Sugar ratios, glutathione redox status and phenols in the resurrection species Haberlea rhodopensis and the closely related non-resurrection species Chirita eberhardtii

Because of their unique tolerance to desiccation, the so‐called resurrection plants can be considered as excellent models for extensive research on plant reactions to environmental stresses. The vegetative tissues of these species are able to withstand long dry periods and to recover very rapidly upon re‐watering. This study follows the dynamics of key components involved in leaf tissue antioxidant systems under desiccation in the resurrection plant Haberlea rhodopensis and the related non‐resurrection species Chirita eberhardtii. In H. rhodopensis these parameters were also followed during recovery after full drying. A well‐defined test system was developed to characterise the different responses of the two species under drought stress. Results show that levels of H₂O₂ decreased significantly both in H. rhodopensis and C. eberhardtii, but that accumulation of malondialdehyde was much more pronounced in the desiccation‐tolerant H. rhodopensis than in the non‐resurrection C. eberhardtii. A putative protective role could be attributed to accumulation of total phenols in H. rhodopensis during the late stages of drying. The total glutathione concentration and GSSG/GSH ratio increased upon complete dehydration of H. rhodopensis. Our data on soluble sugars suggest that sugar ratios might be important for plant desiccation tolerance. An array of different adaptations could thus be responsible for the resurrection phenotype of H. rhodopensis.     

Metabolic profiling of the resurrection plant Haberlea rhodopensis during desiccation and recovery

Desiccation tolerance is among the most important parameters for crop improvement under changing environments. Resurrection plants are useful models for both theoretical and practical studies. We performed metabolite profiling via gas chromatography coupled with mass spectrometry (GC‐MS) and high‐performance liquid chromatography (HPLC) and analyzed the antioxidant capacity of the endemic resurrection plant Haberlea rhodopensis at desiccation and recovery. More than 100 compounds were evaluated. Stress response included changes in both primary and secondary metabolic pathways. The high amounts of the specific glycoside myconoside and some phenolic acids – e.g. syringic and dihydrocaffeic acid under normal conditions tend to show their importance for the priming of H. rhodopensis to withstand severe desiccation and oxidative stress. The accumulation of sucrose (resulting from starch breakdown), total phenols, β‐aminoisobutyric acid, β‐sitosterol and α‐tocopherol increased up to several times at later stages of desiccation. Extracts of H. rhodopensis showed high antioxidant capacity at stress and normal conditions. Myconoside was with the highest antioxidant properties among tested phenolic compounds. Probably, the evolution of resurrection plants under various local environments has resulted in unique desiccation tolerance with specific metabolic background. In our case, it includes the accumulation of a relatively rare compound (myconoside) that contributes alone and together with other common metabolites. Further systems biology studies on the involvement of carbohydrates, phenolic acids and glycosides in the desiccation tolerance and antioxidant capacity of H. rhodopensis will definitely help in achieving the final goal – improving crop drought tolerance.     

Comparison of thylakoid structure and organization in sun and shade Haberlea rhodopensis populations under desiccation and rehydration

The resurrection plant, Haberlea rhodopensis can survive nearly total desiccation only in its usual low irradiation environment. However, populations with similar capacity to recover were discovered recently in several sunny habitats. To reveal what kind of morphological, structural and thylakoid-level alterations play a role in the acclimation of this low-light adapted species to high-light environment and how do they contribute to the desiccation tolerance mechanisms, the structure of the photosynthetic apparatus, the most sensitive component of the chlorophyll-retaining resurrection plants, was analyzed by transmission electron microscopy, steady state low-temperature fluorescence and two-dimensional Blue-Native/SDS PAGE under desiccation and rehydration.     

Desiccation‐induced alterations in surface topography of thylakoids from resurrection plant Haberlea rhodopensis studied by atomic force microscopy,electrokinetic and optical measurements

With their ability to survive complete desiccation, resurrection plants are a suitable model system for studying the mechanisms of drought tolerance. In the present study, we investigated desiccation‐induced alterations in surface topography of thylakoids isolated from well‐hydrated, moderately dehydrated, severely desiccated and rehydrated Haberlea rhodopensis plants by means of atomic force microscopy (AFM), electrokinetic and optical measurements. According to our knowledge, so far, there were no reports on the characterization of surface topography and polydispersity of thylakoid membranes from resurrection plants using AFM and dynamic light scattering. To study the physicochemical properties of thylakoids from well‐hydrated H. rhodopensis plants, we used spinach thylakoids for comparison as a classical model from higher plants. The thylakoids from well‐hydrated H. rhodopensis had a grainy surface, significantly different from the well‐structured spinach thylakoids with distinct grana and lamella, they had twice smaller cross‐sectional area and were 1.5 times less voluminous than that of spinach. Significant differences in their physicochemical properties were observed. The dehydration and subsequent rehydration of plants affected the size, shape, morphology, roughness and therefore the structure of the studied thylakoids. Drought resulted in significant enhancement of negative charges on the outer surface of thylakoid membranes which correlated with the increased roughness of thylakoid surface. This enhancement in surface charge density could be due to the partial unstacking of thylakoids exposing more negatively charged groups from protein complexes on the membrane surface that prevent from possible aggregation upon drought stress.     

