Assessment of leaf micromorphology after full desiccation of resurrection plants

Resurrection plants are unique among higher plants because of their ability to withstand long periods of dehydration without damages. In this study, leaf epidermis and palisade mesophyll of three resurrection species, Haberlea rodopensis, Ramonda serbica and Ramonda myconi, grown under full desiccation and benign conditions, were analyzed by differential interference contrast microscopy. Detailed investigation of adaxial and abaxial leaf surfaces revealed species-specific differences in the size and number of epidermal cells and stomatal density. The applied full desiccation did not cause any significant deviations of these parameters from the controls. There were no changes in the size and number of mesophyll cells as well. Analysis of stomatal patterning displayed essentially hypostomatic leaves, having stomata mainly abaxially positioned. The most significant change detected in the leaves of dehydration-treated plants was the increased formation of adaxially positioned trichomes. This increase was very high in R. myconi, where the adaxial leaf surface was fully covered by trichomes. Despite the existence of small species-specific differences, the results showed uniform desiccation-related responses of the studied resurrection species. The quantified leaf epidermal and mesophyll features are discussed with respect to their possible contribution to the desiccation tolerance of resurrection species.     

Electrolyte leakage differences between poikilohydrous and homoiohydrous species of Gesneriaceae

Comparative analysis of the electrolyte efflux, as a screening test of the membrane tolerance to water stress, was carried out in poikilohydric plants Ramonda serbica Panč. and Ramonda nathaliae Panč. & Petrov. and homoiohydric plant Saintpaulia ionantha Wendl. from the same family Gesneriaceae. Water stress was induced by PEG 600. The high degree of solute leakage in the East-African drought-intolerant Saintpaulia ionantha points to the loss of membrane integrity. In contrast, Balkan endemites Ramonda serbica and R. nathaliae show high resistance to water stress due to the specific constitutional drought tolerance mechanisms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Contents of sugars in leaves of drying desiccation tolerant flowering plants, particularly grasses

Sugar complements were analysed in extracts from leaves of desiccation tolerant species in the angiosperm families Cyperaceae, Gesneriaceae, Liliaceae, Poaceae and Velloziaceae. Total sugar content was higher in live air-dry leaves of all desiccation tolerant species (except the grass Eragrostiella nardoides; 22 µmoles/g dw) than in the dead air-dry leaves of the desiccation sensitive grass Sporobolus pyramidalis (36 µmoles/g dw). Sucrose contents rose to high levels (40–98 µmoles/g dw) in live air-dry leaves of all species (except the grass Eragrostiella nardoides in which it rose to only 11 µmoles/g dw) to become the predominant sugar. Glucose and/or fructose contents frequently were lower after leaf drying but usually these were the sugars of next highest contents in live air-dry leaves. Contents of raffinose (that has been postulated to reduce sucrose crystallization) rose to c. 10% of sucrose contents in air-dry leaves of most desiccation tolerant species (but only c. 4% in Tripogon jacquemontii) compared with c. 2% of sucrose contents in the sensitive grass S. pyramidalis. Trehalose (a rare sugar in seed-plants) was present in all but one desiccation tolerant species (Xerophyta villosa) but only in minor amounts. The results are consistent with the views that sugars play a protective role during drying of desiccation tolerant plants in general but that other factors are also involved indesiccation tolerance, that in desiccation tolerant angiospermae sucrose is generally the predominant protective sugar and that raffinose and trehalose may supplement the role of sucrose.     

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.     

Dynamics of Endogenous Phytohormones during Desiccation and Recovery of the Resurrection Plant Species Haberlea rhodopensis

Drought is one of the most significant threats to world agriculture and hampers the supply of food and energy. The mechanisms of drought responses can be studied using resurrection plants that are able to survive extreme dehydration. As plant hormones function in an intensive cross-talk, playing important regulatory roles in the perception and response to unfavorable environments, the dynamics of phytohormones was followed in the resurrection plant Haberlea rhodopensis Friv. during desiccation and subsequent recovery. Analysis of both leaves and roots revealed that jasmonic acid, along with and even earlier than abscisic acid, serves as a signal triggering the response of the resurrection plants to desiccation. The steady high levels of salicylic acid could be considered an integral part of the specific set of parameters that prime H. rhodopensis desiccation tolerance. The dynamic changes of cytokinins and auxins suggest that these hormones actively participate in the dehydration response and development of desiccation tolerance in the resurrection plants. Our data contribute to the elucidation of a global complex picture of the resurrection plant’s ability to withstand desiccation, which might be successfully utilized in crop improvement.     

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.     

