Liverwort Jungermannia Exsertifolia Research Articles

Transcriptome Analysis and Identification of Sesquiterpene Synthases in Liverwort Jungermannia exsertifolia.

The liverwort Jungermannia exsertifolia is one of the oldest terrestrial plants and rich in structurally specific sesquiterpenes. There are several sesquiterpene synthases (STSs) with non-classical conserved motifs that have been discovered in recent studies on liverworts; these motifs are rich in aspartate and bind with cofactors. However, more detailed sequence information is needed to clarify the biochemical diversity of these atypical STSs. This study mined J. exsertifolia sesquiterpene synthases (JeSTSs) through transcriptome analysis using BGISEQ-500 sequencing technology. A total of 257,133 unigenes was obtained, and the average length was 933 bp. Among them, a total of 36 unigenes participated in the biosynthesis of sesquiterpenes. In addition, the in vitro enzymatic characterization and heterologous expression in Saccharomyces cerevisiae showed that JeSTS1 and JeSTS2 produced nerolidol as the major product, while JeSTS4 could produce bicyclogermacrene and viridiflorol, suggesting a specificity of J. exsertifolia sesquiterpene profiles. Furthermore, the identified JeSTSs had a phylogenetic relationship with a new branch of plant terpene synthases, the microbial terpene synthase-like (MTPSL) STSs. This work contributes to the understanding of the metabolic mechanism for MTPSL-STSs in J. exsertifolia and could provide an efficient alternative to microbial synthesis of these bioactive sesquiterpenes.

Acclimation of Bryophytes to Sun Conditions, in Comparison to Shade Conditions, Is Influenced by Both Photosynthetic and Ultraviolet Radiations.

We studied the acclimation modalities of bryophytes to sun and shade under ambient or close-to-ambient conditions, measuring variables usually influenced by photosynthetically active (PAR) and ultraviolet (UV) radiations. Our aim was to elucidate to what extent the responses to changing radiations were influenced by PAR and UV wavelengths. For this aim, we used three taxonomically and structurally different species: the thalloid liverwort Marchantia polymorpha subsp. polymorpha, the leafy liverwort Jungermannia exsertifolia subsp. cordifolia, and the moss Fontinalis antipyretica. In the field, liverworts were more radiation-responsive than the moss, and the thalloid liverwort was more responsive than the leafy liverwort. Sun plants of M. polymorpha showed, in comparison to shade plants, higher sclerophylly, lower Chl a + b contents, higher Chl a/b ratios, higher (antheraxanthin + zeaxanthin)/(violaxanthin + antheraxanthin + zeaxanthin) ratios (xanthophyll index), lower Fv/Fm values, higher contents of methanol-soluble vacuolar UV-absorbing compounds (soluble UVACs), higher values of the ratio between the contents of methanol-insoluble cell wall-bound UVACs (insoluble UVACs) and soluble UVACs, higher contents of soluble luteolin and apigenin derivatives and riccionidin A, and higher contents of insoluble p-coumaric and ferulic acids. Overall, these responses reduced light absorption, alleviated overexcitation, increased photoprotection through non-photochemical energy dissipation, increased UV protection through UV screening and antioxidant capacity, and denoted photoinhibition. J. exsertifolia showed moderate differences between sun and shade plants, while responses of F. antipyretica were rather diffuse. The increase in the xanthophyll index was the most consistent response to sun conditions, occurring in the three species studied. The responses of soluble UVACs were generally clearer than those of insoluble UVACs, probably because insoluble UVACs are relatively immobilized in the cell wall. These modalities of radiation acclimation were reliably summarized by principal components analysis. Using the most radiation-responsive species in the field (M. polymorpha), we found, under close-to-ambient greenhouse conditions, that sclerophylly and Chl a + b content were only influenced by PAR, Fv/Fm, and luteolin and apigenin derivatives were only determined by UV, and xanthophyll index was influenced by both radiation types. Thus, responses of bryophytes to radiation can be better interpreted considering the influence of both PAR and UV radiation.

