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Changes of microsomal membrane properties in spring wheat leaves (Triticum aestivum L.) exposed to enhanced ultraviolet-B radiation
Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60–65 www.elsevier.nl / locate / jphotobiol
Changes of microsomal membrane properties in spring wheat leaves (Triticum aestivum L.) exposed to enhanced ultraviolet-B radiation a,b
a
a
Lizhe An , Huyuan Feng , Xudong Tang , Xunling Wang
a,c ,
*
a
b
School of Life Science, State Key Laboratory of Arid Agrioecology, Lanzhou University, Lanzhou 730000, PR China State Key Laboratory of Frozen Soil Engineering, Lanzhou Institute of Glyciology and Geocryology, CAS, Lanzhou, 730000, PR China c Department of Biology, Northwest University, Xi’ an, 710069, PR China Received 2 November 1999; accepted 26 June 2000
Abstract The properties of microsomal membranes in spring wheat leaves (Triticum aestivum L. cv. Ganlong No. 92-005) exposed to (0) control, 8.64 (T 1 ) and 11.2 kJ m 22 day 21 (T 2 ) biologically effective UV-B irradiation (UV-B BE ) were studied under greenhouse conditions. These irradiance levels correspond to a decrease in the stratospheric ozone of ¯12.5 and 20%, respectively, for a clear solstice day at Lanzhou (36.048N, 1550 m), China. Compared with controls, the content of malondialdehyde (MDA) increased by 70.8% in T 1 and 83.8% in T 2 on the 7th day of the radiation, and the IUFA (index of unsaturated fatty acids) decreased, indicating peroxidation of lipid acids. Simultaneously, a drastic decrease of phospholipid content after 21 days and an increase of membrane lipid microviscosity on UV-B irradiation were also found, suggesting a reduction in the fluidity of membrane lipids. Ethylene emission by the microsomal membrane, in the presence of exogenous 1-aminocyclopropane-1-carboxylic acid was higher in the wheat seedlings after 7, 14 and 21 days’ irradiation than in the controls. These changes were correlated with a rise in lipoxygenase activity. Membrane-bound enzymes (Ca 21 -ATPase and Mg 21 -ATPase) were promoted by UV radiation in the first 7 days and significantly decreased after 14 and 21 days’ treatment in comparison to control. Our results suggest that UV-B radiation may cause changes in structural complexity and function of microsomal membranes in spring wheat leaves. 2000 Elsevier Science S.A. All rights reserved. Keywords: Ca 21 (Mg 21 )-ATPase; IUFA; Lipid peroxidation; MDA; Microsomal membrane properties; Triticum aestivum; UV-B radiation
1. Introduction Current solar ultraviolet-B (UV-B, 280–315 nm) radiation reaching the earth’s surface is thought to be close to maximum because stratospheric ozone levels are near their lowest point since measurements began and this trend is expected to continue [1]. An increase in UV-B radiation at the earth’s surface will have potentially adverse effects on agricultural production and natural plant ecosystems [2–4]. Numerous studies have demonstrated that UV-B irradiation can affect many aspects of plant growth, development and metabolism. There are three potential targets for UV-B radiation in plant cells, the genetic system, the photosynthetic system and membrane lipids [2–5]. Biomembranes are regarded as a main potential target of UV-B [4,6,7]. Several studies showed that increased UV-B ir*Corresponding author. E-mail address: [email protected] (X. Wang).
radiation could decrease plasma membrane ATPase [6], increase membrane permeability [8], cause damage to unsaturated fatty acids of membrane lipids [7,9,10] and even change the properties of thylakoid membrane [6]. In addition, UV-B increased production of ethylene, ethane and MDA, decreased the content of monogalactosyldiacylglycerol (MGDG), accumulated biamine putrescine and polyamine spermidine in cotyledon and leaf tissues of cucumber and led to lipid peroxidation [7,9– 11,29]. To our knowledge, however, relatively little is known about the effect of UV-B radiation on microsomal membrane characteristics. Caldwell reported that UV-B can induce photodegradation of cucumber microsomal tryptophanyl residues and the generation of free radical or reactive oxygen species in vitro [12] and Bose and Chatterjee observed UV-induced changes in microviscosity, permeability and malondialdialdehyde formation in liposomal membrane [13]. In the present study we assessed
1011-1344 / 00 / $ – see front matter 2000 Elsevier Science S.A. All rights reserved. PII: S1011-1344( 00 )00077-4
L. An et al. / Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60 – 65
the effect of enhanced solar UV-B radiation on plant microsomal membrane properties of spring wheat (Triticum aestivum) in vivo under greenhouse conditions.
