The effects of osmium tetroxide post-fixation and drying steps on leafy liverwort ultrastructure study by scanning electron microscopy.

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO4 ) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post-fixed with 4% OsO4 , while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post-fixation with 4% OsO4 could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO4 post-fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.

The biological response chain to pollution: a case study from the "Italian Triangle of Death" assessed with the liverwort Lunularia cruciata.

The liverwort Lunularia cruciata, known for being a species tolerant to pollution able to colonize urban areas, was collected in the town of Acerra (South Italy) to investigate the biological effects of air pollution in one of the three vertices of the so-called Italian Triangle of Death. The ultrastructural damages observed by transmission electron microscopy in specimens collected in Acerra were compared with samples collected in the city center of Naples and in a small rural site far from sources of air pollution (Riccia, Molise, Southern Italy). The biological response chain to air pollution was investigated considering vitality, photosynthetic efficiency, heat shock protein 70 (Hsp70) induction and gene expression levels, and chlorophyll degradation and related ultrastructural alterations. Particularly, a significant increment in Hsp70 expression and occurrence, and modifications in the chloroplasts' ultrastructure can be strictly related to the environmental pollution conditions in the three sites. The results could be interpreted in relation to the use of these parameters as biomarkers for environmental pollution.

Land plants drive photorespiration as higher electron-sink: comparative study of post-illumination transient O2 -uptake rates from liverworts to angiosperms through ferns and gymnosperms.

In higher plants, the electron-sink capacity of photorespiration contributes to alleviation of photoinhibition by dissipating excess energy under conditions when photosynthesis is limited. We addressed the question at which point in the evolution of photosynthetic organisms photorespiration began to function as electron sink and replaced the flavodiiron proteins which catalyze the reduction of O2 at photosystem I in cyanobacteria. Algae do not have a higher activity of photorespiration when CO2 assimilation is limited, and it can therefore not act as an electron sink. Using land plants (liverworts, ferns, gymnosperms, and angiosperms) we compared photorespiration activity and estimated the electron flux driven by photorespiration to evaluate its electron-sink capacity at CO2 -compensation point. In vivo photorespiration activity was estimated by the simultaneous measurement of O2 -exchange rate and chlorophyll fluorescence yield. All C3-plants leaves showed transient O2 -uptake after actinic light illumination (post-illumination transient O2 -uptake), which reflects photorespiration activity. Post-illumination transient O2 -uptake rates increased in the order from liverworts to angiosperms through ferns and gymnosperms. Furthermore, photorespiration-dependent electron flux in photosynthetic linear electron flow was estimated from post-illumination transient O2 -uptake rate and compared with the electron flux in photosynthetic linear electron flow in order to evaluate the electron-sink capacity of photorespiration. The electron-sink capacity at the CO2 -compensation point also increased in the above order. In gymnosperms photorespiration was determined to be the main electron-sink. C3-C4 intermediate species of Flaveria plants showed photorespiration activity, which intermediate between that of C3- and C4-flaveria species. These results indicate that in the first land plants, liverworts, photorespiration started to function as electron sink. According to our hypothesis, the dramatic increase in partial pressure of O2 in the atmosphere about 0.4 billion years ago made it possible to drive photorespiration with higher activity in liverworts.

Water pollution causes ultrastructural and functional damages in Pellia neesiana (Gottsche) Limpr.

The aim of this work is to evaluate the effects of freshwater pollution in the heavily contaminated Sarno River (Campania, South Italy), using Pellia neesiana (Pelliaceae Metzgeriales) in order to propose this liverwort as a potential bioindicator, able to record the effects of water pollution, particularly the one related to metal (loid) contamination. Samples of P. neesiana in nylon bags were disposed floating for one week on the waters of Sarno River in three sites characterised by an increasing pollution. As control, some specimens were cultured in vitro in Cd- and Pb-added media, at the same pollutants' levels as measured in the most polluted site. P. neesiana cell ultrastructure was modified and severe alterations were observed in chloroplasts from samples exposed in the most polluted site, and Cd- and Pb-cultured samples. Concurrently, a strong increase in the occurrence of Heat shock proteins 70 (HSP70) was detected in gametophytes following the pollution gradient. In conclusion, ultrastructural damages can be directly related to HSP 70 occurrence in liverwort tissues, and proportional to the degree of pollution present in the river; thus our study suggests P. neesiana as an affordable bioindicator of freshwaters pollution.

