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.     

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%).     

Dessication-induced Changes in the Protein Complement of Soluble Extracts from Leaves of Resurrection Plants and Related Desiccation-sensitive Species

Polyacrylamide gel electrophoresis studies were conducted onthe soluble proteins of angiosperm plants whose leaf protoplasmcan revive from complete dehydration (Xerophyta viscosa, Talbotiaelegans, Sporobolus stapfianus, Myrothamnus flabellifolia, Boryanitida) and of desiccation sensitive plants (Sporobolus pyramidalis,Eragrostis tenuifolia, Selaginella kraussiana). Changes in thesoluble protein composition were found in all species afterdehydration, and were extensive in most species, both resurrectionand non-resurrection. Both groups showed loss of protein bands,but there was no consistent pattern of compositional changewithin either type of plant. Net hydrolysis of high molecularweight protein could be deduced, and the possibility of disulphide-mediatedaggregation arose in some species. Induction of tolerance todesiccation in Borya nitida appeared to be associated with retentionor restoration of the control pattern of protein bands in contrastto loss of very low and very high mol. wt protein (loss wasextreme in desiccation-killed leaves). There was evidence of a disproportionately great synthesis ofvery low mol. wt protein during the midphase of rehydrationin X. viscosa. These results point to the possibility of an important roleof protein synthesis for survival of dehydration. Resurrection plants, desiccation-sensitive plants, protein complement, polyacrylamide gel electrophoresis     

Characterization of oxidative and antioxidative events during dehydration and rehydration of resurrection plant <Emphasis Type="Italic">Ramonda nathaliae</Emphasis>

In order to investigate changes of oxidative status in relation to the activity of the various protective mechanisms in resurrection plant Ramonda nathaliae, we have analysed time and relative water content (RWC) related changes in lipid peroxidation and ion leakage, hydrogen peroxide accumulation, changes of pigment content and antioxidative enzyme activity, together with expression of dehydrins. The results indicate that enhanced oxidative status during dehydration, not previously reported for resurrection plants, could play an active role in inducing the desiccation adaptive response in R. nathaliae. A critical phase is shown to exist during dehydration (in the range of RWC between 50 and 70%) during which a significant increase in hydrogen peroxide accumulation, lipid peroxidation and ion leakage, accompanied by a general decline in antioxidative enzyme activity, takes place. This phase is designated as a transition characterized by change in the type of stress response. The initial response, relying mainly on the enzymatic antioxidative system, is suspended but more effective, desiccation specific protective mechanisms, such as expression of dehydrins, are then switched on. The expression of dehydrins in R. nathaliae could be inducible as well as constitutive. In order to cope with the oxidative stress associated with rapid rewatering, R. nathaliae reactivated antioxidative enzymes. We propose that controlled elevation of reactive oxygen species, such as hydrogen peroxide, could be an important mechanism enabling resurrection plants to sense dehydration and to trigger an adaptive programme at an appropriate stage during the dehydration/rehydration cycle.     

Proteomic analysis of cold stress-responsive proteins in <Emphasis Type="Italic">Thellungiella</Emphasis> rosette leaves

Low temperature is one of the most severe environmental factors that impair plant growth and agricultural production. To investigate how Thellungiella halophila, an Arabidopsis-like extremophile, adapts to cold stress, a comparative proteomic approach based on two-dimensional electrophoresis was adopted to identify proteins that changed in abundance in Thellungiella rosette leaves during short term (6 h, 2 and 5 days) and long term (24 days) exposure to cold stress. Sixty-six protein spots exhibited significant change at least at one time point and maximal cold stress induced-proteome change was found in long-term cold stress group while the minimal change was found in 6-h cold treatment group. Fifty protein spots were identified by mass spectrometry analysis. The identified proteins mainly participate in photosynthesis, RNA metabolism, defense response, energy pathway, protein synthesis, folding and degradation, cell wall and cytoskeleton and signal transduction. These proteins might work cooperatively to establish a new homeostasis under cold stress. Nearly half of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology suggesting that the cold stress tolerance of T. halophila is achieved, at least partly, by regulation of chloroplast function. All protein spots involved in RNA metabolism, defense response, protein synthesis, folding and degradation were found to be upregulated markedly by cold treatment, indicating enhanced RNA metabolism, defense and protein metabolism may play crucial roles in cold tolerance mechanism in T. halophila.     

