Endurance of the endolithic desert cyanobacterium Chroococcidiopsis under UVC radiation

Desert cyanobacteria of the genus Chroococcidiopsis are extremely resistant to desiccation and ionizing radiation. When an endolithic strain was exposed to UVC radiation cell lysis, genome damage, photosynthetic pigment bleaching and reduced photochemical performance occurred. Nevertheless, survivors were scored after UVC doses as high as 13?kJ/m² and their endurance ascribed to multicellular aggregates enveloped in thick envelopes, so that attenuated UVC radiation reached the inner cells. In addition, the accumulation of carotenoids contributed to UVC resistance by providing protection against oxidative stress. Finally, in survivors repair mechanisms were responsible for the recovery of the induced damage to genome and photosynthetic apparatus.     

Ultrastructural analysis of the rehydration of desiccatedNostoc commune HUN (cyanobacteria) with particular reference to the immunolabelling of NifH

Summary Vegetative cells and heterocysts of the filamentous desiccation-tolerant cyanobacteriumNostoc commune HUN retain their ultrastructural organisation and the integrity of their intra- and extracellular membranes after two years of desiccation and subsequent rehydration. Immunogold-labelling of thin sections demonstrated the presence of NifH (Fe protein of nitrogenase) in vegetative cells and heterocysts within five minutes of the rehydration of dried colonies. Immunogold label accumulated in discrete areas vegetative cells within 5 minutes of the rewetting of cells, and after 30 minutes a conspicuous association of NifH protein with heterocyst ribosomes was detected. After longer periods of rehydration, the deposition of gold particles became more random within both cell types but occurred with a greater frequency in heterocysts. Up to 24 hours after the rewetting of cells, two morphologically-distinct forms of heterocyst could be discerned. NifH protein was detected through Western blotting (subunit M_r=33,800) in protein extracts from samples ofNostoc commune, collected in different parts of the world and including some which had been desiccated for periods of up to 10 years. The results are discussed in relation to the sequential recovery of metabolic functions, particularly nitrogen fixation, which occurs upon the rehydration of cells after their prolonged storage in the air-dry state.     

Avoidance of protein oxidation correlates with the desiccation and radiation resistance of hot and cold desert strains of the cyanobacterium Chroococcidiopsis

To investigate the relationship between desiccation and the extent of protein oxidation in desert strains of Chroococcidiopsis a selection of 10 isolates from hot and cold deserts and the terrestrial cyanobacterium Chroococcidiopsis thermalis sp. PCC 7203 were exposed to desiccation (air-drying) and analyzed for survival. Strain CCMEE 029 from the Negev desert and the aquatic cyanobacterium Synechocystis sp. PCC 6803 were further investigated for protein oxidation after desiccation (drying over silica gel), treatment with H₂O₂ up to 1 M and exposure to γ-rays up to 25 kGy. Then a selection of desert strains of Chroococcidiopsis with different survival rates after prolonged desiccation, as well as Synechocystis sp. PCC 6803 and Chroococcidiopsis thermalis sp. PCC 7203, were analyzed for protein oxidation after treatment with 10 and 100 mM of H₂O₂. Results suggest that in the investigated strains a tight correlation occurs between desiccation and radiation tolerance and avoidance of protein oxidation.

     

Shifts in the intracellular ATP pools of immobilisedNostoc cells (Cyanobacteria) induced by water stress

Summary Immobilised, desiccated cells ofNostoc commune UTEX 584 have the capacity to increase the size of their extractable intracellular ATP pool upon rewetting. The time taken to recover the pool size depends on the conditions of storage at a particular water potential and the duration of storage. Under the conditions employed, the rewetting of cells induced an increase in ATP pool size at the expense of photophosphorylation or electron transport (oxidative) phosphorylation. The rise in the ATP pool size was instantaneous and was shown to be due to ATP synthesis. This increase did not occur when cells were rewetted in the presence of sodium azide (10 mmol/l), while a partial inhibition was observed with CCCP (carbonyl cyanidem-chlorophenylhydrazone; 2 mol/l). For cells dried at more extreme water potentials, the lag ofc 48 h observed before the ATP pool reached control values is of similar duration to that observed in the recovery of nitrogenase upon rewetting. Chloramphenicol (10 mol/l) stimulated significantly the upshift in the size of the ATP pool ofNostoc cells upon rewetting, yet inhibited completely the rise in nitrogenase activity.     

