Antioxidative defence mechanism contributes to desiccation tolerance in Haberlea rhodopensis population naturally exposed to high irradiation

Drought induced stress is one of the most important among the environmental challenges. Haberlea rhodopensis, a chlorophyll-retaining resurrection plant, can survive desiccation to air-dry stage in its usual low irradiance habitat (“shade” plants). Nevertheless, in the past years, some populations living under high irradiance (“sun” plants) have been also discovered with the same ability to survive dehydration. In order to clarify the adaptation mechanisms to a high irradiation habitat, superoxide dismutase (SOD) activity determined by activity staining on polyacrylamide gels and malondialdehyde (MDA) content of sun and shade plants collected from high and low irradiance environment, respectively, were studied. Desiccation induced a significantly higher induction in SOD activity and thus a smaller increase in the MDA content in sun compared to shade plants. The MDA content and SOD activity was restored in both sun and shade plants after six-day rehydration. Nevertheless, the SOD activity remained higher in rehydrated sun leaves compared to the well-hydrated initial stage. The early enhancement of SOD activity in dehydrating sun plants contributes to the higher stress tolerance of these populations

Reactivation of the Photosynthetic Apparatus of Resurrection Plant Haberlea rhodopensis during the Early Phase of Recovery from Drought- and Freezing-Induced Desiccation

Haberlea rhodopensis is a unique desiccation-tolerant angiosperm that also survives winter frost. As, upon freezing temperatures, H. rhodopensis desiccates, the taxon is proposed to survive low temperature stress using its desiccation tolerance mechanisms. To reveal the validity of this hypothesis, we analyzed the structural alterations and organization of photosynthetic apparatus during the first hours of recovery after drought- and freezing-induced desiccation. The dynamics of the ultrastructure remodeling in the mesophyll cells and the restoration of the thylakoid membranes shared similarities independent of the reason for desiccation. Among the most obvious changes in thylakoid complexes, the proportion of the PSI-LHCII complex strongly increased around 70% relative water content (RWC), whereas the proportion of Lhc monomers decreased from the beginning of rehydration. We identified enhanced levels of cyt b6f complex proteins that contributed to the enhanced electron flow. The high abundance of proteins related to excitation energy dissipation, PsbS, Lhcb5, Lhcb6 and ELIPs, together with the increased content of dehydrins contributed to the preservation of cellular integrity. ELIP expression was maintained at high levels up to 9 h into recovery. Although the recovery processes from drought- and freezing-induced desiccation were found to be similar in progress and time scale, slight variations indicate that they are not identical

Antioxidative Defense, Suppressed Nitric Oxide Accumulation, and Synthesis of Protective Proteins in Roots and Leaves Contribute to the Desiccation Tolerance of the Resurrection Plant Haberlea rhodopensis

The desiccation tolerance of plants relies on defense mechanisms that enable the protection of macromolecules, biological structures, and metabolism. Although the defense of leaf tissues exposed to solar irradiation is challenging, mechanisms that protect the viability of the roots, yet largely unexplored, are equally important for survival. Although the photosynthetic apparatus in leaves contributes to the generation of oxidative stress under drought stress, we hypothesized that oxidative stress and thus antioxidative defense is also predominant in the roots. Thus, we aimed for a comparative analysis of the protective mechanisms in leaves and roots during the desiccation of Haberlea rhodopensis. Consequently, a high content of non-enzymatic antioxidants and high activity of antioxidant enzymes together with the activation of specific isoenzymes were found in both leaves and roots during the final stages of desiccation of H. rhodopensis. Among others, catalase and glutathione reductase activity showed a similar tendency of changes in roots and leaves, whereas, unlike that in the leaves, superoxide dismutase activity was enhanced under severe but not under medium desiccation in roots. Nitric oxide accumulation in the root tips was found to be sensitive to water restriction but suppressed under severe desiccation. In addition to the antioxidative defense, desiccation induced an enhanced abundance of dehydrins, ELIPs, and sHSP 17.7 in leaves, but this was significantly better in roots. In contrast to leaf cells, starch remained in the cells of the central cylinder of desiccated roots. Taken together, protective compounds and antioxidative defense mechanisms are equally important in protecting the roots to survive desiccation. Since drought-induced damage to the root system fundamentally affects the survival of plants, a better understanding of root desiccation tolerance mechanisms is essential to compensate for the challenges of prolonged dry periods

Differential Accumulation of sHSPs Isoforms during Desiccation of the Resurrection Plant <i>Haberlea rhodopensis</i> Friv. under Optimal and High Temperature

