Biology Bulletin, Vol. 32, No. 2, 2005, pp. 188–195. Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2005, pp. 232–239. Original Russian Text Copyright © 2005 by Rozentsvet, Murzaeva, Gushchina.
ECOLOGY
The Role of Membrane Lipids in the Resistance of Clapsing-Leaved Pondweed (Potamogeton perfoliatus L.) to Excess of Cadmium in Water O. A. Rozentsvet, S. V. Murzaeva, and I. A. Gushchina Institute of Ecology, Volga Basin, Russian Academy of Sciences, ul. Komzina 10, Tol’yatti, 445003 Russia e-mail:
[email protected] Received June 18, 2003
Abstract—The effect of cadmium on clapsinng-leaved pondweed (Potamogeton perfoliatus L.) within the concentration range of 1–1000 µM was studied. It was shown that P. perfoliatus leaves accumulated cadmium during three days. This process was accompanied by changes in leaf morphology. The sensitivity of biochemical metabolites to cadmium was different. Low concentrations of cadmium (1 and 10 µM) increased the content of protein, total lipids, and photosynthetic pigments, whereas high concentrations (100 and 1000 µM) decreased the content of total lipids and pigments but increased protein content. Based on cadmium sensitivity, structural lipids were divided into three groups—resistant (neutral, phosphatidylglycerol, digalactosyldiacylglycerol, and sulfolipid), the content of which increased in the presence of cadmium; labile (monogalactosyldiacylglycerol, phosphatidylcholine, and phosphatidylinositol); and nonresistant (phosphatidylethanolamine). It is concluded that the lipid component determines the resistance of P. perfoliatus to cadmium.
INTRODUCTION The ingress of cadmium into the environment with industrial discharges and soil washing results in severe pollution of the environment. Cadmium concentration in water may exceed the maximum permissible concentration in dozens of times. This metal gets into bodies of animals and humans along food chains, through plants, and may cause chronic and epizootic diseases (Levina, 1972; Il’in, 1991). The physiological effect of excess cadmium on plants has been studied sufficiently well. It affects water exchange and biogenesis and suppresses the growth and development of plants (Mel’nuchyk, 1990; Breckle, 1991; Drazkiewicz et al., 2003). As cadmium ions exhibit a high affinity for thiolcontaining groups in organic molecules, they may form strong metal–thiol complexes and substitute ions of other metals linked to these groups, which results in various disturbances in cell metabolism. In response to the toxic effect of cadmium, several programs aimed at survival and adaptation are realized in plants. They include limitation of cadmium entry, activation of the systems that ensure its excretion, as well as insulation of this cation in compartments with low metabolic activity and changes of metabolism that allow the toxic effect of cadmium to be decreased or the consequences of cadmium exposure to be eliminated (Seregin and Ivanov, 2001). Realization of one or another mechanism determines the ability of plants to accumulate cadmium and resistance to its excess. Aquatic plants, macrophytes, exhibit an increased ability to accumulate heavy metals (Gavrilenko and Zolotukhina, 1989; Guilizzoni, 1991). However, the mechanisms of detox-
ication in them remain poorly understood compared to those of terrestrial plants. A study of changes occurring in the biochemical composition of cells under exposure to different concentrations of cadmium allows the role of cell components in the origination of resistance of aquatic plants to heavy metals to be assessed and provides for a possibility of early diagnostics of aquatic environment pollution. The goal of this work was to study the effect of cadmium nitrate within a concentration range of 1–1000 µM on the aquatic plant clasping-leaved pondweed (Potamogeton perfoliatus) growing in natural environment. The level of Cd2+ accumulation and the content of total proteins, lipids, and chlorophylls in exposed plants were measured, and the ratio between individual lipid components that determine the structure and function of biological membranes was determined. MATERIALS AND METHODS The study was performed with Potamogeton perfoliatus L.—attached flowering aquatic plants that are widespread in freshwater bodies. These plants, growing under various conditions, are of economic importance (serve as food for fish and birds) and are involved in self-purification of water (Kokin, 1982). Reservoirs isolating 10–12 plants with roots down to the bottom were placed in a shallow area of the Saratov Reservoir overgrown with thickets of P. perfoliatus. The reservoirs with plants contained 135–140 l of natural water. Cadmium nitrate was added during three days (every 24 h) to final concentrations of 1, 10, 100, and 1000 µM.
