Effects of dehydration rate on physiological responses and survival after rehydration in larvae of the anhydrobiotic chironomid

Strategies to combat desiccation are critical for organisms living in arid and semi-arid areas. Larvae of the Australian chironomid Paraborniella tonnoiri resist desiccation by reducing water loss. In contrast, larvae of the African species Polypedilum vanderplanki can withstand almost complete dehydration, referred to as anhydrobiosis. For successful anhydrobiosis, the dehydration rate of P. vanderplanki larvae has to be controlled. Here, we desiccated larvae by exposing them to different drying regimes, each progressing from high to low relative humidity, and examined survival after rehydration. In larvae of P. vanderplanki, reactions following desiccation can be categorized as follows: (I) no recovery at all (direct death), (II) dying by unrepairable damages after rehydration (delayed death), and (III) full recovery (successful anhydrobiosis). Initial conditions of desiccation severely affected survival following rehydration, i.e. P. vanderplanki preferred 100% relative humidity where body water content decreased slightly. In subsequent conditions, unfavorable dehydration rate, such as more than 0.7 mg water lost per day, resulted in markedly decreased survival rate of rehydrated larvae. Slow dehydration may be required for the synthesis and distribution of essential molecules for anhydrobiosis. Larvae desiccated at or above maximum tolerable rates sometimes showed temporary recovery but died soon after.     

Analysis of the nuclear proteome of the resurrection plant Xerophyta viscosa in response to dehydration stress using iTRAQ with 2DLC and tandem mass spectrometry

Xerophyta viscosa Baker (family Velloziaceae) is a desiccation tolerant plant which survives extremes of dehydration down to 5% relative water content (RWC) and resumes full physiological activity within 80h of rehydration. The nuclear proteome of Xerophyta viscosa and its response to dehydration at 35% RWC as compared to fully hydrated plants was analysed using iTRAQ together with 2DLC and ESI-MS/MS. RWC at 35% is unique for desiccation tolerant species as it represents a distinct phase of the dehydration process where induction of late protection mechanisms are initiated. We reproducibly identified 122 proteins with confidence≥95% (ρ<0.05). In response to dehydration, 65% of the identified proteins had the same protein abundance as the hydrated, 22% were shown to be more abundant while 9.8% were less abundant. Classification of the nuclear proteins according to GO annotation showed that most proteins were part of cellular processes (77.43%) and had binding activity (85.47%) respectively. Ontological classification according to Interpro and Pfam databases categorized most nuclear proteins as part of gene regulation (21%) while the functions of the mapped proteins using MapMan showed involvement in protein synthesis (22%), degradation (9%), DNA structure (8%) and regulation (8%).     

Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation

Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.     

Desiccation tolerance of Hymenophyllacea filmy ferns is mediated by constitutive and non-inducible cellular mechanisms

The Hymenophyllaceae is a primitive family within the Filicopsidae. One of the most exceptional features of this family of ferns is the presence of fronds with one or just a few cell layers (hence their name of filmy ferns), and the absence of stomata. Hymenophyllum caudiculatum and Hymenophyllum dentatum are able to lose more than 82% of their fully hydrated water content, to remain dry for extended periods of time (days or weeks), and to survive and remain viable following rehydration. The aim of this work was to understand whether the adaptive strategy of the Hymenophyllaceae for desiccation tolerance is constitutive or inducible. A proteomic approach was adopted in combination with physiological parameters to assess whether there were changes in the protein content during dehydration and following rehydration. Detached fronds were used to monitor the rates of photosynthesis in desiccation experiments, sugar accumulation, and high-resolution 2-DE to analyze proteome variation during a desiccation–rehydration cycle. The analyzed proteome exhibited little variation (3–4%) between hydrated and desiccated states, while variation was greater between the desiccated and rehydrated states (8.7–10%). Eighty-two discrete proteins were analyzed by MS/MS, and 65 were identified. About 21% of the analyzed proteins (17) were mixtures of two or more different polypeptides. Of the identified proteins, more than a half (33 spots, 55%) had functions related to energy-photosynthesis. The second largest category with known function (five spots, 8%) was related to cell rescue, defense, and virulence. More than one in every four proteins analyzed belonged to a group of hypothetical proteins (18 spots, 28%). The results suggest that the Hymenophyllaceae represent an example of a change in adaptive strategy from a typical vascular to the poikilohydric homoiochlorophyllous adaptation, which they share with the bryophytes that grow in profusion in the same habitats. The speed at which desiccation takes place therefore precludes the induction of protective systems, suggesting a constitutive mechanism of cellular protection.     

