Branka Stevanović

Red Data List of Charophytes in the Balkans

The occurrence of 47 species of all the six recent genera of charophytes recorded in the Balkans is demonstrated. Our aim was to assess their status of threat according to the 2001 IUCN threat categories and criteria, in order to establish the Red List of Charophytes distributed in the Balkan Peninsula. The List underlines the diversity of charophytes in this part of the world and provides guidelines for its conservation. Analysis of the charophytes in each category of threat has provided a general overview of their distribution, species richness, population features and possibility of survival throughout the various regions of the Balkan Peninsula.

Characterization of oxidative and antioxidative events during dehydration and rehydration of resurrection plant Ramonda nathaliae

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.

Chlorophyll Metabolism in Leaves of Higher Poikilohydric Plants Ramonda serbica Panč, and Ramonda nathaliae Panč, et Petrov. during Dehydration and Rehydration

Summary Ramonda serbica and R. nathaliae (Gesneriaceae) are endemo-relic species of the Balkan peninsula and the desiccation-tolerant homoiochlorophyllous flowering plants. In order to follow the pattern of chlorophyll metabolism during dehydration and after rewatering, the chlorophyll content was studied. Its change during the dehydration was monitored in intact plants and during the recovery in intact specimens as well as in cut off leaves. The regreening rate depended on previous hydration state of plants collected from their natural habitat. Eight to 12 h after the start of the rehydration of air-dried detached leaves the resynthesis of chlorophyll began. Rehydration and chlorophyll resynthesis were not light depended. Levulinic acid inhibited completely the chlorophyll resynthesis indicating that the regulatory site on the biosynthetic pathway was located before protoporphyrin formation. Chlorophyllase activity changed during the dehydration and rehydration, but the chlorophyll content was not in direct correlation with this enzyme activity.

Anatomical injury induced by the eriophyid mite Aceria anthocoptes on the leaves of Cirsium arvense

Anatomical injury of the leaves of the invasive species, Cirsium arvense (L.) Scop., caused by the eriophyid mite Aceria anthocoptes (Nal.), which is the only eriophyid mite that has been recorded on C. arvense worldwide, is described. The injury induced by the mite feeding on the leaves of C. arvense results in visible russeting and bronzing of the leaves. Other conspicuous deformations are folding and distortion of the leaf blade and curling of leaf edge, as well as gradual drying of leaves. The anatomical injury of the mature leaves of field-collected plants was limited to the epidermis of the lower leaf surface. However, on young leaves of experimentally infested plants, rust mite injuries extend to epidermal cells on both leaf surfaces and to those of deeper mesophyll layers. On these leaves, lesions on the lower leaf surface even affected the phloem of the vascular bundles. Leaf damage induced by A. anthocoptes is discussed with regard to the mite’s potential as a biological control agent of C. arvense.

Resurrection plants of the genus Ramonda: prospective survival strategies – unlock further capacity of adaptation, or embark on the path of evolution?

Paleoendemic species of the monophyletic genus Ramonda (R. myconi, R. serbica and R.~nathaliae) are the remnants of the Tertiary tropical and subtropical flora in Europe. They are the rare resurrection plants of Northern Hemisphere temperate zone. Ramonda serbica and R. nathaliae are chorologically differentiated in the Balkan Peninsula and occupy similar habitats in calcareous, northward slopes in canyons and mountainsides. They remain well-hydrated during spring, late autumn and even in winter. In summer and early autumn when plants are subjected to drought and thermal stress, their desiccation tolerance comes into operation and they fall into anabiosis. Investigations revealed the permanent presence of ubiquitine and its conjugates, high amounts of oxalic acid and proline. Both species are homoiochlorophyllous. It enables them to rapidly resume photosynthesis upon rehydration, but also makes them susceptible to reactive oxygen species formation. Dehydration induces activation of antioxidative enzymes (ascorbate peroxidase, glutathione reductase, polyphenol oxidase), increase in amounts of AsA and GSH, phenolic acids, dehydrins, sucrose, and inorganic ions. Plasma membranes, characterized by high amount of cholesterol, are subjected to decrease in membrane fluidity mostly on account of increased level of lipid saturation. Cytogenetic analysis revealed that R. nathaliae is a diploid (2n = 48) and probably evolutionary older species, while R. serbica is a hexaploid (2n = 144). Two species live together in only two localities forming hybrid individuals (2n = 96). Polyploidization is the major evolutionary mechanism in the genus Ramonda that together with hybridization ability indicates that these relict species which have preserved an ancient survival strategy are not the evolutionary “dead end.”The species of the genus Ramonda are promising sources of data important for understanding the complex strategy of resurrection plants’ survival, appraised through a prism of their evolutionary and adaptive potential for multiple environmental stresses.