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.     

Group 3 late embryogenesis abundant protein in <Emphasis Type="Italic">Arabidopsis</Emphasis>: structure,regulation, and function

Group 3 late embryogenesis abundant proteins accumulate in maturing seeds, in which their expression correlates with desiccation tolerance. Group 3 proteins are also strongly associated with tolerance for abiotic stresses, such as high salinity, drought, cold, and osmotic stress in vegetative tissues. However, the precise function of these proteins remained obscure for more than 20 years. In this study, the structure of and available regulation information on Group 3 genes/proteins in Arabidopsis are reviewed. The function of Group 3 proteins in response to desiccation and the relationship between protein structure and function are also discussed.     

Identification of two hydrophilins that contribute to the desiccation and freezing tolerance of yeast (Saccharomyces cerevisiae) cells

Hydrophilins are a group of proteins that are present in all organisms and that have been defined as being highly hydrophilic and rich in glycine. They are assumed to play important roles in cellular dehydration tolerance. There are 12 genes in the yeast Saccharomyces cerevisiae that encode hydrophilins and most of these genes are stress responsive. However, the functional role of yeast hydrophilins, especially in desiccation and freezing tolerance, is largely unknown. Here, we selected six candidate hydrophilins for further analysis. All six proteins were predicted to be intrinsically disordered, i.e. to have no stable structure in solution. The contribution of these proteins to the desiccation and freezing tolerance of yeast was investigated in the respective knock-out strains. Only the disruption of the genes YJL144W and YMR175W (SIP18) resulted in significantly reduced desiccation tolerance, while none of the strains was affected in its freezing tolerance under our experimental conditions. Complementation experiments showed that yeast cells overexpressing these two genes were both more desiccation and freezing tolerant, confirming the role of these two hydrophilins in yeast dehydration stress tolerance.     

Ultrastructural responses of the desiccation tolerant plants Xerophyta viscosa and X. retinervis to dehydration and rehydration

This paper compares the changes in water content, chlorophyll a fluorescence and leaf ultrastructure during dehydration and rehydration in two desiccation tolerant plants Xerophyta viscosa and X. retinervis. Both species showed decreasing quantum efficiency of photosystem 2 (F_v/F_m) with decreasing water content. Extreme water loss observed after 25 d of dehydration resulted in considerable damage of leaf tissue ultrastructure. After rehydration, both species need several days to reconstitute their photosynthetic machinery.     

Protein Synthesis in the Desiccation Tolerant Angiosperm Xerophyta villosa during Dehydration

Leaves of the resurrection plant Xerophyta villosa appear tobecome desiccation tolerant while they dry on the intact plant.After a small decline during moderate water stress, the polyribosomecontent of attached leaves appears to rise at 50% relative watercontent (RWC) to almost double the content in controls, beforeit finally declines to zero at 20% RWC. Partition of leaf solubleprotein by polyacrylamide gel electrophoresis indicates an increasein protein with low mobility in dehydrated leaves. Studies onthe incorporation of ¹⁴C-valine and ³H-valine, and of proteinsdissociated into their component polypeptddes suggest that proteinsynthesis during dehydration is at least partly responsiblefor the changes in soluble protein.     

