CZECH POLAR REPORTS 4 (2): 149-157, 2014 Comparative analysis of heterogeneity of primary photosynthe-tic processes within fruticose lichen thalii: Preliminary study of interspecific differences Short Communication Stefano Conti1, Jana Hazdrová2, Josef Hájek2, Petra Očenášová2, Miloš Barták2, Kateřina Skácelová2, Paola Adamo1 1 University of Naples Federico II, Department of Agricultural Sciences, Via Universita 100, 80055 Portici (NA), Italy 2Department of Plant Physiology and Anatomy, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic Abstract Two species of fruticose lichens from different habitats and of distinct color, Usnea antarctica and Stereocaulon vesuvianum, were compared using chlorophyll fluorescence imaging in order to study the distribution of primary photosynthetic processes within the thalli. The thallus of U. antarctica is yellow with black tips: in this species chlorophyll containing cells were mostly located in the middle region of the thallus and the highest PS II efficiency was detected in the middle to basal region, as shown by the FV/FM and €>psn values. No chlorophyll fluorescence was detected in the apical part of the thallus, indicating that little or no photosynthesis takes place in these tissues. The lichen S. vesuvianum is homogeneously pale grayish green and chlorophyll containing cells are distributed along the thallus with maximum concentration in the middle region. In S. vesuvianum, the highest PS II efficiency was detected in the apical to middle region of the thallus, while the basal portion was found to have the lowest efficiency of primary photochemical reactions. Quenching analysis data confirmed the uneven patterns of primary photosynthetic processes within the thalli of these fruticose lichens. Key words: chlorophyll fluorescence, Kautsky kinetics, quenching analysis, Usnea antarctica, Stereocaulon vesuvianum DOI: 10.5817/CPR2014-2-15 Received December 1, 2014, accepted December 31, 2014. Corresponding author: Miloš Barták Acknowledgements: The authors thank the CzechPolar project for research infrastructure and the PASSEB project for funding traveling to co-ordination meetings of team members. 149 S. CONTI et al. Introduction In experimental plant biology, the technique of chlorophyll fluorescence imaging is used for the evaluation of plant primary photosynthetic processes and it allows the researcher to study their distribution within the photosynthesizing organs. This technique has a wide range of applications (Govindjee et Nedbal 2000) and it has been used to study the peculiarities of anatomical structures in lichens, such as e.g. fruiting bodies (Jensen et al. 1999), or the sensitivity of different parts of the thallus to photoinhibition (e.g. Singh et al. 2013), as well as the effects of low or freezing temperature on photosynthesis (Barták et al. 2007). Visualization of residual photosynthetic activity in dehydrated lichen thalii is another typical application (see e.g. Barták et al. 2000, 2005). It is well established that the intrathalline heterogeneity of primary photosynthetic processes, as defined by chlorophyll fluorescence parameters, is dependent on the particular growth pattern of each species, as well as on the age, physiological activity and distribution of symbiotic algae within the thallus. Several studies have been published on the heterogeneity of chlorophyll fluorescence in foliose lichens (e.g. Barták et al. 2000), whose planar thalii are relatively easy to handle but little research has investigated the spatial heterogeneity within the thallus of fruticose lichens. A recent study (Balarinová et al. 2014) reported interspecific differences in distribution of photosynthetic activity within the thalii of two Antarctic species of Usnea, as well as different sensitivities to photoinhibition. In this study, primary photosynthetic processes were found to be more efficient in the basal and middle part of branching thalii (yellow colored) while very low values were detected in the apical (dark colored) parts. Interestingly, unlike most of the other fruticose lichens, Usnea sp. lichens are characterized by contrastingly colored apical and basal parts of the thallus. Therefore, it is also possible that different patterns of primary photosynthetic processes may be observed when comparing Usnea to other fruticose lichen species. In order to test this hypothesis, comparative measurements of chlorophyll fluorescence parameters were done in this study. Two fruticose lichen species were selected for such a purpose: Usnea