2. Materials and methods
2.1. Plant growth and UV-B radiation treatment Grains of spring wheat (Triticum aestivum L. cv. Ganlong No. 92-005) were sterilized by 0.5% NaOCl solution for 20 min, then pre-germinated for 24 h in moistened plates. They were sown in plastic pots (30 cm340 cm) with a 14-h photoperiod in an unshaded-glass greenhouse. Thirty seeds were planted in each pot, when the second leaf had emerged, the seedlings were thinned to 18 per pot. Seedlings were watered daily and fertilized every 2 days with half Hoaglands’ solution. Pots were randomized every few days to diminish the differences. After emergence of the 3rd leaf, seedlings were divided into three groups, ten pots per group. One was used as control (without UV-B but identical light and temperature regimes as for UV-B treatment) and the others as UV-B radiation test samples. Enhanced UV-B radiation was provided by filtered Qin brand (Baoji Lamp Factory, China) 30-W fluorescence sun lamps [14]. The lamps were suspended above and perpendicular to the canopy of seedlings and filtered with either 0.13-mm thick cellulose diacetate (transmission down to 290 nm) for UV-B irradiance or 0.13-mm polyester plastic films (absorbs all radiation below 320 nm) as a control. Cellulose diacetate filters were presolarized. The desired irradiation was obtained by changing the distance between the lamps and the top of seedlings. The spectral irradiance from the lamps was determined with an Optronics Model 742 (Optronics Labs., Orlando, FL, USA) spectroradiometer and weighted with the generalized plant action spectrum [15] normalized to 300 nm to obtain the biologically effective UV-B radiation (UV-B BE ). Two levels of UV-B 22 21 22 irradiation were 8.64 kJ m day (T 1 ) and 11.2 kJ m 21 day (T 2 ) which simulated |12.5% and 20% stratospheric ozone depletion, respectively, on clear summer solstice (Lanzhou, 36.048N, 1550 m) China, using the model of Green et al. [16]. In addition to UV-B radiation, visible radiation (photosynthetically active radiation, PAR 400–700 nm) (400–700 mmol m 22 s 21 ) was also supplied to avoid exaggerating the effects of increased UV-B radiation at lower PAR [3]. UV-B radiation was supplemented for 6 h daily centered at solar noon. The air temperature and relative humidity in the greenhouse were maintained at 25 / 158C and 55 / 75% (day / night), respectively. After 1, 2 and 3 weeks of UV-B radiation the youngest fully expanded leaves were collected at 09:00 a.m. and immediately frozen in liquid nitrogen. Tissues were then subjected to biochemical analyses.
61
2.2. Microsomal membrane preparation Microsomal membranes were prepared by following the process of Caldwell [12] with some modifications. Leaves were cut into pieces and immediately homogenized in blender with cold isolation medium (in a ratio of 3 ml to 1 g fresh weight tissue) containing 0.25 mol / l sucrose, 5% (w / v) soluble polyvinylpyrrolidone (PVP) 2.5 mmol / l EDTA, 250 mmol / l phenylenethyl sulfonyl fluoride (PMSF), 2 mmol / l dithiothreitol (DDT), 0.2% bovine serum albumin (BSA) and 25 mmol / l Tris–HEPES buffer (pH 7.6). The homogenate was filtered through four layers of cheesecloth and centrifuged at 15 000 g for 20 min. The supernatant was again centrifuged for 1 h at 100 000 g to obtain a microsomal pellet, which was resuspended in a buffer containing 0.25 mol / l sucrose, 1 mmol / l EDTA and 25 mmol / l HEPES–KOH buffer (pH 7.2) and recentrifugated at 100 000 g for 1 h. Finally the pellets were suspended in the same buffer. All procedures were conducted below 48C.
2.3. Protein determination and MDA analysis Membrane protein concentration was determined by the method of Bradford [17] using BSA as standard. MDA content was measured according to Buege and Aust [18].
2.4. Lipid analysis and measurement of phospholipid concentration The lipid composition was analyzed according to Norberg and Liljenberg [19] using gas chromatography (GC9A, Shimadzu, Columbia, MD, USA) and the phospholipid content was determined as described by Hagege et al. [20] with a UV–vis spectrophotometer.
2.5. Measurements of ethylene evolution The assay system of ethylene formation formed from exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) contained the following in a volume of 1 ml: (a) 0.2 ml of microsomal membrane suspension, (b) 0.7 ml of 2 mmol / l Hepes–KOH buffer (pH 7.2), (c) 0.1 ml of 10 mmol / l ACC to initiate the reaction. The reaction system was immediately capped in the tubes and incubated at 298C for 1 h. Ethylene was measured using a Shimadzu GC-9A gas chromatograph equipped with a flame ionization detection system (FID) and an Al 2 O 3 column (60 cm30.32 cm). A 1-ml headspace gas was used for ethylene determination.
2.6. Determination of lipid fluidity Microsomal membrane lipid fluidity was measured by steady-state fluorescence depolarization of l-anilino-8naphthalene sulfonic acid (ANS)-labeled membranes in a
L. An et al. / Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60 – 65
62
Hitachi 850 fluorescence spectrofluorometer as described by Thompson et al. [21].
2.7. Activity assay of lipoxygenase ( LOX), Ca 21 -ATPase and Mg 21 -ATPase Membrane-bound LOX activity was measured at pH 5.5 as oxygen consumption according to the method of Macri [22]. Activity of Ca 21 -ATPase and Mg 21 -ATPase were assayed according to Hao and Yu [23].
2.8. Statistical analysis UV-B treatment and sampling dates were arranged in completely randomized designs with three independent experiments with three replications except some explained in the text. Significant difference (at P,0.05 or P,0.01 level) was determined by one-way analysis of variance (ANOVA).
