Rafael Tomás

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.

Influence of Temperature on the Effects of Artificially Enhanced UV-B Radiation on Aquatic Bryophytes Under Laboratory Conditions

We examined, under laboratory conditions, the influence of temperature (2 °C vs. 10 °C) on the physiological responses of two aquatic bryophytes from a mountain stream to artificially enhanced UV-B radiation for 82 d. These organisms may be exposed naturally to relatively low temperatures and high levels of UV-B radiation, and this combination is believed to increase the adverse effects of UV-B radiation. In the moss Fontinalis antipyretica, UV-B-treated samples showed severe physiological damages, including significant decreases in chlorophyll (Chl) and carotenoid (Car) contents, Chl a/b and Chl/phaeopigment ratios, Chl a fluorescence parameters Fv/Fm and ΦPS2, electron transport rate (ETRmax), and growth. In the liverwort Jungermannia cordifolia, UV-B radiation hardly caused any physiological change except for growth reduction. Thus, this liverwort seemed to be more tolerant to UV-B radiation than the moss under the specific experimental conditions used, maybe partly due to the accumulation of UV-B absorbing compounds. The influence of temperature on the effects of UV-B radiation depended on the species: the higher the UV-B tolerance, the lower the influence of temperature. Also, different physiological variables showed varied responses to this influence. Particularly, the lower temperature used in our study enhanced the adverse effects of UV-B radiation on important physiological variables such as Fv/Fm, growth, and Chl/phaeopigment ratios in the UV-B-sensitive F. antipyretica, but not in the more UV-B-tolerant J. cordifolia. Thus, the adverse effects of cold and UV-B radiation were apparently additive in the moss, but this additiveness was lacking in the liverwort. The Principal Components Analyses (PCA) conducted for both species with the physiological data obtained after 36 and 82 d of culture confirmed the above results. Under natural conditions, the relatively high water temperatures in summer might facilitate the acclimation of aquatic bryophytes from mountain streams to high levels of UV-B radiation. This may be relevant to predict the consequences of concomitant global warming and increasing UV-B 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.

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.

A Survey of the Distribution of UV-Absorbing Compounds in Aquatic Bryophytes from a Mountain Stream

Abstract Bryophytes are important primary producers in mountain streams and they should be protected against the adverse effects of the high levels of UV-B radiation typical of these environments. The accumulation of UV-absorbing compounds could be one of the mechanisms of protection. A survey of methanol-extractable UV-absorbing compounds (MEUVAC) was conducted for 14 aquatic bryophytes, 10 mosses, and four liverworts, from a mountain stream located at 2,000 m elevation. For four species, several populations were investigated. Absorbance spectra between 250 and 400 nm were obtained and the amount of MEUVAC was calculated per unit of both dry mass (DM) and surface area. Levels of MEUVAC and the absorbance spectra of UV radiation were significantly affected by species. The high levels of MEUVAC and the clearly hump-shaped spectra in the UV-B and UV-A wavelengths (280–400 nm) that were found in the liverworts contrasted with the low levels and non hump-shaped spectra generally found in the mosses (except for Polytrichum commune). Sclerophylly and intraspecific variability only had a modest influence on the results. It may be concluded that the accumulation of MEUVAC might represent a protecting mechanism against UV-B radiation in the four liverworts studied, but rarely in the mosses of this stream.

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 the moss. These results reproduce the differences between both species that were found in previous more prolonged (36– 82 days) experiments. These types of short-term tests may therefore be used instead of long-duration tests to evaluate the UV-B tolerance of bryophytes. Shade samples were more sensitive to UV-B treatment than sun samples, but only in the more UV-B-sensitive species (Fontinalis antipyretica). Fv/Fm was the physiological variable that better discriminated both types of samples in the moss, since it decreased 42% in the shade samples and only 27% in the sun samples at the end of the culture period. In Jungermannia exsertifolia subsp. cordifolia, controls and UV-B-treated samples were not significantly different in either the sun or the shade samples. Principal Components Analyses (PCA) for each species, ranking the physiological results along the culture period for each radiation regime and field exposure strongly supported these conclusions and also showed that the loading factors of PCAs may be helpful for establishing the combination of key variables responsible for the differences between controls and UV-B-treated samples, or between UV-B-treated sun and shade samples.

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.

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.

Cyclic environmental factors only partially explain the seasonal variability of photoprotection and physiology in two mosses from an unforested headwater stream

Abstract We studied the relationships between environmental variables and the physiology of two (semi-)aquatic mosses (Bryum pseudotriquetrum and Fontinalis antipyretica) in an unforested headwater stream over a three year period. Neither environmental or physiological variables showed significant interannual variations. Most environmental variables (water temperature, stratospheric ozone, and photosynthetic, UV-A and UV-B radiation) showed distinct seasonal variations, but only a few physiological ones did. In both species, photoprotection variables (the activity of the xanthophyll cycle and the bulk UV absorbance of the methanol-extractable UV-absorbing compounds, MEUVAC) varied more seasonally than variables related to physiological activity, such as the sclerophylly index and chlorophyll fluorescence parameters (Fv/Fm and ΦPSII). Changes in physiological activity would be attenuated by the buffering capacity of water with respect to the influence of environmental factors, and dynamic variables like Fv/Fm and ΦPSII would be little determined by cyclic environmental factors. In B. pseudotriquetrum, both MEUVAC and kaempferol 3,7-O-diglycoside (a potentially UV-protective flavonoid) were positively associated with radiation levels, whereas in F. antipyretica photoprotection mechanisms were not correlated with any environmental variable. In addition, MEUVAC was 3–4 fold higher in B. pseudotriquetrum than in F. antipyretica. Thus, different photoprotection mechanisms, with a different environmental regulation, can be suggested for these two species. DNA damage was not found in any sample, probably because both species displayed efficient DNA repair mechanisms.

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

Abstract 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.