Prompt response of superoxide dismutase and peroxidase to dehydration and rehydration of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The relic endemic nature of Haberlea rhodopensis, which grows in Balkan Peninsula, in combination with its high vegetative desiccation-tolerance, makes this species a good model to study mechanisms behind plant adaptation to severe drought stress. The aim of this study was to evaluate the antioxidant protection provided by Superoxide dismutase (SOD) and Peroxidase (PO) in H. rhodopensis after exposure to and recovery from dehydration at different developmental stages. During dehydration the electrolyte leakage from leaf tissue increased more significantly in post-flowering plants than in flowering plants, while upon subsequent rehydration this parameter showed a very fast decrease to the basic value of fresh leaves and did not depend on developmental stage. Like other higher plant species, SOD and PO demonstrated in H. rhodopensis an ability to adjust their activity very promptly to changing water supply. In addition, the leaves of this resurrection species retained significant activities of SOD and PO even in air-dried state, considered as the most severe form of water stress. The enhanced activity of antioxidant enzymes may either enable the scavenging of the active oxygen species produced at very severe water deficit, and/or carry a potential for resurrection on subsequent rehydration. Upon stress treatment total activities of both enzymes were higher in flowering than post-flowering plants which reveals that developmental stage might be a factor affecting plant stress tolerance. This work identified for the first time SOD isoforms of H. rhodopensis. Native PAGE showed at least six multiple isoforms in the protein extract from leaf tissue of flowering plants, and the differential visualization revealed that four of them were Cu, Zn-SOD isoforms, one was Mn-SOD and one Fe-SOD. These findings provide a good starting point for future study of the SOD gene family of this rare resurrection plant at the molecular level.     

Desiccation Tolerance Studied in the Resurrection Plant Craterostigma plantagineum

This review will focus on the acquisition of desiccation tolerancein the resurrection plant Craterostigma plantagineum. Molecularaspects of desiccation tolerance in this plant will be comparedwith the response of non-tolerant plants to dehydration. Uniquefeatures of C. plantagineum are described like the CDT-1 (Craterostigmadesiccation tolerance gene-1) gene and the carbohydrate metabolism.Abundant proteins which are associated with the desiccationtolerance phenomenon are the late embryogenesis abundant (=LEA)proteins. These proteins are very hydrophilic and occur in severalother species which have acquired desiccation tolerance.     

The role of antioxidant defense in freezing tolerance of resurrection plant Haberlea rhodopensis

Haberlea rhodopensis Friv. is unique with its ability to survive two extreme environmental stresses—desiccation to air-dry state and subzero temperatures. In contrast to desiccation tolerance, the mechanisms of freezing tolerance of resurrection plants are scarcely investigated. In the present study, the role of antioxidant defense in the acquisition of cold acclimation and freezing tolerance in this resurrection plant was investigated comparing the results of two sets of experiments—short term freezing stress after cold acclimation in controlled conditions and long term freezing stress as a part of seasonal temperature fluctuations in an outdoor ex situ experiment. Significant enhancement in flavonoids and anthocyanin content was observed only as a result of freezing-induced desiccation. The total amount of polyphenols increased upon cold acclimation and it was similar to the control in post freezing stress and freezing-induced desiccation. The main role of phenylethanoid glucoside, myconoside and hispidulin 8-C-(2-O-syringoyl-b-glucopyranoside) in cold acclimation and freezing tolerance was elucidated. The treatments under controlled conditions in a growth chamber showed enhancement in antioxidant enzymes activity upon cold acclimation but it declined after subsequent exposure to −10 °C. Although it varied under ex situ conditions, the activity of antioxidant enzymes was high, indicating their important role in overcoming oxidative stress under all treatments. In addition, the activity of specific isoenzymes was upregulated as compared to the control plants, which could be more useful for stress counteraction compared to changes in the total enzyme activity, due to the action of these isoforms in the specific cellular compartments.Supplementary informationThe online version contains supplementary material available at 10.1007/s12298-021-00998-0.     

