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.     

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.     

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.     

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.     

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.     

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.

     

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.     

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.     

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.     

Glutathione Status and Protein Synthesis during Drought and Subsequent Rehydration in Tortula ruralis

Glutathione status and its relationship to protein synthesis during water deficit and subsequent rehydration have been examined in the drought-tolerant moss, Tortula ruralis. During slow drying there is a small decrease in total glutathione but the percentage of oxidized glutathione (GSSG) increases. During rapid drying there is little change in total glutathione but a small increase in GSSG. On rehydration of slowly dried moss, GSSG rapidly declines to normal level. But when rapidly dried moss is rehydrated, there is an immediate, sharp increase in GSSG as a percentage of total glutathione. After 2 hours of rehydration GSSG starts declining and reaches a normal level in about 6 hours. When an increasing degree of steady state water deficit is imposed on the moss tissue with polyethylene glycol 6000, there is a progressive decrease in protein synthesis but an increase in oxidized glutathione. When 5 millimolar GSSG is supplied exogenously during rehydration of rapidly dried or slowly dried moss, protein synthesis is strongly inhibited. In vitro protein synthesis supported by moss mRNA is also inhibited by more than 85% by 150 micromolar GSSG. The role of glutathione status in water deficit-induced inhibition of protein synthesis is discussed.     

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.     

Protein Synthesis Related to Cold Temperature Stress in the Desiccation-Tolerant Moss Tortula ruralis

Incubation of hydrated Tortula ruralis (Hedw.) Gaertn., Meyer. Scherb. at temperatures down to 2°C resulted in an accumulation of polyribosomes and a decrease in single ribosomes. No changes in the levels of ribosomal subunits were detected. On rehydration of slowly dried moss, which contains no polyribosomes, these were reormed at 2, 8 and 20°C. Rapid incorporation of labelled leucine into protein was observed on reintroduction of the desiccated plant o water at 20°C and there was significant, but much reduced, ncorporation at 2°C. Previously undesiccated moss was also able o take up radioactive leucine and to synthesize protein at 2 and -2.5°C. Changes in the rate of protein synthesis at low temperature were not detected in cold hardened (winter collected or incubated at 2°C) T. ruralis. The moss appears to be adapted to survive freezing wear round and even summer-collected moss can conduct protein synthesis at low temperatures: seasonal cold hardiness changes do lot appear to take place.     

Drought Stress, Enzymes of Glutathione Metabolism, Oxidation Injury, and Protein Synthesis in Tortula ruralis

The activities of glutathione reductase (EC 1.6.4.2), glutathione peroxidase (EC 1.11.1.9), and glutathione S-transferase (EC 2.5.1.18) were found to increase during slow drying or during rehydration following rapid drying of the drought-tolerant moss Tortula ruralis. Little change was observed in the activity of malate deydrogenase (NAD⁺ oxidoreductase, EC 1.1.1.37) during dehydration or subsequent rehydration. When the tissue was treated with cycloheximide, actinomycin D, or cordycepin, the increase in the activities of glutathione reductase and glutathione S-transferase was largely prevented while effect on glutathione peroxidase was much smaller. Concomitantly, oxidized glutathione (GSSG) as percentage of total glutathione increased. GSSG level was correlated positively with the levels of lipid peroxidation and solute leakage and negatively with the rate of protein synthesis. The results show that GSSG level is a good indicator of oxidation stress and provide support to the suggestion that GSSG mediates, at least in part, the drought stress-induced inhibition of protein synthesis.     

Characterization of Protein Synthetic Changes in a Desiccation-Tolerant Fern, Polypodium virginianum. Comparison of the Effects of Drying, Rehydration and Abscisic Acid

The desiccation-tolerant fern, Polypodium virginianum, was ableto lose more than 60% of its fresh weight during periods ofprolonged water stress and fully recover during periods of wateravailability. Rehydration from the air-dry state occurred rapidly(within 24 h) with a concurrent initiation of protein synthesis.A low-molecular-weight doublet was synthesized during waterstress and was stable for at least 24 h after rehydration. Theseproteins were expressed also when the fully hydrated frondswere subjected to exogenous ABA even though drying resultedin a 10-fold reduction in ABA content. The large subunit ofRubisco was synthesized during drying, but despite temporaryaccumulation, the de novo synthesized component was degradedduring rehydration. During rehydration from the air-dry state,unique rehydration-specific polypeptides were produced. Thesepolypeptides were synthesized at different water contents andtimes of rehydration, and were transiently expressed. This transientexpression may mean that they are only necessary during theearly stages of rehydration when the rapid initiation of physiologicaland repair processes is essential. The response of this fernto desiccation has features which are similar to those reportedfor desiccation-tolerant mosses, and others which are reminiscentof desiccation-tolerant angiosperms. Key words: Desiccation-tolerance, fern, Polypodium virginianum, protein synthesis, abscisic acid     

