Vol. 59, No 1/2012

The effect of carotenoids on stability of model photosyn-thetic pigment-protein complexes subjected to chemical oxidation with hydrogen peroxide or potassium ferricya-nide was investigated. The oxidation of carotenoid-less and carotenoid-containing complexes was conducted in the presence or absence of ascorbic acid. The progress of the reactions was monitored by use of absorption and fluorescence spectroscopy. Our results show that carot-enoids may significantly enhance the stability of photo-synthetic complexes against oxidation and their protective (antioxidant) effect depends on the type of the oxi-dant.


INTRODUCTION
Light is the most important environmental factor for plants.As energy source, light is converted to chemical energy through photosynthesis by which nearly all energy enters our biosphere.Photosynthetic rate depends on photo flux density (PFD).But not all light energy absorbed by pigments is used for photosynthetic carbon dioxide fixation.Light stress is the normal situation for the photosynthesis in plants even in the temperate region.Various factors (temperature, drought, nutrient deficiency, etc.) are known to have consequences on the photosynthetic apparatus, and photoinhibition may be often observed under these environmental conditions.To cope with absorption of excessive light and its consequences, the photosynthesizing organisms have evolved a series of photoprotective mechanisms.Light absorption reduction and excess light energy dissipation by photosystems are the major aspects of plant photoprotective mechanisms.This energy dissipation becomes obvious by the non-photochemical quenching, which is a mixture of different underlying processes.In many cases the degree of non-photochemical energy dissipation is closely correlated with the activity of xanthophyll cycle (XC) (Demmig-Adams, 2003).The purpose of this study is to examine simultaneous changes in photosynthesis, energy used in photochemical and non-photochemical processes and photoprotection through the regulation of XC in hoary plantain plants (Plantago media) in nature.

MATERIAL AND METHODS
Plantago media is a perennial, short-rhizome taprooted herb.Plantain is common in floodplain meadows and sparse forests.The study site (62°45'N, 55°49'E) was located in the South Timan on the left bank of the river Soiva.Experiments were performed in the first half of July in 2007 and 2010.Two groups of plants selected for experiments.The first group grew on the southeast-facing scree slope with sparse vegetation (sun plants).The second group grew on the narrow terrace at the foot of a slope in the dense herbage (shade plants).Measurements of microclimate parameters were performed with portable station (Data Logger LI-1400, USA).Leaf CO 2 -exchange rate were measured with an infrared gas analyzers (LCPro+ (ADC, UK).Chlorophyll a (Chl a) fluorescence was measured with a portable fluorometer (PAM-2100, Walz, Germany).Leaf samples were frozen in liquid nitrogen and stored at -76°C.Photosynthetic pigments were extracted with acetone.The pigment content was determined spectrophotometrically (UV-1700, Shimadsu, Japan).Individual Car were separated using the reversed-phase high-performance liquid chromatography (HPLC) system (Knauer, Germany) equipped with a column 4.0×250 mm Diasphere-110-C18NT.The pigments were eluted for 34 min with gradient solvent systems A (acetonitrile:methanol:water, 75:12:4, v/v) and B (methanol:ethyl acetate, 68:32, v/v) at a flow rate of 2 ml/min.Identification of Car was carried out by comparing of HPLC retention times with corresponding standarts.The de-epoxidation level of XC pigments was calculated according to equation: DEPS = (Z+0.5A)/(V+A+Z), where Z, A, and V designate zeaxanthin, antheraxanthin, violaxanthin, respectively.inhabiting in the dense herbage.The highest irradiance was 200-300 and 1300-1600 μmol/ (m 2 s) in shade and sun sites, respectively.Table 1 summarizes the results of morphological and physiological acclimation of plantain to irradiance.Sun-adapted plants (S-plant) formed leaves with lower area and higher dry mass per area as compared to shade-adapted plants (SH-plant).The content of the photosynthetic pigments strongly depends on growth conditions.Although leaves that were developed on full sunlight had reduced pigment concentration, the ratio Car/Chl was greater than in SH-plants.
The maximal photosynthesis rate in S-plant leaves was 10-12 μmol CO 2 / (m 2 s); while in SH-plants value of this index did not exceed 4 μmol/ (m 2 s).The photosynthetic rate (Pn) in the S-plant leaves was highest in the morning and the nearly full depression of Pn was observed at noon hours.The decreasing of Pn was not so significant in SH-plant leaves.HPLC-analysis revealed the presence of β-carotene and xanthophylls in Car pool.Lutein was predominated among xanthophylls and took 70% of total Car.The content and the conversion state of XC pigments increased significantly from morning to midday in S-plants (Table 2).The percentage of V in VAZ pool was the greatest, 85-90%, at midnight and decreased by 4 and 2 times in S-and SH-plant leaves at noon hours, respectively.An increase in Z content occurred concomitantly with the V decrease.Maximum part of Z in VAZ pool was 60% in the S-plant leaves.DEPS was signifi-cantly lower in SH-plants than that in S-plants, especially in the midday hours.We used epoxidase inhibitorsalicylaldoxime to evaluate the part of V, which was not involved in conversion.It was about 25% and 15% of total V pool in SH-and S-plant leaves respectively.
The ratio F v /F m , which represents the PSII maximum photochemical activity, remained relatively high (approx.0.8) through the day in July 2007.A moderate decrease of F v /F m at midday for S-plant leaves and in the afternoon for SH-plant leaves was noted in warmer July 2010 (Table 3).The coefficient of photochemical (qP) and non-photochemical (qN) quenching of Chl a fluorescence in S-plant leaves changed strongly as irradiance increased.The value of qN was smaller and changed less drastically in SH-plant leaves.The qP coefficient in SH-plant leaves was maintained on the high level during the whole day.
These results, together with earlier reported data (Golovko et al, 2011), showed that under excessive light, up to 90% of the absorbed energy may be dissipated via the qN pathway in hoary plantain plants, which were grown on the open slope and were adapted to high light conditions for long-time.The exact role of zeaxanthin in the qN-mechanisms is still under debate (Janhs et al, 2009).The close relation between heat dissipation and the conversion state of XC pigments in plantain leaves was revealed.The part of light energy dissipated as heat in LHC PSII of plantain hoary S-and SH-plant leaves in midday hours amounted to 75% and 30%, respectively.In conclusion, our data support the idea, that carotenoids play an important role in protection of photosynthetic apparatus and adaptation of Plantago media plants under natural conditions.

Table 1 . Area (S), leaf mass per area (LMA) and content of pigments in Plantago plants
Data are mean ± SE of 15-20 leaves for S and LMA, and of 5 samples for pigments

Table 3 . The leaf chlorophyll fluorescence parameters and heat dissipation (D) (July, 2010)
Data are mean ±S.E. of 25-30 measurements for each point