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.     

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.     

Sip18 hydrophilin prevents yeast cell death during desiccation stress

Aims: For this study, we performed a genetic screen of S. cerevisiae’s deletion library for mutants sensitive to dehydration stress, with which we aimed to discover cell dehydration–tolerant genes. Methods and Results: We used a yeast gene deletion set (YGDS) of 4850 viable mutant haploid strains to perform a genome‐wide screen for the identification of desiccation stress modifiers. SIP18 is among the genes identified as essential for cells surviving to drying/rehydration process. Deletion of SIP18 promotes the accumulation of reactive oxygen species and enhances apoptotic and necrotic cell death in response to dehydration/rehydration process. Conclusions: SIP18p acts as an inhibitor of apoptosis in yeast under dehydration stress, as suggested by its antioxidative capacity through the ROS accumulation reduction after an H₂O₂ attack. Significance and Impact of the Study: To our knowledge, this is the first systematic screen for the identification of putative genes essential to overcoming cell dehydration process. A broad range of identified genes could help to understand why some strains of high biotechnological interest cannot cope with the drying and rehydration treatments. Dehydration sensitivity makes these strains not suitable to be commercialized by yeast manufactures.     

Differential proteomics of dehydration and rehydration in bryophytes: evidence towards a common desiccation tolerance mechanism

All bryophytes evolved desiccation tolerance (DT) mechanisms during the invasion of terrestrial habitats by early land plants. Are these DT mechanisms still present in bryophytes that colonize aquatic habitats? The aquatic bryophyte Fontinalis antipyretica Hedw. was subjected to two drying regimes and alterations in protein profiles and sucrose accumulation during dehydration and rehydration were investigated. Results show that during fast dehydration, there is very little variation in protein profiles, and upon rehydration proteins are leaked. On the other hand, slow dehydration induces changes in both dehydration and rehydration protein profiles, being similar to the protein profiles displayed by the terrestrial bryophytes Physcomitrella patens (Hedw.) Bruch and Schimp. and, to what is comparable with Syntrichia ruralis (Hedw.) F. Weber and D. Mohr. During dehydration there was a reduction in proteins associated with photosynthesis and the cytoskeleton, and an associated accumulation of proteins involved in sugar metabolism and plant defence mechanisms. Upon rehydration, protein accumulation patterns return to control values for both photosynthesis and cytoskeleton whereas proteins associated with sugar metabolism and defence proteins remain high. The current results suggest that bryophytes from different ecological adaptations may share common DT mechanisms.     

Increased activities of superoxide dismutase and catalase are not the mechanism of desiccation tolerance induced by hardening in the moss Atrichum androgynum

Abstract

The effects of treatments that increase desiccation tolerance were tested on the activity of the enzymes superoxide dismutase (SOD) and catalase (CAT) in the moss Atrichum androgynum subjected to a drying/wetting cycle. Hardening by both abscisic acid (ABA) pretreatment and partial dehydration significantly increased the rate of recovery of photosynthesis during rehydration following desiccation. Hardening treatments had little effect on SOD activity. In non-hardened plants, SOD activity increased three-fold during desiccation for 32 h at 52% rh, but hardened material tended to display smaller increases in activity. During rehydration, SOD activities rapidly declined to their initial values in all treatments. Hardening by partial dehydration, but not ABA, reduced CAT activity. After desiccation for 32 h, material from all treatments displayed about half the initial CAT activity, and activity did not change during subsequent rehydration. Results show that, while the induction of SOD appears to play a role in desiccation tolerance, a similar induction occurred in both hardened and non-hardened mosses. Induction of greater activities of enzymes that scavenge reactive oxygen species is not responsible for the added tolerance induced by hardening treatments.     

Fungal-associated NO is involved in the regulation of oxidative stress during rehydration in lichen symbiosis

Background  

Reactive oxygen species (ROS) are normally produced in respiratory and photosynthetic electron chains and their production is enhanced during desiccation/rehydration. Nitric oxide (NO) is a ubiquitous and multifaceted molecule involved in cell signaling and abiotic stress. Lichens are poikilohydrous organisms that can survive continuous cycles of desiccation and rehydration. Although the production of ROS and NO was recently demonstrated during lichen rehydration, the functions of these compounds are unknown. The aim of this study was to analyze the role of NO during rehydration of the lichen Ramalina farinacea (L.) Ach., its isolated photobiont partner Trebouxia sp. and Asterochloris erici (Ahmadjian) Skaloud et Peksa (SAG 32.85 = UTEX 911).     

