Photosynthetic efficiency of endosymbiotic algae of Paramecium bursaria originating from locations with cold and warm climates

Paramecium bursaria (Ehrenberg 1831) is a unicellular organism belonging to ciliates that play a significant role in aquatic ecosystems. Ciliates are considered to be cosmopolitan. They can be easily found in various types of watercourses and bodies of stagnant water. The cytoplasm of a single cell of P. bursaria contains about 700 symbiotic algal cells representing different species (Hoshina, Imamura 2009). Algae cells containing green photosynthetic pigments (chlorophyll a and b) provide protozoan with products of photosynthesis: maltose and oxygen and, in return, they receive carbon dioxide and nitrogen compounds (Reisser 1980). Moreover, P. bursaria protects algal cells from other protozoans and viruses, and directs them to brightly illuminated areas for optimum photosynthesis conditions (Yamada et al. 2006; Reisser 1980).

The growth of photosynthetic organisms is a dynamic process continuously shaped by environmental conditions. The most rapidly changing environmental factor affecting the photosynthesis is light which is a source of energy for carbon fixation (Gilstad et al. 1993; Flameling, Kromkamp 1997; Walters, Horton 1995). During the day, the quality and quantity of photosynthetically active radiation (PAR) changes. Photosynthetic organisms maintain a balance between the photosynthetic energy supply in thylakoid membranes and the energy consumption within the Calvin cycle (Carvalho et al. 2011; Bailey et al. 2008). When algae and some species of higher plants are moved from light to dark conditions, the PSII activity is reduced, probably due to the low rate of reduction of primary electron acceptors (Dau 1994). Furthermore, when the light intensity is too high, the photosynthetic apparatus can be damaged due to the chlorophyll photooxidation.

Photobiological responses and adaptations of P. bursaria endosymbionts exposed to PAR have become an important research subject (Berk et al. 1991; Summerer et al. 2009). Pado (1965) showed that light conditions determine the number of algal cells inside the host cell.

Chlorophyll fluorescence provides information about the physiological state of the organism, the amount of energy absorbed by the chlorophyll molecule and dissipated in the form of heat (Murchie, Lawson 2013). Stress factors lead to increased power dissipation in the PSII center and may in time lead to irreversible damage. Changes in chlorophyll a fluorescence provide insight into the processes of photoprotection and photodamage with respect to the reduction of photosynthesis efficiency (Franklin et al. 1992).

Interactions between temperature and light have a significant impact on photosynthesis and the growth rate of algae (Butterwick et al. 2005). Low temperature may induce structural and functional changes in the photosynthetic apparatus and RuBisCo activity allowing to maintain the energy balance (Mortain-Bertrand et al. 1988). Levasseur et al. (1990) showed that a temperature-induced decrease in the carbon fixation rate was correlated with a decrease in the energy-transfer efficiency between the antenna and the reaction center of PSII. Thompson et al. (1992) reported that the chlorophyll content in algae decreases with decreasing temperature. Measurement of chlorophyll a fluorescence of Chlorella vulgaris incubated at 5°C indicated that the yield of PSII electron transport was less temperature sensitive than in organisms incubated at 2°C (Maxwell et al. 1994). Under the culture conditions, the temperature should oscillate between 20 and 30°C. Temperature lower than 16°C can slow down the growth of algae and temperature higher than 35°C can be lethal to many strains of algae (Coutteau 1996). According to Murata et al. (2007), low temperature stress inhibits the repair of PSII but does not affect the photodamage to PSII. Many studies describe effects of short- and long-term exposure to low temperature on the photosynthetic activity of algae (Morgan-Kiss et al. 2002; Wen et al. 2005). The symbiotic Chlorella of P. bursaria differs significantly from free-living algae at the higher activity of RuBisCo, regardless of the CO₂-concentration and carbonic anhydrase activity (Reisser, Benseler 1981).

The aim of the present study was to determine the efficiency of the photosynthetic apparatus of P. bursaria symbiotic C. vulgaris by measuring the chlorophyll a fluorescence kinetics under low-temperature and constant dark stress. Differences in responses between strain Ard7 and KD64 were determined. Ard7 is originated from Ardmore, OK (USA) with an annual average temperature of 18°C, 235 days of sunshine per year and annual precipitation of 930 mm and strain KD64 originated from Kamchatka with an annual average temperature of 1.6°C, 67 days of sunshine per year and annual precipitation of 2700 mm.

The results obtained in the present study revealed significant changes in PSII in algae strains incubated at 18°C compared to algae from lower temperatures in the light and dark conditions.

The maximum quantum yield of PSII (F_v/F_m) is used to estimate changes in the functioning of reaction centers, as well as to determine the photosynthetic activity (Büchel, Wilhelm 1993). According to Masojidek et al. (2011), the PSII efficiency varies depending on the time of day, which is related to the intensity of light. The photoinhibition is induced not only due to the excess of unused energy by chlorophyll (Osmond et al. 1999), but also by low temperature stress (Germino, Smith 2000a,b). The research conducted by Gómez et al. (2001) revealed that Gelidium pulchellum in high light showed a greater photoinhibition ratio and slower ability to rebuild structures of PSII compared to plants growing under low light conditions. In the present study, the reduction in the F_v/F_m parameter was observed with decreasing temperature regardless of the light conditions. The highest F_v/F_m values were determined in cultures kept under 18°C and 12L/12D conditions. This parameter had the lowest values compared to the control, regardless of the light conditions at a temperature of 6°C (Table 2). Zhang et al. (2012) reported that F_v/F_m of cyanobacteria and green algae was higher at a temperature of 15°C than at 4°C. According to Teoh et al. (2013), the optimal temperature for the growth of Chlorella strains is 18°C. On the other hand, Wilson & Huner (2000) showed that F_v/F_m in C. vulgaris incubated at 5°C under low light intensity was higher than at a temperature of 27°C. In this study, the F_v/F_m values of P. bursaria strains incubated under dark conditions did not change significantly (Table 2). Changes in F_v/F_m result from the reduced production of photosynthesis. They depend on protein D1 and photooxidation of PSII (Vass et al. 1992; Krzemińska et al. 2015). The decrease in F_v/F_m may result from the increase in protective non-radiative energy dissipation or photo-inhibitory damage to the PSII reaction center (Maxwell, Johnson 2000). Under changing light intensity and low temperature, the CO₂ fixation slows down, causing the reduction in electron transport. The low temperature inhibits the scavenging of reactive oxygen species (ROS) that may damage PSII and also inhibits the PSII repair cycle (Torzillo et al. 1998).

Table 2

Some chlorophyll a fluorescence parameters of two strains (Ard7 and KD64) of Paramecium bursaria incubated in temperatures (T): 18, 15, 12, 9, 6°C in the daily cycle of 12 light hours and 12 dark hours (12L/12D), and in constant darkness (24D) conditions

The decrease in the F_v/F_m ratio under varying light intensity can be due to changes in the level of F₀ and F_m. This indicates the loss of energy dissipated in the form of heat, which may result from the inability of PSII to reduce the complete electron acceptors (Magnusson 1997; Saakov 2002). The F₀ fluctuations include changes at the level of the main antenna as light-harvesting complexes, from the trimeric form to the monomeric form. They also include physical dissociation of the core part of LHCII in PSII. The highest intensity of chlorophyll fluorescence F₀, when all PSII reaction centers are open, was observed under 12L/12D conditions at 15°C in both P. bursaria strains. At a temperature of 18°C and 15°C, the F₀ values were significantly higher in Ard7 compared to KD64, which can be due to the adaptation to the optimal temperature of Ard7 originating from a place with a warm climate. When the temperature decreased, the F₀ significantly decreased, regardless of the light conditions. The lowest F₀ values were determined at 6°C (Table 2). F₀ of Chlorella pyrenoidosa incubated in dark conditions largely depends on temperature. These reactions indicate a direct effect of thermal shock associated with PSII (Chemeris et al. 2004b). The accumulation of PSII complexes under dark conditions is characterized by low electron reduction, which depends on the reduced plastoquinone. Processes regulating the photosynthetic apparatus include phosphorylation of thylakoid membrane proteins by protein kinases, which reversibly enhance the rate of plastoquinone reduction. These processes include: (i) the energy distribution between PSI and PSII, (ii) the ratio of cyclic and non-cyclic electron transport, (iii) the synthesis and degradation of thylakoid membrane proteins, (iv) the control of PSII reaction center stability, and (v) the deactivation of the main energy bands in thylakoid membranes (Allen et al. 1995). Chemeris et al. (2004a) revealed the close relationship between the state of reduced plastoquinone and the relative content of inactive PSII complexes.

In the present study, the highest F_m value was determined at 15°C under 12L/12D. These significant differences between two strains may be due to different conditions in which they live. The F_m values for the strains kept under 12L/12D were higher compared to the strains incubated in constant darkness. The lowest values of F_m for both strains were observed at 6°C, regardless of the light conditions (Table 2).

Slight differences between F_m and F₀ represent low F_v values, which indicate the low PSII activity and the energy dissipation in the form of heat. This parameter is determined by the maximum quantum yield of PSII. In the present study, the highest F_v was observed at 18°C and 15°C for Ard7 kept under 12L/12D conditions. The significantly highest F_v values were observed in KD64 incubated under similar light conditions. F_v of symbiotic algae cultured under 24D conditions were statistically lower compared to the cultures grown under 12L/12D conditions. When the temperature decreased, the F_v decreased significantly. The lowest F_v values were determined at a temperature of 6°C under both light conditions (Table 2). It seems that photoinhibition includes at least two inactivation steps. The first phase is considered reversible and develops for about one hour without causing any damage to the PSII (Leitsch et al. 1994). The second stage probably begins with damage in the PSII reaction centers (Aro et al. 1993; Leitsch et al. 1994; Mishra, Singhal 1992; Vass et al. 1992). The highest F_v/F₀ values were noticed in algae strains incubated at a temperature of 18°C, regardless of the light conditions. At temperatures of 12°C, 9°C and 6°C, these values ranged from 0.49 to 0.83 under 12L/12D conditions and from 0.43 to 0.77 under 24D conditions (Table 2). Changes in F_v/F₀ values reveal disturbances in the water dissipating complex, which is very sensitive to environmental changes and determines the resistance of plants (Jiang et al. 2006). Similar changes in F_v/F₀ were observed in barley (Kalaji et al. 2012).

The specific parameters of fluorescence, such as RC/ABS, TRo/CSo, Eto/CSo, DIo/CSo and PI, are used to explain the gradual flow of energy through single PSII reaction centers (Force et al. 2003; Xu et al. 2014). These parameters allow to determine the photosynthetic activity of endosymbiotic algae of P. bursaria in conditions of varying temperature and light. In the present study, the lowest values of RC/ABS, TRo/CSo, ETo/CSo, DIo/CSo were reported for both strains incubated at a temperature of 6°C (Fig. 1). The highest RC/ABS values at 15°C in dark conditions (24D) were revealed for both strains compared to other temperatures. TRo/CSo was temperature-dependent. The highest values were observed in the control group for both strains, regardless of the light conditions. When the temperature dropped, the TRo/CSo significantly decreased (Fig. 1). The highest DIo/CSo values compared to the control (18°C) were observed for both strains of P. bursaria at a temperature of 15°C under 12L/12D conditions (Fig. 1). A decrease or an increase in the DIo/CSo values indicates that part of the energy is converted into heat (Strasser, Strasser 1995; Force et al. 2003). In the present study, the ETo/CSo values were lower at all temperatures compared to the control (18°C) regardless of the light conditions. The reduced electron transport observed in the leaves of barley plants growing in the shade was probably due to energy being trapped by reaction centers (TRo/CSo) and higher loss of energy in the form of heat (DIo/CSo) (Kalaji et al. 2012). The decrease in TRo/CSo and ETo/CSo values indicates that the active reaction centers are converted into inactive centers, which reduces the efficiency of energy capture.

Figure 1

PI is a biophysical parameter indicating changes in PSII, which is calculated by multiplying ABS/Cs × TRo/Cs × ETo (Strasser, Strasser 1995; Force et al. 2003; Stefanov et al. 2011). PI is one of the fluorescence parameters that provides useful information about the state of the vitality of photosynthetic organisms. The PI values were lower under 12L/12D conditions compared to the control. At a temperature of 15°C, the PI values were similar to the control for Ard7 and higher than the control for KD64 under 24D conditions (Fig. 1). According to Ferrante & Maggiore (2007), however, PSII centers are not damaged at low temperatures but at high temperatures. On the other hand, low temperature may indicate a decrease in the accumulation of electron acceptors and in the activity of PSII reaction centers (Vonshak, Novoplansky 2008). Low temperature stress and light stress have synergistic effects and clearly increase the PSII photodamage (Allakhverdieva, Murataa 2004; Nishiyama et al. 2008). These reactions are similar to those observed in algae exposed to various types of stress, such as high salinity (Vonshak 2002), photoinhibition (Lu, Vonshak 1999; Sonoike et al. 2001), bleaching (Hill et al. 2004) or insecticides (Jena et al. 2012).

The time needed to reach the maximum fluorescence (Tf_(max)) may be useful to identify early changes in photosynthetic activity. For P. bursaria endosymbionts, the longest Tf_(max) was recorded for the Ard7 strains compared to the KD64 strain. Tf_(max) was significantly shorter under 12L/12D conditions. The lowest Tf_(max) was observed in cultures incubated at a temperature of 6°C for both strains (Fig. 1).

In natural aquatic ecosystems, the toxic effect of environmental factors on algae depends on the depth of their occurrence and the temperature conditions (Oukarroum et al. 2012). The temperature differences connected with the light factor affect the photosynthetic activity. In some organisms, these factors cause a decrease in the PSII activity, while in others they do not cause significant changes or they are quickly regenerated. This tolerance may result from various repair mechanisms, including the ability to quench the ROS activity or to produce photoprotective compounds, such as mycosporine amino acids (MAAs) and carotenoids (Teoh et al. 2013).

The time of stressor treatment is the most important factor that affects the photosynthetic activity. The parameters of chlorophyll fluorescence used in the present study allow to visualize early changes in PSII under the influence of temperature and light. The ability of endosymbionts from two P. bursaria strains to acclimate to low temperature was observed to improve our understanding of the mechanisms involved in the photosynthetic apparatus. Moreover, these measurements may provide a better insight into the problem of cross adaptation to changing environmental conditions.

Based on the obtained results, we can conclude that all of the chlorophyll fluorescence parameters were related to the decreasing temperature and light conditions. In the case of Paramecium bursaria, the strain originating from geographical places with a warm climate was more sensitive to low temperature stress than the strain originating from a place with a cold climate. The darkness had a negative impact on the activity of PSII and prolonged the time needed to reach the high efficiency of photosynthesis.