Functional characterization of a Mg(2+)-dependent O-methyltransferase with coumarin as preferred substrate from the liverwort Plagiochasma appendiculatum.

Coumarins (1,2-benzopyrones), which originate via the phenylpropanoid pathway, are found ubiquitously in plants and make an essential contribution to the health of the plant. Some natural coumarins have been used as human therapeutics. However, the details of their biosynthesis are still largely unknown. Scopoletin is derived from either esculetin or feruloyl CoA according to the plant species involved. Here, a gene encoding a O-methyltransferase (PaOMT2) was isolated from the liverwort species Plagiochasma appendiculatum (Aytoniaceae) through transcriptome sequencing. The purified recombinant enzyme catalyzed the methylation of esculetin, generating scopoletin and isoscopoletin. Kinetic analysis shows that the construct from the second Met in PaOMT2 had a catalytic efficiency for esculetin (Kcat/Km) of about half that of the full length PaOMT2, while the Kms of two enzymes were similar. The catalytic capacities of the studied protein suggest that two routes to scopoletin might co-exist in liverworts in that the enzyme involved in the methylation process participates in both paths, but especially the route from esculetin. The transient expression of a PaOMT2-GFP fusion in tobacco demonstrated that PaOMT2 is directed to the cytoplasm.

Functional analysis of liverworts in dual symbiosis with Glomeromycota and Mucoromycotina fungi under a simulated Palaeozoic CO2 decline.

Most land plants form mutualistic associations with arbuscular mycorrhizal fungi of the Glomeromycota, but recent studies have found that ancient plant lineages form mutualisms with Mucoromycotina fungi. Simultaneous associations with both fungal lineages have now been found in some plants, necessitating studies to understand the functional and evolutionary significance of these tripartite associations for the first time. We investigate the physiology and cytology of dual fungal symbioses in the early-diverging liverworts Allisonia and Neohodgsonia at modern and Palaeozoic-like elevated atmospheric CO2 concentrations under which they are thought to have evolved. We found enhanced carbon cost to liverworts with simultaneous Mucoromycotina and Glomeromycota associations, greater nutrient gain compared with those symbiotic with only one fungal group in previous experiments and contrasting responses to atmospheric CO2 among liverwort-fungal symbioses. In liverwort-Mucoromycotina symbioses, there is increased P-for-C and N-for-C exchange efficiency at 440 p.p.m. compared with 1500 p.p.m. CO2. In liverwort-Glomeromycota symbioses, P-for-C exchange is lower at ambient CO2 compared with elevated CO2. No characteristic cytologies of dual symbiosis were identified. We provide evidence of a distinct physiological niche for plant symbioses with Mucoromycotina fungi, giving novel insight into why dual symbioses with Mucoromycotina and Glomeromycota fungi persist to the present day.

Functional characterization of a chalcone synthase from the liverwort Plagiochasma appendiculatum.

KEY MESSAGE: A chalcone synthase gene ( PaCHS ) was isolated and functionally characterized from liverwort. The ectopic expression of PaCHS in Marchantia paleacea callus raised the flavonoids content. Chalcone synthase (CHS; EC 2.3.1.74) is pivotal for the biosynthesis of flavonoid and anthocyanin pigments in plants. It produces naringenin chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A thioesters through a polyketide intermediate that is cyclized by intramolecular Claisen condensation. Although CHSs of higher plants have been extensively studied, enzyme properties of the CHSs in liverworts have been scarcely characterized. In this study, we report the cloning and characterization of CHS (designated as PaCHS) from the liverwort Plagiochasma appendiculatum. The gene product was 60-70 % identical with chalcone synthases from other species, and contained the characteristic conserved Cys-His-Asn catalytic triad. The recombinant PaCHS was able to catalyze p-coumaroyl-CoA and malonyl-CoA to generate naringenin in vitro. Heterologously expressed PaCHS protein showed similar kinetic properties to those of higher plant CHS. The ectopic expression of PaCHS in Marchantia paleacea callus raised the content of the total flavonoids. These results suggested that PaCHS played a key role in the flavonoids biosynthesis in liverworts. Furthermore, when the thallus of P. appendiculatum was treated with abiotic stress inducers methyl jasmonate, salicylic acid and abscisic acid, PaCHS expression was enhanced. This is the first time that a CHS in liverworts has been functionally characterized.