Spatial variability of ultraviolet-absorbing compounds in an aquatic liverwort and their usefulness as biomarkers of current and past UV radiation: A case study in the Atlantic–Mediterranean transition

The spatial variability of ultraviolet-absorbing compounds (UVACs) in the freshwater liverwort Jungermannia exsertifolia subsp. cordifolia was studied in mid-latitudes (the Atlantic–Mediterranean transition) across a wide lati-altitudinal gradient, with the aim of testing the usefulness of UVACs as biomarkers of current ambient levels of UV radiation. We analysed 17 samples from streams located in the main mountain ranges of the Iberian Peninsula, differentiating methanol-soluble (SUVACs, mainly located in the vacuoles) and methanol-insoluble (IUVACs, bound to cell walls) compounds, since they represent different manners to cope with UV radiation. In both fractions, the bulk level of UVACs and the concentrations of several individual compounds were measured. In addition, we measured Fv/Fm, DNA damage and sclerophylly index (SI) as possible additional UV biomarkers. UVACs showed a high variability, probably due not only to the gradients of macroenvironmental factors (UV radiation, PAR, and water temperature), but also to microenvironmental factors inherent to the dynamic nature of mountain streams. Two soluble coumarins were positively correlated with UV levels and could be used for ambient UV biomonitoring in the spatial scale. In contrast to the variability in UVACs, the relatively homogeneous values of Fv/Fm and the lack of any DNA damage made these variables useless for ambient UV biomonitoring, but suggested a strong acclimation capacity of this liverwort to changing environmental conditions (in particular, to UV levels). Finally, UVACs of fresh samples of the liverwort were compared to those of herbarium samples collected in the same lati-altitudinal gradient. SUVACs were significantly higher in fresh samples, whereas IUVACs generally showed the contrary. Thus, IUVACs were more stable than SUVACs and hence more adequate for retrospective UV biomonitoring. In conclusion, UVAC compartmentation should be taken into account for bryophyte-based UV biomonitoring in future studies.

UV radiation biomonitoring using cell compartmentation of UV-absorbing compounds in herbarium samples of a liverwort

Ultraviolet-absorbing compounds (UVACs) were analyzed in 90 herbarium samples of the liverwort Jungermannia exsertifolia subsp. cordifolia collected throughout Spain in the period 1913–2006, in order to evaluate their usefulness in UV radiation biomonitoring. This was assessed, for the first time in bryophytes, differentiating the soluble (mainly vacuolar) and insoluble (cell wall-bound) UVACs (respectively, SUVACs and IUVACs), which may represent different mechanisms to cope with UV radiation. The bulk levels of SUVACs and IUVACs, and the concentrations of seven individual compounds (five SUVACs and two IUVACs) were analyzed, and several environmental variables of the sampling points were obtained: longitude, latitude, altitude, ozone column, and modelled UV levels.Diverse positive relationships of UVAC variables with UV radiation levels were identified in both the temporal and spatial scales. The soluble compound p-coumaroylmalic acid showed significantly higher concentrations in the years after the onset of stratospheric ozone degradation (around 1975) with respect to the previous years. The bulk level and the concentrations of three individual SUVACs were higher in the Spanish samples analyzed in the present study than in the northern European samples analyzed in a previous study in which IUVACs were not considered. The bulk level of IUVACs was the biological variable best spatially correlated with the UV exposure of the samples and with important factors determining it, such as altitude and latitude. Furthermore, the bulk level of IUVACs was the biological variable selected by a stepwise multiple linear regression analysis to reconstruct the past levels of UV-B radiation. The reconstructed UV-B did not show a clear trend in the period 1913–2006, which was coincident with other climatic and biological UV reconstructions. Both SUVACs and IUVACs were significant loading factors differentiating summer and autumn samples in the Principal Components Analysis conducted using both UVACs and the environmental variables, but again the bulk level of IUVACs was the main loading factor differentiating the samples collected from the highest and most UV-exposed localities (Sierra Nevada). In general, these findings suggest that UVACs were stimulated by increased UV levels in time or space, and that IUVACs would be better UV protectants than SUVACs, probably due to their more efficient screening capacity.Overall, UVACs of J. exsertifolia subsp. cordifolia were good UV biomarkers, and their location in the soluble or cell wall-bound fractions determined their usefulness in the different aspects of UV biomonitoring, including the assessment of the UV-B increase associated to anthropogenic ozone degradation.

