogenous Adenosine Triphosphate Levels and Protein-synthesizing Capacity

Rehydration of Tortula ruralis in 2,4-dinitrophenol inhibits protein synthesis, polysome formation, and ATP production. Polysomes are conserved intact and are active in vitro in hydrated Tortula placed in this chemical, although in vivo protein synthesis is inhibited. Hydrated moss placed under nitrogen in the dark shows a reduced capacity for ATP and protein synthesis, but polysomes are conserved. During anaerobiosis in light, ATP and protein synthesis are unaffected. Rehydration of slow-dried Tortula in nitrogen in the dark results in reduced in vivo protein synthesis, but not polysome formation; this reduction is much less in the light. Slow-dried moss, but not fast-dried, has a greatly reduced ATP content in the dry state, but this rapidly returns to normal levels on rehydration. The prolonged burst in respiration observed previously on rehydration of Tortula is not paralleled by ATP accumulation. Changes in energy charge in all treatments tested follow the changes in ATP. The aquatic moss, Hygrohypnum luridum, which is intolerant to drought, loses ATP during fast drying and this is not replenished on subsequent rehydration.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

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.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

Poly(A)+ RNA metabolism during oogenesis in Xenopus laevis.

In this study, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA [poly(A)⁺ RNA] in Xenopus laevis oocytes at the maximal lampbrush chromosome stage (stage 3) and at the completion of oocyte growth (stage 6). Oocytes at both stages are shown to be active in the synthesis of poly(A)⁺ RNA. In stage 6 oocytes, the mean rate of synthesis of stable poly(A)⁺ RNA is 15% the instantaneous rate of synthesis, while the mean half-life of the unstable component is 1.6 hr. In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Studies on the nuclear versus the cytoplasmic distribution of the newly synthesized poly(A)⁺ RNA demonstrated that by the end of a 12-hr labeling period for stage 3 oocytes and a 24-hr labeling period for stage 6 oocytes, approximately half of the material was cytoplasmic. This cytoplasmic material had the same electrophoretic mobility as bulk poly(A)⁺ RNA. Similarly, as with bulk poly(A)⁺ RNA, little, if any, of the newly synthesized material was found to be polysomal. Also, poly(A) labeling studies indicated that the newly synthesized poly(A)⁺ RNA was associated with the synthesis of poly(A) of the same length as that appearing on bulk poly(A)⁺ RNA. Studies on the content of bulk oligo(dT)cellulose-bound RNA indicated that about 86 ng is present in both stage 3 and stage 6 oocytes. The continual synthesis of poly(A)⁺ RNA throughout oogenesis in the absence of its accumulation led to the conclusion that it must be turning over. These data are discussed in relation to the hypothesis that bulk levels of poly(A)⁺ RNA are maintained by continually changing rates of synthesis and degradation.     

Characterization of poly(A+)RNA in free messenger ribonucleoprotein and polysomes of mouse Taper ascites cells

Cytoplasmic extracts of mouse Taper ascites cells were centrifuged on sucrose gradients to give 0–80 S, monosome, and polysome fractions. CsCl equilibrium density centrifugation of formaldehyde-fixed material from the 0–80 S fraction demonstrated that the messenger RNA in the 0–80 S fraction was in the form of free ribonucleoprotein. The size of the poly(A⁺)RNA and the size of the poly(A) segments of these molecules were shown to be very similar in both the free mRNP² and polysome fractions. The labeling kinetics of the free mRNP poly(A⁺)RNA was similar to that of the polysomal poly(A⁺)RNA.The free mRNP poly(A⁺)RNA efficiently stimulated protein synthesis in the wheat germ cell-free system, supporting the view that it was mRNA. Two-dimensional gel electrophoresis was used to analyze the proteins whose synthesis was directed by free mRNP and polysomal poly(A⁺)RNA. The free mRNP poly(A⁺)RNA directed the synthesis of a simpler set of abundant protein products than did the polysomal poly(A⁺)RNA. Most of the free mRNP abundant protein products were also present in the polysomal products, though obvious quantitative differences were evident, indicating that each individual mRNA had its own characteristic distribution between polysomes and the translationally inactive RNP form.     