A Cd/Fe/Zn-responsive phytochelatin synthase is constitutively present in the ancient liverwort Lunularia cruciata (L.) dumort.

Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase.

Origin of strigolactones in the green lineage.

The aims of this study were to investigate the appearance of strigolactones in the green lineage and to determine the primitive function of these molecules. We measured the strigolactone content of several isolated liverworts, mosses, charophyte and chlorophyte green algae using a sensitive biological assay and LC-MS/MS analyses. In parallel, sequence comparison of strigolactone-related genes and phylogenetic analyses were performed using available genomic data and newly sequenced expressed sequence tags. The primitive function of strigolactones was determined by exogenous application of the synthetic strigolactone analog, GR24, and by mutant phenotyping. Liverworts, the most basal Embryophytes and Charales, one of the closest green algal relatives to Embryophytes, produce strigolactones, whereas several other species of green algae do not. We showed that GR24 stimulates rhizoid elongation of Charales, liverworts and mosses, and rescues the phenotype of the strigolactone-deficient Ppccd8 mutant of Physcomitrella patens. These findings demonstrate that the first function of strigolactones was not to promote arbuscular mycorrhizal symbiosis. Rather, they suggest that the strigolactones appeared earlier in the streptophyte lineage to control rhizoid elongation. They may have been conserved in basal Embryophytes for this role and then recruited for the stimulation of colonization by glomeromycotan fungi.

Electrophysiological approach to examine a putative cold- and menthol- sensitive channel in the liverwort Conocephalum conicum.

The mechanism of cold perception by plants is still poorly understood. It was found that temperature drop evokes changes in the activity of ion pumps and channels, which leads to plasma membrane depolarization. The nature of the primary step of its action (alteration in membrane composition, transient influx of Ca2+ etc.,) has not been elicited yet. Our electrophysiological experiments conducted on the liverwort Conocephalum conicum showed that its cells respond not only to sudden cooling but also to menthol, generating depolarization of the plasma membrane and action potentials (APs). Similar results are well documented in mammals; cold or "cooling compounds" including menthol cause activation of thermosenstitive channel TRPM8 permeable to Ca2+ and generation of AP series. TRP receptors are detected, among others, in green and brown algae. Possible existence of TRPM8-like channel-receptor in Conocephalum conicum is discussed here.

Effect of cold and menthol on membrane potential in plants.

In animals, cooling substances such as menthol are perceived as cold sensation because they bind to the same receptor TRPM8 (transient receptor potential melastatin) that activates upon temperature drops. We investigated the effect of menthol on the plant membrane potential to search for analogies between animal and plant perception systems. The study was conducted on the liverwort Conocephalum conicum- a non-vascular plant generating action potentials (APs) in response to different stimuli including cold. (+)Menthol, (-)menthol and (+/-)menthol induced one or more APs, depending on the concentration. In contrast to animal reactions to menthol, threshold concentrations of these isomers were the same (1 mM). The presence of menthol in medium shortened cold-induced APs, whereas low temperature prolonged the repolarization phase of AP evoked by menthol. Cells of C. conicum with anion and potassium channels blocked by anthracene-9-carboxylic acid (A9C) and tetraethylammonium chloride (TEACl) generate short spike-like voltage transients (VTs) in response to cold and light stimulation. Membrane potential changes evoked by menthol in A9C- and TEACl-treated plants differed significantly from VTs - lasted much longer and frequently occurred in series. 5 mM LaCl(3) , 1 mM EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (0 Ca(2+) ) but not 0.2 mM verapamil blocked the putative calcium component of AP induced by menthol. Similar inhibitory effect was observed after the application of proton pump inhibitors: 0.05 mM N,N-dicyclohexylcarbodiimide (DCCD), 0.05 mM diethylstilbestrol (DES) or 0.01 mM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). Our results indicate that cold and menthol act independently, activating different membrane transporters in C. conicum cells.