3. Results The fatty acid composition of the total lipid of microsomal membranes from spring wheat seedlings is shown in Table 1. After UV-B treatment, the IUFA (unsaturated: saturated fatty acids ratio) decreased, mainly due to decreases in the amounts of 18:2 (linoleic) and 18:3 (linolenic). Meanwhile, the content of MDA, as an indicator of lipid peroxidation, was significantly higher under the two levels of UV-B treatment than in the controls (Fig. 1). After 7 days radiation, there was an increase in MDA of 70.8% (T 1 ) and 83.8% (T 2 ) compared with control (Fig. 1). But lower MDA accumulation (still higher than the control) and higher IUFA were observed after 14 days of irradiation. This may be related to a temporary adaptation to UV-B and / or UV-B-stimulated protective responses, such as increases in free radical-scavenging enzymes and UV-B absorbing compounds (unpublished
Fig. 1. Effects of supplementary UV-B radiation on MDA content of microsomal membranes in spring wheat seedlings. Bars represent standard deviations of three independent experiments with three replicates, Circles, squares and triangles represent controls, T 1 and T 2 , respectively.
data). Although the content of MDA on the 21st day was lower than on the 7th and 14th days, the large decrease in phospholipid content after 3 weeks treatment (Fig. 2) and the increase in membrane lipid microviscosity (Table 2) indicated the membrane lipid peroxidation and membrane damage. Phospholipid content was significantly increased by the
Fig. 2. Effects of enhanced UV-B radiation on phospholipid content of microsomal membranes in spring wheat seedlings. Bars represent standard deviations of three independent experiments with three replicates; the letters a, b and c represent control, T 1 and T 2 , respectively.
Table 1 Effects of enhanced UV-B radiation on the fatty acid composition of the total lipid extract from microsomal membranes in spring wheat seedlings; the values are the means of three independent experiments with three replicates IUFA5(18:1 mol%118:2 mol%32118:3 mol%33)3100 Treatment
Days
IUFAa
Composition of fatty acids (mol%) 16:0
18:0
18:1
18:2
18:3
Control T1 T2
7
23.160.7 21.560.3 3.260.3
1.560.3 2.160.5 5.860.9
20.760.5 32.061.0 73.462.4
48.162.2 39.261.0 11.060.2
7.160.4 5.260.6 6.560.7
138.266.1 a 126.064.8 b 114.965.0 c
Control T1 T2
14
20.061.0 19.360.4 19.160.4
1.960.3 1.760.4 1.460.2
20.760.8 18.860.9 18.660.3
50.360.9 52.562.0 55.162.0
7.060.4 7.760.2 5.960.3
142.363.8 ns 146.961.9ns 146.561.6ns
Control T1 T2
21
24.560.6 35.860.1 26.061.1
9.360.5 1.560.2 3.160.4
12.860.4 14.060.4 18.761.2
52.861.7 47.761.7 42.961.5
10.660.5 1.060.1 9.360.8
140.263.4a 102.164.1c 132.464.6b
a Different letters in the column of every treatment time shows different significance levels at P,0.05 using ANOVA; ns, means not significant at P,0.05 level.
L. An et al. / Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60 – 65 Table 2 Effects of enhanced UV-B radiation on microviscosity of microsomal membranes in spring wheat leaves; data are the means of three independent experiments Treatment
Microviscosity a (poise) 7 days
Control T1 T2
14 days c
1.3760.08 1.4260.07 b 1.8360.07 a
21 days b
1.8260.04 c 1.9760.02 b 2.0860.02 a
1.4060.01 1.7660.05 a 1.4560.04 b
a
Different superscript letters in each column show different significance levels at P,0.05 using ANOVA.
63
7th and 14th days and a sharp reduction as seen on the 21st day under UV-B radiation. On the 14th and 21st day, the microviscosity of microsomal membranes in spring wheat seedlings increased significantly under enhanced UV-B radiation (Table 2). Ethylene production was significantly higher in the microsomal membrane system compared to the controls (Fig. 3). With the duration of UV-B treatment, evolution of ethylene was gradually increasing. In addition, there seemed to be a dose effect in response to increased UV-B radiation (Fig. 3). The activity of LOX also increased under UV-B radiation after 7, 14 and 21 days of treatment as compared with controls (Fig. 4). Ca 21 - and Mg 21 -ATPase, which are important enzymes bound to microsomal membranes, were influenced by increased UV-B irradiation (Table 3). On the 7th day, these two enzymes were stimulated by UV-B, after 14th and 21st day, however, they were significantly inhibited in comparison to the controls.
4. Discussion
Fig. 3. Effects of enhanced UV-B radiation on ethylene evolution of microsomal membranes, which was conversed from exogenous ACC in spring wheat seedlings. Bars represent standard deviations of three independent experiments with three replicates; the letters a, b and c represent control, T 1 and T 2 , respectively.
Fig. 4. Effects of enhanced UV-B radiation on lipoxygenase activity of microsomal membranes in spring wheat seedlings. Bars represent standard deviations of three independent experiments with three replicates; triangles, squares and circles represent control, T 1 and T 2 , respectively.