Drought Tolerance in Two Mosses: Correlated with Enzymatic Defence Against Lipid Peroxidation

Drought-induced changes in the activities of superoxide dismutase(SOD) and catalase, level of lipid peroxidation, and membranepermeability (solute leakage) have been studied in two mosses,the drought-tolerant Tortula ruralis and the drought-sensitiveCratoneuron filicinum. In T. ruralis the activities of SOD andcatalase increase during slow drying. The level of lipid peroxidationconsequently declines. On subsequent rehydration the enzymeactivities decline and the level of lipid peroxidation risesgradually to normal levels. The leakage of preloaded ⁸⁶Rb onrehydration of slowly dried T. ruralis is similar to that inturgid moss, i.e. leakage of about 20% of tissue ⁸⁶Rb. WhenT. ruralis is subjected to rapid drying there is no change inthe enzyme activities or in lipid peroxidation. However, whenthis moss is rehydrated there is a large immediate increasein lipid peroxidation. Half of the tissue ⁸⁶Rb is leaked intothe bathing medium during the first hour of rehydration. Butwithin the next hour, when SOD and catalase activities haveincreased to high levels, lipid peroxidation quickly declinesto a level lower than that in the turgid control moss, and the⁸⁶Rb leaked earlier is partly reabsorbed indicating that membranerepair is well underway. On prolonged rehydration the enzymeactivities decline and the level of lipid peroxidation risesgradually to reach normal levels found in control turgid moss.In the case of drought-sensitive C. filicinum the activitiesof SOD and catalase decline during drying as well as duringsubsequent rehydration. There is a rapid increase in lipid peroxidationduring rehydration and most of the preloaded ⁸⁶Rb leaks intothe bathing medium irreversibly. The changes in lipid peroxidationduring drying and subsequent rehydration of both the mossesappear to coincide in time with the reported changes in O₂ uptake,indicating that the drought-induced membrane damage may be dueto free radical-induced lipid peroxidation which is known torequire active O₂ uptake. Furthermore, there appears to be agood correlation between an ability of the tissue to controllipid peroxidation and its ability to retain solutes. It issuggested that ability of plant tissues to mobilize enzymaticdefence against uncontrolled lipid peroxidation may be an importantfacet of their drought tolerance.     

On the Metabolism of Tortula ruralis Following Desiccation and Freezing: Respiration and Carbohydrate Oxidation

Recovery from desiccation by Tortula ruralis (Hedw.) Gaertn., Meyer and Scherb was accompanied by an immediate, rapid increase in respiration (measured as oxygen uptake) at 25.5°C or 3.5°C. The respiratory burst was greater on rehydration of moss which had been rapidly desiccated over silica gel than that which had been more slowly desiccated in atmospheres of high relative humidity. No respiration was observed in dry moss. Dried moss which had been placed in liquid nitrogen resumed respiration on rewarming and rehydration but moss which had been frozen in the hydrated state respired to a lesser extent and showed signs of freeze damage. In the initial stages of slow drying a slight increase in respiration was noted, followed by a gradual decrease as drought became more severe. In contrast to observations made on many higher plants under drought stress, this moss did not exhibit any changes in its starch and sugar content during or following desiccation, nor were there any changes in free proline levels. Using (1-¹⁴C)-glucose and (6-¹⁴C)-glucose, the relative activities of the Embden–Meyerhof–Parnas and pentose phosphate pathways in hydrated and rehydrated moss were determined, as were the activities of specific enzymes involved in these pathways. An increased activity of the Embden–Meyerhof–Parnas pathway of glucose oxidation on rehydration of Tortula was observed. The possible significance of this latter observation is outlined.     

The Conservation of Polyribosomes in the Moss Tortula ruralis during Total Desiccation

Total desiccation of the moss Tortula ruralis was achieved byplacing it in a dry atmosphere for 90 min. Reintroduction ofthe moss to water resulted in the recovery of its normal morphologicalform within 15–30 s. The sedimentation profile on a sucrosegradient of the ribosomal content of the totally dry moss showsthe presence of distinct polyribosomal peaks. The levels ofthese polyribosomes rise upon rehydration of the moss. The differencebetween the tolerance to water deficit by this moss and by higherplants is outlined.     

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.     

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).