Ultraviolet-absorbing compounds from the cell walls of an aquatic liverwort are more efficiently extracted by alkaline than by enzymatic digestion

The accumulation of ultraviolet-absorbing compounds (UVACs) within the cell walls of bryophytes provides a spatially uniform filter of ultraviolet-B (UV-B) radiation, and thus represents an important protective mechanism against its adverse effects. Here, the abilities of several cell-wall-degrading enzymes and of alkali (NaOH) in extracting cell-wall-bound UVACs from the aquatic liverwort Jungermannia exsertifolia Steph. subsp. cordifolia (Dumort.) Váňa were compared, in order to select the most appropriate extraction method and to achieve the most reliable assessment of the degree of protection afforded by UVACs against UV-B. We analysed both the overall level of UVACs and the concentrations of two hydroxycinnamic acids (p-coumaric acid and ferulic acid). The most effective extraction (statistically significant) of cell-wall-bound UVACs, in terms both of their overall levels and of the concentrations of individual compounds, was achieved using alkaline digestion, which was more efficient than any of the enzymatic digestions trialled. This may be attributable to the ability of the alkali to break simultaneously both hydrogen bonds and covalent bonds within the cell wall, including ester linkages between phenolics and carbohydrates. In addition, alkaline digestion was more rapid than enzymatic digestions, and was not excessively aggressive, because the molecular integrity of the individual compounds analysed was preserved. Thus, alkaline digestion can be recommended for the extraction of cell-wall-bound UVACs in J. exsertifolia subsp. cordifolia, whether these compounds are to be evaluated overall or individually.

Dynamic response of UV-absorbing compounds, quantum yield and the xanthophyll cycle to diel changes in UV-B and photosynthetic radiations in an aquatic liverwort

We studied the diel responses of the liverwort Jungermannia exsertifolia subsp. cordifolia to radiation changes under laboratory conditions. The samples were exposed to three radiation regimes: P (only PAR), PA (PAR + UV-A), and PAB (PAR + UV-A + UV-B). The day was divided in four periods: darkness, a first low-PAR period, the high-PAR plus UV period, and a second low-PAR period. After 15 days of culture, we measured photosynthetic pigments, chlorophyll fluorescence and UV-absorbing compounds in the four periods of the day on two consecutive days. With respect to UV-absorbing compounds, we analyzed their global amount (as the bulk UV absorbance of methanolic extracts) and the concentration of seven hydroxycinnamic acid derivatives, both in the soluble (mainly vacuolar) and insoluble (cell wall-bound) fractions of the plant extracts. PAB samples increased the bulk UV absorbance of the soluble and insoluble fractions, and the concentrations of p-coumaroylmalic acid in the soluble fraction and p-coumaric acid in the cell wall. Most of these variables showed significant diel changes and responded within a few hours to radiation changes (more strongly to UV-B), increasing at the end of the period of high-PAR plus UV. F v/ F m, Φ PSII, NPQ and the components of the xanthophyll cycle showed significant and quick diel changes in response to high PAR, UV-A and UV-B radiation, indicating dynamic photoinhibition and protection of PSII from excess radiation through the xanthophyll cycle. Thus, the liverwort showed a dynamic protection and acclimation capacity to the irradiance level and spectral characteristics of the radiation received.