ABA Increases the Desiccation Tolerance of Photosynthesis in the Afromontane Understorey Moss Atrichum androgynum

The effect of pretreatment with abscisic acid (ABA) on the physiologyof the moss Atrichum androgynum during a desiccation–rehydrationcycle was examined. During rehydration following desiccationfor 16 h, net CO₂fixation recovered much more slowly than photosystemII (PSII) activity, conditions conducive to the formation ofreactive oxygen species (ROS) in the photosynthetic apparatus.Pretreatment with ABA increased the rate of recovery of photosynthesisand PSII activity, and also doubled non-photochemical quenching(NPQ). Increased NPQ activity will reduce ROS formation, andmay explain in part how ABA hardens the moss to desiccation.In ABA-pretreated, but not untreated mosses, desiccation significantlyincreased the concentration of soluble sugars. Sugar accumulationmay promote vitrification of the cytoplasm and protect membranesduring desiccation. Starch concentrations in freshly collectedA. androgynum were only approx. 40 mg g^-1dry mass; they roseslightly during desiccation but were only slightly affectedby ABA pretreatment. ABA did not reduce chlorophyll breakdownduring desiccation. Copyright 2001 Annals of Botany Company Moss, desiccation, abscisic acid, photosynthesis, chlorophyll fluorescence     

CHARACTERIZATION OF POLY(A) SEQUENCES IN BRAIN RNA

—Nuclear and polysomal brain RNA from the rabbit bind to Millipore filters and oligo(dT)-cellulose suggesting the presence of poly(A) sequences. The residual polynucleotide produced after RNase digestion of ³²P pulse-labelled brain RNA is 95% adenylic acid and 200-250 nucleotides in length. After longer isotope pulses the polysomal poly(A) sequence appears heterodisperse in size and shorter than the nuclear poly (A). Poly(A) sequences of brain RNA are located at the 3′-OH termini as determined by the periodate-[³H]NaBH₄ labelling technique. Cordycepin interferes with the processing of brain mRNA as it inhibits in vivo poly(A) synthesis by about 80% and decreases the appearance of rapidly labelled RNA in polysomes by about 45%. A small poly(A) molecule 10-30 nucleotides in length is present in rapidly labelled RNA. It appears to be less sensitive to cordycepin than the larger poly(A) and is not found in polysomal RNA.     

Use of a cloned library for the study of abundant poly(A)+RNA during Xenopus laevis development

Over 200 cloned sequences from recombinant DNA libraries prepared from Xenopus laevis embryonic poly(A)⁺RNA have been analyzed by colony hybridization with [³²P]cDNA prepared from poly(A)⁺RNA from several stages of development. The period of early embryogenesis extending through the beginning of gastrulation (stage 10) is marked by the relative constancy of the abundant poly(A)⁺RNA population. Between the gastrula and tailbud stages (stage 24) there is a dramatic change in the pattern of abundant poly(A)⁺RNA species; the new pattern remains fairly constant for at least 2 days of development to the late prefeeding tadpole stages (stage 41). We have also compared nonpolysomal and polysomal poly(A)⁺RNA populations at two different stages. In stage 10 (early gastrula) postribosomal (free ribonucleoprotein) and polysomal poly(A)⁺RNA populations partly overlap; however, many cloned sequences occur in quite different concentrations in one fraction or the other. Among the sequences that are predominantly nonpolysomal at gastrula few become predominantly polysomal at tailbud stages. Thus, we have no evidence for a major recruitment of abundant nonpolysomal RNAs into polysomes with progressing development. We rather observe a general pattern in which a cloned sequence that is nonpolysomal in one stage of development tends to be nonpolysomal (if detectable at all) in other stages as well.