Lipids and pigment-protein complexes of photosynthetic apparatus of Deschampsia antarctica Desv . plants under UV-B radiation

Aim. To investigate structural and functional modifications of major components of photosynthetic membranes of endemic antarctic species D. antarctica under UV-B radiation. Methods. For quantitative determination of photosynthetic membrane components we used Arnon’s method (for chlorophylls and carotenoids); separation of carotenoids was carried out by Merzlyak’s method; polar lipids were isolated by Zill and Harmon method in modification of Yakovenko and Mihno; glycolipids separation and identification we carried out by Yamamoto method; and sulfoquinovosyl diacylglycerol content was determined by Kean method. The separation, disintegration and determination of pigment-protein complexes of chloroplasts were carried out by Anderson method. Authenticity of differences between the mean arithmetic values of indices was set after the Student criterion. Differences were considered as reliable at p ≤ 0.05. Results. We determined structural and functional changes in lipids, carotenoids and pigment-protein complexes at the photosyntetic apparatus level in D. antarctica plants under UV-B radiation. Conclusions. Adaptation of D. antarctica plants to UV-B radiation is accompanied by a cascade of physiological and biochemical rearrangements at the level of photosynthetic apparatus, manifested as the changes in pigment, lipid and pigment-protein complexes content.

Introduction.D. antarctica Desv. is the only native species of Poaceae found in Antarctica.These plants are naturally adapted to the cold maritime Antarctic climate and high level of natural UV-B [1][2][3][4].The radiation may result in the damage of the genetic system and cell membranes, and affect several metabolic processes.Many authors noted that the action of UV-B radiation on plants caused the reduction of biomass, leaf area, growth rate, and photosynthesis [5,6].UV-B radiation influenced the plant development not only at the physiological and biochemical levels, but also at the genetic one [7].
There was observed the growth inhibition of D. antarctica plants at Palmer station at the Antarctic Peninsula under conditions of Antarctic solar UV-B in com-parison with the plots, where solar UV-B radiation was filtered out [8].Low temperature, high light (illumination) increased the degradation of ozonosphere.It caused enhancement of UV-B radiation level and induced the intensive formation of reactive oxygen species (ROS).Carotenoids, tocopherols and ascorbate are known to protect the photosynthetic apparatus from ROS [9][10][11][12][13].
Besides, there were noted the changes of fluorescence parameters and content of pigments [6], lipids [14] and pigment-proteins complexes [15] in plants under UV-B radiation.Therefore we that investigation of these parameters is expedient to explain the mechanisms of D. antarctica plant resistance to UV-B radiation.
Materials and methods.Photosynthetic tissues of D. antarctica plants selected from territories of Antarctic islands were investigated.Control and experimental plants were grown under conditions of constant temperature (10 o C), relative humidity (60 %), CO 2 concentration of 330 ppm and daily illumination by luminescent lamps (L 30W/77, Fluora, 450-650 nm) for 10 days (23 W/m 2 , photoperiod 16 h).
The experimental plants were grown under the same conditions, but on the fifth day of growing they were irradiated by UV-B for 20 h in five expositions (4 h in every light period).The UV-B lamp with absorption filter (TL 20BB/12RS («Philips», USA), 280-300 nm) was used for illumination of plants.The dose of UV-B radiation was 6.17 kJ× m -2 d -1 .The distance from the source of illumination to plants was 10 cm.The pigment content in leaves was determined according to Arnon [16].The separation of carotenoids was carried out by TLC method [17].Polar lipids were isolated according to Zill and Harmon method [18] in modification of Yakovenko and Mihno [19].Glycolipids were separated by TLC and content of monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) was determined by densitometring TLC plates [20].Content of sulfoquinovosyl diacylglycerol (SQDG) was determined according to Kean [21].
Separation of thylakoid membranes was carried out in nondenaturing polyacrylamide gel for their disintegration [22].Content of chloroplast pigment-protein complexes was determined by Anderson method [23].
The experiments were fourfold reproduced; authenticity of differences between the mean arithmetic values of indices was set after the Student's criterion.Differences were considered as reliable at p £ 0.05.
Results and discussion.The significant changes in photosynthetic pigment composition in leaves of D. antarctica plants under UV-B radiation were noted.It induced degradation of chlorophylls (Chls) a, b and b-carotene.However, the Chl a/b ratio, lutein + zeaxanthin (Lu + Zea), violaxanthin (Vio) and neoxanthin (Neo) pools were unchanged.The total content of carotenoids (Cars) in D. antarctica plants under UV-B light was also stable.
The molar Chls/Cars ratio was lower in plants under UV-B radiation (1.62) in comparison with control one (2.36) (Fig. 1, a, b).The Chls/Cars ratio is an important characteristic of the pigment complex.Our data agree with the results of other authors which showed that total Chl concentration was significantly lower in the plants grown under UV-B radiation [24].
UV-B radiation induces the decrease in content of two galactolipids, MGDG and DGDG, in photosynthetic membranes by 40 and 22 %, respectively.At the same time MGDG/DGDG ratio decreased by 28 % (Fig. 1, c).SQDG content was unchanged in these conditions.The ratios between MGDG and xanthophylls in leaves are important characteristics of adaptation of lipid-pigment complexes.In particular, the MGDG/(Lu + + Zea) and MGDG/Vio ratios decreased by 38 % and 45 %, respectively, under UV-B action (Fig. 2).
The distribution of Chl in pigment-protein complexes (CP) of plants under UV-B radiation was presented in Table .The percentage of Chl in pigment-protein complexes of photosystem I (PSI) (CP1a + СР1) increased by 22 % in chloroplasts of irradiated plants.UV-B radiation induced the decrease in percentage of Chl by 34 % in photosystem II (PSII) reaction centre complexes.It was proved, that PSII in photosynthetic apparatus is the most susceptible part to UV-B radiation [25].These data indicate a direct disruption of PSII re-action  ).We obtained experimental results, which argue for degradation of b-carotene in carotenoid pool of leaves of irradiated plants (Fig. 1, b). b-Carotene is localized in antenna and reaction centers of PSII.Quenching of triplet state of the primary donor ( 3 P 680 ) and deoxidation of the oxidized form (P 680 + ) play an important role in photoprotection.Singlet oxygen ( 1 O 2 ) and triplet excited chlorophyll ( 3 Chl*) are quenched also by b-carotene [26][27][28][29][30].The protection mechanism of photosynthetic apparatus against 1 O 2 leads to the destruction of b-carotene molecules [31][32][33][34].
Under UV-B radiation the decrease in non-bilayer lipid content induces the decrease of MGDG/DGDG ratio in chloroplast membranes and chlorophyll-protein complexes.Therefore, photosynthetic membranes are more susceptible to UV-B radiation.The similar data were obtained in our previous investigations of wheat plants response to heat stress [35].In our previous publications the dependence between the decrease in MGDG content and the stable content of Vio and Zea in leaves of drought resistant cultivars of Triticum aestivum L. under conditions of drought stress was described for wheat plants [36].
Stable SQDG content against the background of galactolipids decrease in photosynthetic membranes can be considered as an adaptive reaction.It is known, that SQDG provides the maintenance of normal functional state of the PSII reaction center [37].Therefore, it is possible to presume, that under conditions of UV-B radiation SQDG may support optimum conditions for light phase of photosynthesis.It is established, that the changes in the ratios of different lipid groups, induced by various stress factors are the reflection of a passive adaptation.An active adaptation is associated with the intensity of fatty acids desaturation [33,38,39].
Under UV-B radiation MGDG content decreased whereas xanthophyll pool was unchanged.The decrease in b-carotene content may influence the thylakoid membrane fluidity and activate free radical oxidation of lipids that leads to a decrease in MGDG and DGDG content.
Our data coincide with the results of Sakaki [38], who found out the pigment and lipid (mainly MGDG) decrease at the oxidative stress caused by ozone action.However, the anionic lipid content was stable during the whole exposition period.UV-B action upon D. antarctica plants induced the photoinhibition process (degradation of D1 protein of PSII reaction centre) [14].It is possible that SQDG localized on the surface of the native D1/D2 heterodimers stabilizes it at stress conditions [40,42].
UV-B action causes different responses including functional changes and changes of pigment-protein content in photosynthetic apparatus [43].Pigment-protein composition in chloroplasts treated by UV-B radiation changed.Especially, the content of Chl in pigment-protein complexes of PSI (CP1a + СР1) increased (by 22 %), while it decreased (by 34 %) in pigment-proteins complexes of PSII reaction centre.As it has been above mentioned PSII is known as the most susceptible part of the photosynthetic apparatus to UV-B radiation [25] that affects both the donor and acceptor sides of PSII.Inhibition of PSII electron transport after UV-B irradiation was studied in isolated spinach PSII membrane particles, using low-temperature EPR spectroscopy and chlorophyll fluorescence measurements.There were noted inhibition of oxygen evolution, decline of variable chlorophyll fluorescence and degradation of D1 protein in reaction center after UV-B radiation action [43].Thus, we described the structural changes at the level of photosynthetic apparatus of endemic antarctical plant D. antarctica under the action of UV-B radiation.The results of our work are essential for deeper understanding of adaptation mechanisms of this unique antarctical plant under stress conditions.