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.     

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 : 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.

     

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.     

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.     

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.     

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.     

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.     

Ribosome-thylakoid association in peas: influence of anoxia

Isolated pea chloroplast thylakoids ordinarily have ribosomes attached which survive sequential washes. Extensive in vivo loss of these thylakoidbound ribosomes occurred if the pea plants were placed in the dark without O₂ for 2 or more hours. This loss was indicated from measurements of both the total thylakoid-bound RNA levels, and the capacity for amino acid incorporation into proteins on the addition of soluble enzymes for protein synthesis. Stroma ribosome profiles lost any indication of polysome structure due to the same anoxic treatment in vivo. The return of ribosomes to the thylakoids when plants were placed in the light in air occurred over an 8-hour time course. This return was prevented by lincomycin, spectinomycin, and chloramphenicol, indicating a requirement for protein synthesis steps in the stroma at some point in the reassociation process.     

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.     

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.     

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.     

Non-Autotrophic CO2 Fixation and Drought Tolerance in Mosses

Cratoneuron filicinum, a drought-sensitive moss, and Tortularuralis, a drought-tolerant moss, fix CO₂ non-autotrophicallyat a rate of about 1.2 and 2.2 µmol h^–1 g^–1dry wt. respectively. During drying, T. ruralis fixes CO₂ atan undiminished rate until the tissue loses about 60% of theinitial fresh weight. Thereafter, CO₂ fixation rapidly declinesto zero. Dark CO₂ fixation by C.filicinum declines steadilyduring the dehydration period. On rehydration, dark CO₂ fixationis resumed immediately in T. ruralis but not in C.filicinum.When dried T. ruralis is equilibrated with an atmosphere ofnearly 100% relative humidity, its weight increases to about40% of the original fresh weight and dark CO₂ fixation resumesat a rate about 60% of the fresh moss. In C.filicinum thereis only a small increase in weight and little CO₂ fixation inthe dark. The non-autotrophically fixed carbon, in both mossesstudied, is incorporated into amino acids (more than 60% ofthe total, mainly into aspartate, alanine and glutamate) andorganic acids (less than 40% of the total, mainly into malate).It is suggested that on rehydration immediate availability ofNADPH, known to be produced by transhydrogenation from NADHduring dark CO₂ fixation, may be an important factor in therepair of drought-induced cellular damage by reductive biosynthesisof membrane components and other cellular constituents. Key words: Mosses, Dehydration, Rehydration, Dark CO₂ fixation, Amino acids, Organic acids, NADPH, Drought tolerance.     

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).     

Rehydration of the Lichen Ramalina lacera Results in Production of Reactive Oxygen Species and Nitric Oxide and a Decrease in Antioxidants

Lichens are slow-growing associations of fungi and unicellular green algae or cyanobacteria. They are poikilohydric organisms whose lifestyle in many cases consists of alternating periods of desiccation, with low metabolic activity, and hydration, which induces increase in their metabolism. Lichens have apparently adapted to such extreme transitions between desiccation and rehydration, but the mechanisms that govern these adaptations are still poorly understood. In this study, the effect of rehydration on the production of reactive oxygen species and nitric oxide as well as low-molecular-weight antioxidants was investigated with the lichen Ramalina lacera. Rehydration of R. lacera resulted in the initiation of and a rapid increase in photosynthetic activity. Recovery of photosynthesis was accompanied by bursts of intracellular production of reactive oxygen species and nitric oxide. Laser-scanning confocal microscopy using dichlorofluorescein fluorescence revealed that formation of reactive oxygen species following rehydration was associated with both symbiotic partners of the lichen. The rate and extent of reactive oxygen species production were similar in the light and in the dark, suggesting a minor contribution of photosynthesis. Diaminofluorescein fluorescence, indicating nitric oxide formation, was detected only in fungal hyphae. Activities associated with rehydration did not have a deleterious effect on membrane integrity as assessed by measurement of electrolyte leakage, but water-soluble low-molecular-weight antioxidants decreased significantly.     

Reduction of Adenosine Triphosphate Levels in Susceptible Maize Mesophyll Protoplasts by Helminthosporium maydis Race T Toxin

Helminthosporium maydis race T (HMT) toxin caused a reduction in the steady-state ATP levels when leaf mesophyll protoplasts isolated from maize containing Texas male-sterile (T) but not male-fertile (N) cytoplasm were incubated in the dark. At a toxin concentration 10 times the mean effectived dose for inhibition of root growth, the ATP levels began to fall in 30 to 90 seconds, fell by 50% in about 4 minutes, and reached 23% of the original levels in 100 minutes. This is faster than any previously observed response of whole cells or tissues to HMT toxin. In protoplasts incubated in the light, ATP levels were 25% higher than in the dark and were either unaffected or only slightly diminished by toxin. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, overcame the effect of light on both toxin-treated and control protoplasts. Oligomycin, an inhibitor of mitochondrial ATP synthesis, mimicked the effects of toxin in the dark, in the light, and in the light plus DCMU, but it was not specific for T cytoplasm. During the first 24 hours of culture, ATP levels in control protoplasts increased in both the light and dark. In the dark, ATP was not detectable after 24-hour incubation in the presence of toxin, whereas in the light a substantial amount of ATP remained. Our results are compatible with the hypothesis that mitochondria in vivo are inhibited by HMT toxin. Other responses of cells and tissues to toxin can be explained in terms of reduced ATP levels.     

Polysome Formation in Light-controlled Dormancy

Lettuce (Lactuca sativa) seeds var. Grand Rapids could be maintained many weeks in the dark without germination. Following illumination with white light, a gradual increase in polyribosome population up to the time of germination was demonstrated by sucrose gradient centrifugation. Polysomes could not be detected in imbibed seeds maintained continuously in the dark. Thus, polysome formation and therefore the capacity for a high rate of protein synthesis required for germination and growth, is not associated with the process of imbibition, but is dependent upon the seeds having received the dormancy-breaking stimulus of illumination.     

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.     

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