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.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

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.     

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.