antarc-tica and Stereocaulon vesuvianum. These two species differ in thallus color: while the thallus of U. antarctica is yellow with black tips, S. vesuvianum is uniformly pale grayish green. The different colors of these two species are attributed to secondary metabolites located mainly in the upper cortex of the lichen: usnic acid in the case of U. antarctica or atranorin, norstictic acid, stictic acid in S. Vesuvianum (e.g. Walker et Lintott 1997). In the latter species several secondary metabolites were found (e.g. Caccamese et al. 1986), some of them with antimicrobial activity (Ingólfsdóttir et al. 1985, Kim et al. 2013). Usnea antarctica is widely distributed over coastal ecosystems in maritime Antarctica (see e.g. Lamb 1964, Osyczka et Olech 2005) and several photosynthetic studies based on chlorophyll fluorescence parameters in this species have already been published. Potential quantum yield (FV/FM) and effective quantum yield (0PSn) of photosystem II photochemistry, are sensitive indicators of efficiency of the photosynthetic apparatus. As such, these parameters are readily affected as a result of excess (Barták et al. 2004, Barták et al. 2012) or medium physiological light doses (Balarinová et al. 2014) or by freezing temperatures (Barták 2014). Contrastingly, only a few reports on the physiology of Stereocaulon vesuvianum have been published to date. These studies have been devoted to the photobiont classification using molecular biology approaches (Anuforo 2012), to the chemical weathering of mineral substrates due to the 150 CHLOROPHYLL FLUORESCENCE IMAGING excretion of lichen metabolites (Adamo et al. 1997, Adamo et Violante 2000, Vingi-ani et al. 2013) or to radionuclide accumulation in the thallus (Adamo et al. 2004). Some particular ecophysiological aspects of species of the Stereocaulon genus have been studied such as e.g. tolerance to long-term submersion in melt water ponds Material and Methods (Sadowsky et al. 2012). Peksa et Skaloud (2011) reported a specific association with algae of the genus Asterochloris (reclassified Trebouxia) as the primary photo-biont, while a secondary stigonemoid photobiont was reported in cephalodia by Boekhout (1982). Lichen species For a comparative study of the intrathalline distribution of primary photosynthetic processes, two lichens species with fruticose morphology of the thallus were chosen. Usnea antarctica is a typical representative of fruticose lichens in coastal regions of Antarctica with characteristic black hairy tips of individual thalii. This lichen grows on the upper parts of stones and rocky outgrowths forming a shrub-like structure. Stereocaulon vesuvianum has a widespread distribution at all latitudes on Earth. The thallus is pale grey with tufts of ascending to erect branches (pseudopodetia) and numerous globose clusters of fruiting bodies (soredia) are formed along the axis and at the tips of the pseudopodetia. Collection of samples In February 2013, lichen thalii of Usnea antarctica were collected at the James Ross Island (Antarctica) at several sites in Halozetes Valley (63°48'57" S, 57°50'30" W, 260 m a.s.l.). The collection sites were distributed within a 300 x 100 m area located at hyaloclastite breccia boulders forming an upper layer of ice-cored moraine. Thalii of Stereocaulon vesuvianum (Pers) were collected in November 2014 from a species-rich site on lavas of Mount Vesuvio, 12 km south-east of Naples (southern Italy). The samples reported in this study were collected from a monospecific lichen cover growing on recent tefritic leucitic lava flows (1944 eruption) in the Valley of the Giant (40°49'59" N 14°25'45" E, 885 m a.s.l.). After collection, lichen thalii were air dried under local climatic conditions, they were subsequently transferred to the laboratory and stored at 5°C in the dark. Chlorophyll fluorescence imaging Prior to the chlorophyll fluorescence measurements, dry thalii were allowed to rehydrate for 48 h over wet filter paper in Petri dishes under dim light at 5°C. After rehydration and full activation of primary photochemical processes (maximum FV/FM - data not shown), the lichen samples were subjected to the measurements of chlorophyll fluorescence imaging so that heterogeneity of chlorophyll fluorescence parameters could be determined. Lichen thalii were pre-darkened for 10 min in the measuring compartment of a FluorCam HFC-010 fluorometer (Photon Systems Instruments, Czech Republic). Then Kautsky kinetics supplemented with quenching analysis were measured (Fig. 1). The method