The impact of dehydration rate on the production and cellular location of reactive oxygen species in an aquatic moss

Background and Aims

The aquatic moss Fontinalis antipyretica requires a slow rate of dehydration to survive a desiccation event. The present work examined whether differences in the dehydration rate resulted in corresponding differences in the production of reactive oxygen species (ROS) and therefore in the amount of cell damage.

Methods

Intracellular ROS production by the aquatic moss was assessed with confocal laser microscopy and the ROS-specific chemical probe 2,7-dichlorodihydrofluorescein diacetate. The production of hydrogen peroxide was also quantified and its cellular location was assessed.

Key Results

The rehydration of slowly dried cells was associated with lower ROS production, thereby reducing the amount of cellular damage and increasing cell survival. A high oxygen consumption burst accompanied the initial stages of rehydration, perhaps due to the burst of ROS production.

Conclusions

A slow dehydration rate may induce cell protection mechanisms that serve to limit ROS production and reduce the oxidative burst, decreasing the number of damaged and dead cells due upon rehydration.     

Carbohydrate formation in rewetted terrestrial cyanobacteria

Summary In the terrestrial cyanobacterium Nostoc commune Vauch. formation of carbohydrate polymers was measured upon rewetting the mats in a light-dark regime. To discriminate between carbohydrates of different physiological function, total carbohydrate was determined as anthrone-reactive material (ARM) and storage carbohydrate (glycogen) assayed by an enzymic test. In the dry thalli glycogen was found to represent less than one tenth of the ARM. After rewetting an increase of total carbohydrate was observed in illuminated samples. Only glycogen, however, showed a regular pattern of synthesis and degradation during a 12:12 h light-dark cycle. This indicates that most carbohydrates detected by anthrone belong to the metabolically inert sheath material.When illuminated colonies were kept submerged after rewetting glycogen was hydrolyzed indicative of being used in the rapid recovery of cellular functions as observed in rewetted colonies. Apparently, photosynthesis allowed for net glycogen synthesis only, provided the mats were sufficiently aerated. These findings give evidence that the (carbohydrate) sheath plays an important role in water retention in an organism bound to a terrestrial habitat.     

The effect of soil desiccation on the nutrient status of Eucalyptus regnans F. Muell seedlings

The poor growth of young Eucalyptus regnans seedlings in undried soil from the mature forest of E. regnans can be overcome by previously air-drying the soil or by adding sufficient amounts of complete soluble fertilizer or equivalent concentrations of P (as NaH₂PO₄) and N (as NaNO₃). A factorial pot experiment in which phosphate and nitrate were added to undried soil indicated that P was the primary deficiency for young seedlings and that response to N did not occur until this lack was satisfied. In dried soil, seedlings also responded to additions of complete fertilizer but most of this effect was due to N rather than P. Field trials in the mature forest also indicated greater growth in dried soil than undried soil and confirmed a response of young seedlings to superphosphate. In pot experiments, the concentration of P and N per g plant dry weight after four months was relatively constant irrespective of the final size of the plant. Seedlings in dried soil extracted up to 15 times more P than did those grown in undried soil. In general, chemical analysis of soil indicated more extractable P and N from dried soil although this was not always consistently so. Soil desiccation resulted in an increase in soil surface area due to the fragmentation of larger peds and to an increase in the number of microfractures which remained in the soil crumbs after rewetting. Mycorrhiza are likely to be important since the differentiation of the growth response of seedlings in dried and undried soil, which occurred at 5–6 weeks, corresponded with the establishment of full ectomycorrhizal development (80% root tips). The factors concerned with the increase in fertility after air-drying are discussed.Abbreviations GR Growth Ratio     

Preferential distribution of excitation energy into photosystem I of desiccated samples of the lichen Cladonia impexa and the isolated lichen-alga Trebouxia pyriformis