Effects of temperature regime on primary producers in Icelandic geothermal streams

Eight adjacent sub-arctic streams with consistently different temperatures but broadly similar chemistry, other than some differences in conductivity and linked pH, were used to investigate effects of temperature regime on the structure of primary producer communities. Grazing by invertebrates was also taken into account to detect possible effects on the primary producers. The moss species Fontinalis antipyretica Hedw. (Bryophyta) was only observed in the warmer streams and its cover was positively linked with temperature. The liverwort Jungermannia exsertifolia (Steph.) was absent from the warmer streams and scarce in the colder streams. The biomass of F. antipyretica was inversely correlated with concentration of epilithic chlorophyll a, possibly due to shading from the bryophyte and/or competition between algae and bryophytes for nutrients. Epilithic algae and bryophyte biomass were not related to density of the main grazer, Radix peregra Müller (Gastropoda). A high biovolume of green algae (Chlorophyta) occurred in all streams. The biovolume Cyanobacteria was greatest in the coldest stream and in the two intermediately warm streams. Diatom biovolume was low compared with the biovolumes of Cyanobacteria and green algae in all streams, except in the two warmest streams, where the highest biovolume of diatom occurred. Regularized Canonical Correlation Analysis (RCCA) classification based on all primary producer assemblages and invertebrates indicated that warm streams (16.7–22.2 °C) were similar to each other. It also showed that the colder streams (6.6–13.2 °C) were different from the warmer streams and from each other because of differences in conductivity. Temperature and conductivity were the most influential variables in determining overall stream diversity.

Effects of enhanced UV-B radiation on hydroxycinnamic acid derivatives extracted from different cell compartments in the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia

We examined the responses of ultraviolet-absorbing compounds (UVAC) to enhanced UV-B radiation in the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia for 31 days under laboratory conditions. Samples were exposed to three radiation regimes: P (only photosynthetic radiation), PA (photosynthetic + UV-A radiation) and PAB (photosynthetic + UV-A + UV-B radiation). We measured both the bulk UV absorbance of the methanolic extracts and the levels of individual UVAC. In both cases, the methanol-soluble and the methanol-insoluble, alkali-extractable cell wall-bound fractions were analyzed. The bulk UV absorbance of the soluble fraction was higher than that of the cell wall-bound fraction. The bulk UV absorbances of both fractions increased under enhanced UV-B (PAB regime). Five different hydroxycinnamic acid (HCA) derivatives were found in the soluble fraction and two additional ones in the cell wall-bound fraction, among which only p-coumaroylmalic acid in the soluble fraction and p-coumaric acid in the cell wall-bound fraction increased under enhanced UV-B. The maximum quantum yield of PSII (F(v) /F(m)) decreased and DNA damage (amount of thymine dimers) strongly increased under enhanced UV-B, showing UV-B-induced damage. We conclude that methanol-soluble and cell wall-bound fractions of the liverwort studied have different UVAC, and each individual compound may respond in a different way to UV-B radiation. Thus, the analysis of individual UVAC in both the methanol-soluble and cell wall-bound fractions is advisable to better evaluate the protection mechanisms of liverworts against UV-B radiation. In particular, p-coumaric acid and p-coumaroylmalic acid seem to be especially UV-B responsive and merit further investigation.

Structure and Anti-TB Activity of Trachylobanes from the Liverwort Jungermannia exsertifolia ssp. cordifolia

In the critical search for new antituberculosis lead compounds, bryophytes represent a largely untapped resource of chemically diverse structures. From the liverwort Jungermannia exsertifolia subsp. cordifolia, 11 new trachylobane diterpene derivatives, as well as three known compounds, were isolated. Their structures were elucidated by spectroscopic means, and full (1)H NMR spin analysis of one model compound confirmed the relative configurational assignments of the congeners. Four of the isolates exhibited noticeable activity against virulent Mycobacterium tuberculosis H(37)Rv with minimal inhibitory concentrations of 61-24 microg/mL. This finding suggests that bryophytes in general and trachylobanes in particular deserve further attention in the search for new antimycobacterial leads.