Thermoluminescence study of photosystem II activity in Haberlea rhodopensis and spinach leaves during desiccation

Thermoluminescence glow curve parameters were used to access the functional features of PS II in the Balkan endemic Haberlea rhodopensis. This representative of the higher desiccation-tolerant plants is unique for the European flora. An unusual high temperature of TL emission from Haberlea leaves after excitation by one flash at 5 degrees C was observed. The position of the main TL B band (S (2)Q (B)(-)) was at 45 - 47 degrees C, while this temperature was 30 - 32 degrees C in drought-sensitive mesophytic spinach. Consistent with the up-shift in TL emission, the lifetime of the S (2) state was also increased, showing a stabilization of charge storage in PS II complex in this resurrection plant. In addition, a part of PS II centres was less susceptible to DCMU. We consider the observed unusual TL characteristics of Haberlea rhodopensis reflect some structural modifications in PS II (especially in D1 protein), which could be related to the desiccation tolerance of this plant. This suggestion was supported by the different manner in which dehydration affected the TL properties in desiccation-tolerant Haberlea and desiccation-sensitive spinach plants.     

Characterization of polyphenol oxidase changes induced by desiccation of Ramonda serbica leaves

Resurrection plants are able to dehydrate/rehydrate rapidly without cell damage by a mechanism, the understanding of which may be of ecological importance in the adaptation of crop plants to dry conditions. The o -diphenol oxidase in Ramonda serbica Pan. & Petrov, a rare resurrection plant of the Balkan Peninsula, was characterized in respect to different isoforms, preferable substrates and specific inhibitors. Two anionic isoforms with pI 4.6 and 4.7 were separated from turgid leaves. Three additional anionic isoforms (pI 5.1, 5.3 and 5.6) and three neutral isoforms (pI from 6.8 to 7.4) were induced in desiccated leaves. Based on apparent K_m values, the affinity for reducing substrates decreased as follows: methyl catechol > chlorogenic acid > 3,4-dihydroxyphenylalanine > caffeic acid > pyrogallol. Polyphenol oxidase (PPO) activity was specifically sensitive to diethyldithiocarbamate and also inhibited by KCN, DTT and salicylic hydroxamic acid but with no inhibitory effect of Na₃N. Plants were subjected to drought-to-near complete water loss (approximately 2% relative water content, RWC) and several fold higher PPO activity was detected in desiccated leaves. Ramonda leaves contain high levels of phenolics, which decreased during drought. Rehydration of dry leaves from 2% RWC to 95% RWC led to transient inhibition of PPO in the first few hours. Within a day, the levels completely recovered to those determined in desiccated leaves. The finding of desiccation-induced high activity of PPO and new isoforms, which were also present in rehydrated turgid leaves, indicates a substantial role for PPO in the adaptation mechanism of resurrection plants to desiccation and also to the oxidative stress during rehydration.     

Carbohydrate metabolism in the developing and maturing wheat embryo in relation to its desiccation tolerance

Embryos of wheat (Triticum aestivum L. cv. Sappo) were studiedthroughout their development and maturation to investigate therelationships among starch, sucrose and raffinose and the onsetof desiccation tolerance. Starch accumulated in axes and scutellafrom about 20 d post anthesis (dpa) to reach a maximum at approximately35 d. The starch content then declined to a very low value inlate maturation. Extractable -amylase activity increased inembryos throughout the period of starch deposition and showeda substantial rise coincident with starch breakdown. In earlymaturation (approximately 26 dpa) sucrose and raffinose appeared,and continued to increase. The rise in the amount of sucroseparalleled the accumulation of starch, but the major increasein raffinose approximated to the fall in starch content. Embryoswere desiccation intolerant prior to the age when free sucroseand raffinose accumulated: the development of desiccation tolerancewas associated with increasing raffinose: sucrose ratios. Possiblemetabolic and physiological relationships among starch, raffinose,sucrose and the onset of desiccation tolerance are discussed. Key words: Wheat embryos, development, maturation, starch, raffinose, sucrose, desiccation tolerance     

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.     

The role of carbohydrates in the responses of chilling-sensitive plants to short- and long-term low-temperature treatments

The content of particular components of water-soluble carbohydrates and cold tolerance of cucumber (Cucumis sativus L.) cotyledonary leaves were studied at early developmental stages in dynamics during 6-day-long treatment with a temperature reduced to 12°C at two regimes: short-term cooling (2 h in the end of the night period, DROP) or permanent low-temperature treatment (PLT). PLT cucumber plants were characterized by the accumulation of oligosaccharides, whereas DROP plants contained increased amounts of glucose, fructose, and raffinose, indicating their higher metabolic status. When changes in carbohydrate fractions were compared with the dynamics of cold tolerance, it was found that these changes were synchronous in PLT plants but asynchronous for glucose and oligosaccharides in DROP plants. We suppose that, in cotyledonary leaves of DROP plants, two pools of sugars are produced; one of them used for tolerance development and another one — in active metabolism. This provides for the combination of activated metabolism and high cold tolerance of these plants. In PLT plants, all components of water-soluble carbohydrates are involved in cold tolerance development.     