A field portable method for the semi‐quantitative estimation of dehydration tolerance of photosynthetic tissues across distantly related land plants

Desiccation tolerant (DT) plants withstand complete cellular dehydration, reaching relative water contents (RWC) below 30% in their photosynthetic tissues. Desiccation sensitive (DS) plants exhibit different degrees of dehydration tolerance (DHT), never surviving water loss >70%. To date, no procedure for the quantitative evaluation of DHT extent exists that is able to discriminate DS species with differing degrees of DHT from truly DT plants. We developed a simple, feasible and portable protocol to differentiate between DT and different degrees of DHT in the photosynthetic tissues of seed plants and between fast desiccation (< 24 h) tolerant (FDT) and sensitive (FDS) bryophytes. The protocol is based on (1) controlled desiccation inside Falcon tubes equilibrated at three different relative humidities that, consequently, induce three different speeds and extents of dehydration and (2) an evaluation of the average percentage of maximal photochemical efficiency of PSII (F_v/fm) recovery after rehydration. Applying the method to 10 bryophytes and 28 tracheophytes from various locations, we found that (1) imbibition of absorbent material with concentrated salt‐solutions inside the tubes provides stable relative humidity and avoids direct contact with samples; (2) for 50 ml capacity tubes, the optimal plant amount is 50–200 mg fresh weight; (3) the method is useful in remote locations due to minimal instrumental requirements; and (4) a threshold of 30% recovery of the initial F_v/fm upon reaching RWC ≤ 30% correctly categorises DT species, with three exceptions: two poikilochlorophyllous species and one gymnosperm. The protocol provides a semi‐quantitative expression of DHT that facilitates comparisons of species with different morpho‐physiological traits and/or ecological attributes.     

Comparative proteomics of dehydration response in the rice nucleus: New insights into the molecular basis of genotype‐specific adaptation

Dehydration is the most crucial environmental factor that considerably reduces the crop harvest index, and thus has become a concern for global agriculture. To better understand the role of nuclear proteins in water‐deficit condition, a nuclear proteome was developed from a dehydration‐sensitive rice cultivar IR‐64 followed by its comparison with that of a dehydration‐tolerant c.v. Rasi. The 2DE protein profiling of c.v. IR‐64 coupled with MS/MS analysis led to the identification of 93 dehydration‐responsive proteins (DRPs). Among those identified proteins, 78 were predicted to be destined to the nucleus, accounting for more than 80% of the dataset. While the detected number of protein spots in c.v. IR‐64 was higher when compared with that of Rasi, the number of DRPs was found to be less. Fifty‐seven percent of the DRPs were found to be common to both sensitive and tolerant cultivars, indicating significant differences between the two nuclear proteomes. Further, we constructed a functional association network of the DRPs of c.v. IR‐64, which suggests that a significant number of the proteins are capable of interacting with each other. The combination of nuclear proteome and interactome analyses would elucidate stress‐responsive signaling and the molecular basis of dehydration tolerance in plants.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> is highly tolerant against drought,salt and osmotic stress

In order to determine the degree of tolerance of the moss Physcomitrella patens to different abiotic stress conditions, we examined its tolerance against salt, osmotic and dehydration stress. Compared to other plants like Arabidopsis thaliana, P. patens exhibits a high degree of abiotic stress tolerance, making it a valuable source for the identification of genes effecting the stress adaptation. Plants that had been treated with NaCl tolerated concentrations up to 350 mM. Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM. Furthermore, plants that had lost 92% water on a fresh-weight basis were able to recover successfully. For molecular analyses, a P. patens expressed sequence tag (EST) database was searched for cDNA sequences showing homology to stress-associated genes of seed plants and bacteria. 45 novel P. patens genes were identified and subjected to cDNA macroarray analyses to define their expression pattern in response to water deficit. Among the selected cDNAs, we were able to identify a set of genes that is specifically up-regulated upon dehydration. These genes encode proteins exerting their function in maintaining the integrity of the plant cell as well as proteins that are known to be members of signaling networks. The identified genes will serve as molecular markers and potential targets for future functional analyses.