Influence of soil fumigation by methyl bromide and methyl iodide on rhizosphere and phyllosphere microbial community structure.

Rhizosphere and phyllosphere microbial communities were evaluated on roots and leaves of growth chamber-grown lettuce (Lactuca sativa (L.) cv. Green Forest) plants by culture-dependent and -independent methods after soil fumigation. Denaturing gradient gel electrophoresis (DGGE) with 16S rRNA primers followed by cloning and sequencing was used to identify major rRNA bands from the rhizosphere and phyllosphere. Three weeks after fumigation, there were no differences (P = 0.16) in rhizosphere microbial communities between the fumigated treatments and the control. The same effect was observed during week seven after fumigation (P=0.49). Also, no significant differences (P=0.49) were found in the phyllosphere microbial communities between the fumigated treatments and the control during the growth period of the plant. A majority of the bands in the rhizosphere were related to known bacterial sequences with a 96 to 100 % sequence similarity. Some of the derived sequences were related to Pseudomonas syringae pv. tomato DC300 and Bradyrhizobium japonicum USDA 110. A total of 23 isolates were identified from leaf surface by both culture-dependent and independent methods, and only Photorhabdus luminescens was found on leaf surface using both techniques. All the Biolog isolates from phyllosphere were from the Proteobacteria phylum compared to the culture-independent bands from the leaves that were from different bacterial phyla. Based on our data, methyl bromide (MeBr) and methyl iodide (MeI) did not have any significant negative effects on rhizosphere and phyllosphere microbial communities throughout the growing period of lettuce.

Retrospective bioindication of stratospheric ozone and ultraviolet radiation using hydroxycinnamic acid derivatives of herbarium samples of an aquatic liverwort.

We analyzed bulk UV absorbance of methanolic extracts and levels of five UV-absorbing compounds (hydroxycinnamic acid derivatives) in 135 herbarium samples of the liverwort Jungermannia exsertifolia subsp. cordifolia from northern Europe. Samples had been collected in 1850-2006 (96% in June-August). Both UV absorbance and compound levels were correlated positively with collection year. p-Coumaroylmalic acid (C1) was the only compound showing a significant (and negative) correlation with stratospheric ozone and UV irradiance in the period that real data of these variables existed. Stratospheric ozone reconstruction (1850-2006) based on C1 showed higher values in June than in July and August, which coincides with the normal monthly variation of ozone. Combining all the data, there was no long-term temporal trend from 1850 to 2006. Reconstructed UV showed higher values in June-July than in August, but again no temporal trend was detected in 1918-2006 using the joint data. This agrees with previous UV reconstructions.

Genistein and changes of resting and action potentials in Conocephalum conicum.

Genistein (4',5,7-trixydroxyflavone) is a member of the family of plant flavonoids that widely occurs in crop plants. It is involved in a wide spectrum of pharmacological activities, and is suggested to have anti-cancer dietary properties. Cell membranes are one of the targets of anti-cancer drugs. In the present study, we used the liverwort Conocephalum conicum as a model plant in an electrophysiological study. Intracellular microelectrode measurements were carried out to examine the effects of genistein alone and in combination with verapamil on resting and action potentials. The application of isoflavone genistein resulted in a statistically significant elevation in action potential amplitudes. An increase of 13-62% compared with the control was noted. An increase was also found in the membrane resting potentials in genistein-treated plants. Verapamil, the known calcium channel inhibitor, caused a gradual decline of AP amplitudes, whereas preincubation of Conocephalum thalli with genistein prevented inhibition of APs by verapamil. It is concluded that genistein strongly affects the membranes, and the effect of genistein in canceling the activity of verapamil is discussed.

The polycyclic aromatic hydrocarbon phenanthrene causes oxidative stress and alters polyamine metabolism in the aquatic liverwort Riccia fluitans L.

