The green alga Ulvaria obscura (Kütz) Gayral was an object of the study. In the Barents Sea, this alga occupies the lower littoral zone descending to the sub-.
Doklady Biological Sciences, Vol. 394, 2004, pp. 51–54. Translated from Doklady Akademii Nauk, Vol. 394, No. 3, 2004, pp. 423–426. Original Russian Text Copyright © 2004 by Voskoboinikov, Makarov, Maslova, Sherstneva.
GENERAL BIOLOGY
Ultrastructure and Pigment Composition of the Photosynthetic Apparatus of the Green Alga Ulvaria obscura during the Polar Day and Polar Night G. M. Voskoboinikov*, M. V. Makarov*, T. G. Maslova**, and O. A. Sherstneva** Presented by Academician G.G. Matishov August 5, 2003 Received August 14, 2003
The ability of marine macrophytes to survive in Arctic regions with extreme environmental conditions (long-term exposure to negative temperature, changes in illuminance ranging from polar night to polar day, etc.) has always been a subject of considerable interest of researchers. It was found recently that photoperiod played a decisive role in changes in the growth rate of algae [1–3]. We earlier demonstrated that the onset of intense growth of most algae at the Barents Sea coast coincided with the increase in photoperiod in February and March at negative temperatures of the water. The rate of this process reaches its maximum in May and declines to a minimum level by November. The growth of algae is completely ceased in December [3]. The effect of these contrasting environmental conditions on the state of the algal photosynthetic apparatus is obscure. Probably, the adaptation of the photosynthetic apparatus allows the plants to survive in the Arctic environment. MATERIALS AND METHODS This work was performed at the Dal’nie Zelentsy Marine Biological Station of the Murmansk Marine Biological Institute, Kola Scientific Center, Russian Academy of Sciences (the Murmansk coast of the Barents Sea). The green alga Ulvaria obscura (Kütz) Gayral was an object of the study. In the Barents Sea, this alga occupies the lower littoral zone descending to the sublittoral zone. The parts of the plate without signs of gametogenesis or sporogenesis were used. We used for analysis the central part of the thallus, which, in algae of the order Ulvales, is characterized by the highest
*Murmansk Marine Biological Institute, Kola Scientific Center, Russian Academy of Sciences, ul. Vladimirskaya 17, Murmansk, 183010 Russia **Komarov Botanical Institute, Russian Academy of Sciences, ul. Professora Popova 2, St. Petersburg, 190000 Russia
content of photosynthetic pigments and the highest rate of photosynthesis [4]. Experimental samples for pigment assay were collected at noon in July, November, and December. The photoperiods in these months were 24, 12, and 24 h, respectively. The material for the cytological assay was fixed in July and December. To study the ultrastructure of the photosynthetic apparatus, sections of the central part of the thallus about 1 cm2 in size were fixed for 12 h in a 2.5% solution of glutaraldehyde in a 0.1 M cacodylate buffer solution (pH 7.2), washed two times in the buffer solution, and subjected to postfixed for 12 h in 1% OsO4. Fixation solutions were made isotonic to seawater by adding sucrose. The resulting preparations were dehydrated with ethanol and acetone and embedded in a mixture of araldite and Epon. Ultrathin sections were contrasted with uranyl acetate and lead and viewed using a JEM-100B electron microscope. Both qualitative and quantitative pigment compositions of U. obscura were assayed using the method described in [5, 6] with some modifications. The pigment mixture contained in the ethanol extract was separated by paper chromatography. The spectra of the pigments and their concentrations were measured using a Specord UV-VIS spectrophotometer (Carl Zeiss, Jena). The following pigments were assayed: chlorophyll a, chlorophyll b, and carotenoids (β-carotene, lutein, violaxanthin, and neoxanthin). Pigment distributions among photosynthetic pools were calculated [7]. RESULTS Electron microscopy of U. obscura cells showed that the fine structures of chloroplasts in summer and winter substantially differed (figure). There was no degradation of cell structures during the polar night. Embedded pyrenoids and starch granules were observed in chloroplast stroma throughout the year. The partial volume of the starch granules in summer was significantly larger than in winter. The winter season was characterized by a denser packing of thylakoids, a larger number of photosynthetic membranes
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SG
Py
Chp
1 µm
(a)
SG
Py í Chp
(b)
1 µm
Fig. 1. The ultrastructure of the photosynthetic apparatus of U. obscura during (a) a polar day and (b) a polar night. Designations: Chp, chloroplast; Py, pyrenoid; SG, starch granule; T, thylakoid.
per unit area of the chloroplast stroma, and a smaller number of ribosomes in the stroma compared to that in summer. The central part of the pyrenoid in summer was filled with diffuse granular contents. Conversely, in winter, fibrillate netted material was dominant, whereas granular material was segregated as small bodies. The area occupied by the starch sheath of the pyrenoid in microscopic sections in summer was two to three times larger than in winter.
The total content of pigments in summer was 2.5 mg per gram dry weight. In winter, this value increased seven to eight times (table). There were almost no seasonal changes in the chlorophyll a to chlorophyll b ratio. The chlorophyll-to-carotenoid ratio was maximum (10.2) in summer season; in other seasons, this ratio was, on average, 4.8 (4.4 and 5.5 in November and December, respectively). The proportion of chlorophyll DOKLADY BIOLOGICAL SCIENCES
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incorporated in the light-harvesting complex was 80– 90% of the total chlorophyll content.
Table 1. Concentration of pigments (mg per gram dry weight) in U. obscura in summer and winter Pigment
DISCUSSION AND CONCLUSIONS A very high content of chlorophyll b is a specific feature of the pigment composition of U. obscura from the Arctic region. The chlorophyll a to chlorophyll b ratio in this alga is about 1.7 (in terrestrial plants, this ratio is about 3). In addition to the large proportion of chlorophyll incorporated in the light-harvesting complex, these parameters are typical of shade-enduring plants and plants growing at low light intensity. The ratio of the total chlorophyll content to the total carotenoid content (table) is about 4–5 (in summer, it sometimes reaches 10). This can be regarded as evidence for a generally low content of carotenoids and an increased relative content of carotenoids upon the decrease in light intensity. Carotenoids fulfil many functions in plant cells. On the one hand, they increase the efficiency of photosynthesis by absorbing light in the blue and green spectral ranges (the chlorophyll absorption at these wavelengths is minimum). On the other hand, carotenoids protect chlorophylls from photooxidation. A decrease in light intensity induces a rapid increase in the content of carotenoids in the thalli. Probably, this ensures a more effective utilization of incident light. During the spring–autumn season, the chlorophyll-to-carotenoid ratio was almost constant. However, this season is characterized by substantial increase in the contents of all pigments per unit weight. Therefore, this can be regarded as a mechanism of adaptation of plants to the reduced light intensity. The causes of the conservation of the photosynthetic apparatus in an undamaged state during the long polar night remain insufficiently understood. It has been reported that some unicellular algae are able to survive for a long time (as long as 1.5 months and even two years in the cases of Euglena gracilis and Chlorella vulgaris, respectively) in a mineral medium in complete darkness (carbon starvation) [8, 9]. This ability was explained by the transition of algal metabolism to a lower level (the state of mesabiosis) [10]. This was accompanied by gradual degradation of chlorophyll b, a decrease in the concentration of chlorophyll a (pheophytinization in some cases), disturbance in the photosynthetic membrane arrangement, and formation of vacuoles and myelinlike structures in the chloroplast stroma. This transformation of the chloroplast membrane system was accompanied by a decrease in the number of starch granules (or a complete disappearance of these granules) and reduction of pyrenoids. Exposure of unicellular algae to the dark in an organic medium resulted in the transition of chloroplasts into protoplasts. A higher rate of degradation was observed in the macrophytes from the Far Eastern seas exposed to the darkness at positive temperatures. The most darkresistant alga (Ahnfelia tobuchinsis) was capable of DOKLADY BIOLOGICAL SCIENCES
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Chlorophyll a Chlorophyll b Chlorophyll a to chlorophyll b ratio Neoxanthin Violaxanthin Lutein β-Carotene
July
November December
1.5 0.9 1.66
9.0 6.25 1.44
10.5 5.75 1.8
0.02 0.01 0.15 0.1
2.0 0.6 1.2 0.5
2.0 0.5 1.0 0.4
enduring such an alternating exposure for no longer than two weeks [11]. There was a decrease in the photosynthetic activity of algae at the Barents Sea coast during winter when the water temperature was close to zero and ambient illuminance was minimum. Many researchers studied the processes of photosynthesis and respiration using the method of Vincler [12] and reported that there was a decrease in the rate of photosynthesis in autumn and its complete cessation in winter. The decrease in the number of ribosomes in chloroplasts and in the total pool of ribosomes in cells observed in our experiments can also be regarded as evidence for reduction in the protein biosynthesis activity. Degradation changes were observed neither in chloroplasts nor in cells of Ulvaria. Therefore, the processes of adaptation of the U. obscura cells to low-intensity light differ from the processes of degradation observed in the aforementioned marine macrophytes exposed to the darkness at positive temperatures. The changes in the pyrenoid structure of U. obscura can be regarded only as evidence for decrease in the photosynthetic activity and in the rate of starch biosynthesis. Analyzing the ability of some unicellular algae to survive in the dark, we could suggest that Ulvaria transits into the state of mesabiosis in early winter and comes out of this state in early February, when the photoperiod begins to increase. However, in contrast to the state of mesabiosis in unicellular algae, no symptoms of degradation were observed in Ulvaria. Another possible mechanism of these processes seems to be most probable. According to this mechanism, 2- to 3-h-long exposure to low-intensity light, which is observed at these latitudes from late November to late January, is sufficient to maintain the rate of photosynthesis at the minimum level. It is beyond doubt that the rate of metabolism of the algae during this period of time is significantly lower than during the rest of the year, and respiration prevails over photosynthesis. Nevertheless, it is conceivable that light of minimum intensity is also used, along with dark fixation of CO2, in reactions of metabolite transformation. This suggestion is confirmed by (1) the presence of starch granules in algal
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cells in late December and (2) increase in the concentration of pigments and growth of the photosynthetic membrane system. The results obtained in this work on the concentrations and concentration ratio of pigments and ultrastructure of algae in winter are consistent with similar data obtained in algae grown under the conditions of natural shading (in a grotto) and fit the model of the socalled ulva-type adaptation of photosynthetic apparatus to shade described in [13]. It follows from this model that an increase in the potential photosynthetic activity under the conditions of shading is provided by an increase in the concentration of photosynthetic units in chloroplasts and activity of photosynthetic enzymes. ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research (project no. 02-04-49887). REFERENCES 1. Van den Hoek, C. and Breeman, A.M., in Evolutionary Biogeography of the Marine Algae of the North Atlantic (NATO ASI Ser., G22), Berlin: Springer, 1991, pp. 55–87. 2. Luning, K., Seaweeds. Their Environment, Biogeography and Ecophysiology, New York: Wiley, 1990.
3. Shoshina, E.V., Makarov, V.N., Voskoboinikov, G.M., et al., Bot. Mar., 1996, vol. 39, no. 1, pp. 83–93. 4. Li, B.D. and Titlyanov, E.A., Biol. Morya, 1978, no. 2, pp. 47–55. 5. Li, B.D., Ekologicheskie aspekty fotosinteza (Ecological Aspects of Photosynthesis), Vladivostok, 1978, pp. 38–55. 6. Maslova, T.G., Popova, I.A., and Popova, O.F., Fiziol. Rast., 1986, vol. 33, issue 3, pp. 615–619. 7. Maslova, T.G. and Popova, I.A., Photosynthetica, 1993, vol. 29, no. 2, pp. 195–203. 8. Leedale, G.F., Euglenoid Flagellates, New York, 1967. 9. Voskoboinikov, G.M., Morphological and Functional alterations in the Unicellular Alga Euglena gracilis Klebs Kept in a Mineral Medium in the Dark for a Long Time, Cand. Sci. (Biol.) Dissertation, Leningrad, 1980. 10. Goldovskii, A.M., Usp. Sovrem. Biol., 1977, vol. 74, issue 3(6), pp. 461–472. 11. Voskoboinikov, G.M. and Kamnev, A.N., Morfofunktsional’nye izmeneniya khloroplastov v ontogeneze vodoroslei (Morphological and Functional Alterations in Chloroplasts during the Ontogeny of Algae), St. Petersburg: Nauka, 1991. 12. Tikhovskaya, Z.P., Bot. Zh., 1960, vol. 45, no. 8, pp. 1149–1160. 13. Titlyanov, E.A., Kolmakov, P.V., Leletkin, V.A., et al., Biol. Morya, 1987, no. 2, pp. 48–57.
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