Abstract The effect of desiccation on distribution of excitation energy between the two photosystems has been studied in the lichen Cladonia impexa Harm., in the green alga Trebouxia pyriformis Archibald, isolated from Cladonia squamosa; and in the non-lichen green alga Scenedesmus obliquus, strain D₃. The method used was to compare the low temperature fluorescence emission of samples equilibrated with air with different humidity prior to freezing in liquid nitrogen. Desiccation of Cladonia and Trebouxia caused a pronounced increase of the height of the far red fluorescence emission band, F 715, over the short wave bands, F685 and F697; the ratio between the two short wave bands remained essentially constant. Upon rewetting, these species regained normal fluorescence emission properties, indicating that they are desiccation-tolerant. Scenedesmus, which was used as a desiccation intolerant species, also showed an increase of the far red fluorescence band over the two short wave bands upon desiccation, but the original fluorescence spectrum was not restored upon rewetting. These results are interpreted as showing that desiccation of tolerant species such as Cladonia and Trebouxia causes a preferential energy distribution into photosystem I. We tentatively believe that desiccation induces conformational changes within the chloroplast thylakoids, thereby controlling distribution of energy between the two photosystems. Furthermore, this change in energy distribution may be of ecological significance as the mechanism by which desiccated lichens or algae avoid photo-dynamic destruction of the photosynthetic apparatus when photosynthesis is inhibited under dry conditions. By a preferential distribution of absorbed energy into photosystem I, the organisms avoid the formation of strong, harmful oxidants in photosystem II when photosynthesis is inhibited. It is suggested that β-carotene associated with the far red-absorbing chlorophyll a fraction of the reaction center antenna of photosystem I is the final sink for excess excitation energy in dry, desiccation-tolerant lichens and algae.     

Post-translational modification of RNA m6A demethylase ALKBH5 regulates ROS-induced DNA damage response

Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N⁶-methyladenosine (m⁶A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m⁶A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m⁶A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m⁶A levels that protect genomic integrity of cells in response to ROS.     

Desiccation tolerance in red and green gametophytes of Jamesoniella colorata in relation to photoprotection

This study tests the hypothesis that red-leaved gametophytes of the liverwort Jamesoniella colorata (Lehm.) Schiffn., which are found in relatively dry habitats, are more desiccation tolerant than their green counterparts, which are found in moister environments, through superior photoprotective systems. The potential role of red foliar pigments in relation to water deficits is investigated by measuring cell water-relations, oxidative damage and photosynthetic responses. The presence of red pigments, or other cellular constituents, did not affect cell water-relations during dehydration and thus appear not to be involved in cell osmotic regulation. During drying, both colour morphs showed a similar non-photochemical quenching activity and did not experience significant oxidative damage, as measured by the amounts of ascorbate, malondialdehyde and photosynthetic pigments. However, the levels of oxidative damage increased directly upon rewetting the gametophytes, especially in low light conditions (25 μmol m⁻² s⁻¹). The efficiency of photosystem II only recovered partially after severe water deficits in both phenotypes. However, the red gametophytes recovered faster and more completely from mild water deficits than did the greens. Moreover, they experienced significantly less photobleaching after rehydration in low light. It is suggested that red pigments and/or carotenoids in these gametophytes improve desiccation tolerance by alleviating photooxidative damage.     

Oxidative stress management in the filamentous, heterocystous, diazotrophic cyanobacterium, Anabaena PCC7120

Reactive oxygen species (ROS) are inevitably generated as by-products of respiratory/photosynthetic electron transport in oxygenic photoautotrophs. Unless effectively scavenged, these ROS can damage all cellular components. The filamentous, heterocystous, nitrogen-fixing strains of the cyanobacterium, Anabaena, serve as naturally abundant contributors of nitrogen biofertilizers in tropical rice paddy fields. Anabaena strains are known to tolerate several abiotic stresses, such as heat, UV, gamma radiation, desiccation, etc., that are known to generate ROS. ROS are detoxified by specific antioxidant enzymes like superoxide dismutases (SOD), catalases and peroxiredoxins. The genome of Anabaena PCC7120 encodes two SODs, two catalases and seven peroxiredoxins, indicating the presence of an elaborate antioxidant enzymatic machinery to defend its cellular components from ROS. This article summarizes recent findings and depicts important perspectives in oxidative stress management in Anabaena PCC7120.     