Exploring the Mechanism of Physcomitrella patens Desiccation Tolerance through a Proteomic Strategy

The moss Physcomitrella patens has been shown to tolerate abiotic stresses, including salinity, cold, and desiccation. To better understand this plant's mechanism of desiccation tolerance, we have applied cellular and proteomic analyses. Gametophores were desiccated over 1 month to 10% of their original fresh weight. We report that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling, and microtubule depolymerization. Despite the severe desiccation, the membrane system maintains integrity. Through two-dimensional gel electrophoresis and image analysis, we identified 71 proteins as desiccation responsive. Following identification and functional categorization, we found that a majority of the desiccation-responsive proteins were involved in metabolism, cytoskeleton, defense, and signaling. Degradation of cytoskeletal proteins might result in cytoskeletal disassembly and consequent changes in the cell structure. Late embryogenesis abundant proteins and reactive oxygen species-scavenging enzymes are both prominently induced, and they might help to diminish the damage brought by desiccation.     

Energetic consequences of repeated and prolonged dehydration in the Antarctic midge, Belgica antarctica

Larvae of the Antarctic midge, Belgica antarctica, routinely face periods of limited water availability in their natural environments on the Antarctic Peninsula. As a result, B. antarctica is one of the most dehydration-tolerant insects studied, surviving up to 70% loss of its body water. While previous studies have characterized the physiological effects of a single bout of dehydration, in nature larvae are likely to experience multiple bouts of dehydration throughout their lifetime. Thus, we examined the physiological consequences of repeated dehydration and compared results to larvae exposed to a single, prolonged period of dehydration. For the repeated dehydration experiment, larvae were exposed to 1-5 cycles of 24 h dehydration at 75% RH followed by 24 h rehydration. Each bout of dehydration resulted in 30-40% loss of body water, with a concomitant 2- to 3-fold increase in body fluid osmolality. While nearly 100% of larvae survived a single bout of dehydration, <65% of larvae survived five such cycles. Larvae subjected to multiple bouts of dehydration also experienced severe depletion of carbohydrate energy reserves; glycogen and trehalose content decreased with each successive cycle, with larvae losing 89% and 48% of their glycogen and trehalose, respectively, after five cycles of dehydration/rehydration. Larvae exposed to prolonged dehydration (99% RH for 10d) had 26% less water, 43% less glycogen, and 27% less lipid content than controls, but did not experience any mortality. Thus, both repeated and prolonged dehydration results in substantial energetic costs that are likely to negatively impact fitness.     

干燥-再吸水条件下爪哇伪枝藻生理特性和超微结构特征

以典型荒漠丝状蓝藻爪哇伪枝藻为材料,在温室中设置水合(对照)、轻微干燥、中度干燥和极度干燥4种处理,研究干燥胁迫对藻体光合活性、膜脂过氧化、细胞可溶性物质含量、抗氧化酶活性以及细胞超微结构的影响,并采用不同促进剂和抑制剂对干燥藻体进行再吸水处理,测定藻体光合活性的恢复情况。结果显示:(1)爪哇伪枝藻在干燥胁迫下PSⅡ最大光化学效率(Fv/Fm)显著降低,并与其藻体水分含量之间呈极显著性相关(r=0.97、P<0.000 1);(2)随着干燥胁迫程度增加,藻体MDA含量、SOD和CAT活性随之升高,细胞可溶性蛋白和可溶性糖含量增加;(3)在藻体再吸水条件下,培养液(BG-110)、胞外多糖和蔗糖对藻体Fv/Fm的恢复具有重要作用,N-乙酰半胱氨酸和脯氨酸对Fv/Fm有一定的恢复效果,氯霉素和敌草隆则抑制Fv/Fm;(4)与水合状态下的细胞结构相比,干燥藻体细胞结构发生明显的变化,如细胞壁增厚,原生质粘稠浓缩、呈紧密分层排列,细胞内出现大量细小黑色颗粒物等。(5)采用不同外源物质对干燥藻体进行再吸水时,藻体的光合活性呈现不同的恢复效果。研究表明,干燥胁迫下爪哇伪枝藻的光合活性受到明显抑制,细胞质膜过氧化程度加剧,细胞出现可溶性小分子物质积累,抗氧化酶活性增强,并造成细胞结构出现适应性变化。     

