Gergana Mihailova

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. In contrast to the great differences in the morphology of plants, the ultrastructure and the organization of thylakoids were surprisingly similar in well-hydrated shade and sun populations. A high ratio of photosystem (PS)I binding light harvesting complex (LHC)II, important in low- and fluctuating light environment, was characteristic to both shade and sun plant, and the ratios of the main chlorophyll-protein complexes were also similar. The intensive protective mechanisms, such as shading by steep leaf angle and accumulation of protective substances, probably reduced the light intensity at the chloroplast level. The significantly increased ratio of monomer to oligomer antennae in well-hydrated sun plants may be connected with the temporary high light exposure of chloroplasts. During desiccation, LHCII was removed from PSI and part of PSII supercomplexes disassembled with some loss of PSII core and LHCII. The different reorganization of antennae, possibly connected with different quenching mechanisms, involved an increased amount of monomers in shade plants but unchanged proportion of oligomers in sun plants. Desiccation-induced responses were more pronounced in sun plants which also had a greater capacity to recover due to their stress-acclimated attitude.

Response of sun- and shade-adapted plants of Haberlea rhodopensis to desiccation

The differences in some morphological and physiological characteristics of sun- and shade-adapted Haberlea rhodopensis plants were compared. Changes in the photosynthetic activity, electrolyte leakage from leaf tissues, malondialdehyde content (MDA) and leaf anatomy were studied at different degrees of desiccation as well as after rehydration of plants. The MDA content in well-watered sun Haberlea plants was higher compared to shade plants suggesting higher lipid peroxidation, which is commonly regarded as an indicator of oxidative stress, but desiccation of plants at high light did not cause additional oxidative damage as judged by the unaffected MDA content. The electrolyte leakage from dried leaves (8% RWC) from both shade and sun plants increased fourfold indicating similar membrane damage. However, the recovery after rehydration showed that this damage was reversible. Well-watered sun plants had higher photosynthetic activity probably due to the larger thickness of the mesophyll layer in such plants. On the other hand, desiccation at high light reduced CO2 assimilation which was in accordance with the stronger reduction of stomatal conductance. Stomata were visible only on the abaxial side of sun leaves having also higher abundance of non-glandular trichomes. Increased trichomes density and epicuticular waxes and filaments upon desiccation could help plants to increase reflection, reduce net radiation income, slow down the rate of water loss and survive adverse conditions.

Effects of habitat light conditions on the excitation quenching pathways in desiccating Haberlea rhodopensis leaves: an Intelligent FluoroSensor study.

Resurrection plants can survive dehydration to air-dry state, thus they are excellent models of understanding drought and dehydration tolerance mechanisms. Haberlea rhodopensis, a chlorophyll-retaining resurrection plant, can survive desiccation to relative water content below 10%. Leaves, detached from plants of sun and shade habitats, were moderately (∼50%) dehydrated in darkness. During desiccation, chlorophyll a fluorescence was detected by the recently innovated wireless Intelligent FluoroSensor (IFS) chlorophyll fluorometer, working with three different detectors: a pulse-amplitude-modulated (PAM) broadband channel and two channels to measure non-modulated red and far-red fluorescence. No change in area-based chlorophyll content of leaves was observed. The maximal quantum efficiency of photosystem II decreased gradually in both shade and sun leaves. Shade leaves could not increase antennae-based quenching, thus inactivated photosystem II took part in quenching of excess irradiation. Sun leaves seemed to be pre-adapted to quench excess light as they established an intensive increase in antennae-based non-photochemical quenching parallel to desiccation. The higher far-red to red antennae-based quenching may sign light-harvesting complex reorganization. Thus, compared to PAM, IFS chlorophyll fluorometer has additional benefits including (i) parallel estimation of changes in the Chl content and (ii) prediction of underlying processes of excitation energy quenching.

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.

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.