UV-B is a stress factor, and impacts on plant photomorphogensis, growth and development [2]. Membrane lipid, DNA and protein have proved to be susceptible to direct UV-B-induced photoxidation or indirect degradation [3,4]. Therefore, an in vivo microsomal membrane effect in spring wheat seedlings on enhanced UV-B irradiation was expected to be observed in this experiment. Our results presented here are in accord with those of Caldwell [12] showing that the protein of cucumber microsomal membranes can be modified in vitro by UV-B radiation and that the tryptophanyl residues can be photodegraded, and supporting the hypothesis that membrane is a target for UV-B radiation [6,7]. The increase of MDA, an indicator of membrane lipid peroxidation, is related to decrease of unsaturated fatty lipid content (Fig. 1 and Table 1), which mainly resulted from a reduction of linoleic (18:2) and linolenic (18:3) (Fig. 2). A pronounced increase in membrane microviscosity under enhanced UV-B was found, indicating decrease of membrane fluidity (Table 2). These results showed peroxidation of microsmoal membrane lipids in wheat seedlings under greenhouse conditions. Several reports
Table 3 Effects of enhanced UV-B radiation on ATPase activity of microsomal membrane in wheat leaves; data are means of two independent experiments with three replicates a Day
Ca 21 -ATPase (mmol Pi (mg protein 21 ) min 21 ) Control
T1 c
7 14 21
0.31660.05 0.44760.01 a 0.50460.07 a a
Mg 21 -ATPase (mmol Pi (mg protein 21 ) min 21 ) T2
b
0.33060.03 0.31460.02 c 0.28160.04 b
Control a
0.38160.02 0.32260.02 b 0.20760.09 c
T1 b
0.50860.02 0.59260.06 a 0.64060.01 a
Different superscript letters in a row show different significance levels at P,0.05 using ANOVA.
T2 c
0.35060.02 0.39760.07 b 0.25860.03 b
0.61160.04 a 0.27260.02 c 0.21060.05 c
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L. An et al. / Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60 – 65
which investigated other biomembranes such as plasma membrane, thylakoid membrane, liposomal membrane, etc., found they were sensitive to supplemental solar UV-B radiation [9,10,13]. For example, Bose and Chatterjee [13] observed that the susceptibility of liposomal membrane to UV light radiation was correlated with changes in microviscosity, permeability and MDA formation. Membrane lipid unsaturated acyl chains were sensitive to direct UV-B and indirect processes [24]. Direct UV-B effect may come from direct absorption of conjugated double bond system of unsaturated fatty acids, which may trigger a chain reaction resulting in peroxidation of unsaturated fatty acids [4] and indirect degradative effect was as a result of UV-B-mediated free radicals [25]. Other stress factors (e.g. salt, drought) could lead to lipid peroxidation of microsomal membranes in wheat [26,27]. The indications are that lipid peroxidation and related processes are a general response to various stresses including increased UV-B radiation. Membrane-binding proteins of spring wheat microsomal membrane, LOX (Fig. 4) and Mg 21 -ATPase and Ca 21 ATPase (Table 3) were also sensitive like membrane lipids to increased UV-B radiation. The changes of Ca 21 -ATPase, Mg 21 -ATPase activity and LOX indicated the alteration of microsomal membrane functions. Ethylene was thought to be generated by the process of peroxidation at the membrane level [28] through attacking the membrane phospholipids by LOX [29,30]. Thus, UV-B-induced increases LOX activity (Fig. 4) and ethylene evolution by in the presence of exogenous ACC (Fig. 3), indicated LOX may be involved in the process of lipid peroxidation by catalyzing the conversion of unsaturated acids to saturated ones. In a recent study, Long and Jenkins [31] observed that UV-B radiation could promote Ca 21 efflux from the cytosol and regulate calcium levels in a cytosol pool, partly via the action of specific Ca 21 -ATPase. The action spectrum for inactivation of plasma membrane bound ATPase peaks at 290 nm [8,12]. This paper has elucidated some effects of enhanced UV-B irradiance on the properties of microsomal membranes in spring wheat seedlings under greenhouse conditions. Our results, the lipid peroxidation of microsomal membrane and the changes of membrane-bound ATPase, LOX activities and ethylene emission in UV-B irradiated wheat seedlings, suggest an alteration of structure and function of microsome membranes in vivo. In addition, our data indicate that spring wheat cv. Ganlong 92-005 cultivar is sensitive to supplemental UV-B radiation, although there was some evidence that wheat, as a monocotyledonous species, was relatively resistant to UV-B compared to dicotyledonous ones (e.g. pulses) [32].
Acknowledgements This work was funded by the National Natural Science Foundation of China (No: 39670132, 39970126), State
Key Laboratory of Frozen Soil Engineering, Lanzhou Institute of Glaciology and Geocryology, CAS (9808), by key project-B, CAS (KZ952-S1-216) and by Fukang desert ecosystem observation and experiment station of Xinjiang, CAS.