Seasonal variations in UV‐absorbing compounds and physiological characteristics in the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia over a 3‐year period

Temporal physiological variations in relation to environmental factors, in particular to ultraviolet (UV) radiation, have been studied in bryophytes from circumpolar latitudes, but not in mid-latitudes with longer growing seasons. In addition, seasonal and interannual changes in individual UV-absorbing compounds (UVAC) have not been previously studied in bryophytes. To fill these gaps, samples of the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia were collected on a monthly basis during 3 years from a mountain stream in northern Spain. Sclerophylly index, chlorophyll fluorescence, DNA damage, the bulk UV absorbance of methanolic extracts and the concentration of five UVAC (hydroxycinnamic acid derivatives) were measured. Interannual changes were little marked, probably because the 3 years studied were environmentally similar. In summer-autumn, with respect to seasonal variations, newly grown tender young shoots with high F(v)/F(m) accumulated higher amounts of several hydroxycinnamic acid derivatives than in winter-spring. DNA damage was not detected in any of the samples analyzed. p-Coumaroylmalic acid was the compound best associated with radiation changes, and the best model explaining UV-B took into consideration the concentration of this compound and the ozone level. The specific effects of UV radiation could not be separated from the effects caused by other environmental factors, such as global radiation or temperature, because all these variables were correlated. However, indirect evidence strongly suggests that seasonal changes in bulk UV absorbance and p-coumaroylmalic acid are mainly driven by UV radiation. This compound may be a promising physiological variable to be used for UV bioindication.

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.

High-Level Phosphate Addition Does Not Modify UV Effects in Two Aquatic Bryophytes

The interaction between UV radiation and mineral availability has not been investigated in bryophytes, despite the important peculiarities of their mineral nutrition. The question under investigation was whether an improvement in phosphorus (P) availability, and the consequent increase in tissue P concentration, modified the responses to enhanced UV radiation in two aquatic bryophytes (the liverwort Jungermannia exsertifolia subsp. cordifolia and the moss Fontinalis antipyretica) under laboratory conditions. These responses were evaluated in terms of photosynthetic pigment composition, chlorophyll fluorescence, photosynthesis and respiration rates and the accumulation of protecting UV-absorbing compounds (both the commonly used bulk UV-absorbance of methanol extracts in both species and the concentrations of five hydroxycinnamic acid derivatives in the liverwort). The culture period (36 days) affected significantly most variables of both species. However, no variable in either species showed significant differences between the P-supplemented and non-supplemented samples at the end of the experiment, except the vitality index OD430/OD410 in the liverwort. This was probably due to the fact that the bryophytes studied have low nutritional requirements, and thus increasing tissue P may have only increased luxury consumption, without any modification in the responses to enhanced UV radiation.

Hydroxycinnamic acid derivatives in an aquatic liverwort as possible bioindicators of enhanced UV radiation

We examined, under laboratory conditions, the physiological responses of the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia to artificially enhanced ultraviolet (UV) radiation for 82 days, especially considering the responses of five hydroxycinnamic acid derivatives. This species lives in mountain streams, where it is exposed to low temperatures and high UV levels, and this combination is believed to increase the adverse effects of UV. Enhanced UV radiation hardly caused any change in several physiological variables indicative of vitality, such as F v/ F m and chlorophylls/phaeopigments ratio (OD430/OD410). Thus, this liverwort seemed to be tolerant to UV radiation, probably due to the accumulation of three UV-absorbing hydroxycinnamic acid derivatives: p-coumaroylmalic acid, 5″-(7″,8″-dihydroxycoumaroyl)-2-caffeoylmalic acid, and 5″-(7″,8″-dihydroxy-7- O-β-glucosyl-coumaroyl)-2-caffeoylmalic acid. These compounds might serve as bioindicators of enhanced UV radiation.

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.

Physiological changes and UV protection in the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia along an altitudinal gradient of UV-B radiation.

