Biologia 65/4: 587—594, 2010 Section Botany DOI: 10.2478/s11756-010-0058-y Chromatic adaptation in lichen phyco- and photobionts Bazyli Czeczuga,EwaCzeczuga-Semeniuk & Adrianna Semeniuk Department of General Biology, Medical University, Kili´nskiego 1,PL-15-089 Bialystok, Poland; e-mail: [email protected] Abstract: The effect of light quality on the photosynthetic pigments as chromatic adaptation in 8 species of lichens were examined. The chlorophylls, carotenoids in 5 species with green algae as phycobionts (Cladonia mitis, Hypogymnia physodes, H. tubulosa var. tubulosa and subtilis, Flavoparmelia caperata, Xanthoria parietina) and the chlorophyll a, carotenoids and phycobiliprotein pigments in 3 species with cyanobacteria as photobionts (Peltigera canina, P. polydactyla, P. rufescens) were determined. The total content of photosynthetic pigments was calculated according to the formule and particular pigments were determined by means CC, TLC, HPLC and IEC chromatography. The total content of the photosynthetic pigments (chlorophylls, carotenoids) in the thalli was highest in red light (genus Peltigera), yellow light (Xanthoria parietina), green light (Cladonia mitis) and at blue light (Flavoparmelia caperata and both species of Hypogymnia). The biggest content of the biliprotein pigments at red and blue lights was observed. The concentration of C-phycocyanin increased at red light, whereas C-phycoerythrin at green light. In Trebouxia phycobiont of Hypogymnia and Nostoc photobiont of Peltigera species the presence of the phytochromes was observed. Key words: carotenoids; chlorophylls; chromatic adaptation; lichens; photobionts; phycobiliproteins; phycobionts; phy- tochromes. Introduction Taking the above into account, we decided to in- vestigate the chromatic adaptation of common lichen Lichens can be found all over our planet, inhabiting species in the Knyszy´nska Forest to various light con- diverse biotopes and tolerating even extreme environ- ditions during the vegetative period. mental conditions (Kershaw 1985). A number of adap- tive mechanisms allow them to exist in these condi- tions. As autotrophic organisms they need solar energy Material and methods used by phyco- and photobionts in the process of photo- synthesis. In different ecological niches, intensity of the Material light factor may vary depending on the season of the The study was conducted on 10 species of lichens (Table 5), year or the time of day (Czeczuga et al. 2006a,b, 2007), including three belonging to cyanolichens. The specimens not mentioning the differences observed between open were collected from ecological niches varying in the type of and shadowed areas of a respective ecosystem (Smith substratum, shade degree and substratum humidity, which included ground flora of the coniferous forest (spruce), leafy 1981). Moreover, the type of shadowing itself differenti- forest (hombeam), tree bark (epiphytes on poplar) and for- ates the spectral composition of the visible light in the est scarp by the road. shadow (Czeczuga et al. 2007). The spectral composi- Pieces of soil(5 × 10 cm) or branch covered with the tion of light is found to vary according to the shadow particular lichens were collected in sommer (August) from formed by various tree species in the vegetative period. the Knyszy´nska Forest, and glued to small pieces of card- The influence of the air pollution on content of board which were similar to that operating on the lichens in chlorophylls have been examined by Arb et al. (1990) seasonal under field conditions. The experimental beakers and on chlorophylls and carotenoids by Czeczuga & were stored in boxes equipped with appropriate glass fil- Krukowska (2001), and influence of high temperature ters (Czeczuga 1986b), manufactured by the FPN-Bytom Pisani et al. (2007). Whereas, the influence of the heavy Works, wave-lengths being indicated by the producers. Four λ λ metals on the content of chlorophylls in lichen species basic colours were used: red ( = 700 nm), yellow ( = 590 nm), green (λ = 500 nm), and blue (λ = 450 nm). A culture have been determined by Chettri et al. (1998), Bačkor & of lichens grown in a box provided with normal, “colourless” Zetikova (2003) and on carotenoids Bačkor et al. (2003). (white), glass served for the control. The boxes were placed The light intensity investigated Czeczuga et al. (2004b) on a table situated 1 metre from a window. In addition, the and Bačkor et al. (2006), and osmotic stress Vaczi & boxes containing the lichens were exposed to light of 2.7 Bartak (2006). Wm−2 from a glow-tube lamp for 12 hours. After passing c 2010 Institute of Botany, Slovak Academy of Sciences 588 B. Czeczuga et al. through the different filters, light intensities were: colour- RF-535 fluorescence detector. CC, TLC, and HPLC are de- less light – 95.5% (6.3 W m−2), red – 52.6% (4.1 W m−2), scribed in detail in Czeczuga et al. (2006b). yellow – 21.1% (1.4 W m−2), green – 13.2% (0.9 W m−2), Carotenoids were identified by comparison with stan- −2 and blue – 7.9% (0.5 W m ) of the all light in front of dards from: a) the behavior in CC; b) their UV-VIS spec- every filter. From time to time each lichens was moistened tra; c) their partition between n-hexane and 95% ethanol; with a sprayer. After 4 weeks, the chlorophylls, particular d) their Rf -values in TLC; e) the presence of the allylic OH carotenoids and biliproteins were measured. group determined by the acid CHCl3 test; f) the epoxide test; g) the mass spectrum. Analysis of lichen pigments Carotenoid pigment standards were purchased from the Hoffman-La Roche Co., Switzerland; the International The total amount of chlorophylls and carotenoids was calcu- 14 lated from the following formulae (E standing for extinction Agency for C Determinations, Denmark, and Sigma at the given wavelengts) (Czeczuga et al. 2006b): Chemical Co., USA. Chlorophyll a = 11.63 × E665 –2.39× E649 The structure of particular carotenoids was described by Straub (1987) and Czeczuga (1988). Chlorophyll b = 20.11 × E649 –5.18× E665 Total carotenoids = 4.695 × E440 – 0.268 × Ch a + b The presence of the respective carotenoids in the specimens Protein analysis of particular species of lichens assayed was identified by The phytochrome protein were isolated using method ac- column (CC) and thin-layer chromatography (TLC) with cording to Tokuhisa et al. (1985) described by López- different solvent systems (Czeczuga 1981) as well as high- Figueroa et al. (1989). The extraction of the phytochrome performance liquid chromatography (HPLC). protein was performed ether according to Lindemann Prior to chromatography, the material was homoge- et al. (1989) with 50% ethylene glycol and 2 nM Tri- nized and hydrolized in nitrogen, at room temperature. The ton X-100. After removal of contaminating material with extract was subsequently placed on an Al2O3 – filled Quick- polyethyleneimine concentration of the desired protein frac- fit Co. column. The individual fractions were eluted using tion was done using ammonium sulfate (45% saturation). various solvent systems. The eluent was evaporated, and Relative amounts of the photoreversible protein were de- the residue was dissolved in an appropriate solvent to draw termined by measurement of the absorbtion of difference the maximum of absorption. In addition to CC, an acetone spectrum of A660 nm – A730 nm determined after saturat- extract was divided into fractions with TLC Silicon gel cov- ing far-red irradiation, minus the difference A660 nm – A730 ered glass plates (Merck Co.) and various solvent systems nm determined after saturatin red irradiation. In the Nostoc were used. The Rf values were established according to com- punctiforme photobiont of Peltigera investigated species of monly accepted criteria (Kraus and Koch 1996). the photoreversible protein were determined by measure- Pigments were also determined by ion-pairing in ment of the changes in the absorbance at A540 nm and reverse-phase HPLC accordingtoMantoura&Llewellyn A650 nm (MacColl & Guard-Friar 1987). Absorption spec- (1983). The HPLC equipment consisted of a Shimadzu SCL- tra was recorded with a Beckman spectrophotometr model 6B gradient programmer and a Rheodyne 7125 injector. 2400 DU. Further purification on a CC from hydroxyap- Detection was achieved in a Schimadzu SPD–6AV UV-VIS atite was achieved with the extract from investigated mate- spectrophotometric detector set at 440 nm and a Shimadzu rial. Sodium dodecyl sulphate gel electrophoresis with 10% Table 1. Percentage contain of different coloured light. Type of light (in %) Specification Intensity of sunlight (in W m−2) Red Yellow Green Blue 1. In the months (10–11h) January 0.75 25.4 30.2 34.4 10.0 June 6.45 23.1 28.5 33.5 14.9 December 0.47 26.8 30.6 32.0 10.6 2. Sunny day (July 9, 10 h) 6.36 23.8 29.9 37.6 8.7 3. Cloudy day (July 11, 10 h) 3.91 18.4 26.1 38.6 16.9 4. In the different time of day (July 10) 500 31.0 32.3 28.9 7.8 1400 22.0 31.9 31.5 14.6 2030 30.8 36.0 26.5 6.7 5. In the shade of particular trees (July 7–10, 10–14 h) 3.32 – 4.38 Alnus glutinosa (L.) Gear. 21.0 31.7 26.3 21.0 Betula verrucosa Ehrh. 22.6 26.1 37.4 13.9 Carpinus betulus L. 21.0 28.2 36.8 14.0 Picea excelsa (Lam.) Lk. 22.7 27.2 36.5 13.6 Pinus sylvestris L. 20.5 30.8 34.2 14.5 Populus tremula L. 25.0 28.2 34.3 12.5 Quercus robur L. 23.5 29.5 35.3 11.7 Tilia cordata Mill. sunny day 31.0 25.0 31.4 12.6 cloudy day 21.2 27.2 36.5 15.1 Acer pseudoplatanus L.