Morphological injury of cut-leaf teasel, Dipsacus laciniatus L. (Dipsacaceae) induced by the eriophyid mite Leipothrix dipsacivagus Petanovic et Rector (Acari: Eriophyoidea)

Abstract The eriophyid mite Leipothrix dipsacivagus Petanovic et Rector provokes severe malformations to its host plant, cut-leaf teasel (Dipsacus laciniatus L.), in the field. These injuries were examined at the morpho-anatomical level in infested plants collected in the field and experimentally infested plants kept under controlled laboratory conditions. A number of symptoms were observed including reduced growth, internode shortening, leaf rolling and wrinkling, and shrunken inflorescences. After severe attack, the leaves of bolted plants became chlorotic with necrotic spots and started to wilt. Young rosettes died. Morphometric analysis revealed significant differences between infested and healthy field-collected bolting plants. Infested plants were significantly shorter than healthy plants, with smaller leaves and flower heads. Striking injuries were also observed on young leaves of experimentally infested plants. It is expected that further investigations will elucidate the full extent of the damage to aboveground parts of D. laciniatus due to infestation by L. dipsacivagus.

Desiccation tolerance of the resurrection plant Ramonda serbica is associated with dehydration-dependent changes in levels of proteolytic activities

The unique response of desiccation-tolerant, or resurrection plants, to extreme drought is accompanied by major changes in the protein pool, raising the possibility of the involvement of proteases. We detected and characterized proteases present in their active state in leaf extracts of desiccated Ramonda serbica Panč., a resurrection plant from the Balkan Peninsula. Plants desiccated under laboratory conditions and maintained in anhydrobiosis for 4 and 14 months revived upon rehydration. Protease activities were determined spectrophotometrically in solution and by zymography on gels. Several endo- and aminopeptidases were detected and characterized by their pH profiles. Their enzyme class was determined using specific inhibitors. Those with higher activities were a serine endopeptidase active against Bz-Arg-pNA with a pH optimum around 9, and aminopeptidases optimally active at pHs from 7 to 9 against Leu-pNA, Met-pNA, Phe-pNA, Pro-pNA and Ala-pNA. The levels of their activities in leaf extracts from desiccated plants were significantly higher than those from rehydrated plants and from regularly watered plants, implying their involvement in the recovery of vegetative tissues from desiccation.

ECOPHYSIOLOGICAL AND ANATOMICAL CHARACTERISTICS OF THE SUBTROPICAL SHRUB ZANTHOXYLUM ACANTHOPODIUM (RUTACEAE) IN CONDITIONS OF A TEMPERATE CONTINENTAL CLIMATE (SERBIA)

The evergreen shrub Zanthoxylum acanthopodium DC. (Rutaceae), originating from warm temperate and subtropical Asia, has existed successfully in the Jevremovac Botanical Garden in Belgrade for more than 80 years. The seasonal pattern of water management in leaves, electrolyte leakage, essential oil composition, and leaf anatomy were examined in order to understand the resistance and viability of this subtropical shrub in the temperate continental cli- mate of Belgrade, Serbia.

Chemical Composition of the Essential Oil of the Species Artemisia eriantha Ten. (Asteraceae) from Yugoslavia

Abstract The essential oil of Artemisia eriantha Ten. (Asteraceae) from two localities on the mountain Durmitor in Montenegro (Yugoslavia) was studied using GC/MS. The oil yield was 0.7% and the major constituents were identified as α-thujone (77.5%, 60.4%) and β-thujone (17.6%, 14.6%), respectively.

PHYTOCHEMICAL AND ANTIMICROBIAL SCREENING OF LEAVES AND STEMS OF BALKAN ENDEMIC SPECIES DAPHNE MALYANA BLEČIĆ

ABSTRACT Daphne malyana Blečić (Thymelaeaceae) is an endemic species of the western part of the Balkan Peninsula. No previous detailed studies exist on D. malyana Blečić. The aim of this study was to provide information on the anatomy of the leaf and stem as well as to perform phytochemical screening and to assay the antimicrobial activity of methanol extracts of the leaves and stems of D. malyana Blečić. The phytochemical analysis showed that coumarins and flavonoids are the major classes of secondary metabolites in both leaves and stems. The antimicrobial activity of the methanol extracts from leaves and stems of D. malyana was also evaluated against six Gram-positive and six Gram-negative bacteria by employing both microdilution and disc diffusion methods. The results from the well-diffusion assay showed significant antimicrobial activity at all tested concentrations of the extracts. The results showed that methanol extracts of leaves and twigs have similar chemical compositions and similar antimicrobial activity. To our knowledge this is the first report on the phytochemistry and antimicrobial activity of this plant species.