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effects of selenium stress on photosynthetic pigment contents and

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					Journal of Plant Physiology and Molecular Biology 2005, 31 (4): 369 373

369

Effects of Selenium Stress on Photosynthetic Pigment Contents and Growth of Chlorella vulgaris
CHEN Tian-Feng1, ZHENG Wen-Jie1*, LUO Yong2, YANG Fang1, BAI Yan1, TU Fang1
(1Department of Chemistry, Jinan University; 2Guangzhou Jinan Biomedicine Research and Development Base, Guangzhou 510632, China)

Abstract: Changes in photosynthetic pigment and protein contents, growth and the spectral characteristics in Chlorella vulgaris in response to selenium stress were investigated. Carotenoids (β-carotene and xanthophylls) and chlorophyll (Chl a and Chl b) contents in cells exposed to Se 50 mg/L increased primarily and decreased afterwards, while photosynthetic pigments in cells exposed to Se 800 mg/L decreased significantly. Chlorophyll (Chl) absorption peak at 693 nm and prominent Chl emission peak at 700 nm, weakened significantly after Se stress. The excitation spectra showed a decrease in excitation energy transfer efficiency in Se-stressed cells. Total soluble protein decreased after Se stress. The changes in total Se, Mg2+ , Ca2+, K+ and Na+ concentrations in culture medium and cells were also determined by ICP-AES.
Key words: selenium stress; Chlorella vulgaris; photosynthetic pigment; spectral characteristics

cellular phototrophic marine alga. Our previous works have shown that it possesses a good tolerance to high levels of Se, which indicated that C. vulgaris is a good carrier for Se transformation. In this paper, we report the changes in biochemical composition (pigments including chlorophylls and carotenoids, and proteins), and the spectral characteristics of C. vulgaris exposed to different Se stress (50 mg/L and 800 mg/L). The absorption and biotransformation of Se and changes in concentrations of Mg2+ , Ca2+, K+ and Na+ in culture medium and test cells were also studied, which enhanced our understanding of the interactions between Se and marine microalgae.

1

Materials and Methods

The trace mineral selenium (Se) is an essential nutrient of importance to human biology. Se deficiency may cause such diseases as cardiomyopathy, cancer, endemic osteoarthropathy, anemia, etc. The effectiveness of Se supplementation in the prevention of chronic selenium deficiency symptoms such as those in Keshan disease in China has been well recognized (Zheng and Ouyang 2001). Considerable efforts have been performed to investigate the relationship between selenium and marine phytoplankton (Wang et al. 2001a, b). Most of the efforts have been focused on the accumula tion a nd uptake of selenium in different conditions. The effects of selenium on physiological, biochemical, and biophysical aspects of phytoplankton properties have been investigated to a smaller extent. Chlorella vulgaris is a widely used typical uni-

1.1 Test organism, medium and stress treatments The microalgal species used in this study was C. vulgaris, kindly provided by the Research Center of Hydrobiology of Jinan University (Guangzhou, China), and the axenic treatment was carried out in our laboratory. The cultivation of C. vulgaris was carried out in 250-mL Erlenmeyer flasks containing 100 mL UC-1 medium (pH 6.0) at 30 with a light illumination of 88 MJ m-2 s-1 and a 14 h/10 h light/dark cycle. The experiment was started with a 10% (V/V) inoculation from the stock cultures. Se was added on the 5th day in the form of sodium selenite (Na 2SeO3, AR) to get different final concentrations. 1.2 Growth and protein measurements Growth of the microalgal cultures was measured
Received 2004-07-20, Accepted 2005-06-27. This work was supported by the Key Project of Education Ministry of China (No.01141), and Natural Science and Technology Project of Guangzhou (No.2001-J-010-01).
* Corresponding au thor (E-mail: tzhwj@jnu.edu.cn; Tel: 02 0-

85226360).

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daily as dry weight. Soluble protein content was measured by a UV absorbance method (Murphy and Kies 1960). 1.3 Pigment extraction and HPLC analysis Cell suspensions (10 mL) were centrifuged at 1 200×g for 10 min at 4 . The pellets were resuspended in 10 mL acetone, and then disrupted by sonication on ice (4 s, interval: 5 s; 30 times). The mixtures were centrifuged at 8 000×g for 10 min at 4 to remove debris. Pigments in the extract were measured by an isocratic RP-HPLC (HP1100) method for simultaneous determination of pigments, using a C18 colu mn a n d a mo b i l e p h a s e c o n s i s t i n g o f dichloromethane acetonitrile methanol water (22.5 9.5 67.5 0.5) (Yuan et al. 1997). The flow rate was 1.0 mL/min. The pigments were detected by a UV-Vis absorbance detector set at 450 nm with a reference wavelength of 730 nm. 1.4 Absorption spectra and fluorescence measurements The UV-Vis spectra of the intact and the Sestressed cells were recorded on a UV500 spectrophotometer between 400 and 750 nm. Room-temperature fluorescence spectra were recorded on a 970CRT fluorescent spectrophotometer with different determination condition. 1.5 Determination of total Se (IV) and Mg2+, Ca2+, K+ and Na+ concentrations Cells exposed to different concentrations of Se (IV) for 6 days were harvested. The filtrate was prepared for determination of total Se (IV), Mg2+, Ca 2+ , K+ and Na+ in the culture medium. Washed pellets were resuspended in 3 mL of concentrated nitric acid and digested in a intelligent digestion system for 2 h (250 ), then the volume was reconstituted to 10 mL using HCl 1 mol/L. The Se concentration of the digested solutions and the media was analysed by ICPAES method on an Optime 2 000 DV spectrometer. All the determinations were performed at least in triplicates and the average values were presented.

ence in biomass concentration, protein and Se contents between control cultures and Se-stressed C. vulgaris. Low Se concentrations (1–25 mg/L) caused slight increase in biomass concentrations. The Se 50–200 mg/ L resulted in lower biomass concentrations and protein contents, with the significant increase in Se contents. The much higher Se concentrations (400– 1 000 mg/L) led to the significant decrease in biomass concentrations. As shown in Fig.2, the addition of Se 50 mg/L provoked a significant increase in biomass concentration from the 6th to the 8th day, while the adverse effect on biomass concentration was observed under Se concentration of 800 mg/L.

Fig.1

Effects of Se on the biomass, protein and Se contents of

C. vulgaris

Fig.2

Effects of selenium on the growth of C. vulgaris

2
2.1

Results
Changes in growth and protein content As shown in Fig.1, there were significant differ-

2.2 Changes in photosynthetic pigments content of C. vulgaris after Se stress After 6 days of Se stress, chlorophyll contents were affected more seriously than carotenoid contents. An significant increase in carotenoids/Chl ratio in response to Se stress was observed. As shown in Fig.3, under high Se stress (800 mg/L), significant declines

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Fig. 3

Changes in photosynthetic pigment concentration in C. vulgaris cell suspensions exposed to different Se stress

in the overall photosynthetic pigment concentrations in C. vulgaris cells were evident compared with control culture. The total chlorophyll content showed decline, while carotenoids including β-carotene and xanthophyll increased gradually with the growth of cells. However, the low Se concentration (50 mg/L) assayed provoked a significant increase of carotenoids for a short time, which began to decrease after the 9th day. Similarly, the total chlorophylls increased nearly 1 time in the first day after being subject to Se stress and decreased afterwards. 2.3 Changes in total Se(IV), Mg2+, Ca2+, K + and Na+ concentrations in media and C. vulgaris cells The Mg2+, Ca 2+, K+ and Na+ contents in the cells can directly affect the membrane permeability and chloroplast ultrastructure of C. vulgaris. As shown in table
Table 1 Ions Se 0 mg/L Mg K
+ 2+

1, according to the LSD test on the level of P<0.05, there was significant decrease in overall concentrations of Mg2+, Ca2+, K+ and Na+ between control culture and Se-stressed cultures of C. vulgaris. However, there was a converse trend in culture media, concentrations of these ions increased significantly with the increase in Se concentr a tion. Se content wa s up to 2.71 mg/g for C. vulgaris cultured in Se (IV) 50 mg/L. While cultured in Se (IV) 800 mg/L medium, C. vulgaris had a final Se content of 17.26 mg/g. The high Se-enrichment for C. vulgaris may be due to the adsorption by dea d cells under letha l Se (IV) concentration. 2.4 Absorption spectra Significant changes in absorption spectra between control and Se-stressed cells were observed. The PSII

Change in total concentrations of Se (IV), Mg2+, Ca2+, K+and Na+* C. vulgaris cells (mg/g DW) Se 50 mg/L 115.57±0.85 3.79±0.12 3.72±0.05 0.22±0.01 2.71±0.16 Se 800 mg/L 66.49±2.20 3.73±0.05 2.66±0.07 0.36±0.02 17.26±0.44 Se 0 mg/L 8.90±0.32 5.65±0.04 195.00±0.31 288.01±0.79 0.00±0.00 Culture media (mg/L) Se 50 mg/L 11.27±0.16 6.64±0.17 188.02±0.27 283.00±1.52 37.61±1.24 Se 800 mg/L 102.26±1.29 8.76±0.21 242.03±0.43 265.07±1.48 682.72±2.81

117.34±1.64 4.23±0.04 8.97±0.08 0.33±0.01 0.00±0.00

Ca2+ Na+ Se (IV)

Data are expressed as mean±SD of three cultures.