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[26] F. Zeng, Y. An, H. Zhang, M. Zhang, The effects of La 31 on the peroxidation of membrane lipids in wheat seedlings leaves under osmotic stress, Biol. Trace Elem. Res. 69 (1999) 141–150. [27] X.X. Tang, J.F. Jia, G.C. Zheng, Damage responses to salt stress in salt-tolerant cell line of wheat, Acta Bot. Sin. 41 (1999) 757–760. [28] H.W. Gardner, J.W. Newton, Lipid hydroperoxides in the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene, Phytochemistry 26 (1987) 621–626. [29] D.T. Krizek, G.F. Kramer, A. Upadhyaya, R.M. Mirecki, UV-B response of cucumber seedlings grown under metal halide and high pressure sodium deluxe lamps, Physiol. Plant. 88 (1993) 350–358. [30] T.T. Wang, S.F. Yang, The physiological role of lipoxygenase in ethylene formation from 1-aminocyclopropane-1-carboxylic acid in oat leaves, Planta 170 (1987) 190–196. [31] J.C. Long, G.I. Jenkins, Involvement of plasma membrane redox activity and calcium homeostasis in the UV-B and UV-A / blue light induction of gene expression in Arabidopsis, Plant Cell 10 (1998) 2077–2086. [32] M. Tevini, A.H. Teramura, UV-B effects on terrestrial plants, Photochem. Photobiol. 50 (1989) 479–487.
Changes of microsomal membrane properties in spring wheat leaves (Triticum aestivum L.) exposed to enhanced ultraviolet-B radiation a,b
a
a
Lizhe An , Huyuan Feng , Xudong Tang , Xunling Wang
a,c ,
*
a
b
School of Life Science, State Key Laboratory of Arid Agrioecology, Lanzhou University, Lanzhou 730000, PR China State Key Laboratory of Frozen Soil Engineering, Lanzhou Institute of Glyciology and Geocryology, CAS, Lanzhou, 730000, PR China c Department of Biology, Northwest University, Xi’ an, 710069, PR China Received 2 November 1999; accepted 26 June 2000
Abstract The properties of microsomal membranes in spring wheat leaves (Triticum aestivum L. cv. Ganlong No. 92-005) exposed to (0) control, 8.64 (T 1 ) and 11.2 kJ m 22 day 21 (T 2 ) biologically effective UV-B irradiation (UV-B BE ) were studied under greenhouse conditions. These irradiance levels correspond to a decrease in the stratospheric ozone of ¯12.5 and 20%, respectively, for a clear solstice day at Lanzhou (36.048N, 1550 m), China. Compared with controls, the content of malondialdehyde (MDA) increased by 70.8% in T 1 and 83.8% in T 2 on the 7th day of the radiation, and the IUFA (index of unsaturated fatty acids) decreased, indicating peroxidation of lipid acids. Simultaneously, a drastic decrease of phospholipid content after 21 days and an increase of membrane lipid microviscosity on UV-B irradiation were also found, suggesting a reduction in the fluidity of membrane lipids. Ethylene emission by the microsomal membrane, in the presence of exogenous 1-aminocyclopropane-1-carboxylic acid was higher in the wheat seedlings after 7, 14 and 21 days’ irradiation than in the controls. These changes were correlated with a rise in lipoxygenase activity. Membrane-bound enzymes (Ca 21 -ATPase and Mg 21 -ATPase) were promoted by UV radiation in the first 7 days and significantly decreased after 14 and 21 days’ treatment in comparison to control. Our results suggest that UV-B radiation may cause changes in structural complexity and function of microsomal membranes in spring wheat leaves. 2000 Elsevier Science S.A. All rights reserved. Keywords: Ca 21 (Mg 21 )-ATPase; IUFA; Lipid peroxidation; MDA; Microsomal membrane properties; Triticum aestivum; UV-B radiation
1. Introduction Current solar ultraviolet-B (UV-B, 280–315 nm) radiation reaching the earth’s surface is thought to be close to maximum because stratospheric ozone levels are near their lowest point since measurements began and this trend is expected to continue [1]. An increase in UV-B radiation at the earth’s surface will have potentially adverse effects on agricultural production and natural plant ecosystems [2–4]. Numerous studies have demonstrated that UV-B irradiation can affect many aspects of plant growth, development and metabolism. There are three potential targets for UV-B radiation in plant cells, the genetic system, the photosynthetic system and membrane lipids [2–5]. Biomembranes are regarded as a main potential target of UV-B [4,6,7]. Several studies showed that increased UV-B ir*Corresponding author. E-mail address: [email protected] (X. Wang).
radiation could decrease plasma membrane ATPase [6], increase membrane permeability [8], cause damage to unsaturated fatty acids of membrane lipids [7,9,10] and even change the properties of thylakoid membrane [6]. In addition, UV-B increased production of ethylene, ethane and MDA, decreased the content of monogalactosyldiacylglycerol (MGDG), accumulated biamine putrescine and polyamine spermidine in cotyledon and leaf tissues of cucumber and led to lipid peroxidation [7,9– 11,29]. To our knowledge, however, relatively little is known about the effect of UV-B radiation on microsomal membrane characteristics. Caldwell reported that UV-B can induce photodegradation of cucumber microsomal tryptophanyl residues and the generation of free radical or reactive oxygen species in vitro [12] and Bose and Chatterjee observed UV-induced changes in microviscosity, permeability and malondialdialdehyde formation in liposomal membrane [13]. In the present study we assessed
1011-1344 / 00 / $ – see front matter 2000 Elsevier Science S.A. All rights reserved. PII: S1011-1344( 00 )00077-4
L. An et al. / Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60 – 65
the effect of enhanced solar UV-B radiation on plant microsomal membrane properties of spring wheat (Triticum aestivum) in vivo under greenhouse conditions.