Moisture Stress Effects on Survival of Infective Trichostrongylus colubriformis Larvae

Water was evaporated from infective Trichostrongylus colubriformis larvae suspended in tap, distilled, and triple-distilled water, and the nematodes were then exposed to 50% and 70% relative humidity (RH) at 20 and 30 C. Sample groups were rehydrated for 4 h daily in similar quality water, observed for motility and counted, then returned to the same RH and temp and re-desiccated. Desiccation and rehydration were repeated until all motility ceased. Longest survival was 30 days at 20 C and 70% RH. In all temp and RH combinations, control (nondesiccated) and desiccated larvae survived longer in distilled or triple-distilled water than in tap water.     

Photosynthetic activity of homoiochlorophyllous desiccation tolerant plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> during dehydration and rehydration

The functional state of the photosynthetic apparatus of flowering homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and subsequent rehydration was investigated in order to characterize some of the mechanisms by which resurrection plants survive drought stress. The changes in the CO₂ assimilation rate, chlorophyll fluorescence parameters, thermoluminescence, fluorescence imaging and electrophoretic characteristics of the chloroplast proteins were measured in control, moderately dehydrated (50% water content), desiccated (5% water content) and rehydrated plants. During the first phase of desiccation the net CO₂ assimilation decline was influenced by stomatal closure. Further lowering of net CO₂ assimilation was caused by both the decrease in stomatal conductance and in the photochemical activity of photosystem II. Severe dehydration caused inhibition of quantum yield of PSII electron transport, disappearance of thermoluminescence B band and mainly charge recombination related to S₂Q_A⁻ takes place. The blue and green fluorescence emission in desiccated leaves strongly increased. It could be suggested that unchanged chlorophyll content and amounts of chlorophyll–proteins, reversible modifications in PSII electron transport and enhanced probability for non-radiative energy dissipation as well as increased polyphenolic synthesis during desiccation of Haberlea contribute to drought resistance and fast recovery after rehydration.     

Antioxidant metabolism in the intertidal red seaweed Stictosiphonia arbuscula following desiccation

Seaweeds grow in distinct vertical bands on the seashore and it is well known that their ability to recover physiological processes following desiccation is correlated to their shore position. Despite this, little is known of the cellular mechanisms by which intertidal seaweeds limit membrane damage during desiccation and subsequent rehydration. In this study, specimens of the intertidal red seaweed Stictosiphonia arbuscula were placed in sealed tanks and maintained at different relative humidities (control, RH 90-100%; moderate desiccation, RH 70-80% and severe desiccation, RH 40-50%) for 12, 24 or 48 h. Membrane damage and antioxidant metabolism was examined immediately following specimen rehydration. Amino acid leakage, through the plasmalemma, was greater for desiccated low-band specimens than high-band specimens, indicating greater membrane damage. In addition, low-band specimens produced more hydrogen peroxide and lipid hydroperoxides than high-band specimens. This indicates that, upon rehydration, high-band populations have a greater ability to reduce the build-up of hydrogen peroxide, limit lipid peroxidation and hence membrane and protein damage, than low-band populations. The greater ability to prevent or reduce the production of reactive oxygen species was not due to a larger antioxidant pool, but rather increased activity of the enzymes required to regenerate ascorbate and glutathione. These findings suggest that antioxidant metabolism is one of the defence mechanisms that protect S. arbuscula from cellular damage due to desiccation.     

