Low genetic diversity in small leading edge populations of the European paleoendemic Ramonda serbica (Gesneriaceae) in Bulgaria

Ramonda serbica Pan?. (Gesneriaceae) is an endangered endemic species of the Balkan Peninsula which has been the subject of several studies in the past, but has not yet been investigated in terms of its genetic variation. Ramonda serbica is one of only five European representatives of the mainly subtropical family Gesneriaceae categorized as paleoendemics or Tertiary relicts sequestered in southern Europe by Quaternary climate oscillations. Here, an inter simple sequence repeat (ISSR) analysis was performed to determine the genetic diversity of five populations sampled from the geographically eastern fringe of its range in northwestern Bulgaria. We found relatively low levels of genetic diversity and significant genetic differentiation among the investigated populations, typical of a leading edge scenario. From a conservation point of view, the low genetic diversity, together with the presence of only few extant localities stress the need for urgent in situ and ex situ conservation actions to ensure the long‐term survival of R. serbica in Bulgaria.     

Desiccation increases sucrose levels in Ramonda and Haberlea, two genera of resurrection plants in the Gesneriaceae

A few genera of angiosperms are known as 'resurrection plants' since their leaves withstand complete desiccation. In many organisms, including some resurrection plants, desiccation tolerance is associated with the accumulation of special carbohydrates. We examined whether this is also true for the two European angiosperm genera of resurrection plants, Ramonda and Haberlea in the Gesneriaceae. Using gas chromatography, non-structural carbohydrates were determined as a percentage of the dry weight in leaves of Ramonda nathaliae subjected to various desiccation regimes. Sucrose was the predominant soluble carbohydrate in all samples, and its level steadily increased from 2 to 10% during desiccation. Starch amounted to ca 2% in control leaves and disappeared completely within 8 days of desiccation. Considerable amounts (1–2.5%) of raffinose and smaller amounts of its precursor galactinol (1-a-galactosyl- myo -inositol) were present in control leaves; these carbohydrates showed only minor changes upon desiccation. Similar results were obtained when excised leaves of Ramonda nathaliae, Ramonda myconi and Haberlea rhodopensis were subjected to desiccation. These data indicate that sucrose accumulation is connected to desiccation tolerance in Gesneriaceae; the presence of raffinose may be a pre-adaptation since this sugar prevents crystallization of sucrose during drying.     

CO2 fixation and chlorophyll a fluorescence in leaves of Ramonda serbica during a dehydration-rehydration cycle

Changes in CO(2) photo-assimilation and PSII photochemical efficiency in Ramonda serbica leaves during a dehydration-rehydration cycle were examined. The rate of CO(2) photo-assimilation was greatly reduced during dehydration, but recovery was complete with rehydration when the relative water content of leaves reached values similar to those of well-hydrated, control leaves. The results showed that the response of R. serbica leaves to severe water stress involves two different mechanisms. In the first, CO(2) assimilation is limited by stomata closure that creates an excess proton concentration in the lumen and activates non-photochemical quenching. This plays an important role in the mechanism of photoprotection by dissipation of excitation energy. When dehydration became severe and leaf RWC reached very low values, the electron transport rate (ETR) decreased markedly, while the capacity for regulatory mechanisms such as q(NP) (non-photochemical quenching) was greatly reduced. For severely dehydrated leaves of R. serbica, it appears that reactive oxygen species (ROS) formation is better prevented by mechanisms that quench chlorophyll triplet formation via lutein.     

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.     

Photocontrol of in vitro bud differentiation in Saintpaulia ionantha leaves and Lycopersicon esculentum cotyledons

The regeneration of buds from leaves of Saintpaulia ionantha Wendl cv. Sona and from cotyledons of Lycopersicon esculentum Mill. cv. UC 105 was studied in response to continuous light and daily light pulses. The regeneration of buds from cotyledons of Lycopersicon esculentum is high under continuous red and white light. Trie effect of light may be related to phytochrome. In Saintpaulia ionantha blue light appears to be very effective. The experimental results suggest that the blue light effects involve both a blue light absorbing photoreceptor and phytochrome     

Cold-induced sudden reversible lowering of in vivo chlorophyll fluorescence after saturating light pulses : a sensitive marker for chilling susceptibility

In chilling-sensitive plants (Glycine max, Saintpaulia ionantha, Saccharum officinarum) a sudden reversible drop in chlorophyll fluorescence occurs during photosynthetic induction immediately following saturating light pulses at low temperatures in the range 4 to 8°C. A comparison of two soybean cultivars of different chilling sensitivities revealed that this phenomenon, termed lowwave, indicates specific thresholds of low temperature stress. Its occurrence under controlled chilling can be regarded as a quantitative marker for screening chilling susceptibility in angiosperms.     