Here we report the effects of a natural altitudinal gradient of UV-B radiation, from 1140 to 1816 m altitude, on the physiology of the aquatic liverwort Jungermannia exsertifolia Steph. subsp. cordifolia (Dumort.) Váña collected in mountain streams. Photosynthetic pigments, net photosynthesis and dark respiration rates, chlorophyll fluorescence, protein concentration, sclerophylly, and UV-absorbing compounds [both global UV absorbance of methanol-extractable UV-absorbing compounds (MEUVAC) and concentrations of five individual compounds] were measured. Two new caffeic acid derivatives were discovered: 5″-(7″,8″-dihydroxycoumaroyl)-2-caffeoylmalic acid and 5″-(7″,8″-dihydroxy-7-O-β-glucosyl-coumaroyl)-2-caffeoylmalic acid, whereas three additional compounds were already known in other species: p-coumaroylmalic acid, phaselic acid (both compounds in their cis- and trans- forms) and feruloylmalic acid. Most physiological variables changed considerably along the altitudinal gradient, but only six showed significant linear relationships with altitude: MEUVAC levels, the concentrations of the two new secondary compounds, the maximal apparent electron transport rate through PSII (ETRmax) and the maximal non-photochemical quenching (NPQmax) increased with altitude, whereas photoinhibition percentage decreased. A principal components analysis (PCA) was conducted to rank the values of the physiological and ecological variables obtained along the altitudinal transect, showing that those variables correlated with altitude were responsible for the ordination of the sampling points. The liverwort was not adversely affected by the changing conditions along the altitudinal gradient and, in particular, by the increasing UV-B irradiance, probably because the characteristics shown by high-altitude populations may confer tolerance to high UV-B levels. The response to UV-B of the two new compounds suggests that they could be used as indicators of the spatial changes in UV-B radiation.

Assessing the UV-B Tolerance of Sun and Shade Samples of Two Aquatic Bryophytes Using Short-term Tests

Abstract Sun and shade samples of two aquatic bryophytes, the moss Fontinalis antipyretica and the liverwort Jungermannia exsertifolia subsp. cordifolia, were collected from a mountain stream and cultivated in the laboratory under two artificially imposed radiation regimes: control (only photosynthetically active radiation, PAR) and UV-B (PAR + UV-B). Samples were cultured at 2°C for 78 hr under continuous radiation to determine whether the physiological responses of the two bryophytes to UV-B radiation depended on their previous field acclimation to sun or shade conditions. We also aimed to study whether the short-term effects of UV-B were similar to those caused by longer exposure. Fontinalis antipyretica was more sensitive to UV-B treatment than Jungermannia exsertifolia subsp. cordifolia, showing significant decreases in several physiological variables indicative of vitality: PN rates, OD430/OD410, OD665/OD665a and especially Fv/ Fm. This higher sensitivity occurred in both sun and shade samples of th...

Different physiological responses of two aquatic bryophytes to enhanced ultraviolet-B radiation

Two aquatic bryophytes, the moss Fontinalis antipyretica Hedw. and the foliose liverwort Jungermannia exsertifolia Steph. subsp. cordifolia (Dumort.) Váña were analysed with regard to their ultraviolet-B (UV-B) tolerance. The plants were cultivated in the laboratory for 36 days under three different radiation regimes which were set up using appropriate lamps and filters: P (PAR alone, control), PA (PAR + UV-A) and PAB (PAR + UV-A + UV-B). The two bryophytes responded differently to the enhancement in UV-B radiation, while UV-A radiation had only a slight biological effect. The samples of the moss which were irradiated with UV-B showed, with respect to the control, decreases in the chlorophyll and carotenoids concentration, the chlorophyll a/b quotient, the chlorophylls/phaeopigments ratios, the net photosynthesis rates, the light saturation point, the maximum quantum yield of photosystem II (Fv/Fm) and the apparent electron transport rate (ETR). They also showed increases in the sclerophylly index and the dark respiration rates. The majority of these changes are indicative of plant stress and some of them had been found previously in bryophytes exposed to enhanced UV-B radiation. However, the UV-B-irradiated samples of the liverwort only showed a decrease in Fv/Fm, which might be the most sensitive physiological variable to UV-B, together with an increase in the concentration of UV-absorbing compounds. This defence mechanism, rarely described in bryophytes, would enable the liverwort to have a higher tolerance than the moss against UV-B radiation, at least under the specific experimental conditions used in this study. Also, the increment of UV-absorbing compounds in the liverwort, which seems to be a specific response to the enhancement in UV-B radiation, might be a useful ecophysiological tool in the bioindication of this phenomenon; long-term field research is needed to confirm this usefulness. The different UV-B tolerances of the species studied may be related, to a certain extent, with their different distribution and ecology. Given that UV-B tolerance depends on the species, bryophytes should not be grouped as a single functional type in the prediction of the consequences of a hypothetic enhancement in UV-B radiation.