Application of a diffusion model to measure ion leakage of resurrection plant leaves undergoing desiccation

Haberlea rhodopensis is a chlorophyll-retaining resurrection plant, which can survive desiccation to air dry state under both low light and sunny environments. Maintaining the integrity of the membrane during dehydration of resurrection plants is extremely important. In the present study, the diffusion model was improved and used for a first time to evaluate the changes in ion leakage through different cellular compartments upon desiccation of H. rhodopensis and to clarify the reasons for significant increase of electrolyte leakage from dry leaves. The applied diffusion approach allowed us to distinguish the performance of plants subjected to dehydration and subsequent rehydration under different light intensities. Well-hydrated (control) shade plants had lower and slower electrolyte leakage compared to control sun plants as revealed by lower values of phase amplitudes, lower rate constants and ion concentration. In well-hydrated and moderately dehydrated plants (50% relative water content, RWC) ion efflux was mainly due to leakage from apoplast. The electrolyte leakage sharply increased in severely desiccated leaves (8% RWC) from both sun and shade plants mainly due to ion efflux from symplast. After 1 day of rehydration the electrolyte leakage was close to control values, indicating fast recovery of plants. We suggest that the enhanced leakage in air-dried leaves should not be considered as damage but rather as a survival mechanism based on a reversible modification in the structure of cell wall, plasma membrane and alterations in vacuolar system of the cells. However, further studies should be conducted to investigate the changes in cell wall/plasma membrane to support this conclusion.

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.

Characterization of Energy Transfer Processes and Flash Oxygen Yields of Thylakoid Membranes Isolated from Resurrection Plant Haberlea Rhodopensis Subjected to Different Extent of Desiccation

The resurrection plants are unique with their extra desiccation tolerance. The physico-chemical properties of photosynthetic apparatus are of crucial importance for survival of plants upon water stress. In present work the effect of different extent of desiccation on the energy transfer properties and oxygen evolving capacity of isolated thylakoid membranes from resurrection plant Haberlea Rhodopensis are investigated. The plants from different habitats in Bulgaria are compared. Energy distribution and spillover between both photosystems are studied by means of 77 K chlorophyll fluorescence. The dependence of fluorescence ratio F735/F685 on the degree of desiccation of plants is also followed. Functionality of PSII and especially of oxygen-evolving apparatus under water deficit is estimated by flash oxygen yields and initial oxygen burst of thylakoid membranes isolated from plants desiccated up to 50% and 8% relative water content (RWC). Population of Si states as well as the misses and the double hits are calculated according non-cooperative Kok’s model and compared for plants from different habitats and different RWC. The results are discussed in terms of involvement of “fast” and “slow” centers from grana and stroma regions in oxygen evolution and alteration of their contribution as a result of desiccation.

Effect of Light on the Photosynthetic Activity during Desiccation of the Resurrection Plant Haberlea Rhodopensis

The effect of light during desiccation of the resurrection plant Haberlea rhodopensis on the photosynthetic activity and some morphological parameters was evaluated using plants growing at low or high irradiance in natural habitat. Chlorophyll content was not only lower in sun plants compared to shade plants, but it declined to a higher extent when desiccation was carried out at high light irradiance. Regardless of lower chlorophyll content in sun plants their photosynthetic activity (PN) was about 30% higher compared to shade plants. However, during dehydration PN declined more rapidly in sun plants. The mean leaf thickness of fully hydrated leaves from sun plants was larger when compared with shade plants, which was due to higher thickness of the mesophyll. Following rehydration plants rapidly recovered and PN was higher by about 70% in sun than in shade plants. The results showed that the sun-exposed Haberlea plants exhibited good adaptation to desiccation under high irradiance.

Effect of Desiccation of the Resurrection Plant Haberlea Rhodopensis at High Temperature on the Photochemical Activity of PSI and PSII

Changes in the photochemical activity of the homoiochlorophyllous poikilohydric plant Haberlea rhodopensis under dehydration at high temperature was investigated. Plants, growing under low irradiance in their natural habitat, were desiccated to air-dry state at a similar light intensity (about 30 μmol m−2 s−1 PPFD) under optimal (23/20 °C) or high (38/30 °C) day/night temperature. Water deficit reduced photochemical activity of PSII and PSI. The results showed that desiccation of Haberlea rhodopensis at high temperature had more limiting effects than desiccation at optimal temperature. However, the damage was limited to a level where repair was still possible and thus plants fully recovered after 7 days of rehydration.