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.     

Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration

Plants of Ramonda serbica were dehydrated to 3.6% relative water content (RWC) by withholding water for 3 weeks, afterwards the plants were rehydrated for 1 week to 93.8% RWC. Plasma membranes were isolated from leaves using a two-phase aqueous polymer partition system. Compared with well-hydrated (control) leaves, dehydrated leaves suffered a reduction of about 75% in their plasma membrane lipid content, which returned to the control level following rewatering. Also the lipid to protein ratio decreased after dehydration, almost regaining the initial value after rehydration. Lipids extracted from the plasma membrane of fully-hydrated leaves were characterized by a high level of free sterols and a much lower level of phospholipids. Smaller amounts of cerebrosides, acylated steryl glycosides and steryl glycosides were also detected. The main phospholipids of control leaves were phosphatidylcholine and phosphatidylethanolamine, whereas sitosterol was the free sterol present in the highest amount. Following dehydration, leaf plasma membrane lipids showed a constant level of free sterols and a reduction in phospholipids compared with the well-hydrated leaves. Both phosphatidylcholine and phosphatidylethanolamine decreased following dehydration, their molar ratio remaining unchanged. Among free sterols, the remarkably high cholesterol level present in the control leaves (about 14 mol%) increased 2-fold as a result of dehydration. Dehydration caused a general decrease in the unsaturation level of individual phospholipids and total lipids as well. Upon rehydration the lipid composition of leaf plasma membranes restored very quickly approaching the levels of well-hydrated leaves.     

Role of phenolics in the antioxidative status of the resurrection plant Ramonda serbica during dehydration and rehydration

Ramonda serbica plants dehydrated for 14 days reached a relative water content of 4.2% and entered into anabiosis prior to being rehydrated for 48 h. Total ascorbate (AsA + DHA) and glutathione (GSH + GSSG) contents increased during dehydration and approached control values by the end of rehydration. Reduced ascorbate (AsA) and glutathione (GSH) were consumed during the first 13 days of dehydration when guaiacol-, syringaldazine- and phenolic peroxidases (EC 1.11.1.7) increased. At the end of dehydration AsA and GSH accumulated whereas peroxidases decreased to half the value of controls. In this period, plants of R. serbica face a phase of reduced metabolism and, thus, of reduced consumption of antioxidants. During rehydration, both AsA and GSH were utilized reaching, after 48 h, about 20 and 40% of their total pools, respectively; moreover peroxidases increased showing the recovery of metabolic activities. In the dehydration process total phenolic acids decreased, but accumulated after 5 h of rehydration and returned to control values at the end of rehydration. In R. serbica leaves, the most representative phenolic acids were protocatechuic, p -hydroxybenzoic and chlorogenic acids. Most concentrated phenolic acids, such as protocatechuic and chlorogenic acids, accumulated during the first period of rehydration when AsA decreased. These results suggest a role of ascorbate in inhibiting oxidation when phenolic peroxidases remain at low levels. As a consequence of this inhibition, ascorbate was oxidized and when most of it was consumed, oxidation of phenols resumed.     

The effect of air humidity on nutrient uptake of some greenhouse plants

Young plants of nine different greenhouse species were grown for 24 to 100 days at 55–60, 70–75, and 90–95% relative air humidity (RH) in growth rooms. They were given a complete nutrient solution twice a week. Transpiration rate decreased significantly by increasing RH from the lowest to the highest level inEuphorbia pulcherrima (60%), Begonia × hiemalis (54%), Saintpaulia ionantha (54%), Nephrolepis exaltata (48%), andLycopersicon esculentum (44%). The content of macro nutrient elements in the plant leaves decreased by increasing RH. This decrease was significant only for K and Ca in Lycopersicon and Kjeldahl-N in Nephrolepis. The content of the macro nutrient elements in the growth medium at the end of the experiment was lowest when the plants had been growing at high RH. The elements mainly affected were N and K. Report No. 342     

Phylogeny and disjunct distribution: evolution of Saintpaulia (Gesneriaceae).