Short-Term Physiological Responses of the Aquatic Liverwort Jungermannia exsertifolia subsp. cordifolia to KH2PO4and Anoxia

The effects of four increasing levels of KH2PO4 on the physiology of the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia were analyzed in the laboratory in the short term (15 d). The accumulation of P and K in the liverwort tissues was influenced (ANOVA) by the level of KH2PO4-enrichment and was significantly higher in the more enriched culture solutions. However, only the concentration of P was influenced by the effect of time (ANOVA), and the gradual P accumulation throughout the culture period contrasted with the fluctuations observed in K accumulation; these were presumably due to the higher liability of K to be leaked from the cells. Our results suggest that the analysis of P in transplants of J. cordifolia may be a useful bioindicator of short term water eutrophication both in the spatial and the temporal scales, although the highest PO43− concentration used in this study (20 mg liter−1) may induce a P saturation in J. cordifolia tissue (0.53% DM). The rates of net photosynthesis showed a significant quadratic regression with the tissue P concentration, which might resemble the action curve of mineral nutrients. Using this regression as an indicator, there was no clear deficiency zone, probably due to the relatively high tissue P concentration initially found in the liverwort. The lack of stimulation of net photosynthesis with increasing tissue P could be due either to a deficiency in other mineral elements such as N, or to an intrinsic inability to use the excess of nutrients. The decline in net photosynthesis when the tissue P concentration exceeded 0.45% DM could be interpreted as a toxicity process. Chlorophyll concentration was not affected by P enrichment, but the decline in the chlorophyll a/b ratio and in the proportions of chlorophylls to phaeopigments, together with the increase in the proportion of carotenoids to chlorophylls, suggested also P toxicity. This phenomenon needs further investigation to be confirmed in other species and conditions, but it may help to explain the disappearance of certain aquatic bryophytes in eutrophicated water courses. The physiological effects of increasing tissue K concentration (decreases in the rate of dark respiration and in the chlorophyll a/b ratio) were slighter than those of P, probably because the accumulation of K was lower than that of P (1.44 and 1.96 times the initial value, respectively). In a different experiment in which J. cordifolia was cultured in P-enriched aerated and non-aerated solutions, anoxia caused a strongly diminished P accumulation in the first three days, probably because the mitochondrial respiration was blocked. Then, a clear net loss of P from the liverwort tissues was observed, maybe caused by membrane damage.

Ent-kaurane-type diterpenoids from the liverwort Jungermannia exsertifolia ssp. cordifolia

A new bis- ent-kaurane-type, exsertifolin A, two new 6,7-seco- ent-kaurane-type, secoexsertifolins A and B, and seven new ent-kaurane-type diterpenoids, exsertifolins B, C, D, E, F, G, and H, were isolated from the French liverwort Jungermannia exsertifolia Steph. ssp. cordifolia (Dum.) Va'ňa, together with the previously known seven ent-kaurane-type diterpenoids. These structures were determined by means of extensive NMR techniques, chemical degradation and X-ray crystallographic analysis.

3α,18-Dihydroxytrachyloban-19-oic acid from the liverwort Jungermannia exsertifolia subsp. cordifolia

Extraction of the liverwort Jungermannia exsertifolia subsp. cordifolia afforded 3α,18-dihydroxytrachyloban-19-oic acid, isolated as its diacetate methyl ester.