The polycyclic aromatic hydrocarbon (PAH) phenanthrene (PHEN) is a highly toxic pollutant, commonly found in aquatic environments, the effects of which on aquatic plants have not been studied in depth. As PAHs are known to induce oxidative stress and recent studies have shown that polyamines (PAs) participate in the defence reactions protecting plants against environmental stresses, PA metabolism and oxidative damage were investigated in the aquatic form of the liverwort Riccia fluitans L. exposed to PHEN. Exposure of Riccia fluitans plants to PHEN at concentrations of 0.5 microm or less induced oxidative stress, but at a level from which plants could recover. Despite increased levels of enzymatic and non-enzymatic antioxidants, recovery appeared, at least in part, due to increased synthesis of PAs, achieved via increased activities of the enzymes arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC). Chemical inhibition of these enzymes inhibited plant recovery, while treatment with PAs aided recovery. Finally, as chloroplasts and the plasma membrane appeared to be key targets for PHEN-induced damage, the potential roles of PAs in protecting these cellular components were considered. How PAs could protect plant cells from serious environmental pollutants such as PHEN and could prevent oxidative stress is discussed.

Impact of heavy metals (copper, zinc, and lead) on the chlorophyll content of some mosses.

The effects of the heavy metals copper (Cu), zinc (Zn), and lead (Pb) on the chlorophyll content of two mosses Thuidium delicatulum (L.) Mitt. and T. sparsifolium (Mitt.) Jaeg., as well as leafy liverwort Ptychanthus striatus (Lehm. & Linderb.) were examined to understand the impact of metal accumulation on the chlorophyll content of mosses and leafy liverwort, which are found only in uncontaminated sites of the Kathmandu valley. These plants were treated with different concentrations of CuCl(2, )ZnCl(2), and Pb(NO(3))(2), ranging from 10(-10) M to 10(-2) M, in isolation and in combination under experimental conditions. Metal accumulation in the plant bodies increased with metal concentrations. Cu accumulation showed a significant inhibitory effect on chlorophyll-a, chlorophyll-b, and total chlorophyll in the mosses and the leafy liverwort. An insignificant decrease in chlorophyll content in both Thuidium species, but a significant decrease in leafy liverwort, was observed after Zn and Pb accumulation. Chlorophyll-a decreased significantly in T. sparsifolium; chlorophyll-b and total chlorophyll decreased significantly in T. delicatulum; and all chlorophyll contents decreased insignificantly in P. striatus after accumulation of Cu+Zn+Pb ions together from mixed metal solution. The ratio of chlorophyll-a to -b decreased more rapidly in both Thuidium species, with higher concentrations occurring when Cu+Zn+Pb ions were together than when Cu, Zn, or Pb ions were alone. This indicated a more destructive effect of Cu metals on the chlorophyll contents of both Thuidium species. High concentrations of Cu are known to activate oxidative damage and alter cell-membrane properties by lipid peroxidation, thereby demonstrating the inhibitory effect on the enzymes involved in chlorophyll production. The greater loss of chlorophyll from heavy-metal accumulation in P. striatus than in T. delicatulum and T. sparsifolium may have been caused by relatively more K(+) efflux in leafy liverwort than in the mosses, thus indicating their differences in membrane integrity.

The influence of glutamic and aminoacetic acids on the excitability of the liverwort Conocephalum conicum.

Intracellular microelectrode measurements revealed that a resting potential (RP), an action potential (AP) and a calcium component of AP (named voltage transient, VT) can be influenced by glutamic acid (Glu) and aminoacetic acid (glycine, Gly) in the liverwort Conocephalum conicum. In the continuous presence of 5mM Glu or 5mM Gly, the RP hyperpolarized constantly and the plants became desensitized to the excitatory amino acids (Glu or Gly). Under such circumstances, the amplitudes of APs evoked by stimuli other than Glu or Gly grew, as did their calcium components (VTs). The sudden application of 1-15 mM Glu or Gly to a thallus not yet desensitized resulted in an excitation, i.e. a single AP or AP series. Aspartate (Asp) could not substitute for Glu in any way. Simultaneous action of both amino acids acted synergically to trigger APs. The same phenomenon was observed when glycine solution was enriched with N-methyl-D-aspartic acid (NMDA). Gly-induced APs were totally hindered by 1mM D-amino-5-phosphonopentanoic acid (AP5)--an inhibitor of ionotropic glutamate receptors of the NMDA kind. Glu-induced APs could be totally suppressed by 1mM AP5 as well as by 1mM 6,7-dinitroquinoxaline-2,3-dione (DNQX)--an inhibitor of AMPA/KA receptors. DNQX also completely blocked the calcium component of Glu-evoked APs. After DNQX treatment, the only response to Glu was a membrane potential hyperpolarization (like the Glu response in a desensitized plant). It was concluded that the Glu-induced depolarization and hyperpolarization are separate phenomena. The stimulatory effects of both Glu and Gly on liverwort excitability may be the consequences of an activation of a variety of ionotropic Glu receptor subtypes.