is described elsewhere (e.g. Barták et al. 2004) and consists of measurements of chlorophyll fluorescence curves induced by an actinic light supplemented with saturation pulses during the period when the light is on (FM') or off (FM"X respectively. Using a FluorCam 7 software, effective quantum yield (0PSn) and non-photochemical 151 S. CONTI et al. quenching (NPQ) were calculated and visualized. Subareas of high and low photosynthetic activity were then identified within the thallus projection area and their particular shares were calculated. Statistical analysis For each species, at least 16 replicate measurements of chlorophyll fluorescence parameters were recorded and means and standard deviations were calculated. The Student's t-test was used to determine the significance of the differences between FV/FM or 0PSn values in the apical and basal regions of the thalli. Results and Discussion The Kautsky kinetics differed between the two lichen species, both in the absolute values of chlorophyll fluorescence and in the curve shapes (Fig. 1). The higher values observed in S. vesuvianum may be due to its higher chlorophyll content and/or to lower content of light absorbing compounds in the upper cortex of the thallus. Unlike U. antarctica, in S. vesuvianum an apparent secondary peak of chlorophyll fluorescence, higher than FP, was recorded during actinic light period (Fig. 1, 70 s). In S. vesuvianum this phenomenon could be attributed either to less effective reoxida-tion of plastochinone pool or to contribution of F0 (during actinic light) to the overall chlorophyll fluorescence signal (Roháček et Barták 1999) in S. vesuvianum. Further study will, therefore, be focused on F0 changes in this species during actinic light period. -10 40 90 140 1§0 240 290 340 390 440 -10 40 90 140 190 240 290 340 390 440 Fig. 1. Slow Kautsky kinetics of chlorophyll fluorescence supplemented with maximum values reached after saturation pulses. The curves are means of 12 replicates, normalized to background chlorophyll fluorescence (F0). The following chlorophyll fluorescence parameters are indicated: FM - maximum fluorescence on dark adapted thalli, FM' - maximum fluorescence on light-adapted thalli, FM" - maximum fluorescence measured in dark when actinic light is off, F0, F0' - background chlorophyll fluorescence, FP - peak fluorescence during actinic light period, Fs - steady state fluorescence. 152 CHLOROPHYLL FLUORESCENCE IMAGING Fig. 2. Intrathalline distribution of chlorophyll fluorescence parameters in Usnea antarctica. Basic chlorophyll fluorescence (F0, left panel) showed that chlorophyll containing cells were mostly located in the middle part of the thalli. Potential (FV/FM, central panel) and effective (psii, right panel) efficiency of PS II were found to have the same values in the middle and in the basal parts of thalli. No chlorophyll fluorescence was detected in the apical parts of thalli, thus resulting in the absence of imaging false colors. Fig. 3. Intrathalline distribution of chlorophyll fluorescence parameters in Stereocaulon vesuvianum. Basic chlorophyll fluorescence (F0, left panel) indicates that the highest concentration of chlorophyll containing cells was observed in the middle part of the thalli, while a lower signal could be measured in the apical and basal portions. Potential (FV/FM, central panel) and effective (•J^snX right panel) yield of PS II were high in the apical and middle portions of thalli and they were found to decrease in the basal parts. Chlorophyll fluorescence imaging showed species-specific intrathalline distribution of primary photochemical processes. In U. antarctica {see Fig. 2), the middle and basal parts of the thallus were found to be the most photosynthetically effective (0PSn within a range of 0.405-0.375). This result was in accordance with earlier studies (Ocenäsovä et al. 2014, Balarinovä et al. 2014), which also found the highest photo- synthetic activity to be located within the basal portion of the thallus. As expected, little or no fluorescence could be detected in the dark colored apical portion of thalli. This could be due to the abundance of pigments which interfered with the measurements and/or to the small number of the symbiotic algal cells in these tissues. The basal part of the thallus exhibited a lower Fv/Fm (0.375) value compared with the 153 S. CONTI et al. middle part (0.522) (Fig. 4). Fluorescence signals were found to be stronger in the middle and basal region of the thallus, resulting in higher FV/FM and 0PSn values. This