Haberlea rhodopensis belongs to the small group of angiosperms that can survive desiccation to air-dry state and quickly restore their metabolism upon rehydration. In the present study, we investigated the accumulation of sHSPs and the extent of non-photochemical quenching during the downregulation of photosynthesis in H. rhodopensis leaves under desiccation at optimum (23 °C) and high temperature (38 °C). Desiccation of plants at 38 °C caused a stronger reduction in photosynthetic activity and corresponding enhancement in thermal energy dissipation. The accumulation of sHSPs was investigated by Western blot. While no expression of sHPSs was detected in the unstressed control sample, exposure of well-hydrated plants to high temperature induced an accumulation of sHSPs. Only a faint signal was observed at 50% RWC when dehydration was applied at 23 °C. Several cross-reacting polypeptide bands in the range of 16.5–19 kDa were observed in plants desiccated at high temperature. Two-dimensional electrophoresis and immunoblotting revealed the presence of several sHSPs with close molecular masses and pIs in the range of 5–8.0 that differed for each stage of treatment. At the latest stages of desiccation, fourteen different sHSPs could be distinguished, indicating that sHSPs might play a crucial role in H. rhodopensis under dehydration at high temperatures

Acquisition of Freezing Tolerance of Resurrection Species from Gesneriaceae, a Comparative Study

Resurrection plants have the unique ability to restore normal physiological activity after desiccation to an air-dry state. In addition to their desiccation tolerance, some of them, such as Haberlea rhodopensis and Ramonda myconi, are also freezing-tolerant species, as they survive subzero temperatures during winter. Here, we compared the response of the photosynthetic apparatus of two other Gesneriaceae species, Ramonda serbica and Ramonda nathaliae, together with H. rhodopensis, to cold and freezing temperatures. The role of some protective proteins in freezing tolerance was also investigated. The water content of leaves was not affected during cold acclimation but exposure of plants to −10 °C induced dehydration of plants. Freezing stress strongly reduced the quantum yield of PSII photochemistry (Y(II)) and stomatal conductance (gs) on the abaxial leaf side. In addition, the decreased ratio of Fv/Fm suggested photoinhibition or sustained quenching. Freezing-induced desiccation resulted in the inhibition of PSII activity, which was accompanied by increased thermal energy dissipation. In addition, an increase of dehydrins and ELIPs was detected, but the protein pattern differed between species. During recovery, the protein abundance decreased and plants completely recovered their photosynthetic activity. Thus, our results showed that R. serbica, R. nathaliae, and H. rhodopensis survive freezing stress due to some resurrection-linked traits and confirmed their freezing tolerance

Protein Changes in Shade and Sun <i>Haberlea rhodopensis</i> Leaves during Dehydration at Optimal and Low Temperatures

Haberlea rhodopensis is a unique resurrection plant of high phenotypic plasticity, colonizing both shady habitats and sun-exposed rock clefts. H. rhodopensis also survives freezing winter temperatures in temperate climates. Although survival in conditions of desiccation and survival in conditions of frost share high morphological and physiological similarities, proteomic changes lying behind these mechanisms are hardly studied. Thus, we aimed to reveal ecotype-level and temperature-dependent variations in the protective mechanisms by applying both targeted and untargeted proteomic approaches. Drought-induced desiccation enhanced superoxide dismutase (SOD) activity, but FeSOD and Cu/ZnSOD-III were significantly better triggered in sun plants. Desiccation resulted in the accumulation of enzymes involved in carbohydrate/phenylpropanoid metabolism (enolase, triosephosphate isomerase, UDP-D-apiose/UDP-D-xylose synthase 2, 81E8-like cytochrome P450 monooxygenase) and protective proteins such as vicinal oxygen chelate metalloenzyme superfamily and early light-induced proteins, dehydrins, and small heat shock proteins, the latter two typically being found in the latest phases of dehydration and being more pronounced in sun plants. Although low temperature and drought stress-induced desiccation trigger similar responses, the natural variation of these responses in shade and sun plants calls for attention to the pre-conditioning/priming effects that have high importance both in the desiccation responses and successful stress recovery

Antioxidant Defense during Recovery of Resurrection Plant <i>Haberlea rhodopensis</i> from Drought- and Freezing-Induced Desiccation

In this study, the contribution of nonenzymatic (ascorbate, glutathione) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase) in the first hours of recovery of the resurrection plant Haberlea rhodopensis from drought- and freezing-induced desiccation was assessed. The initial stage of recovery after desiccation is critical for plants, but less investigated. To better understand the alterations in the activity of antioxidant enzymes, their isoenzyme patterns were determined. Our results showed that ascorbate content remained high during the first 9 h of rehydration of desiccated plants and declined when the leaves′ water content significantly increased. The glutathione content remained high at the first hour of rehydration and then strongly decreased. The changes in ascorbate and glutathione content during recovery from drought- and freezing-induced desiccation showed great similarity. At the beginning of rehydration (1–5 h), the activities of antioxidant enzymes were significantly increased or remained as in dry plants. During 7–24 h of rehydration, certain differences in the enzymatic responses between the two plant groups were registered. The maintenance of a high antioxidant activity and upregulation of individual enzyme isoforms indicated their essential role in protecting plants from oxidative damage during the onset of recovery