Effects of cadmium and enhanced UV radiation on the physiology and the concentration of UV-absorbing compounds of the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia.

The aquatic liverwort Jungermannia exsertifolia subsp. cordifolia was cultivated for 15 d under controlled conditions to study the single and combined effects of cadmium and enhanced ultraviolet (UV) radiation. Both cadmium and UV radiation caused chlorophyll degradation and a decrease in the maximum quantum yield of photosystem II (PSII), together with an increase in the mechanisms of non-photochemical dissipation of energy (increase in the xanthophyll index). Cadmium was more stressing than UV radiation, since the metal also influenced photosynthesis globally and caused a decrease in net photosynthetic rates, in the effective quantum yield of photosynthetic energy conversion of PSII, and in the maximal apparent electron transport rate through PSII. Ultraviolet radiation increased the level of trans-p-coumaroylmalic acid and cadmium increased trans-phaselic and feruloylmalic acids. The increase in these compounds was probably related to both a more efficient absorption of harmful UV radiation and an enhanced protection against oxidative stress. DNA damage was specifically caused by UV-B radiation, but was intensified under the presence of cadmium, probably because the metal impairs the DNA enzymatic repair mechanisms. Ultraviolet radiation and cadmium seemed to operate additively on some physiological processes, while other responses were probably due to either factor alone.

Effect of cadmium on gene expression in the liverwort Lunularia cruciata.

Bryophytes are valuable bioaccumulators, because they have high absorbing and ion exchange capacities. Cadmium (Cd) is a heavy metal naturally present in soil; it is non-essential and highly toxic to most organisms, having toxicity 2 to 20 times higher than many other heavy metals. The presence of elevated levels of Cd ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To investigate the ability of Cd to affect gene transcription, the messenger RNA (mRNA) differential display technique was applied to the identification and isolation of genes whose transcription was altered in cultured Lunularia cruciata plants that were grown in the presence of cadmium salts. Four genes whose mRNA levels significantly changed in response to cadmium exposure were isolated and identified. The first gene identified in our analysis is up-regulated by Cd: it encodes the enzyme cystathionine gamma-synthase. The other genes are down-regulated by cadmium. These genes encode a methyltransferase, a tyrosine phosphatase and the EST 408 of the diatom Fragilariopsis cylindrus, whose function is unknown. Our findings demonstrate the usefulness of mRNA differential display technique for the detection of plant metabolic pathways affected by cadmium stress.

Accumulation, localisation, and toxic effects of cadmium in the liverwort Lunularia cruciata.

Accumulation, tissue and intracellular localisation, and toxic effects of cadmium were investigated in the liverwort Lunularia cruciata. The results of analyses carried out by atomic absorption spectrometry on single plants showed that the cadmium accumulation was dose- and time-dependent. Cadmium localisation was assessed by X-ray scanning electron microscopy microanalysis in gemmalings and in the different tissues of the thallus and by X-ray transmission electron microscopy microanalysis at the cellular level. The metal preferentially accumulated in the hyaline parenchyma and at the base of the gemma cups. Inside the cell, cadmium accumulated in the vacuoles and the cell wall. Metal accumulation was accompanied by a concomitant increase in sulphur content within the vacuoles of stressed cells. Gel-permeation chromatography showed that most of the cadmium was associated with a low-molecular-mass fraction eluting at a ratio of elution volume to void volume corresponding to that of phytochelatins. The excess of sulphur deposited in the vacuoles may well have been caused by the stress-induced synthesis of phytochelatins. At the ultrastructural level, sublethal concentrations of cadmium caused alterations of the fine structure of the cells, inducing marked alterations of the chloroplast structure. Cadmium also induced a dose-dependent inhibition of apical thallus growth and gemma germination.