In situ localization of glucose and sucrose in dehydrating leaves of Sporobolus stapfianus

Accumulation of soluble carbohydrates during dehydration stress is thought to be a very important mechanism for the acquisition of desiccation tolerance. Despite the proposed importance of soluble carbohydrate accumulation (especially sucrose), nothing is known about the cellular localization of carbohydrates in desiccation-tolerant plants. The present study proposes a novel and selective method for the in situ localization of sucrose and glucose in the desiccation-tolerant plant Sporobolus stapfianus. The detection of sucrose and glucose is based on a series of coupled enzymatic reactions leading to the formation of NADH. Iodonitrotetrazolium (INT) reacts with NADH, thereby providing the red-colored insoluble INT-formazan. Stained tissue sections were immediately visualized using light microscopy. Localization of the respective sugars was site specific. Sucrose was visualized in all leaf cell types during dehydration: vascular bundles, bundle sheath cells, mesophyll cells and epidermal cells. Similarly, glucose was shown to be localized in the same leaf compartments as reported for sucrose. This is the first report that describes sucrose localization in dehydrating leaf tissues of a "resurrection" plant. We conclude that, during dehydration stress, sucrose accumulates in all viable tissues; these results are in agreement with the previously proposed theories about its function as a cellular protectant.     

Preservation of integrity and activity of Haberlea rhodopensis photosynthetic apparatus during prolonged light deprivation

The effect of prolonged light deprivation on ultrastructure, pigment composition and functions of photosynthetic apparatus of the resurrection plant Haberlea rhodopensis Friv. (Gesneriaceae) was studied. For this purpose, intact plants were kept in darkness for up to 6 months. Haberlea rhodopensis demonstrated extraordinary ability to preserve its photosynthetic machinery intact despite complete absence of light. During the first 4 weeks of light deprivation, we observed preservation of pigment content, chloroplast ultrastructure and a decrease in the rate of CO(2) assimilation. The signs of dark-induced senescence were observed only after the fourth week. This phase was characterized by decrease of pigment content, partial disintegration of chloroplast ultrastructure and by the development of photosystem II down regulation that includes the increases in non-photochemical fluorescence quenching, qN. In comparison with other plants like common bean and Arabidopsis, the processes of dark-induced senescence were very slow and the plants still can recover even after 6 months of light deprivation. We think these findings can open new opportunities for studying not only dark-induced senescence but also to investigate mechanisms determining tolerance to multiple stresses affecting integrity of photosynthetic apparatus.     

Changes in soluble sugars in relation to desiccation tolerance and effects of dehydration on freezing characteristics of Acer platanoides and Acer pseudoplatanus seeds

The role of soluble sugars in desiccation tolerance was investigated in seeds of two species from the genus Acer: Norway maple (Acer platanoides L.) — tolerant and sycamore (Acer pseudoplatanus L.) — intolerant to dehydration. During two years of observations it was found that seeds of Norway maple acquire desiccation tolerance at the end of August i.e. about 125 days after flowering (DAF). During seed development, the transition from intolerant to tolerant state in Norway maple seeds was accompanied by the accumulation in seed tissues of raffinose, stachyose and sucrose. The sucrose/raffinose ratio in Norway maple seeds was lower than in sycamore. In mature Norway maple seeds sucrose and raffinose contents were higher than in sycamore. It was concluded, that soluble sugars such as sucrose, raffinose and stachyose may play an important role in desiccation tolerance and/or intolerance of Norway maple and sycamore seeds. Differential thermal analysis (DTA) was used to study the relationship between desiccation sensitivity and the state of water in seed tissues. The level of non-freezable water was the same in both analysed seed species, but the temperature of water crystallization during desiccation was lower in sycamore seeds.     

Compound-specific differences in (13)C of soluble carbohydrates in leaves and phloem of 6-month-old Eucalyptus globulus (Labill)

Movement of photoassimilates from leaves to phloem is an important step for the flux of carbon through plants. Fractionation of carbon isotopes during this process may influence their abundance in heterotrophic tissues. We subjected Eucalyptus globulus to 20, 25 and 28 °C ambient growth temperatures and measured compound-specific δ(13)C of carbohydrates obtained from leaves and bled phloem sap. We compared δ(13)C of sucrose and raffinose obtained from leaf or phloem and of total leaf soluble carbon, with modelled values predicted by leaf gas exchange. Changes in δ(13)C of sucrose and raffinose obtained from either leaves or phloem sap were more tightly coupled to changes in c(i)/c(a) than was δ(13)C of leaf soluble carbon. At 25 and 28 °C, sucrose and raffinose were enriched in (13)C compared to leaf soluble carbon and predicted values - irrespective of tissue type. Phloem sucrose was depleted and raffinose enriched in (13)C compared to leaf extracts. Intermolecular and tissue-specific δ(13)C reveal that multiple systematic factors influence (13)C composition during export to phloem. Predicting sensitivity of these factors to changes in plant physiological status will improve our ability to infer plant function at a range of temporal and spatial scales.