Desiccation tolerance in Physcomitrella patens: Rate of dehydration and the involvement of endogenous abscisic acid (ABA)

The moss Physcomitrella patens , a model system for basal land plants, tolerates several abiotic stresses, including dehydration. We previously reported that Physcomitrella patens survives equilibrium dehydration to ?13 MPa in a closed system at 91% RH. Tolerance of desiccation to water potentials below ?100 MPa was only achieved by pretreatment with exogenous abscisic acid (ABA). We report here that gametophores, but not protonemata, can survive desiccation below ?100 MPa after a gradual drying regime in an open system, without exogenous ABA. In contrast, faster equilibrium drying at 90% RH for 3–5 days did not induce desiccation tolerance in either tissue. Endogenous ABA accumulated in protonemata and gametophores under both drying regimes, so did not correlate directly with desiccation tolerance. Gametophores of a Ppabi3a/b/c triple knock out transgenic line also survived the gradual dehydration regime, despite impaired ABA signaling. Our results suggest that the initial drying rate, and not the amount of endogenous ABA, may be critical in the acquisition of desiccation tolerance. Results from this work will provide insight into ongoing studies to uncover the role of ABA in the dehydration response and the underlying mechanisms of desiccation tolerance in this bryophyte.     

Reversible cellular and metabolic changes induced by dehydration in desiccation-tolerant wheat seedling shoots

Wheat seedlings obtained after 2 or 3 days of seed germination in darkness at 20°C (i.e. with a 0.5–0.7 cm long coleoptile) were still viable after drying in darkness in ambient conditions which reduced the shoot moisture content to about 0.30 g H₂O g^?1 dry mass (DM). Coleoptile and primary leaf growth resumed upon rehydration, but primary roots died and new roots regenerated. In the present work we have investigated whether desiccation tolerance of the shoot (coleoptile and primary leaf combined) was related to some reversible cellular or metabolic changes induced by dehydration. Non‐dehydrated shoots were high in moisture content (4.0–5.0 g H₂O g^?1 DM) and exhibited an active metabolism as indicated by a high energy charge (EC = 0.85) and cells with well developed mitochondria, endoplasmic reticulum, polysomes and Golgi bodies. Dehydration induced changes in cell membrane properties since it reduced in vivo capacity of the shoot to convert 1‐aminocyclopropane 1‐carboxylic acid (ACC) to ethylene (i.e. ACC oxidase activity). This effect was already observed at 4–5 h of dehydration, namely when shoot moisture content dropped down below about 3.0 g H₂O g^?1 DM, and ACC‐dependent ethylene production became almost nil when shoot moisture content reached 1.0 g H₂O g^?1 DM. Dehydration also resulted in decreases in ATP and non‐adenylic triphosphate nucleotide (NTP) contents down to 1–2% of their initial values, and in EC value to 0.20. Concomitant with water loss, sucrose content of the shoot increased and was maximal (about 330 mg g^?1 DM, namely three‐fold that of non‐dehydrated organs) after 2 days of drying. Upon rehydration, shoots regained their original moisture content within 3 days, during which they progressively recovered apparent normal metabolism. Reversal of extensive dehydration‐associated cell wall folding occurred between 2 and 3 days of rehydration, when the ultrastructure of coleoptile and primary leaf cells also provided evidence of intensive autophagic activity, indicative of the removal of damaged cell components. Concomitantly, apparently undamaged organelles and endomembranes persisted in the cytoplasm. Restoration of 60–70% of ACC oxidase activity and 80–90% of EC value occurred within 48 and 18 h, respectively. However, the values of the ATP/ADP and NTP/ATP ratios remained lower than in control non‐dehydrated shoots, indicating that not all metabolic deterioration induced by dehydration was completely repaired. Differences in relationships between shoot moisture content and ACC‐oxidase activity or energy metabolism during dehydration and upon rehydration, and cell ultrastruture analyses suggest that desiccation tolerance of wheat seedling shoot is related to mechanisms involved in the maintenance of cell structure during water loss and the cell capacity to repair the dehydration damage.     

