CZECH POLAR REPORTS 8 (1): 84-93, 2018 Adaptations of cyanobacterium Nostoc commune to environ- mental stress: Comparison of morphological and physiological markers between European and Antarctic populations after re- hydration Dajana Ručová1, Michal Goga1, Marek Matik2, Martin Bačkor1* 1Department of Botany, Institute of Biology and Ecology, Faculty of Science, University of Pavol Jozef Šafárik, Mánesova 23, 041 67 Košice, Slovakia 2Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, 040 01 Košice, Slovakia Abstract Availability of water may influence activities of all living organisms, including cyano- bacterial communities. Filamentous cyanobacterium Nostoc commune is well adapted to wide spectrum of ecosystems. For this reason, N. commune had to develop diverse pro- tection strategies due to exposition to regular rewetting and drying processes. Few studies have been conducted on activities, by which cyanobacteria are trying to avoid water deficit. Therefore, the present study using physiological and morphological param- eters is focused on comparison between European and Antarctic ecotypes of N. commune during rewetting. Gradual increase of FV/FM ratios, as the markers of active PS II, demonstrated the recovery processes of N. commune colonies from Europe as well as from Antarctica after time dependent rehydration. During the initial hours of rewetting, there was lower content of soluble proteins in colonies from Antarctica in comparison to those from Europe. Total content of nitrogen was higher in European ecotypes of N. commune. Significantly higher frequency of occurrence of heterocysts in Antarctic ecotypes was observed. The heterocyst cells were significantly longer in Antarctic ecotypes rather than European ecotypes of N. commune. Key words: Antarctica, soluble proteins, cyanobacteria, chlorophyll fluorescence, heterocysts, nitrogen, James Ross Island DOI: 10.5817/CPR2018-1-6 ——— Received December 19, 2017, accepted April 4, 2018. *Corresponding author: M. Bačkor <[email protected]> Acknowledgements: The authors are grateful to CzechPolar2 (Czech Polar Research Infrastructure, project No. LM2015078) for an opportunity to conduct research during the Antarctic expedition in 2017. This study was partly supported by projects KEGA 012UPJŠ-4/2016 and project from the Slovak Grant Agency (VEGA 1/0792/16). 84 D. RUČOVÁ et al. Introduction Nostoc commune is a filamentous cyano- photoinhibition as well. For photosynthesis, bacterium which forms macroscopic colo- the light is essential, but under dry con- nies. Hydrated colonies have an olive-green ditions when photosynthesis is inhibited, colour with the consistency of a solid gel, the light becomes harmful. Deactivation of while in dry state they resemble black photosystem II and photosynthetic activi- crusts (Novis et al. 2007). In N. commune ties would be appropriate because when several cell types can be differentiated: photosynthesis is stopped while photo- vegetative cells, which are photosynthetic chemical reactions are active due to inhi- cells that fix carbon (Meeks et Elhai 2002) bition of Calvin- Benson cycle by dehy- and heterocysts, which are specialized ni- dration, strong reductants or oxidants could trogen-fixing cells containing nitrogenase be created by PS I or PS II (Hirai et al. enzymes. They perform many functions, 2004). Physiological changes in N. com- but one of the main roles is to provide ni- mune during the cycles of wetting and trogen and nutrients (phosphorus etc.) to drying were investigated in several studies. cells in filament. Cells of N. commune are According to Scherer et al. (1984) N. com- ringed by gelatinous matrix, consisting pri- mune can perform photosynthetic CO2 fix- marily of polysaccharides. These polymers ation and nitrogen fixation under wet con- are responsible for protection against proto- ditions. Satoh et al. (2002) measured recov- zoan predation, antimicrobial agents, but ery processes during rewetting in colonies mainly against desiccation (Potts 1994, De of N. commune, focused on energy transfer Phillippis et Vincenzini 1998). In general, and photochemical reactions. It has been Nostoc can survive for 100 years in almost shown that phycobiliproteins and photo- complete dehydration in the dark (Cameron system I (PS I) complexes activate their 1962). forms very quickly as soon as 1 min after N. commune is well adapted to terres- rehydration, but recovery of the photosys- trial ecosystems from the tropics to polar tem II (PS II) was slower. Respiration regions of the Earth. Activities of cyano- processes recovered 30 minutes after ini- bacterial communities are influenced by tiation of rehydration. However, the details the availability of water, and pressure of a of these processes of recovery and activa- water deficit apparently affected the cells tion of photosynthesis remain unknown. at the early stage of their evolution (Potts In this study, we tested populations of 1994). Some species of cyanobacteria de- cyanobacterium N. commune from Antarc- veloped the way for desiccation tolerance tica and from Europe. We have focused on (Kennedy et al. 1994). Filamentous cyano- the physiological parameters as well as the bacterium N. commune developed the pro- morphological differences of the studied tective strategies against water deficit be- species after time dependent rehydration. cause of exposition to regular rewetting and We evaluated the chlorophyll fluorescence drying processes. Water content of desic- measurements for assessment of changes cated (anhydrobiotic) cyanobacteria cells in photosynthetic processes, content of sol- is very low. In this dry state, N. commune uble proteins, frequency and size of heter- as photosynthetic organism must have ocysts and nitrogen content in rehydrated strategy to avoid not only desiccation but colonies of N. commune. 