was possibly due to the abundance of algal cells in the subcortical photobiont layer, whose thickness was reported to be in the order of as much as 101 to 102 micrometers (Gielwanowska et Olech 2012, Barták 2014). In S. vesuvianum (Fig. 3), the apical to middle regions of the thallus were found to have the highest maximum theoretical efficiency of PS II, with a measured FV/FM value of 0.616 (Fig. 4). The maximum efficiency was recorded in the basal region of the thallus, with a mean FV/FM value of 0.500. The overall efficiency of PS II followed the same pattern, with a measured Opsn value of 0.498 in the apical thalii regions and a lower efficiency in the basal parts of the thalii, with a mean 0PSn value of 0.344. These data closely matched the chlorophyll content imaging results (Fig. 3) if the intrathalline distribution of basal chlorophyll fluorescence (F0) and steady-state chlorophyll fluorescence were considered (data not shown here). Thus, most of the chlorophyll content could be expected to be found in the middle region of S. vesuvianum thalli. Since information on S. vesuvianum cross sections are missing in literature, further research on thallus anatomy and on the intrathalline distribution of symbiotic algae is needed, in order to interpret the chlorophyll fluorescence data in the light of the anatomical structure of this lichen. On the basis of these data, it could also be possible to understand why lower values of FV/FM and Opsn were recorded in the basal part compared to the middle and apical region of the thallus. PSII Fig. 4. Potential (FV/FM) and effective (OpSn) quantum yield of photochemical processes in the apical (white bars) and basal regions (black bars) of Stereocaulon vesuvianum thalli. In the case of Usnea antarctica, data refer to middle (white bars) and basal region of thalli (black bars), instead. Means were calculated from at least 16 replicate measurements, error bars represent standard deviations. 154 CHLOROPHYLL FLUORESCENCE IMAGING Results of quenching analysis are summarized in Table 1. In U. antarctica, photochemical quenching (qP) and photochemical quenching recorded in the dark (qP-d) did not differ between middle and basal portions of the thalli, indicating that primary photochemical processes were similar between these tissues. Such conclusion might be supported by the 0PSn values that did not differ either (Fig. 4). Both non-photochemical quenching (NPQ) and non-photochemical quenching recorded in dark (NPQ-d) when actinic light was switched off, were higher in the basal than in the middle region of the thalli, although these differences did not appear to be statistically significant due to high data variability. In S. vesuvianum NPQ did not differ significantly between the apical and the basal region of the thallus. These data suggest that an effective quenching system is present in both regions of the thallus, which may protect the photosynthetic apparatus from adverse environmental condition, as previously reported e.g. by Nayaka et Saxena (2014) in the case of light stress, or by Singh et al. (2013) in the case of water stress. Photochemical quenching (qP), however, was higher in the apical region, indicating that photosynthetic performance in these tissue may be higher than at the base the thalli, confirming the FV/FM and Opsn data. The NPQ-d parameter was found to be significantly higher in the basal compared with the apical part of the thalli. Similarly to qP, the qP-d parameter was found to be significantly higher in the apical part of S. vesuvianum thalli. NPQ qP NPQ-d qP-d U. antarctica middle 0.228±0.060 0.856±0.058 0.257±0.061 0.971±0.040 basal 0.452±0.203 0.825±0.049 0.432±0.171 0.952±0.038 S. vesuvianum apical 0.150±0.044 0.855±0.069 0.226±0.064 0.498±0.029 basal 0.132±0.042 0.723±0.061 0.500±0.065 0.344±0.063 Table 1. Quenching parameters derived from chlorophyll fluorescence analysis for Stereocaulon vesuvianum and Usnea antarctica. The differences between apical/middle and basal parts of the thalli. Means and standard deviations were calculated from at least 16 replicates. Concluding remarks In this preliminary study, chlorophyll fluorescence imaging and quenching analysis data confirmed that the efficiency of primary photosynthetic processes was unevenly distributed within the thallus of each lichen species. Moreover, this study provided evidence that the patterns of distributions of primary photosynthetic proc- esses within the thallus may differ among species. 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