Extreme Desiccation Research Articles

Cryopreservation of conditionally dormant orthodox seeds of Betula pendula

To evaluate the feasibility of long-term cryopreservation of Polish provenances of silver birch (Betula pendula), the sensitivity of conditionally dormant seeds to extreme desiccation and/or the ultra-low tem- perature of liquid nitrogen (LN; -196C) was evaluated. The critical water content (WC) of desiccated seeds and the high-moisture freezing limit of seeds desiccated or moistened to various WCs and frozen for 24 h or for 2 years in LN was also determined. Germination tests revealed no critical WC for seeds (to 0.02 g H2 Og -1 dry mass (g g -1 )). Seeds tolerated freezing in LN within specific safe range of WC 0.02-0.23 g g -1 (nuts). Seeds desiccated to the safe WC and stored in LN for 2 years had similar or higher germination as seeds stored at -3C for 2 years, depending on provenance. Therefore, long-term cryopreservation of B. pendula seeds in gene banks is feasible.

Towards research-based approaches for solving body composition problems in sports: ski jumping as a heuristic example

Extremely low body weight and associated eating and body composition disorders are becoming prevalent in many sports, and can be summarised into three groups: I. Sports in which weight restricts...

A role for a cell wall localized glycine‐rich protein in dehydration and rehydration of the resurrection plant Boea hygrometrica

The acquisition of desiccation tolerance in dicotyledonous angiosperms requires the induction of a co-ordinated programme of genetic and biochemical processes during drying and the adaptive mechanisms are primarily protoplasmic in nature. Recent studies have shown that changes in cell wall structure and composition are also important for recovery after drying, however, the molecular mechanisms that underpin these adaptive responses are largely unknown. Here, the desiccation-tolerant plant Boea hygrometrica was used as a model system to investigate the changes in gene expression and cell wall adaptation that take place during extreme dehydration. A cDNA macroarry analysis of dehydration-inducible genes led to the identification of a gene encoding a glycine-rich protein (BhGRP1). The corresponding transcript was up-regulated during drying in B. hygrometrica leaves. In silico analysis revealed that BhGRP1 is targeted to the cell wall and this was confirmed in planta. Morphological changes in the cell wall architecture were also observed during the process of drying and re-watering. Concomitant with this observation, cell wall profiling by Fourier transform infrared spectroscopy indicated that protein levels increased upon desiccation and remained broadly similar upon re-watering. These findings suggest that the deposition of BhGRP1 may play a role in cell wall maintenance and repair during dehydration and rehydration in B. hygrometrica.

Cryopreservation of dormant European ash (Fraxinus excelsior) orthodox seeds

We investigated the sensitivity of dormant Polish-provenance Fraxinus excelsior L. seeds to extreme desiccation and liquid nitrogen (LN, -196 degrees C). Germination and seedling emergence tests revealed that the critical water content (WC) of the desiccated seeds was 0.04-0.06 g H(2)O g(-1) dry mass, g g(-1) (0.04 g g(-1) in the germination test and 0.06 g g(-1) in the seedling emergence test). The seeds that were within the safe WC range (0.06-0.24 g g(-1) in the germination test and 0.08-0.24 g g(-1) in the seedling emergence test) tolerated freezing in LN. A 2-year seed storage period in LN after desiccation to a safe WC did not decrease the germination after thawing as compared with a 2-year seed storage at -3 degrees C. When the seeds were stored in LN after dormancy breaking, the germination rate after storage was lower, because the seeds had to be desiccated to the lower level (~ 0.11 g g(-1)) within the seeds' safe WC range after stratification (before storage). Secondary dormancy was induced in seeds that were desiccated after stratification. The results of this study confirm the feasibility of long-term cryopreservation of European ash seeds in forest gene banks.

Water-stress induced trehalose accumulation and control of trehalase in the cyanobacterium Nostoc punctiforme IAM M-15

We investigated the biochemical properties of the enzymes involved in trehalose metabolism in the cyanobacterium Nostoc punctiforme strain IAM M-15 to elucidate the mechanism of trehalose accumulation in response to desiccation and salt stress. There was no detectable trehalose in fully hydrated N. punctiforme cells; however, these cells accumulated trehalose upon desiccation. Moreover, NaCl treatment also induced trehalose accumulation. The three genes for trehalose metabolism, treZ (maltooligosyltrehalose trehalohydrolase, Mth), treY (maltooligosyltrehalose synthase, Mts), and treH (trehalase), were found as a gene cluster, and the mRNAs for these genes were detectable at similar levels during desiccation. Trehalase of N. punctiforme was heterologously expressed in E. coli cells in an active form with a molecular mass of 52 kDa. Trehalase activity was strongly inhibited in the presence of 10 mM NaCl while trehalose synthesis activity remained active in the presence of salt. These data suggest that the rate of trehalose production exceeds that of trehalose hydrolysis under water-stress conditions characterized by increased cellular solute concentrations. In the proposed mechanism, control of trehalase plays an important role in trehalose accumulation in terrestrial cyanobacteria under conditions of extreme desiccation.

La cryoconservation des semences dormantes orthodoxes des arbres forestiers : le merisier (Prunus avium L.)

• The sensitivity of dormant seeds of mazzard cherry (Prunus avium L.) of Polish provenances to extreme desiccation and/or the ultra-low temperature of liquid nitrogen, LN (−196 ◦ C), was investigated to identify the optimum seed water content (w.c.) at this temperature. • Germination and seedling emergence tests in this study showed that there is no critical seed w.c. for deeply desiccated seeds (stone w.c. 1.6%), although such desiccated seeds were sensitive to the temperature of LN in the seedling emergence test. For seeds frozen in LN, the highest germinability was observed at w.c. of 9.0–16.9%, but seedling emergence was then significantly lower than in nonfrozen seeds. The 2-year of storage in LN of seeds desiccated to 7.8% w.c. did not decrease germinability after thawing, in comparison with 2-year of storage at −3 ◦ C. Storage in LN showed that if seeds were stored after breaking of their dormancy, germinability after storage was lower because of the necessity of seed desiccation to the lower level (∼ 8%) of w.c. after stratification (before storage). Secondary dormancy was induced in seeds desiccated after stratification. • The results of this study demonstrate the potential for long-term cryopreservation of mazzard cherry seeds in forest gene banks.

Cloning, characterisation, and expression analysis of an oleosin gene in coconut (Cocos nucifera L.) pulp

SummaryOleosins are structural proteins found in oil bodies, organelles found in the cells of plant tissues with a high oil content that undergo extreme desiccation as part of their maturation process. Oleosins stabilise oil bodies. In this paper, a full-length cDNA sequence homologous to oleosin, a seed-storage oil-body protein, from coconut (Cocos nucifera L.) was identified from cDNA libraries during fruit development and characterised. The gene, termed Coco-Ole, contained an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. The predicted amino acid sequence of coconut oleosin had a molecular mass of 13.0 kDa, and showed 92% (AAF76238.1) and 67% (AAC02239.1) sequence similarity to oil palm (Elaeis guineensis Jacq.) and rice (Oryza sativa) oleosin proteins, respectively. The amino acid sequence clustered in the same branch as oil palm in the cladogram, but was distant from other species. The results of semi-quantitative RT-PCR indicated that the Coco-Ole gene was expressed only in the pulp, and its expression increased significantly during pulp development. Compared with fluctuations in oil content, expression of the Coco-Ole gene was consistent with the anabolism of oil during pulp development. The cloning and sequencing of the Coco-ole gene provides a new marker for studies on oil body biogenesis and fruit development in coconut.

How do terrestrial Antarctic organisms survive in their harsh environment?

Anhydrobiosis, or extreme desiccation tolerance, is one of the strategies that allows terrestrial Antarctic organisms to survive in a harsh environment. A new study in BMC Genomics analyses gene expression in an Antarctic nematode during desiccation, and sheds new light on this phenomenon.

Cloning and expression analysis of a dirigent protein gene from the resurrection plant Boea hygrometrica

Resurrection plants are tolerant to extreme dehydration and are useful model systems to study genes that play a role in drought tolerance. A gene fragment encoding a dirigent protein that is predicted to function in lignin biosynthesis was identified from leaves of the resurrection plant Boea hygrometrica via cDNA microarray screening. A cDNA, designated BhDIR1, containing the complete predicted open reading frame, was obtained by 5′-RACE. BhDIR1 transcripts were found to be accumulated in response to changes in plant dehydration status, exogenously applied phytohormones and signaling molecules, and temperature stresses. BhDIR1 encodes a protein of 199 amino acids, which shows 20–40% similarity to dirigent proteins reported from other plants. BhDIR1 is predicted to contain a cleavable signal peptide at the N-terminal, and its plasma membrane/cell wall localization was confirmed using a GFP fusion protein assay. Consistent with this discovery, the acid-soluble lignin content decreased in dehydrated B. hygrometrica leaves . Taken together, our results indicate a protective role for a dirigent protein in response to drought stress by changing the physical characters of lignin which in turn is predicted to affect the mechanical strength and flexibility of the plant cell wall.

CDT retroelement

The resurrection plant Craterostigma plantagineum can tolerate up to 96% loss of its water content and recover from such extreme dehydration within several hours. This property is not shared by callus which has a strict requirement for exogenous abscissic acid (ABA) to survive severe water loss. ABA treatment and dehydration result in the induction of similar drought-responsive genes. Activation tagging led to the isolation of CDT-1 gene which renders callus desiccation tolerant bypassing the ABA requirement. This gene belongs to a retroelement family, members of which are induced by ABA and dehydration in callus, supporting its role in desiccation tolerance. Indeed, CDT genes have been detected in other desiccation tolerant Craterostigma species. CDT-1 RNA of both strands was identified by in situ hybridization and a CDT-1-derived short interfering RNA was detected in desiccation tolerant tissues and was able to induce dehydration genes in transfected protoplasts to the same extent as an ABA treatment. Thus, under environmental stress the induced transposition, over generations, directs the amplification of CDT-copy number in the genome and increases the desiccation tolerance phenomenon.Addendum to: Hilbricht T, Varotto S, Sgaramella V, Bartels D, Salamini F, Furini A. Retrotransposons and siRNA have a role in the evolution of desiccation tolerance leading to resurrection of the plant Craterostigma plantagineum. New Phytol 2008; 179:877-87.

LEA 4 group genes from the resurrection plant Boea hygrometrica confer dehydration tolerance in transgenic tobacco

The resurrection plant Boea hygrometrica can survive extreme dehydration and is used as a model system to study desiccation tolerance. Screening of a cDNA library prepared from desiccated leaves via a macroarray technique has resulted in the identification of two dehydration responsive genes that encode group 4 late embryogenesis abundant (LEA) proteins, designated as BhLEA1 and BhLEA2, respectively. BhLEA1 and BhLEA2 were induced by dehydration and signaling molecules, including abscisic acid (ABA). Transgenic tobacco that ectopically express BhLEA1 and BhLEA2 were generated and used to study the role of LEA proteins in dehydration tolerance. After a period of drought, the relative water content of leaves and photosystem II activity in transgenic tobacco were higher than wild-type plants. Furthermore the membrane permeability was lower in selected transgenic lines that expressed BhLEA1 and BhLEA2 than in wild-type plants. Superoxide dismutase and peroxidase activities were increased in transgenic plants relative to that observed in the wild-type control and proteins including ribulose-bisphosphate carboxylase (large subunit), light-harvesting complex II and photosystem II extrinsic protein were stabilized in transgenic plants compared to wild-type plants. Surprisingly, the steady state levels of BhLEA1 and BhLEA2 protein substantially increased in response drying, despite being under the transcriptional control of the CaMV 35S promoter. Data presented here suggests that BhLEA genes are likely to play a role in the general protection of the plant cell during dehydration and affect membrane and protein stability.

Mechanisms and molecular adaptation to extreme dehydration in tardigrades: Hsp gene expression in Milnesium tardigradum

The tardigrade Milnesium tardigradum Doyere 1840 is a well-known cosmopolitan species and a typical inhabitant of moist environments which facilitate the animal's gaseous exchange and avoid desiccation. However, such habitats frequently undergo seasonal changes that impact animal life. M. tardigradum is able to survive these periods of adverse conditions due to its ability to enter into a anhydrobiotic state. In adverse environments, all terrestrial and freshwater tardigades arrest their metabolic activity and get dehydrated to form the tun state. In the anhydrobiotic stage, tardigrades showextraordinary tolerance tophysical extremes includinghigh-energy radiation, immersion inorganic solvents, brief exposure to high temperatures and prolonged exposure to indefinitely low temperatures. When environmental conditions are adequate, tuns rehydrate and the animals resume metabolic activity. Drying of cells generally leads to massive damage to cellular membranes and proteins, which eventually results in cell death and, consequently, in the death of the entire organism.Heat shock proteins and theirmolecular partners are known to play diverse roles, even in unstressed cells, in successful folding, assembly, intracellular localization, secretion, regulation, and degradation of other proteins. In this study, a complementary focus is on tardigrades undergoing stress in nature and on the roles of stress genes of the hsp10, hsp20/30, hsp40, hsp60, hsp70 and hsp90 family in the stress physiology ofwhole organisms in different life history stages. This study is part of the project www.FUNCRYPTA.de, funded by the German Federal Ministry of Education and Research, BMBF (0313838).

Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki

Anhydrobiosis is an extremely dehydrated state in which organisms show no detectable metabolism but retain the ability to revive after rehydration. Thus far, two hypotheses have been proposed to explain how cells are protected during dehydration: (i) water replacement by compatible solutes and (ii) vitrification. The present study provides direct physiological and physicochemical evidence for these hypotheses in an African chironomid, Polypedilum vanderplanki, which is the largest multicellular animal capable of anhydrobiosis. Differential scanning calorimetry measurements and Fourier-transform infrared (FTIR) analyses indicated that the anhydrobiotic larvae were in a glassy state up to as high as 65 degrees C. Changing from the glassy to the rubbery state by either heating or allowing slight moisture uptake greatly decreased the survival rate of dehydrated larvae. In addition, FTIR spectra showed that sugars formed hydrogen bonds with phospholipids and that membranes remained in the liquid-crystalline state in the anhydrobiotic larvae. These results indicate that larvae of P. vanderplanki survive extreme dehydration by replacing the normal intracellular medium with a biological glass. When entering anhydrobiosis, P. vanderplanki accumulated nonreducing disaccharide trehalose that was uniformly distributed throughout the dehydrated body by FTIR microscopic mapping image. Therefore, we assume that trehalose plays important roles in water replacement and intracellular glass formation, although other compounds are surely involved in these phenomena.

Effects of heat on camel platelet structure and function–a comparative study with humans

Camels and many other desert animals are uniquely adapted to conserve water and other fluids in order to survive intense heat for long periods. Earlier studies have suggested that human platelets may be the trigger for the coagulopathy involved in heat prostration and stroke. The present study has compared the resistance of camel and human platelets to heat in order to see if they might help to protect camels from the effects of high body temperature for prolonged periods. The findings demonstrate that camel platelets are significantly less sensitive to heat than human platelets. Temperatures (43°C–45°C) that cause human cells to undergo marked structural alterations and lose their ability to spread and aggregate have no effect on camel platelets. Even higher temperatures (50°C) that destroy human platelets have minor effects on camel cells and do not seriously compromise their function. Temperatures of 55°C do destroy camel platelets and their functional capability. The resistance of camel platelets to heat may help protect camels from the effects of extreme body temperature and dehydration, which are everyday conditions in the desert.

Novel thermostable glycosidases in the extracellular matrix of the terrestrial cyanobacterium Nostoc commune

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and forms a visible colony in which the cells are embedded in extracellular polysaccharides (EPSs), which play a crucial role in the extreme desiccation tolerance of this organism. When natural colonies were immersed in water, degradation of the colonies occurred within 2 days and N. commune cells were released into the water. The activities that hydrolyze glycoside bonds in various N. commune fractions were examined using artificial nitrophenyl-linked sugars as substrates. A beta-D-glucosidase purified from the water-soluble fraction was resistant to 20 min of boiling. The beta-D-glucosidase, with a molecular mass of 20 kDa, was identified as a cyanobacterial fasciclin protein based on its N-terminal amino-acid sequence. The 36-kDa major protein in the water-soluble fraction was purified, and the N-terminal amino-acid sequence of the protein was found to be identical to that of the water-stress protein (WspA) of N. commune. This WspA protein also showed heat-resistant beta-D-galactosidase activity. The fasciclin protein and WspA in the extracellular matrix may play a role in the hydrolysis of the EPSs surrounding the cells, possibly as an aid in the dispersal of cells, thus expanding the colonies of this cyanobacterium.

Ultraviolet radiation-induced limitation to epilithic microbial growth in arid deserts – Dosimetric experiments in the hyperarid core of the Atacama Desert

Experiments were conducted during November 2003 in the dry core of the Atacama Desert, Yungay, Chile to test the hypothesis that UV radiation, in environments where liquid water is not available, and thus enzymatic repair of UV-induced damage is inhibited, can prevent epilithic colonization. Novel dosimeters made from the cryptoendolithic, desiccation and radiation-resistant cyanobacterium Chroococcidiopsis sp. isolated from the dry Negev desert, Israel, showed that monolayers of this organism were killed within one day. The diurnal profile of microbial loss of viability was investigated with dosimeters of Bacillus subtilis, which similarly showed cell death within one day. Soil grains obtained from south of Yungay where liquid water is more abundant and transported to the hyperarid core showed killing of indigenous vegetative organisms within one day. Gypsum and mineral grain coverings of 1 mm were sufficient to prevent measurable UV-induced damage of Chroococcidiopsis and B. subtilis after 8 d exposure. These results show that under extreme desiccation and an ambient UV flux the surface of rocks can potentially be rendered sterile, but that millimetre thick mineral coverings can protect organisms from UV-induced killing, consistent with the observed patterns of lithophytic colonization in the Atacama Desert. These data further show that UV radiation can be an important limiting factor in surface biological rock weathering in arid regions.

Extreme desiccation- and heat-tolerance of desert seeds: A study on Welwitschia mirabilis

Extreme desiccation- and heat-tolerance of desert seeds: A study on Welwitschia mirabilis

Lichens Survive in Space: Results from the 2005 LICHENS Experiment

This experiment was aimed at establishing, for the first time, the survival capability of lichens exposed to space conditions. In particular, the damaging effect of various wavelengths of extraterrestrial solar UV radiation was studied. The lichens used were the bipolar species Rhizocarpon geographicum and Xanthoria elegans, which were collected above 2000 m in the mountains of central Spain and as endolithic communities inhabiting granites in the Antarctic Dry Valleys. Lichens were exposed to space in the BIOPAN-5 facility of the European Space Agency; BIOPAN-5 is located on the outer shell of the Earth-orbiting FOTON-M2 Russian satellite. The lichen samples were launched from Baikonur by a Soyuz rocket on May 31, 2005, and were returned to Earth after 16 days in space, at which time they were tested for survival. Chlorophyll fluorescence was used for the measurement of photosynthetic parameters. Scanning electron microscopy in back-scattered mode, low temperature scanning electron microscopy, and transmission electron microscopy were used to study the organization and composition of both symbionts. Confocal laser scanning microscopy, in combination with the use of specific fluorescent probes, allowed for the assessment of the physiological state of the cells. All exposed lichens, regardless of the optical filters used, showed nearly the same photosynthetic activity after the flight as measured before the flight. Likewise, the multimicroscopy approach revealed no detectable ultrastructural changes in most of the algal and fungal cells of the lichen thalli, though a greater proportion of cells in the flight samples had compromised membranes, as revealed by the LIVE/DEAD BacLight Bacterial Viability Kit. These findings indicate that most lichenized fungal and algal cells can survive in space after full exposure to massive UV and cosmic radiation, conditions proven to be lethal to bacteria and other microorganisms. The lichen upper cortex seems to provide adequate protection against solar radiation. Moreover, after extreme dehydration induced by high vacuum, the lichens proved to be able to recover, in full, their metabolic activity within 24 hours.

Using Experimental Evolution to Study the Physiological Mechanisms of Desiccation Resistance inDrosophila melanogaster

Data from populations undergoing experimental evolution can be used to make comparisons between physiologically differentiated populations and to determine evolutionary trajectories. Comparisons of long-established laboratory populations of Drosophila melanogaster that are strongly differentiated with respect to desiccation resistance are used to test alternative hypotheses concerning the mechanisms that fruit flies use to survive bouts of extreme desiccation. This comparative study supports the hypothesis that, in at least one case, D. melanogaster can evolve increased resistance to desiccation by decreasing water loss rates and by increasing bulk water content but not by increasing metabolic water content or dehydration tolerance. While glycogen was involved in water storage, its primary role was in water binding, not the production of metabolic water. Measurement of the trajectories of these component mechanisms during selection for desiccation resistance is used to demonstrate that water loss rate quickly plateaus in response to selection, while water content continues to improve. This disparity reveals the value of studying evolutionary trajectories and the need for longer-term selection studies in evolutionary physiology.

A high-resolution diatom-inferred palaeoconductivity and lake level record of the Aral Sea for the last 1600 yr

Formerly the world's fourth largest lake by area, the Aral Sea is presently undergoing extreme desiccation due to large-scale irrigation strategies implemented in the Soviet era. As part of the INTAS-funded CLIMAN project into Holocene climatic variability and the evolution of human settlement in the Aral Sea basin, fossil diatom assemblages contained within a sediment core obtained from the Aral Sea have been applied to a diatom-based inference model of conductivity ( r 2 = 0.767, RMSEP = 0.469 log 10 μS cm − 1). This has provided a high-resolution record of conductivity and lake level change over the last ca. 1600 yr. Three severe episodes of lake level regression are indicated at ca. AD 400, AD 1195–1355 and ca. AD 1780 to the present day. The first two regressions may be linked to the natural diversion of the Amu Darya away from the Aral Sea and the failure of cyclones formed in the Mediterranean to penetrate more continental regions. Human activity, however, and in particular the destruction of irrigation facilities are synchronous with these early regressions and contributed to the severity of the observed low stands.