Physiological consequences of desiccation in the aquatic bryophyte <Emphasis Type="Italic">Fontinalis antipyretica</Emphasis>

The moss Fontinalis antipyretica, an aquatic bryophyte previously described as desiccation-intolerant, is known to survive intermittent desiccation events in Mediterranean rivers. To better understand the mechanisms of desiccation tolerance in this species and to reconcile the apparently conflicting evidence between desiccation tolerance classifications and field observations, gross photosynthesis and chlorophyll a fluorescence were measured in field-desiccated bryophyte tips and in bryophyte tips subjected in the laboratory to slow, fast, and very fast drying followed by either a short (30 min) or prolonged (5 days) recovery. Our results show, for the first time, that the metabolic response of F. antipyretica to desiccation, both under field and laboratory conditions, is consistent with a desiccation-tolerance pattern; however, drying must proceed slowly for the bryophyte to regain its pre-desiccation state following rehydration. In addition, the extent of dehydration was found to influence metabolism whereas the drying rate determined the degree of recovery. Photosystem II (PSII) regulation and structural maintenance may be part of the induced desiccation tolerance mechanism allowing this moss to recover from slow drying. The decrease in the photochemical quenching coefficient (qP) immediately following rehydration may serve to alleviate the effects of excess energy on photosystem I (PSI), while low-level non-photochemical quenching (NPQ) would allow an energy shift enabling recovery subsequent to extended periods of desiccation. The findings were confirmed in field-desiccated samples, whose behavior was similar to that of samples slowly dried in the laboratory.     

快速脱水与复水过程中2种藓类植物的活性氧代谢与脂质过氧化特征

以来自不同水分生境的金发藓和湿地匐灯藓为材料,对二者在脱水与复水胁迫条件下的活性氧代谢、脂质过氧化损伤程度及其抗氧化系统应答的差异进行比较研究。结果显示:在脱水与复水过程中,(1)硅胶快速脱水更接近阳光直射条件下藓类植物的水分丧失。(2)随着含水量的变化,湿地匐灯藓虽然能够在复水后迅速修复细胞的完整性,但变化剧烈;金发藓则能够始终维持较低的膜透性。(3)2种藓类植物的丙二醛(MDA)含量变化均呈先升后降趋势,但金发藓的MDA含量明显低于湿地匐灯藓。(4)2种藓类植物的超氧阴离子自由基(O2.-)产生速率和过氧化氢含量(H2O2)的变化均与MDA含量变化相似,且金发藓活性氧水平明显高于湿地匐灯藓。(5)2种藓类植物的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性受活性氧诱导亦呈先升后降的趋势,但金发藓抗氧化酶对活性氧迸发的应答更快,活性更强。(6)2种藓类植物的抗坏血酸(AsA)含量呈先降后升态势,但金发藓的含量低于湿地匐灯藓。研究表明,来自不同生境的2种藓类植物对脱水胁迫所致的氧化胁迫均具有很强的适应能力,尤其是复水过程中的修复能力,但不同藓类可能通过不同途径和机制来适应脱水所致的氧化胁迫;来自易发生水分亏缺生境的金发藓可能因具有更强抗氧化能力,从而获得比来自水分充沛生境的湿地匐灯藓更高的脱水耐性。     

Analysis of desiccation-induced candidate phosphoproteins from Craterostigma plantagineum isolated with a modified metal oxide affinity chromatography procedure

Reversible protein phosphorylation/dephosphorylation is crucial for regulation of many cellular events, and increasing evidence indicates that this post-translational modification is also involved in the complex process of acquisition of desiccation tolerance. To analyze the phosphoproteome of the desiccation tolerant resurrection plant Craterostigma plantagineum, MOAC-enriched proteins from leaves at different stages of a de-/rehydration cycle were separated by 2-D PAGE and detected by phosphoprotein-specific staining. Using this strategy 20 putative phosphoproteins were identified by MALDI-TOF MS and MS/MS, which were not detected when total proteins were analyzed. The characterized desiccation-related phosphoproteins CDeT11-24 and CDeT6-19 were used as internal markers to validate the specificity of the analyses. For 16 of the identified proteins published evidence suggests that they are phosphoproteins. Comparative analysis of the 2-D gels showed that spot intensities of most identified putative phosphoproteins change during the de-/rehydration cycle. This suggests an involvement of these proteins in desiccation tolerance. Nearly all changes in the phosphoproteome of C. plantagineum, which are triggered by dehydration, are reversed within 4 days of rehydration, which is in agreement with physiological observations. Possible functions of selected proteins are discussed in the context of the de-/rehydration cycle.     