Protection of thylakoids against combined light and drought by a lumenal substance in the resurrection plant Haberlea rhodopensis

Background and Aims

Haberlea rhodopensis is a perennial, herbaceous, saxicolous, poikilohydric flowering plant that is able to survive desiccation to air-dried state under irradiance below 30 µmol m⁻² s⁻¹. However, desiccation at irradiance of 350 µmol m⁻² s⁻¹ induced irreversible changes in the photosynthetic apparatus, and mature leaves did not recover after rehydration. The aim here was to establish the causes and mechanisms of irreversible damage of the photosynthetic apparatus due to dehydration at high irradiance, and to elucidate the mechanisms determining recovery.

Methods

Changes in chloroplast structure, CO₂ assimilation, chlorophyll fluorescence parameters, fluorescence imaging and the polypeptide patterns during desiccation of Haberlea under medium (100 µmol m⁻² s⁻¹; ML) irradiance were compared with those under low (30 µmol m⁻² s⁻¹; LL) irradiance.

Key Results

Well-watered plants (control) at 100 µmol m⁻² s⁻¹ were not damaged. Plants desiccated at LL or ML had similar rates of water loss. Dehydration at ML decreased the quantum efficiency of photosystem II photochemistry, and particularly the CO₂ assimilation rate, more rapidly than at LL. Dehydration induced accumulation of stress proteins in leaves under both LL and ML. Photosynthetic activity and polypeptide composition were completely restored in LL plants after 1 week of rehydration, but changes persisted under ML conditions. Electron microscopy of structural changes in the chloroplast showed that the thylakoid lumen is filled with an electron-dense substance (dense luminal substance, DLS), while the thylakoid membranes are lightly stained. Upon dehydration and rehydration the DLS thinned and disappeared, the time course largely depending on the illumination: whereas DLS persisted during desiccation and started to disappear during late recovery under LL, it disappeared from the onset of dehydration and later was completely lost under ML.

Conclusions

Accumulation of DLS (possibly phenolics) in the thylakoid lumen is demonstrated and is proposed as a mechanism protecting the thylakoid membranes of H. rhodopensis during desiccation and recovery under LL. Disappearance of DLS during desiccation in ML could leave the thylakoid membranes without protection, allowing oxidative damage during dehydration and the initial rehydration, thus preventing recovery of photosynthesis.Key words: Haberlea rhodopensis, resurrection plant, electron microscopy, blue–green fluorescence, chlorophyll fluorescence     

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.     

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.     

Molecular characterization of two alanine-rich Lea genes abundantly expressed in the resurrection plant C. plantagineum in response to osmotic stress and ABA

Expression of many genes is induced during dehydration in vegetative tissues of the desiccation tolerant resurrection plantCraterostigma plantagineum. The most abundant group of desiccation-related gene products belong to the LEA (= Late Embryogenesis Abundant) proteins. Here we describe structures and expression patterns of members of group 3 and group 4Lea genes fromC. plantagineum. The most intriguing observation is the strong conservation of repeat motifs inLea genes found across divers plant species includingC. plantagineum and non-desiccation tolerant plants. This conservation of structural elements leads to speculations about evolution of desiccation tolerance in the resurrection plant.     

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.     

Effect of high temperature on dehydration-induced alterations in photosynthetic characteristics of the resurrection plant Haberlea rhodopensis

The effect of high temperature (HT) and dehydration on the activity of photosynthetic apparatus and its ability to restore membrane properties, oxygen evolution, and energy distribution upon rehydration were investigated in a resurrection plant, Haberlea rhodopensis. Plants growing under low irradiance in their natural habitat were desiccated to air-dry state at a similar light intensity [about 30 μol(photon) m^?2 s^?1] under optimal day/night (23/20°C) or high (38/30°C) temperature. Our results showed that HT alone reduced the photosynthetic activity and desiccation of plants at 38°C and it had more detrimental effect compared with desiccation at 23°C. The study on isolated thylakoids demonstrated increased distribution of excitation energy to PSI as a result of the HT treatment, which was enhanced upon the desiccation. It could be related to partial destacking of thylakoid membranes, which was confirmed by electron microscopy data. In addition, the surface charge density of thylakoid membranes isolated from plants desiccated at 38°C was higher in comparison with those at 23°C, which was in agreement with the decreased membrane stacking. Dehydration led to a decrease of amplitudes of oxygen yields and to a loss of the oscillation pattern. Following rehydration, the recovery of CO₂ assimilation and fluorescence properties were better when desiccation was performed at optimal temperature compared to high temperature. Rehydration resulted in partial recovery of the amplitudes of flash oxygen yields as well as of population of S₀ state in plants desiccated at 23°C. However, it was not observed in plants dehydrated at 38°C.