85 ADAPTATIONS OF NOSTOC COMMUNE TO ENVIRONMENTAL STRESS Material and Methods Plant material Antarctic populations of N. commune 57° 52′ 57″ E). European cyanobacterial colonies were collected randomly (during samples were collected randomly from the January 2017) at James Ross Island, Ant- soil surface at Illmitz (47° 45′ 26″ S, arctica (Fig. 1) in shallow streams where 16° 49′ 01″ E) in the district of Neusiedl the appearance of colonies is widespread am See, Austria. The area is typical by wide (Skácelová et al. 2013, Komárek et al. 2015). open plains and many small salt lakes Selected locality was close to Czech Ant- around. arctic station J. G. Mendel (63° 48′ 02″ S, Fig. 1. Nostoc commune colonies at James Ross Island, Antarctica, sampling site. Rehydration assay Nineteen N. commune colonies with dry they were stored in laboratory conditions at weights around 100 mg were used. Desic- 24 ± 1°C and illuminated with daylight flu- cated colonies were rehydrated in 15 ml orescent tubes (light intensity 25 μE m-2 s-l). plastic tubes through the addition of dis- After these time intervals, water was re- tilled water (5 ml), at time intervals (30 moved by blotting N. commune colonies minutes, 1 hour, 2 hours, 4 hours, 8 hours, with paper tissue. 16 hours and 24 hours). During rehydration Chlorophyll fluorescence measurements Rewetted colonies of N. commune were The chlorophyll fluorescence measure- transferred on Petri dish and dark adapted ments were performed using a FluorCam at least 15 minutes prior to measurement. MF-800 fluorescence-imaging camera (Pho- 86 D. RUČOVÁ et al. ton Systems Instruments, Czech Republic) dark-adapted state and processed by FluorCam 7 software- - FV/FM – quantum yield of PS II photo- (Photon Systems Instruments, Czech Re- chemistry in the dark adapted state public). Chlorophyll a fluorescence ex- For this study, we measured chloro- pressed as FV/FM was calculated phyll fluorescence at time intervals (30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, (FM − F0) / FM = FV/FM Eqn. 1 16 hours and 24 hours) after the rehy- - F0 represent ground fluorescence in dration. Chlorophyll fluorescence parame- the dark-adapted state ters were taken from ten positions on each - FM represent maximum fluorescence Petri dish, and the average value was used caused by saturating radiation pulse in the as one observation. Soluble proteins N. commune colonies were homoge- Inc., Hercules, California) and 100 µl of nized in an ice- cold mortar with added supernatants we obtained a solution of sol- 2 ml of 50mM phosphate buffer (pH 6.5). uble proteins. Absorbance of samples was Homogenized suspension was then centri- spectrophotometrically measured at wave- fuged at 15 000 x g at 4 ºC for 20 min. length 595 nm with the multi-detection mi- After centrifugation, soluble proteins con- croplate reader The Synergy HT, BioTek. tent was measured according to Bradford Bovine serum albumin was used as a cali- (1976) method. Using the 900 µl Bio-Rad bration standard. protein assay kit (Bio- Rad Laboratories CHNS analysis Dry thalli of N. commune around 100 mg Helium and oxygen (purity 99.995%) were were homogenized in an ice cold mortar. used as the carrier combusting gases. Com- Then they were transferred to 2 ml eppen- bustion tube was set up at 1150°C and re- dorfs and used for measurement. Elemen- duction tube at 850°C. Sulfanilamide (C ¼ tal analysis for carbon, hydrogen, nitrogen 41.81%, N ¼ 16.26%, H ¼ 4.65%, S ¼ and sulphur was performed by elementary 18.62%) was used as a CHNS standard. analyzer Vario MACRO cube (Elementar This analysis was measured at Institute of Analysensysteme GmbH, Germany) equip- Geotechnics, Slovak Academy of Sciences ped with a thermal conductivity detector. in Košice, Slovakia. Heterocyst counts Measurements for quantifying hetero- solved in 25 ml of distilled water. We cysts were done in a laboratory (Cell Im- placed a 0.5 ml drop of fluid in the centre aging and Ultrastructure Research, Univer- of the slide avoiding air bubbles and we sity of Vienna, Austria). Colonies were removed excess water with paper towel. maintained in laboratory conditions at 24 ± Images were taken at 40x magnification 1°C and illuminated with daylight fluores- with a microscope. We identified and pho- cent tubes (light intensity 25 μE m-2 s-l). We tographed at least 200 heterocysts of both used well- mixed culture prepared from ecotypes of N.