Desiccation induced changes in osmolytes production and the antioxidative defence in the cyanobacterium Anabaena sp. PCC 7120

Cells of Anabaena sp. PCC 7120, a low desiccation tolerant cyanobacterium, was subjected to prolonged desiccation and effect of loss of water was examined on production of osmolytes, and antioxidant response as well as on overall viability in terms of photosynthetic activity. During dehydration (22 h), the organism maintained about 98.5 % loss of cellular water, yet cells remained viable as about 30 % of photosynthetic O₂-evolution activity resumed upon hydrating (1 h) such cells. In desiccated state, cyanobacterial cells accumulated osmolytes within 1 h though their contents decreased thereafter. The highest levels of trehalose (179 nmol mg⁻¹ protein), sucrose (805 nmol mg⁻¹ protein) and proline (23.2 nmol mg⁻¹ protein) were attained within 1 h. Chlorophyll a and carotenoid contents also increased within 1 h but phycocyanin level showed opposite trend. The oxygen-evolving activity declined in desiccated cyanobacterial biomass while rehydration led to instant recovery, indicating that cells protect the photosynthetic machinery against desiccation. Notwithstanding, activities of antioxidant enzymes (catalase, peroxidase and superoxide dismutase) attained their peaks after 3 h of desiccation, though within 10 min of rehydration, their levels returned back close to basal activities of the cultured cells. We propose that onset of osmolyte production in conjunction with upshift of antioxidant enzymes apparently protects the cyanobacterial cells from desiccation stress.     

Dehydration rate and time of desiccation affect recovery of the lichenic algae <Emphasis Type="Italic">Trebouxia erici</Emphasis>: alternative and classical protective mechanisms

The mechanisms involved in desiccation tolerance of lichens and their photobionts are still poorly understood. To better understand these mechanisms we have studied dehydration rate and desiccation time in Trebouxia, the most abundant chlorophytic photobiont in lichen. Our findings indicate that the drying rate has a profound effect on the recovery of photosynthetic activity of algae after rehydration, greater than the effects of desiccation duration. The basal fluorescence (F′_o) values in desiccated algae were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried Trebouxia erici compared to rapid-dried algae. The main component of non-photochemical quenching after slow dehydration was energy dependent (q _E), whereas after fast dehydration it was photoinhibition (q _I). Although q _E seems to play a role during desiccation recovery, no significant variations were detected in the xanthophyll cycle components. Desiccation did not affect PSI functionality. Classical antioxidant activities like superoxide dismutase or peroxidase decreased during desiccation and early recovery. Dehydrins were detected in the lichen-forming algae T. erici and were constitutively expressed. There is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in this algae. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role, if any. However, our results indicate that there is an alternative mechanism of light energy dissipation during desiccation, where activation is dependent on a sufficiently slow dehydration rate.     

Dehydration,rehydration, and overhydration alter patterns of gene expression in the Antarctic midge, <Emphasis Type="Italic">Belgica antarctica</Emphasis>

We investigated molecular responses elicited by three types of dehydration (fast, slow and cryoprotective), rehydration and overhydration in larvae of the Antarctic midge, Belgica antarctica. The larvae spend most the year encased in ice but during the austral summer are vulnerable to summer storms, osmotic stress from ocean spray and drying conditions due to wind and intense sunlight. Using suppressive subtractive hybridization (SSH), we obtained clones that were potentially responsive to dehydration and then used northern blots to evaluate the gene’s responsiveness to different dehydration rates and hydration states. Among the genes most responsive to changes in the hydration state were those encoding heat shock proteins (smHsp, Hsp70, Hsp90), antioxidants (superoxide dismutase, catalase), detoxification (metallothionein, cytochrome p450), genes involved in altering cell membranes (fatty acid desaturase, phospholipase A2 activating protein, fatty acyl CoA desaturase) and the cytoskeleton (actin, muscle-specific actin), and several additional genes including a zinc-finger protein, pacifastin and VATPase. Among the three types of dehydration evaluated, fast dehydration elicited the strongest response (more genes, higher expression), followed by cryoprotective dehydration and slow dehydration. During rehydration most, but not all, genes that were expressed during dehydration continued to be expressed; fatty acid desaturase was the only gene to be uniquely upregulated in response to rehydration. All genes examined, except VATPase, were upregulated in response to overhydration. The midge larvae are thus responding quickly to water loss and gain by expressing genes that encode proteins contributing to maintenance of proper protein function, protection and overall cell homeostasis during times of osmotic flux, a challenge that is particularly acute in this Antarctic environment.     

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.     

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.     

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     

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.     

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.