2. Materials and methods
2.1. Plant growth and UV-B radiation treatment Grains of spring wheat (Triticum aestivum L. cv. Ganlong No. 92-005) were sterilized by 0.5% NaOCl solution for 20 min, then pre-germinated for 24 h in moistened plates. They were sown in plastic pots (30 cm340 cm) with a 14-h photoperiod in an unshaded-glass greenhouse. Thirty seeds were planted in each pot, when the second leaf had emerged, the seedlings were thinned to 18 per pot. Seedlings were watered daily and fertilized every 2 days with half Hoaglands’ solution. Pots were randomized every few days to diminish the differences. After emergence of the 3rd leaf, seedlings were divided into three groups, ten pots per group. One was used as control (without UV-B but identical light and temperature regimes as for UV-B treatment) and the others as UV-B radiation test samples. Enhanced UV-B radiation was provided by filtered Qin brand (Baoji Lamp Factory, China) 30-W fluorescence sun lamps [14]. The lamps were suspended above and perpendicular to the canopy of seedlings and filtered with either 0.13-mm thick cellulose diacetate (transmission down to 290 nm) for UV-B irradiance or 0.13-mm polyester plastic films (absorbs all radiation below 320 nm) as a control. Cellulose diacetate filters were presolarized. The desired irradiation was obtained by changing the distance between the lamps and the top of seedlings. The spectral irradiance from the lamps was determined with an Optronics Model 742 (Optronics Labs., Orlando, FL, USA) spectroradiometer and weighted with the generalized plant action spectrum [15] normalized to 300 nm to obtain the biologically effective UV-B radiation (UV-B BE ). Two levels of UV-B 22 21 22 irradiation were 8.64 kJ m day (T 1 ) and 11.2 kJ m 21 day (T 2 ) which simulated |12.5% and 20% stratospheric ozone depletion, respectively, on clear summer solstice (Lanzhou, 36.048N, 1550 m) China, using the model of Green et al. [16]. In addition to UV-B radiation, visible radiation (photosynthetically active radiation, PAR 400–700 nm) (400–700 mmol m 22 s 21 ) was also supplied to avoid exaggerating the effects of increased UV-B radiation at lower PAR [3]. UV-B radiation was supplemented for 6 h daily centered at solar noon. The air temperature and relative humidity in the greenhouse were maintained at 25 / 158C and 55 / 75% (day / night), respectively. After 1, 2 and 3 weeks of UV-B radiation the youngest fully expanded leaves were collected at 09:00 a.m. and immediately frozen in liquid nitrogen. Tissues were then subjected to biochemical analyses.
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2.2. Microsomal membrane preparation Microsomal membranes were prepared by following the process of Caldwell [12] with some modifications. Leaves were cut into pieces and immediately homogenized in blender with cold isolation medium (in a ratio of 3 ml to 1 g fresh weight tissue) containing 0.25 mol / l sucrose, 5% (w / v) soluble polyvinylpyrrolidone (PVP) 2.5 mmol / l EDTA, 250 mmol / l phenylenethyl sulfonyl fluoride (PMSF), 2 mmol / l dithiothreitol (DDT), 0.2% bovine serum albumin (BSA) and 25 mmol / l Tris–HEPES buffer (pH 7.6). The homogenate was filtered through four layers of cheesecloth and centrifuged at 15 000 g for 20 min. The supernatant was again centrifuged for 1 h at 100 000 g to obtain a microsomal pellet, which was resuspended in a buffer containing 0.25 mol / l sucrose, 1 mmol / l EDTA and 25 mmol / l HEPES–KOH buffer (pH 7.2) and recentrifugated at 100 000 g for 1 h. Finally the pellets were suspended in the same buffer. All procedures were conducted below 48C.
2.3. Protein determination and MDA analysis Membrane protein concentration was determined by the method of Bradford [17] using BSA as standard. MDA content was measured according to Buege and Aust [18].
2.4. Lipid analysis and measurement of phospholipid concentration The lipid composition was analyzed according to Norberg and Liljenberg [19] using gas chromatography (GC9A, Shimadzu, Columbia, MD, USA) and the phospholipid content was determined as described by Hagege et al. [20] with a UV–vis spectrophotometer.
2.5. Measurements of ethylene evolution The assay system of ethylene formation formed from exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) contained the following in a volume of 1 ml: (a) 0.2 ml of microsomal membrane suspension, (b) 0.7 ml of 2 mmol / l Hepes–KOH buffer (pH 7.2), (c) 0.1 ml of 10 mmol / l ACC to initiate the reaction. The reaction system was immediately capped in the tubes and incubated at 298C for 1 h. Ethylene was measured using a Shimadzu GC-9A gas chromatograph equipped with a flame ionization detection system (FID) and an Al 2 O 3 column (60 cm30.32 cm). A 1-ml headspace gas was used for ethylene determination.
2.6. Determination of lipid fluidity Microsomal membrane lipid fluidity was measured by steady-state fluorescence depolarization of l-anilino-8naphthalene sulfonic acid (ANS)-labeled membranes in a
L. An et al. / Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60 – 65
62
Hitachi 850 fluorescence spectrofluorometer as described by Thompson et al. [21].
2.7. Activity assay of lipoxygenase ( LOX), Ca 21 -ATPase and Mg 21 -ATPase Membrane-bound LOX activity was measured at pH 5.5 as oxygen consumption according to the method of Macri [22]. Activity of Ca 21 -ATPase and Mg 21 -ATPase were assayed according to Hao and Yu [23].