Insights into the chaotropic tolerance of the desert cyanobacterium Chroococcidiopsis sp. 029 (Chroococcidiopsales,Cyanobacteria)

The mechanism of perchlorate resistance of the desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was investigated by assessing whether the pathways associated with its desiccation tolerance might play a role against the destabilizing effects of this chaotropic agent. During 3 weeks of growth in the presence of 2.4 mM perchlorate, an upregulation of trehalose and sucrose biosynthetic pathways was detected. This suggested that in response to the water stress triggered by perchlorate salts, these two compatible solutes play a role in the stabilization of macromolecules and membranes as they do in response to dehydration. During the perchlorate exposure, the production of oxidizing species was observed by using an oxidant-sensing fluorochrome and determining the expression of the antioxidant defense genes, namely superoxide dismutases and catalases, while the presence of oxidative DNA damage was highlighted by the over-expression of genes of the base excision repair. The involvement of desiccation-tolerance mechanisms in the perchlorate resistance of this desert cyanobacterium is interesting since, so far, chaotropic-tolerant bacteria have been identified among halophiles. Hence, it is anticipated that desert microorganisms might possess an unrevealed capability of adapting to perchlorate concentrations exceeding those naturally occurring in dry environments. Furthermore, in the endeavor of supporting future human outposts on Mars, the identified mechanisms might contribute to enhance the perchlorate resistance of microorganisms relevant for biologically driven utilization of the perchlorate-rich soil of the red planet.     

The ultrastructure of immobilised desiccated cells of the cyanobacterium Nostoc commune UTEX 584

Cells of the cyanobacterium Nostoc commune UTEX 584 were immobilised, subjected to acute matric water stress (ψ_m = −128 MPa) and then desiccated. Their ultrastructure was investigated by the use of an anhydrous fixation procedure. Although shrunken and bleached, the integrity of the vegatative cells at the ultrastructural level was apparently preserved. The ease with which certain cyanobacterial cells can recover from desiccation may be consequent upon the maintenance of cellular organisation at the ultrastructural level.     

The protein index ofNostoc commune UTEX 584 (cyanobacteria): changes induced in immobilized cells by water stress

Two-dimensional gel electrophoresis was used to analyze the effects of water stress (immobilization and rapid drying, desiccation, rewetting) on the protein index of the desiccation-tolerant cyanobacteriumNostoc commune UTEX 584. Five major landmark protein constellations were detected in the protein index of control cells (in liquid culture) and were designated A (1 protein), B (7 proteins), C (8 proteins), D (3 proteins) and E (2 proteins). These included proteins which showed different sensitivities to water stress. Upon immobilization and rapid drying of the cells at a water potential ({ie87-1}) of -99.5 MPa (a_w=0.5) for 30 min, few changes took place in the index. Four conspicuous proteins and the majority of proteins in the size range 18 to 97 K diminished in abundance while most proteins of constellations A, B and C were detected in fluorographs with the same intensity as in the control. Although protein synthesis continued during this time of drying, no novel class of proteins was detected. The level of incorporation of³⁵S in protein increased rapidly during the first 60 min of rehydration, and then decreased gradually for a further 2.5 h. Extant proteins that were hardly detectable after 24 h of drying, reappeared and increased in abundance upon rewetting of cells for 60 min while a number of proteins which disappeared after drying did not appear during this time. No novel class of proteins appeared upon rewetting. During further rehydration, extensive proteolysis was observed.ThenifH product (Fe protein of nitrogenase) was detected on Western blots — through cross-reaction with antibody — as an acidic polypeptide with a molecular mass of 33.8 K. Fe-protein was detected in immobilized cells after 30 min of drying, in desiccated material, and in rehydrated cells.Abbreviations PMSF Phenylmethylsulfonyl fluoride - IEF Isoelectric focussing     

Acquisition of desiccation tolerance in developing wheat embryos correlates with appearance of a fluid phase in membranes