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Samples were taken daily at 5:00 p.m. Plants from reservoirs into which Cd(NO3)2 was not added for three days, as well as plants growing outside reservoirs served as a control (K1 and K2, respectively). Tests were performed with leaves of the fourth–seventh nodes from the beginning of shoots. Leaves were thoroughly washed with flowing water, drained with filter paper, weighed, and tested for the content of lipids, proteins, and pigments. Data shown in figures and tables were the mean values of three biological replicates (SD = ±10–20%). The mean value of the two controls (K1 and K2) was taken as a control. Lipids were extracted by the method of Bligh and Dyer (1959) and separated by thin-layer chromatography (TLC) on plates (10 × 10 and 6 × 6 cm) with a fixed layer of silica sol (Haapsalu, Estonia). Neutral lipids were separated by one-dimensional TCL successively using the systems toluene : hexane : formic acid (140 : 60 : 1) and hexane : diethyl ether : formic acid (60 : 40 : 1). Glycolipids were analyzed in the system acetone : benzene : water (91 : 30 : 8). Phospholipids were analyzed by two-dimensional TLC in the systems chloroform : methanol : benzene : ammonia (130 : 60 : 20 : 12; the first direction) and chloroform : methanol : benzene : acetone : acetic acid (140 : 60 : 20 : 10 : 8; the second direction). Total lipids were determined gravimetrically. The content of phospholipids was determined by the method of Vaskovsky (Vaskovsky et al., 1975); neutral lipids, by the method of Kabara and Chen (1976); and glycolipids, by the content of galactose determined using the anthrone reagent (Severin and Solov’eva, 1989). To determine total protein and pigments, a weighed portion of leaves (0.2–0.5 g) was ground in a mortar with quartz sand supplemented with CaCO3 in 30 volumes of distilled water on ice. Homogenate was mixed with 20 volumes of cold 90% acetone. The extract was used to determine the concentration of chlorophylls (Vernon, 1960). The precipitate was resuspended in 0.5 M NaOH, and the concentration of total protein in the resulting solution was determined (Bradford, 1976). To determine Cd2+, a weighed portion of fresh leaves (0.2–0.3 g) was dried, ground, and placed into boiling concentrated nitric acid until complete burning. Mineralizates were tested for cadmium content by atomic adsorption spectrophotometry, as described by Guschina and Harwood (2002).
At the beginning of experiments, at the end of July, plants were at the stage of budding and beginning of flowering. During experiments, water temperature varied from 23 to 26°C (including nights), there were no precipitations and overcast days, and solar illumination was maximal. Visual monitoring of plants during incubations showed that exposure of plants to 100 and 1000 µM cadmium rendered their leaves rigid. Upon further incubation, an increase in the density of leaf plates was accompanied by thickening and increase in weight, with their color varying from brown to fulvous. Similar changes were observed at lower cadmium concentrations (1 and 10 µM); however, they were delayed. The control plants had dark-green leaves throughout the experiment. Exposure to cadmium for one weak did not lead to death of the experimental plants; however, discolored and brown–fulvous vacuole-like particles were detected in their leaves with a light microscope, with the number of these particles increasing proportionally to the concentration of cadmium. These observations showed that Cd(NO3)2 induced alterations in plants, which depended on its concentration and exposure duration and which were expressed as morphological changes of the leaf plate and changes in the microstructure of cells. It was assumed that similar changes in higher plants are due to limitation of cadmium influx across the cell wall and through the plasmalemma (Barcelo and Poschenrieder, 1990). It was also reported that cadmium exerts a structuring effect on lipid bilayer throughout its depth, thereby significantly increasing the rigidity of plasmalemma of plant cells (Fodor et al., 1995).