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.     

The use of aeroponics to investigate antioxidant activity in the roots of <Emphasis Type="Italic">Xerophyta viscosa</Emphasis>

In order to ultimately understand the whole plant mechanism of attaining desiccation tolerance, we undertook to investigate the root tissues of the resurrection plant Xerophyta viscosa, as previous work has only been conducted on the leaf tissues of resurrection plants. An aeroponic plant growth system was designed and optimised to observe the root’s response to desiccation without the restrictions of a soil medium, allowing easy access to roots. Successful culture of both X.viscosa and the control, Zea mays, was achieved and dehydration stress was implemented through reduction of nutrient solution spraying of the roots. After drying to the air dry state (achieved after 7 days for roots and 10 days for shoots), rehydration was achieved by resumption of root spraying. X.viscosa plants survived desiccation and recovered but Z. mays did not. The activity of the antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase and quantities of ascorbate and glutathione were determined during root desiccation. There was an initial decline in activity in all enzymes upon drying to 80% RWC, but activity thereafter remained constant, at rates indicative of potential metabolic activity, to the air-dry state. This data suggests that these enzymes are not denatured by desiccation of the root tissue. Ascorbate and glutathione content remained constant at concentrations of 70 and 100 μM, respectively during drying. Thus root tissues appear to retain antioxidant potential during drying, for use in recovery upon rehydration, as has been reported for leaf tissues of this and other resurrection plants.     

Photosynthetic limitations and volatile and non‐volatile isoprenoids in the poikilochlorophyllous resurrection plant Xerophyta humilis during dehydration and rehydration

We investigated the photosynthetic limitations occurring during dehydration and rehydration of Xerophyta humilis, a poikilochlorophyllous resurrection plant, and whether volatile and non‐volatile isoprenoids might be involved in desiccation tolerance. Photosynthesis declined rapidly after dehydration below 85% relative water content (RWC). Raising intercellular CO₂ concentrations during desiccation suggest that the main photosynthetic limitation was photochemical, affecting energy‐dependent RuBP regeneration. Imaging fluorescence confirmed that both the number of photosystem II (PSII) functional reaction centres and their efficiency were impaired under progressive dehydration, and revealed the occurrence of heterogeneous photosynthesis during desiccation, being the basal leaf area more resistant to the stress. Full recovery in photosynthetic parameters occurred on rehydration, confirming that photosynthetic limitations were fully reversible and that no permanent damage occurred. During desiccation, zeaxanthin and lutein increased only when photosynthesis had ceased, implying that these isoprenoids do not directly scavenge reactive oxygen species, but rather protect photosynthetic membranes from damage and consequent denaturation. X. humilis was found to emit isoprene, a volatile isoprenoid that acts as a membrane strengthener in plants. Isoprene emission was stimulated by drought and peaked at 80% RWC. We surmise that isoprene and non‐volatile isoprenoids cooperate in reducing membrane damage in X. humilis, isoprene being effective when desiccation is moderate while non‐volatile isoprenoids operate when water deficit is more extreme.     

Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to mammalian somatic cells

Late Embryogenesis Abundant (LEA) proteins are commonly found in plants and other organisms capable of undergoing severe and reversible dehydration, a phenomenon termed “anhydrobiosis”. Here, we have produced a tagged version for three different LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, binds to cellular membranes, and pTag-LEA-BFP, Artemia franciscana LEA protein group 3 that targets the mitochondria. Sheep fibroblasts transfected with single or all three LEA proteins were subjected to air drying under controlled conditions. After rehydration, cell viability and functionality of the membrane/mitochondria were assessed. After 4 h of air drying, cells from the un-transfected control group were almost completely nonviable (1% cell alive), while cells expressing LEA proteins showed high viability (more than 30%), with the highest viability (58%) observed in fibroblasts expressing all three LEA proteins. Growth rate was markedly compromised in control cells, while LEA-expressing cells proliferated at a rate comparable to non-air-dried cells. Plasmalemma, cytoskeleton and mitochondria appeared unaffected in LEA-expressing cells, confirming the protection conferred by LEA proteins on these organelles during dehydration stress. This is likely to be an effective strategy when aiming to confer desiccation tolerance to mammalian cells.