2.8. Statistical analysis UV-B treatment and sampling dates were arranged in completely randomized designs with three independent experiments with three replications except some explained in the text. Significant difference (at P,0.05 or P,0.01 level) was determined by one-way analysis of variance (ANOVA).
3. Results The fatty acid composition of the total lipid of microsomal membranes from spring wheat seedlings is shown in Table 1. After UV-B treatment, the IUFA (unsaturated: saturated fatty acids ratio) decreased, mainly due to decreases in the amounts of 18:2 (linoleic) and 18:3 (linolenic). Meanwhile, the content of MDA, as an indicator of lipid peroxidation, was significantly higher under the two levels of UV-B treatment than in the controls (Fig. 1). After 7 days radiation, there was an increase in MDA of 70.8% (T 1 ) and 83.8% (T 2 ) compared with control (Fig. 1). But lower MDA accumulation (still higher than the control) and higher IUFA were observed after 14 days of irradiation. This may be related to a temporary adaptation to UV-B and / or UV-B-stimulated protective responses, such as increases in free radical-scavenging enzymes and UV-B absorbing compounds (unpublished
Fig. 1. Effects of supplementary UV-B radiation on MDA content of microsomal membranes in spring wheat seedlings. Bars represent standard deviations of three independent experiments with three replicates, Circles, squares and triangles represent controls, T 1 and T 2 , respectively.
data). Although the content of MDA on the 21st day was lower than on the 7th and 14th days, the large decrease in phospholipid content after 3 weeks treatment (Fig. 2) and the increase in membrane lipid microviscosity (Table 2) indicated the membrane lipid peroxidation and membrane damage. Phospholipid content was significantly increased by the
Fig. 2. Effects of enhanced UV-B radiation on phospholipid content of microsomal membranes in spring wheat seedlings. Bars represent standard deviations of three independent experiments with three replicates; the letters a, b and c represent control, T 1 and T 2 , respectively.
Table 1 Effects of enhanced UV-B radiation on the fatty acid composition of the total lipid extract from microsomal membranes in spring wheat seedlings; the values are the means of three independent experiments with three replicates IUFA5(18:1 mol%118:2 mol%32118:3 mol%33)3100 Treatment
Days
IUFAa
Composition of fatty acids (mol%) 16:0
18:0
18:1
18:2
18:3
Control T1 T2
7
23.160.7 21.560.3 3.260.3
1.560.3 2.160.5 5.860.9
20.760.5 32.061.0 73.462.4
48.162.2 39.261.0 11.060.2
7.160.4 5.260.6 6.560.7
138.266.1 a 126.064.8 b 114.965.0 c
Control T1 T2
14
20.061.0 19.360.4 19.160.4
1.960.3 1.760.4 1.460.2
20.760.8 18.860.9 18.660.3
50.360.9 52.562.0 55.162.0
7.060.4 7.760.2 5.960.3
142.363.8 ns 146.961.9ns 146.561.6ns
Control T1 T2
21
24.560.6 35.860.1 26.061.1
9.360.5 1.560.2 3.160.4
12.860.4 14.060.4 18.761.2
52.861.7 47.761.7 42.961.5
10.660.5 1.060.1 9.360.8
140.263.4a 102.164.1c 132.464.6b
a Different letters in the column of every treatment time shows different significance levels at P,0.05 using ANOVA; ns, means not significant at P,0.05 level.
L. An et al. / Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60 – 65 Table 2 Effects of enhanced UV-B radiation on microviscosity of microsomal membranes in spring wheat leaves; data are the means of three independent experiments Treatment
Microviscosity a (poise) 7 days
Control T1 T2
14 days c
1.3760.08 1.4260.07 b 1.8360.07 a
21 days b
1.8260.04 c 1.9760.02 b 2.0860.02 a
1.4060.01 1.7660.05 a 1.4560.04 b
a
Different superscript letters in each column show different significance levels at P,0.05 using ANOVA.
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7th and 14th days and a sharp reduction as seen on the 21st day under UV-B radiation. On the 14th and 21st day, the microviscosity of microsomal membranes in spring wheat seedlings increased significantly under enhanced UV-B radiation (Table 2). Ethylene production was significantly higher in the microsomal membrane system compared to the controls (Fig. 3). With the duration of UV-B treatment, evolution of ethylene was gradually increasing. In addition, there seemed to be a dose effect in response to increased UV-B radiation (Fig. 3). The activity of LOX also increased under UV-B radiation after 7, 14 and 21 days of treatment as compared with controls (Fig. 4). Ca 21 - and Mg 21 -ATPase, which are important enzymes bound to microsomal membranes, were influenced by increased UV-B irradiation (Table 3). On the 7th day, these two enzymes were stimulated by UV-B, after 14th and 21st day, however, they were significantly inhibited in comparison to the controls.
4. Discussion
Fig. 3. Effects of enhanced UV-B radiation on ethylene evolution of microsomal membranes, which was conversed from exogenous ACC in spring wheat seedlings. Bars represent standard deviations of three independent experiments with three replicates; the letters a, b and c represent control, T 1 and T 2 , respectively.