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chlorophylls absorption peak of C. vulgaris at 693 nm was significantly weakened after Se stress. Absorption bands of Chl b occurring at 653 nm also decreased, while the carotenoid peaks A449 and A509 rose slightly. The increase in the relative carotenoid abundance compared to Chl a was observed, which can improve energy collection efficiency under a variety of external and internal conditions and at the same time prevent harmful over-excitation and subsequent damage (Grudzinski et al. 2002). 2.5 Room-temperature fluorescence spectra Room-temperature fluorescence emission spectra were obtained when the samples were excited at 445 nm and 476 nm. These spectra only displayed a prominent peak at approximately 685 nm (F685). An important feature of the spectra was the intensity of the fluorescence peaks F685, which showed a major decrease under different Se stress, indicating that there were highly fluorescent Chl-containing complexes in C. vulgaris cells and the Se stress resulted in qualitative changes in the light-harvesting structure (Li et al. 2003). Fluorescence at 685 nm was attributed to the PSII core antenna pigment-proteins. There was significantly higher energy dissipation under Se stress as that part of the energy transferred to the PSII did not flow though the regular way but was transferred to the protein of pigment-protein complexes (PPC) (Jansson 1994). This part of energy was dissipated mainly though enhanced fluorescence in the 685 nm region.

Se, uptake of Se in excess to requirements may cause some metabolic reactions, or possibly some phototoxic responses (Vangronsveld and Clijsters 1994). As seen in Fig.3, significant decreases in the level of total chlorophyll and to a lesser degree in that of carotenoids were evident in C. vulgaris cells in response to Se stress of 50 mg/L and 800 mg/L, suggesting that the chlorophyll synthesizing system or the chlorophyllase activity were affected during Se stress. Substitution of Mg2+ by Se may also be another damage mechanism. Changes in membrane permeability and chloroplast ultrastructure contributed to declines in photosynthetic pigments levels due to lipid peroxidation (Prasad and Strzalka 1999). In our study, the changes in room-temperature fluorescence spectra showed the Se (IV) stress can cause some changes in the light-harvesting structure and higher energy dissipation. Relationships were also observed between the declines of photosynthetic pigment contents and the increase in concentrations of Mg2+, Ca2+, K+ and Na+ inside C. vulgaris cells (Fig. 3 and Table 1). Decreases in pigments would lead to reductions of carbon fixation and possible effects at the whole organism levels (Macfarlane and Burchett 2001). The decrease in protein content and biomass should be the results of this damage. References
Francois DL, Robinson GGC (1990). Indices of triazine toxicity in Chlamydomonas geitleri Ettl. Aquat Toxicol, 16: 205228 Grudzinski W, Krupa Z, Garstka M (2002). Conformational rearran gem en ts in light-h arvestin g com plex II accompanying light-induced chlorophyll a fluorescence quenching. Biochim Biophys Acta, 1554: 108–117 Jansson S (1994). The light-harvesting chlorophyll a/b-binding proteins. Biochim Biophys Acta, 1184: 1–19 Li DX, Cong W, Cai ZL, Shi DJ, Ouyang F (2003). Some physiological and biochemical changes in marine eukaryotic red tide alga Heterosigma akashiwo during the alleviation from iron limitation. Plant Physiol Biochem, 41: 295–301 Macfarlane GR, Burchett MD (2001). Photosynthetic pigments and peroxidase activity as indicators of heavy metal stree in the Grey mangrove, Avicennia marina (Forsk.) Vierh. Mar Pollut Bull, 42(3): 233–240 Murphy JB, Kies MW (1960). Note on spectrophotometric determination of proteins in dilute solutions. Biochim

3

Discussion

C. vulgaris was found to have a high tolerance to Se, though Se may cause some abnormal physiological and biochemical changes in C. vulgaris. As shown in Fig.1, high Se enrichment in C. vulgaris cells was observed, suggesting that C. vulgaris was a good carrier for Se transformation. The stimulative effects, obtained at low Se concentrations, indicate the ability of C. vulgaris to resist and to adapt to the inhibitory effect of external stress, which can also be interpreted as a tolerance mechanism (Francois and Robinson 1990) resulting from a homeostatic mechanism triggered by the exposure to the Se stress. Although C. vulgaris was considered tolerant to

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silicate on Cr (VI) and Se (IV) accumulation in marine phytoplankton. Aquat Toxicol, 52: 39–47 Yuan JP( Gong XD( ), Zhang YM( ), Chen F( ), Shi XM ( ), ) (1997). Simultaneous

Prasad MNV, Strzalka K (1999). Impact of heavy metals on photosynthesis. In: Prasad MNV, Hagemeyer J (eds). Heavy Metal Stress in Plants: from Molecules to Ecosystems. Berlin: Springer, 117–138 Vangronsveld J, Clijsters H (1994). Toxic effects of metals. In: Farargo ME (ed). Plants and the Chemical Elements. Weinheim: VCH, 149–177 Wang WX, Robert CHD (2001a). Effects of major nutrient additions on metal uptake in phytoplankton. Environ Pollut, 111: 233–240 Wang WX, Robert CHD (2001b). Influences of phosphate and

determination of cartenoids and chlorophylls in algae by high performance liq uid ch romagraph y. Ch in J Chromatogr ( Zheng WJ ( ), 15(2): 133–135 (in Chinese) ), Ouyang Z( )(2001). Organic

Selenium Compounds from Plants: Their Chemistry & Applications in Medicine. Guangzhou: Jinan University Press (in Chinese)

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