The molecular phylogeny of African violets (Saintpaulia H. Wendl.), based on ribosomal DNA internal transcribed spacer (ITS) sequences, follows the disjunct biogeography of the genus. Sequence analysis by parsimony of 19 accessions, representing 17 currently recognized Saintpaulia species, resulted in four trees of 182 steps. The first major division is between S. goetzeana, from the Uluguru Mts, Tanzania, and the rest of the genus. The basal position of S. goetzeana, and its putative primitive characters, may indicate an Uluguru origin for Saintpaulia and subsequent colonization of the more northerly mountains. Of the remainder, S. teitensis, from the Teita Hills of Kenya, is sister taxon to the other species (which occur mainly in the Usambara Mts of north-east Tanzania). A group of nine Usambaran species that we call the ''ionantha complex'' show minimal ITS genetic differentiation and are also taxonomically critical. Species diversity in the Usambara Mts appears to be the result of rapid, recent (possibly Pleistocene) radiation. This study reveals the limitations of ITS sequences for elucidating the radiation of poorly differentiated species (the ionantha complex). However, the molecular data strongly suggest that conservation of the Uluguru and Teita populations is essential for the protection of the full range of diversity within the genus.     

非洲紫罗兰叶片体细胞胚培养及快繁技术研究

以非洲紫罗兰叶片为材料进行胚状体诱导及快繁技术研究.结果表明:.在MS NAA0.1mg/L BA0.1 mg/L 2,4-D1.0 mg/L的培养基上培养15d利于诱导胚性细胞分化,起始黑暗培养5～10d可提高胚性细胞分化率;在MS BA0.05～1mg/L的培养基上可诱导胚状体大量发生;在MS NAA0.1mg/L十BA0.1 mg/L的培养基上能够获得茎芽快速增殖;在1/2MS NAA0.01mg/L的培养基上可以生根.     

Photosynthesis parameters and activities of enzymes of oxidative stress in two young ‘Chemlali’ and ‘Chetoui’ olive trees under water deficit

The effects of water deficit on photochemical parameters and activities of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase were investigated in two olive cultivars differing in drought tolerance — ‘Chemlali’ and ‘Chetoui’. After 30 days without irrigation, leaf water potential fell to −5.5 MPa that was accompanied by a marked decrease in net photosynthesis in ‘Chetoui’ olive cultivar. Maximal efficiency of PSII photochemistry (F_v/F_m) decreased slightly in ‘Chemlali’ (28 %) and substantially in ‘Chétoui’ (47 %). Both cultivars showed a similar decline (about 25 %) in the photochemical quenching coefficient, but only the drought-sensitive olive cultivar exhibited an enhancement (31 %) of non-photochemical fluorescence quenching under water deficit conditions. The quantum yield of electron transport decreased in both olive cultivars. ‘Chemlali’ showed a higher protection against oxidative stress, as judged from the lower levels of the malondialdehyde production. Catalase activity was higher in ‘Chetoui’. Glutathione reductase activity was increased similarly in both olive cultivars under water stress. Ascorbate peroxidase activity was enhanced in ‘Chemlali’ under water stress, but was unaffected in ‘Chetoui’. While, superoxide dismutase activity was inhibited in both cultivars under water stress, but higher activity was detected in ‘Chemlali’. Thus, the ability to increase ascorbate peroxidase and a higher superoxide dismutase activity might be an important attribute linked to the drought tolerance in ‘Chemlali’ olive cultivar.     

Flavohaemoglobin HmpX from Erwinia chrysanthemi confers nitrosative stress tolerance and affects the plant hypersensitive reaction by intercepting nitric oxide produced by the host

Host cells respond to infection by generating nitric oxide (NO) as a cytotoxic weapon to facilitate killing of invading microbes. Bacterial flavohaemoglobins are well-known scavengers of NO and play a crucial role in protecting animal pathogens from nitrosative stress during infection. Erwinia chrysanthemi, which causes macerating diseases in a wide variety of plants, possesses a flavohaemoglobin (HmpX) whose function in plant pathogens has remained unclear. Here we show that HmpX consumes NO and prevents inhibition by NO of cell respiration, indicating a role in protection from nitrosative stress. Furthermore, infection of Saintpaulia ionantha plants with an HmpX-deficient mutant of E. chrysanthemi revealed that the lack of NO scavenging activity causes the accumulation of unusually high levels of NO in host tissue and triggers hypersensitive cell death. Introduction of the wild-type hmpX gene in an incompatible strain of Pseudomonas syringae had a dramatic effect on the hypersensitive cell death in soya bean cell suspensions, and markedly reduced the development of macroscopic symptoms in Arabidopsis thaliana plants. These observations indicate that HmpX not only protects against nitrosative stress but also attenuates host hypersensitive reaction during infection by intercepting NO produced by the plant for the execution of the hypersensitive cell death programme.     