Fig. 4. Effects of enhanced UV-B radiation on lipoxygenase activity of microsomal membranes in spring wheat seedlings. Bars represent standard deviations of three independent experiments with three replicates; triangles, squares and circles represent control, T 1 and T 2 , respectively.
UV-B is a stress factor, and impacts on plant photomorphogensis, growth and development [2]. Membrane lipid, DNA and protein have proved to be susceptible to direct UV-B-induced photoxidation or indirect degradation [3,4]. Therefore, an in vivo microsomal membrane effect in spring wheat seedlings on enhanced UV-B irradiation was expected to be observed in this experiment. Our results presented here are in accord with those of Caldwell [12] showing that the protein of cucumber microsomal membranes can be modified in vitro by UV-B radiation and that the tryptophanyl residues can be photodegraded, and supporting the hypothesis that membrane is a target for UV-B radiation [6,7]. The increase of MDA, an indicator of membrane lipid peroxidation, is related to decrease of unsaturated fatty lipid content (Fig. 1 and Table 1), which mainly resulted from a reduction of linoleic (18:2) and linolenic (18:3) (Fig. 2). A pronounced increase in membrane microviscosity under enhanced UV-B was found, indicating decrease of membrane fluidity (Table 2). These results showed peroxidation of microsmoal membrane lipids in wheat seedlings under greenhouse conditions. Several reports
Table 3 Effects of enhanced UV-B radiation on ATPase activity of microsomal membrane in wheat leaves; data are means of two independent experiments with three replicates a Day
Ca 21 -ATPase (mmol Pi (mg protein 21 ) min 21 ) Control
T1 c
7 14 21
0.31660.05 0.44760.01 a 0.50460.07 a a
Mg 21 -ATPase (mmol Pi (mg protein 21 ) min 21 ) T2
b
0.33060.03 0.31460.02 c 0.28160.04 b
Control a
0.38160.02 0.32260.02 b 0.20760.09 c
T1 b
0.50860.02 0.59260.06 a 0.64060.01 a
Different superscript letters in a row show different significance levels at P,0.05 using ANOVA.
T2 c
0.35060.02 0.39760.07 b 0.25860.03 b
0.61160.04 a 0.27260.02 c 0.21060.05 c
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L. An et al. / Journal of Photochemistry and Photobiology B: Biology 57 (2000) 60 – 65
which investigated other biomembranes such as plasma membrane, thylakoid membrane, liposomal membrane, etc., found they were sensitive to supplemental solar UV-B radiation [9,10,13]. For example, Bose and Chatterjee [13] observed that the susceptibility of liposomal membrane to UV light radiation was correlated with changes in microviscosity, permeability and MDA formation. Membrane lipid unsaturated acyl chains were sensitive to direct UV-B and indirect processes [24]. Direct UV-B effect may come from direct absorption of conjugated double bond system of unsaturated fatty acids, which may trigger a chain reaction resulting in peroxidation of unsaturated fatty acids [4] and indirect degradative effect was as a result of UV-B-mediated free radicals [25]. Other stress factors (e.g. salt, drought) could lead to lipid peroxidation of microsomal membranes in wheat [26,27]. The indications are that lipid peroxidation and related processes are a general response to various stresses including increased UV-B radiation. Membrane-binding proteins of spring wheat microsomal membrane, LOX (Fig. 4) and Mg 21 -ATPase and Ca 21 ATPase (Table 3) were also sensitive like membrane lipids to increased UV-B radiation. The changes of Ca 21 -ATPase, Mg 21 -ATPase activity and LOX indicated the alteration of microsomal membrane functions. Ethylene was thought to be generated by the process of peroxidation at the membrane level [28] through attacking the membrane phospholipids by LOX [29,30]. Thus, UV-B-induced increases LOX activity (Fig. 4) and ethylene evolution by in the presence of exogenous ACC (Fig. 3), indicated LOX may be involved in the process of lipid peroxidation by catalyzing the conversion of unsaturated acids to saturated ones. In a recent study, Long and Jenkins [31] observed that UV-B radiation could promote Ca 21 efflux from the cytosol and regulate calcium levels in a cytosol pool, partly via the action of specific Ca 21 -ATPase. The action spectrum for inactivation of plasma membrane bound ATPase peaks at 290 nm [8,12]. This paper has elucidated some effects of enhanced UV-B irradiance on the properties of microsomal membranes in spring wheat seedlings under greenhouse conditions. Our results, the lipid peroxidation of microsomal membrane and the changes of membrane-bound ATPase, LOX activities and ethylene emission in UV-B irradiated wheat seedlings, suggest an alteration of structure and function of microsome membranes in vivo. In addition, our data indicate that spring wheat cv. Ganlong 92-005 cultivar is sensitive to supplemental UV-B radiation, although there was some evidence that wheat, as a monocotyledonous species, was relatively resistant to UV-B compared to dicotyledonous ones (e.g. pulses) [32].
Acknowledgements This work was funded by the National Natural Science Foundation of China (No: 39670132, 39970126), State
Key Laboratory of Frozen Soil Engineering, Lanzhou Institute of Glaciology and Geocryology, CAS (9808), by key project-B, CAS (KZ952-S1-216) and by Fukang desert ecosystem observation and experiment station of Xinjiang, CAS.
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