Membrane behaviour in developing wheat (Triticum aestivum cv Priokskaya) embryos was studied in relation to the acquisition of desiccation tolerance, using spin probe techniques. Fresh embryos were able to develop into seedlings at day 15 after anthesis, but it took 18 d before fast‐dried, isolated embryos could germinate. On the basis of membrane integrity measurements it was estimated that between 14 and 18 d after anthesis the proportion of embryonic cells surviving fast drying increased and the critical moisture content, to which embryonic cells could be dehydrated, decreased. Apparently, embryonic cells do not acquire the same level of desiccation tolerance simultaneously. Only when all cells had become desiccation tolerant was germination of air‐dried embryos possible. Using 5‐doxylstearic acid as the probe molecule, an approximately similar lipid–water interface ordering of membranes was observed in all hydrated embryos, irrespective of age. Dehydration had a dual effect on the lipid interface: further ordering of the major part of the interface and the appearance of additional, disturbed regions. The proportion of these regions correlated with the proportion of desiccation‐tolerant cells. We propose that the membrane surface disturbance be caused by endogenous amphiphiles that partition from the cytoplasm into membranes during drying. The absence of such disturbed regions in dried, desiccation‐sensitive embryos might reflect a lack of sufficient amphiphiles. The relevance of membrane surface disturbance for desiccation tolerance is discussed.     

Ionizing-Radiation Resistance in the Desiccation-Tolerant Cyanobacterium Chroococcidiopsis

The effect of X-ray irradiation on cell survival, induction, and repair of DNA damage was studied by using 10 Chroococcidiopsis strains isolated from desert and hypersaline environments. After exposure to 2.5 kGy, the percentages of survival for the strains ranged from 80 to 35%. In the four most resistant strains, the levels of survival were reduced by 1 or 2 orders of magnitude after irradiation with 5 kGy; viable cells were recovered after exposure to 15 kGy but not after exposure to 20 kGy. The severe DNA damage evident after exposure to 2.5 kGy was repaired within 3 h, and the severe DNA damage evident after exposure to 5 kGy was repaired within 24 h. The increase in trichloroacetic acid-precipitable radioactivity in the culture supernatant after irradiation with 2.5 kGy might have been due to cell lysis and/or an excision process involved in DNA repair. The radiation resistance of Chroococcidiopsis strains may reflect the ability of these cyanobacteria to survive prolonged desiccation through efficient repair of the DNA damage that accumulates during dehydration.     

Accumulation of trehalose in response to desiccation and salt stress in the terrestrial cyanobacterium Nostoc commune

Changes in photosynthetic activity and trehalose levels in field‐isolated, natural colonies of the terrestrial cyanobacterium Nostoc commune responding to desiccation and salt stress were investigated. As the water content decreased in N. commune colonies during desiccation, photosynthetic O₂‐evolving activity decreased and no activity was detected in desiccated colonies. A high level of O₂ evolution was restored in the colonies as they absorbed atmospheric moisture, indicating that only a small amount of water is required for reactivation of photosynthesis. No detectable trehalose was found in fully hydrated N. commune colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air‐dried colonies. Moreover, a 0.2 M NaCl treatment also induced trehalose accumulation to a level equivalent to that by desiccation. Photosynthetic O₂ evolution was inhibited by 0.2 M NaCl, indicating that N. commune can tolerate only low levels of salt. These results suggest that cessation of photosynthesis and trehalose accumulation occur in response to both matric water stress (desiccation) and osmotic water stress (high salt concentration), and that while trehalose may be a less effective osmoprotective compound than others, it is important for the extreme tolerance to desiccation observed in terrestrial cyanobacterium.     

Isolation of two thioredoxins from the cyanobacterium Synechococcus 6301

Two different thioredoxins designated as thioredoxin A and B have been isolated from the cyanobacterium Synechococcus 6301. Methods for large scale purification of these thioredoxins were developed. Thioredoxin B has been purified to homogeneity; it has a molecular weight of 11,800 and an isoelectric point of 4.6. The following K _m data were obtained for this thioredoxin; a) in the PAPS-sulfotransferase assay of Synechococcus 6301: 10.7 M; b) in the fructose-1-6-bisphosphatase assay of Synechococcus 6301: 1.7 M; c) in the APS-sulfotransferase assay of Chroococcidiopsis 7203: 5.4M. Thioredoxin A has an isoelectric point of 4.1 and it is active in the PAPS-sulfotransferase and fructose-1-6-bisphosphatase of Synechococcus 6301; it is not active in the APS-sulfotransferase of Chroococcidiopsis 7203.Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday