ECOPHYSIOLOGICAL PERFORMANCE OF THE AEROTERRESTRIAL GREEN ALGA KLEBSORMIDIUM CRENULATUM (CHAROPHYCEAE,STREPTOPHYTA) ISOLATED FROM AN ALPINE SOIL CRUST WITH AN EMPHASIS ON DESICCATION STRESS1

Aeroterrestrial filamentous green algae of the genus Klebsormidium (Klebsormidiales, Streptophyta) are typical components of biological soil crusts, which occur worldwide in arid and semiarid habitats including alpine regions. In the present study, Klebsormidium crenulatum (Kütz.) Lokhorst was isolated from an alpine soil crust above the timberline of the Austrian Alps. Growth responses, photosynthetic performance, and desiccation tolerance were measured under controlled laboratory conditions. K. crenulatum exhibited optimal growth and the highest photosynthetic efficiency under relatively low photon fluence densities (30 and 21.9 μmol photons · m^?2 · s^?1, respectively), indicating low‐light requirements. It grew in a narrow range of salinities between 1.2 and 15 practical salinity units (psu), pointing to a pronounced stenohaline response pattern. Increasing temperatures from 5°C to 40°C led to different effects on photosynthetic oxygen evolution and respiratory oxygen consumption in K. crenulatum. While at low temperatures (5°C–10°C) photosynthesis was relatively high, respiration was not detectable or was at a very low level. Conversely, at the highest temperature of 40°C, photosynthesis was inhibited, and respiration unaffected, indicating strong differences in temperature sensitivity between both physiological processes. K. crenulatum was capable of photosynthesizing efficiently for up to 2.5 h under desiccation, followed by a decrease to 15% of the initial value after 3 h. Complete recovery took place within 2 h after rehydration. All ecophysiological data explain the widespread abundance of K. crenulatum in soil crusts of the alpine regions of the European Alps.     

Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration

Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2‐DE and nanoscale capillary LC/MS/MS. Twenty‐four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration‐regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.     

Desiccation and cryopreservation of actively-growing cultured plant cells and protoplasts

Actively-growing cultured cells of Pogonatum and Polytrichum were desiccated and cryopreserved. Although Pogonatum was slightly more tolerant to desiccation, both species were cryopreserved with >90% survival rate. An examination of isolated protoplasts revealed that differences in desiccation tolerance were likely dependent on levels of injury of plasma membranes. Trehalose and sucrose provided some protective effects during protoplast desiccation, but mannitol and glucose were less effective when Pogonatum protoplasts were directly desiccated and preserved at various temperatures. The effectiveness of glucose was enhanced when combined with culture medium components.     

PHOTOSYNTHETIC INSENSITIVITY OF THE TERRESTRIAL CYANOBACTERIUM NOSTOC FLAGELLIFORME TO SOLAR UV RADIATION WHILE REHYDRATED OR DESICCATED1

Photosynthetic performance of the terrestrial cyanobacterium Nostoc flagelliforme (M. J. Berkeley et M. A. Curtis) Bornet et Flahault during rehydration and desiccation has been previously characterized, but little is known about the effects of solar UV radiation (280–400 nm) on this species. We investigated the photochemical activity during rehydration and subsequent desiccation while exposing the filamentous colonies to different solar radiation treatments. Photochemical activity could be reactivated by rehydration under full‐spectrum solar radiation, the species being insensitive to both ultraviolet‐A radiation (UVAR; 315–400 nm) and ultraviolet‐B radiation (UVBR). When the rehydrated colonies were exposed for desiccation, the effective PSII photochemical yield was inhibited by visible radiation (PAR) at the initial stage of water loss, then increased with further decrease in water content, and reached its highest value at the water content of 10%–30%. However, no significant difference was observed among the radiation treatments except for the moment when they were desiccated to critical water content of about 2%–3%. At such a critical water content, significant reduction by UVBR of the effective quantum yield was observed in the colonies that were previously rehydrated under indoor light [without ultraviolet radiation (UVR)], but not in those reactivated under scattered or direct solar radiation (with UVR), indicating that preexposure to UVR during rehydration led to higher resistance to UVR during desiccation. The photosynthetic CO₂ uptake by the desiccated colonies was enhanced by elevation of CO₂ but was not affected by both UVAR and UVBR. It increased with enhanced desiccation to reach the maximal values at water content of 40%–50%. The UV‐absorbing compounds and the colony sheath were suggested to play an important role in screening harmful UVR.     

Desiccation-induced Changes in Antioxidant Enzymes,Fatty Acids,and Amino Acids in the Cyanobacterium <Emphasis Type="Italic">Tolypothrix scytonemoides</Emphasis>

Tolypothrix scytonemoides subjected to desiccation exhibited increased antioxidant enzyme activities when compared to fresh cells. The activities of catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) were enhanced in desiccated cells by 42.8% and 8.1%, respectively. The isoforms of catalase and superoxide dismutase were detected by activity staining of crude samples separated on native gels. The isoforms of superoxide dismutase were identified based on their sensitivity to hydrogen peroxide and cyanide. The changes in fatty acids and amino acids in fresh and desiccated cells were also investigated and it was found that the quantity of certain fatty acids and amino acids was greater in desiccated cells. Palmitic acid, palmitoleic acid, heptadecanoic acid, linoleic acid, and myristic acid were more in desiccated cells when compared to fresh cells. Desiccated cells synthesized myristoleic acid, eicosenoic acid and behenic acid, acids which were not synthesized by the fresh cells, whereas tricosanoic acid was synthesized by the fresh cells and not by desiccated cells. The levels of lysine, serine, glycine, proline and cysteine were also comparatively greater in the desiccated cells.     

Desiccation sensitivity and tolerance in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>: assessing limits and damage

The moss Physcomitrella patens is becoming the model of choice for functional genomic studies at the cellular level. Studies report that Physcomitrella survives moderate osmotic and salt stress, and that desiccation tolerance can be induced by exogenous ABA. Our goal was to quantify the extent of dehydration tolerance in wild type moss and to examine the nature of cellular damage caused by desiccation. We exposed Physcomitrella to humidities that generate water potentials from −4 (97% RH) to −273 MPa (13% RH) and monitored water loss until equilibrium. Water contents were measured on a dry matter basis to determine the extent of dehydration because fresh weights (FW) were found to be variable and, therefore, unreliable. We measured electrolyte leakage from rehydrating moss, assessed overall regrowth, and imaged cells to evaluate their response to drying and rehydration. Physcomitrella did not routinely survive water potentials <−13 MPa. Upon rehydration, moss dried to water contents >0.4 g g dm⁻¹ maintained levels of leakage similar to those of hydrated controls. Moss dried to lower water contents leaked extensively, suggesting that plasma membranes were damaged. Moss protonemal cells were shrunken and their walls twisted, even at −13 MPa. Moss cells rehydrated after drying to −273 MPa failed to re-expand completely, again indicating membrane damage. ABA treatment elicited tolerance of desiccation to at least −273 MPa and limited membrane damage. Results of this work will form the basis for ongoing studies on the functional genomics of desiccation tolerance at the cellular level.     

Core-shell encapsulation formulations to stabilize desiccated Bradyrhizobium against high environmental temperature and humidity

Engineered materials to improve the shelf-life of desiccated microbial strains are needed for cost-effective bioaugmentation strategies. High temperatures and humidity of legume-growing regions challenge long-term cell stabilization at the desiccated state. A thermostable xeroprotectant core and hydrophobic water vapour barrier shell encapsulation technique was developed to protect desiccated cells from the environment. A trehalose core matrix increased the stability of desiccated Bradyrhizobium by three orders of magnitude over 20 days at 32°C and 50% relative humidity (RH) compared to buffer alone; however, the improvement was not deemed sufficient for a shelf-stable bioproduct. We tested common additives (skim milk, albumin, gelatin and dextran) to increase the glass transition temperature of the desiccated product to provide further stabilization. Albumin increased the glass transition temperature of the trehalose-based core by 40°C and stabilized desiccated Bradyrhizobium for 4 months during storage at high temperature (32°C) and moderate humidity (50% RH) with only 1 log loss of viability. Although the albumin-trehalose core provided exceptional protection against high temperature, it was ineffective at higher humidity conditions (75%). We therefore incorporated a paraffin shell, which protected desiccated cells against 75% RH providing proof of concept that core and shell encapsulation is an effective strategy to stabilize desiccated cells.     

Effect of Moisture on Ethylene Oxide Sterilization

Bacterial cells dehydrated beyond a critical point no longer react uniformly to ethylene oxide sterilization. The percentage of cells resistant to the lethal effect of ethylene oxide after desiccation is often as small as 0.1 to 0.001%. However, 5% resistant cells were observed with one type of microorganism dried in broth. The presence of organic matter increases the percentage of cells that become resistant to ethylene oxide after dehydration. The phenomenon is produced by exposing cells to a vacuum or a chemically desiccated atmosphere. It is not a permanent change, because the resistant cells rapidly become susceptible if wetted with water. On the other hand, mere exposure to a high relative humidity (RH), i.e., 75 to 98%, after desiccation requires 6 and 4 days, respectively, to overcome this resistance. Moisture studies showed that there is less water in bacterial cells that have been desiccated and then equilibrated to successively high RH values up to 100% RH, than in cells that have not been desiccated, but allowed to dry naturally until equilibrated to the same RH values.     

Dry artificial seeds and desiccation tolerance induction in microspore-derived embryos of broccoli

Desiccation tolerance of broccoli microspore-derived embryos was induced by exogenous application of abscisic acid (ABA). Embryos, which were desiccated to about 10% water content, were estimated for viability after rehydration. Survival was dependent on the ABA concentration and the development stage of embryo, but not on the length of exposure period to ABA or genotype. Cotyledonary stage embryos acquired the highest desiccation tolerance when treated with 1×10^-4M ABA. Under this condition, on average 27–48% of the desiccated embryos could convert into plants. Embryos treated with 1×10^-6M ABA or no ABA or earlier development-staged embryos, such as globular and heart stages, lost viability after desiccation. A one day exposure to ABA had the similar effect on the induction of desiccation tolerance as a 7-day treatment. The dried embryos maintained their ability of plant conversion after three months of storage under room conditions. The plants derived from the desiccated embryos were not different in the morphology or ploidy level from those from non-desiccated ones.Abbreviations ABA abscisic acid - RH relative humidity     

Resurrection kinetics of photosynthesis in desiccation‐tolerant terrestrial green algae (Chlorophyta) on tree bark

The rough bark of orchard trees (Malus) around Darmstadt is predominantly covered in red to purple‐brown layers (biofilms) of epiphytic terrestrial alga of Trentepohlia umbrina. The smooth bark of forest trees (Fagus sylvatica L. and Acer sp.) in the same area is covered by bright green biofilms composed of the green algae Desmococcus, Apatococcus and Trebouxia, with a few cells of Coccomyxa and ‘Chlorella’ trebouxioides between them. These algae are desiccation tolerant. After samples of bark with the biofilms were kept in dry air in darkness for various periods of time, potential quantum yield of PSII, F_v/F_m, recovered during rehydration upon rewetting. The kinetics and degree of recovery depended on the length of time that the algae were kept in dry air in the desiccated state. Recovery was better for green biofilm samples, i.e. quite good even after 80 days of desiccation (F_v/F_m = ca. 50% of initial value), than the red samples, where recovery was only adequate up to ca. 30–40 days of desiccation (F_v/F_m = ca. 20–55% of initial value). It is concluded that the different bark types constitute different ecophysiological niches that can be occupied by the algae and that can be distinguished by their capacity to recover from desiccation after different times in the dry state.     

Peroxidase activity of desiccation-tolerant loblolly pine somatic embryos

Summary Desiccation tolerance can be induced by culturing somatic embryos of loblolly pine (Pinus taeda L.) on medium supplemented with 50 μM abscisic acid (ABA) and/or 8.5% polyethylene glycol (PEG₆₀₀₀). Scanning electron microscopy of desiccated somatic embryos showed that the size and external morphology of the desiccation-tolerant somatic embryos recovered to the pre-desiccation state within 24–36 h, whereas the non-desiccation-tolerant somatic embryos did not recover and remained shriveled, after rehydration. Peroxidase activity of desiccated somatic embryos increased sharply after 1 d of desiccation treatment at 87% relative humidity (RH), and desiccation-tolerant somatic embryos had higher peroxidase activity compared to sensitive somatic embryos. Higher peroxidase activity of desiccation-tolerant somatic embryos may have allowed them to catalyze the reduction of H₂O₂ produced by drought stress, and protected them from oxidative damage.     

Effect of desiccation to low moisture content on germination,synchronization of root emergence,and plantlet regeneration of black spruce somatic embryos

A desiccation protocol was developed to evaluate the effect of different levels of desiccation on germination and plantlet regeneration of black spruce somatic embryos. Large desiccation chambers (80 l) with four liters of saturated salt solutions provided constant relative humidities (RH) of 63, 79, 88, and 97% (± 2%). Under these conditions, an embryo mass of 10 mg always dried fast even at 97% RH. In contrast, an embryo mass of 80 mg generated different kinetics of water loss, from fast drying at 63% RH to slow drying at 97% RH. Drying rates similar to those obtained with 80 mg embryos were also generated by combining 40 mg embryos with 40 mg water. The effects of drying rate and embryo MC on germination rate, root elongation, and plantlet regeneration were examined. A fast drying rate to 4–5% embryo MC, obtained under 63% RH, was detrimental to germination and plantlet development. However slower drying rates, obtained under 79–97% RH and generating 7–19% MC in the embryos, gave developmental responses similar to the control. Synchronization of root emergence was improved only for embryos desiccated to approx. 16% MC under 97% RH. The optimal desiccation protocol using large desiccation chamber at 97% RH and a constant embryo mass of 40 mg embryos plus 40 mg water was applied to five genotypes of black spruce. For all genotypes, desiccated embryos gave plantlet regeneration rates similar to the control undesiccated embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

INFLUENCE OF DESICCATION ON THE MECHANICAL PROPERTIES OF IRIDAEA CORDATA (RHODOPHYTA)1

The influence of desiccation on the mechanical properties of the intertidal macroalga Iridaea cordata (Turner) Bory (Rhodophyta) was investigated over a range of water losses (0–83%) that bracketed in situ levels (26–67%). The tissue modulus (stiffness) remained constant for water losses up to about 70%, but increased sharply with losses between 70–83%. Tissue strength of desiccated samples did not fall below the range measured for undesiccated samples. There was a significant increase in breaking strain up to water losses of about 50%, after which breaking strain decreased. The relationship between toughness and desiccation resembled that for breaking strain. Overall, the mechanical properties of Iridaea cordata did not deteriorate when desiccated to levels consistent with those observed in the field.     

Interspecies interaction extends bacterial survival at solid–air interfaces

Despite the ubiquity of biofilms in natural and man-made environments, research on surface-associated cells has focused primarily on solid–liquid interfaces. This study evaluated the extent to which bacterial cells persist on inanimate solid–air interfaces. The desiccation tolerance of bacterial strains isolated from indoor air, as well as of a test strain (Pseudomonas aeruginosa), was determined at different levels of relative humidity (RH) using the large droplet inoculation method in an aerosol chamber. The cells survived longer at lower (25 and 42%) than at high RH (95%). Four of the seven indoor strains selected for further study showed extended period of survival following deposition as 0.05–0.1?ml of washed culture followed by desiccation, each with different effects on the survival of the test strain, P. aeruginosa. A strain closely related to Arthrobacter species afforded the highest level of protection to the test strain. Even though the desiccation-tolerant strains survived when they were deposited as bioaerosols, the protective role towards the test strain was not observed when the latter was deposited as a bioaerosol. These, which are often-unculturable, bacteria may go undetected during routine monitoring of biofouling, thereby allowing them to act as reservoirs and extending the habitat range of undesired microorganisms.     

Freeze avoidance: a dehydrating moss gathers no ice

Using cryo‐SEM with EDX fundamental structural and mechanical properties of the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to tolerance of freezing temperatures. In contrast to more complex plants, no ice accumulated within the moss during the freezing event. External ice induced desiccation with the response being a function of cell type; water‐filled hydroid cells cavitated and were embolized at ?4 °C while parenchyma cells of the inner cortex exhibited cytorrhysis, decreasing to ～20% of their original volume at a nadir temperature of ?20 °C. Chlorophyll fluorescence showed that these winter acclimated mosses displayed no evidence of damage after thawing from ?20 °C while GCMS showed that sugar concentrations were not sufficient to confer this level of freezing tolerance. In addition, differential scanning calorimetry showed internal ice nucleation occurred in hydrated moss at ～?12 °C while desiccated moss showed no evidence of freezing with lowering of nadir temperature to ?20 °C. Therefore the rapid dehydration of the moss provides an elegantly simple solution to the problem of freezing; remove that which freezes.     

Acclimation and endogenous abscisic acid in the moss Physcomitrella patens during acquisition of desiccation tolerance

The moss Physcomitrella patens has been used as a model organism to study the induction of desiccation tolerance (DT), but links between dehydration rate, the accumulation of endogenous abscisic acid (ABA) and DT remain unclear. In this study, we show that prolonged acclimation of P. patens at 89% relative humidity (RH) [?16 MPa] can induce tolerance of desiccation at 33% RH (?153 MPa) in both protonema and gametophore stages. During acclimation, significant endogenous ABA accumulation occurred after 1 day in gametophores and after 2 days in protonemata. Physcomitrella patens expressing the ABA‐inducible EARLY METHIONINE promoter fused to a cyan fluorescent protein (CFP) reporter gene revealed a mostly uniform distribution of the CFP increasing throughout the tissues during acclimation. DT was measured by day 6 of acclimation in gametophores, but not until 9 days of acclimation for protonemata. These results suggest that endogenous ABA accumulating when moss cells experience moderate water loss requires sufficient time to induce the changes that permit cells to survive more severe desiccation. These results provide insight for ongoing studies of how acclimation induces metabolic changes to enable DT in P. patens.     

Light,Temperature, and Desiccation Effects on Photosynthetic Activity,and Drought-Induced Ultrastructural Changes in the Green Alga <Emphasis Type="Italic">Klebsormidium dissectum</Emphasis> (Streptophyta) from a High Alpine Soil Crust

Members of the cosmopolitan green algal genus Klebsormidium (Klebsormidiales, Streptophyta) are typical components of terrestrial microbiotic communities such as biological soil crusts, which have many important ecological functions. In the present study, Klebsormidium dissectum (Gay) Ettl &; Gärtner was isolated from a high alpine soil crust in the Tyrolean Alps, Austria. Physiological performance in terms of growth and photosynthesis was investigated under different controlled abiotic conditions and compared with ultrastructural changes under the treatments applied. K. dissectum showed very low light requirements as reflected in growth patterns and photosynthetic efficiency. Increasing temperatures from 5°C to 40°C led to different effects on respiratory oxygen consumption and photosynthetic oxygen evolution. While at low temperatures (5–10°C), respiration was not detectable or on a very low level, photosynthesis was relatively high, Reversely, at the highest temperature, respiration was unaffected, and photosynthesis strongly inhibited pointing to strong differences in temperature sensitivity between both physiological processes. Although photosynthetic performance of K. dissectum was strongly affected under short-term desiccation and recovered only partly after rehydration, this species was capable to survive even 3 weeks at 5% relative air humidity. K. dissectum cells have a cell width of 5.6?±?0.3 μm and a cell length of 8.4?±?2.0 μm. Desiccated cells showed a strongly reduced cell width (46% of control) and cell length (65% of control). In addition, in desiccated cells, fewer mitochondria were stained by DIOC₆, and damaged plasma membranes were detected by FM 1–43 staining. High-pressure freeze fixation as well as chemical fixation allowed visualizing ultrastructural changes caused by desiccation. In such cells, the nucleus and chloroplast were still visibly intact, but the extremely thin cell walls (75–180 nm) were substantially deformed. The cytoplasm appeared electron dense and mitochondria were altered. Although K. dissectum can be characterized as euryoecious species, all ecophysiological and ultrastructural data indicate susceptibility to desiccation. However, the steadily occurring fragmentation of filaments into smaller units leads to improved self protection and thus may represent a life strategy to better survive longer periods of drought in exposed alpine soil crusts.     

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.