Oligo-dT anchored cDNA-SRAP and cDNA-SCoT aided identification of transcripts differentially expressed during the early stages of recovery of resurrection plant Haberlea rhodopensis Friv. from freezing-induced desiccation

AbstractResurrection plants are a unique group of flora showing an extraordinary ability to survive extreme water shortages. Among them, the Bulgarian endemic plant Haberlea rhodopensis Friv. has been used as a model to study the mechanisms underlying desiccation tolerance induced by water and low temperature stress. Although extensive transcriptome data, including two RNAseq experiments, are available for drought induced desiccation and recovery, the gene expression during recovery after freezing-induced desiccation (RAF) remains largely unexplored. In this study we performed a differential screening using a new approach, based on oligo-dT anchored cDNA-SRAP (copy DNA sequence-related amplified polymorphism) and a modified oligo-dT anchored cDNA-SCoT (copy DNA start codon targeted) assays to provide new information on genes involved in the tolerance to low temperature stress and the recovery of H. rhodopensis. The putative functions of 19 identified transcript derived fragments were established by searching for homology with known sequences in public databases and the expression of 10 of them during the early stages of recovery was confirmed by quantitative reverse transcription polymerase chain reaction. Some of the identified genes have known functions in the response to abiotic stress in other plant species including resurrection plants, which confirms the effectiveness of the approach used in the present study. The results of this study will contribute to obtaining new information on the defence mechanisms in the recovery process of H. rhodopensis, especially at the early stages of recovery, which is the most vulnerable period for plants

Alterations in the sugar metabolism and in the vacuolar system of mesophyll cells contribute to the desiccation tolerance of Haberlea rhodopensis ecotypes

Haberlea rhodopensis belongs to the small group of resurrection plants having the unique ability to survive desiccation to air dry state retaining most of its chlorophyll content and then resume normal function upon rehydration. It prefers the shady valleys and northward facing slopes of limestone ridges in mountain zones with high average humidity. Nevertheless, it can be found rarely on rocks directly exposed to the sunlight, without the coverage of the canopy. In the present study, we follow the alterations in the subcellular organization of mesophyll cells and sugar metabolism upon desiccation of shade and sun H. rhodopensis plants. Composition and content of soluble carbohydrates during desiccation and rehydration were different in plants grown below the trees or on the sunny rocks. Sucrose, however, was dominating in both ecotypes. The amount of starch grains in chloroplasts was inversely related to that of sugars. Concomitantly with these changes, the number of vacuoles was multiplied in the cells. This can be explained by the development of small (secondary) vacuoles peripherally in the cytoplasm, rather than by the fragmentation of the single vacuole, proposed earlier in the literature. Accordingly, the centripetal movement of chloroplasts and other organelles may be a result of the dynamic changes in the vacuolar system. Upon rehydration, the inner vacuoles enlarged and the organelles returned to their normal position

Different Responses to Water Deficit of Two Common Winter Wheat Varieties: Physiological and Biochemical Characteristics

Since water scarcity is one of the main risks for the future of agriculture, studying the ability of different wheat genotypes to tolerate a water deficit is fundamental. This study examined the responses of two hybrid wheat varieties (Gizda and Fermer) with different drought resistance to moderate (3 days) and severe (7 days) drought stress, as well as their post-stress recovery to understand their underlying defense strategies and adaptive mechanisms in more detail. To this end, the dehydration-induced alterations in the electrolyte leakage, photosynthetic pigment content, membrane fluidity, energy interaction between pigment–protein complexes, primary photosynthetic reactions, photosynthetic and stress-induced proteins, and antioxidant responses were analyzed in order to unravel the different physiological and biochemical strategies of both wheat varieties. The results demonstrated that Gizda plants are more tolerant to severe dehydration compared to Fermer, as evidenced by the lower decrease in leaf water and pigment content, lower inhibition of photosystem II (PSII) photochemistry and dissipation of thermal energy, as well as lower dehydrins’ content. Some of defense mechanisms by which Gizda variety can tolerate drought stress involve the maintenance of decreased chlorophyll content in leaves, increased fluidity of the thylakoid membranes causing structural alterations in the photosynthetic apparatus, as well as dehydration-induced accumulation of early light-induced proteins (ELIPs), an increased capacity for PSI cyclic electron transport and enhanced antioxidant enzyme activity (SOD and APX), thus alleviating oxidative damage. Furthermore, the leaf content of total phenols, flavonoids, and lipid-soluble antioxidant metabolites was higher in Gizda than in Fermer