Role of endoglucanases in Erwinia chrysanthemi 3937 virulence on Saintpaulia ionantha.

The role of endoglucanases (endoglucanases Z and Y) in Erwinia chrysanthemi pathogenicity on Saintpaulia ionantha was assessed by mutagenizing cloned cel genes (celZ and celY) and recombining them with the chromosomal alleles. Strains with an omega interposon in celZ, a deletion in celY, or a double cel mutant were as virulent as the wild-type strain. However, in the strain with a deletion in celY, a delay in the appearance of symptoms was observed, and then maceration progressed as in plants infected with the wild-type strain, suggesting that E. chrysanthemi endoglucanases play a minor role in soft rot disease development.     

Micropropagation and in vitro conservation of the rare and threatened plants Ramonda serbica and Ramonda nathaliae

Ramonda serbica and Ramonda nathaliae are rare and endemo relict plant species from Balkan Peninsula. An efficient micro propagation and in vitro conservation method via direct and indirect organogenesis from seed and leaf explants, respectively, was established in this study. The seed of both Ramonda species were collected from different populations in Kosovo, and were germinated in nutrient media JG-B without any phytohormone. The highest number of shoots and multiplication rate was observed on JG-B medium supplemented with BAP and IAA (0.5 mg l⁻¹ each), whereas the highest number of leaves per plantlets was found on WPM and RA medium supplemented with BAP and IAA (0.1 mg l⁻¹ each). During this stage of micro propagation some significant differences were observed in plantlets from different populations. The indirect organogenesis from parts of leaves of natural plants was not successful due to unavailability of established protocol for disinfections of the plant material. On other hand, parts of leaves from micro propagated plantlets, cultured on MS medium supplemented with different ratio of BAP and NAA, resulted in the highest efficiency for shoot regeneration. In vitro conservation of micro propagated plants at the lower temperature (4 °C) had a significantly positive effect for storage of more than 12 months.     

Exogenously applied polyamines increase drought tolerance of rice by improving leaf water status,photosynthesis and membrane properties

Drought stress hampers rice performance principally by disrupting the plant–water relations and structure of biological membranes. This study appraised the role of polyamines (PAs) in improving drought tolerance in fine grain aromatic rice (Oryza sativa L.). Three PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)] were used each at 10 μM as seed priming (by soaking seeds in solution) and foliar spray. Primed and non-primed seeds were sown in plastic pots with normal irrigation in a phytotron. At four-leaf stage, plants were subjected to drought stress by bringing the soil moisture down to 50% of field capacity by halting water supply. For foliar application, 10 μM solutions each of Put, Spd and Spm were sprayed at five-leaf stage. Results revealed that drought stress severely reduced the rice fresh and dry weights, while PAs application improved net photosynthesis, water use efficiency, leaf water status, production of free proline, anthocyanins and soluble phenolics and improved membrane properties. PAs improved drought tolerance in terms of dry matter yield and net photosynthesis was associated with the maintenance of leaf water status and improved water use efficiency. Among the antioxidants, catalase activity was negatively related to H₂O₂ and membrane permeability, which indicated alleviation of oxidative damage on cellular membranes by PAs application. Foliar application was more effective than the seed priming, and among the PAs, Spm was the most effective in improving drought tolerance.     

Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in tolerance to drought stress. To try to determine the mechanisms of acquisition of tolerance to drought stress by heat shock, the rice small heat-shock protein gene, sHSP17.7, the product of which was shown to act as molecular chaperones in vitro and in vivo in our previous study, was overexpressed in the rice cultivar “Hoshinoyume”. Western and Northern blot analyses showed higher expression levels of sHSP17.7 protein in three transgenic lines than in one transgenic line. Drought tolerance was assessed in these transgenic lines and wild-type plants by withholding water for 6 days for evaluation of the ability of plants to continue growth after water-stress treatments. Although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHSP17.7 protein could regrow after rewatering. Similar results were observed in survival rates after treatments with 30% polyethylene glycol (PEG) 3640 for 3 days. These results suggest that overproduction of sHSP17.7 could increase drought tolerance in transgenic rice seedlings.