RESULTS AND DISCUSSION Accumulation of heavy metals in macrophytes significantly differs from that in higher aquatic plants. In terrestrial plants, accumulation of metals in leaves is determined by the reflux of them from roots (Jarvis et al., 1976). In submersed aquatic plants, dissolved compounds are adsorbed from water by the entire surface (leaves and roots), and accumulation of a metal is determined by its availability and total concentration in
Limited adsorption of cadmium by P. perfoliatus leaves in our experiments was corroborated by atomic absorption spectroscopy data (Table 1). During the first day of cadmium exposure, leaves actively accumulated cadmium ions, with accumulation rate being proportional to the concentration of Cd(NO3)2 in water. On the second and especially third day of exposure, the concentration dependence of cadmium accumulation was altered (even in the presence of 1 µM Cd(NO3)2) in water. Changes in cadmium accumulation by leaves
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water (Lukina and Smirnova, 1988). In our experiments, only leaves were used. The used concentration range of Cd(NO3)2 (1–1000 µM) encompassed both small (subtoxic) (1 and 10 µM) and high (toxic) (100 and 1000 µM) concentrations (Seregin and Ivanov, 2001). In higher plants, subtoxic concentrations of cadmium induce morphological changes in roots, suppress growth, disturb mitosis (Mel’nichuk et al., 1990; Arduini et al., 1994) and lipid metabolism (Quarity et al., 1997) as well as decrease the content of photosynthetic pigments and inhibit photosynthesis (Drazkiewicz et al., 2003). Aquatic macrophytes tolerate a long-term exposure to the same concentrations of cadmium, with their growth and viability being not suppressed (Van der Werff and Pruyt, 1982; Gavrilenko and Zolotukhina, 1989).
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Table 1. Time course of cadmium accumulation in P. perfoliatus leaves (µg per g dry weight) Day of exposure
Cd(NO3)2, µM
1
2
3
0 1 10 100 1000
0.07 0.25 1.0 12.4 15.3
– 0.55 1.1 22.3 33.0
– 0.57 3.7 9.7 33.0
occurring under a long-term exposure of plants to cadmium could result from changes in the permeability of the cell wall for cadmium (Hollenbach et al., 1997), excretion of cadmium from cells (Cumming and Taylor, 1990; Mazen and Maghraby, 1998), or general suppression of metabolism of the plant. Determination of biochemical parameters in photosynthesizing cells showed that the content of total protein, lipids, and chlorophyll increased during two days of exposure to Cd(NO3)2 at all concentrations tested (Table 2). This effect was more pronounced at a low Cd2+ concentration in water (1 µM), especially during the first day of exposure. Therefore, general metabolism in P. perfoliatus leaves was intensified due to Cd2+ influx into cells. Upon further exposure (on the third day), an increased level of lipids was retained at Cd(NO3)2 concentrations of 1 and 10 µM, as opposed to proteins, whose level remained increased in the presence of 100 and 1000 µM Cd(NO3)2. In other cases, the content of proteins, lipids, and chlorophyll decreased. Thus, a prolonged exposure to low and high concentrations of cadmium revealed differences in the response of metabolites to this agent. We discovered that proteins were cadmium-resistant (the content of them continued increasing upon a long-term exposure to toxic concentrations of cadmium), whereas lipids and chlorophyll exhibited a lower resistance to cadmium. An especially rapid decrease in the content of pigments on the third day of cadmium exposure was indicative of depression
of photosynthetic processes, which possibly led to the decrease in the content of total lipids in P. perfoliatus leaves. It was assumed that an increased protein content in higher plants under exposure to cadmium is related to the induction of defensive mechanisms and detoxication of the metal (Grill et al., 1989; Rai et al., 1995). Cadmium induces the synthesis of phytochelatins— low-molecular-weight metal-binding proteins, which are involved in the transport of heavy metals into vacuoles and their subsequent excretion from plant cells (De Knecht et al., 1994), as well as in the stimulation of expression of the ltp gene responsible for the synthesis of nonspecific lipid-transporting proteins in epidermis (Hollenbach et al., 1997). The synthesis of phytochelatins induced by heavy metals was also observed in aquatic macrophytes (Gupta et al., 1995). Thus, the increase in the content of total protein observed in the presence of high Cd2+ concentrations and under a longterm exposure to low Cd2+ concentrations can be regarded as evidence for the induction of defensive mechanisms and detoxication of cadmium in P. perfoliatus leaves. The sensitivity of the lipid component to high Cd2+ concentrations in the environment, observed in our experiments, is consistent with the published data. It was shown that Cd2+ causes dramatic changes in the composition of membranes, which applies to all classes of lipids (Ouariti et al., 1997). Some authors believe that structural and functional modifications of plasma membranes may play the key role in the development of tolerance of plants to excess of heavy metals (Demidchik et al., 2001). Figure 1 shows the ratio between lipids belonging to the main classes: neutral lipids (NLs), glycolipids (GLs), and phospholipids (PLs). In the control, slight fluctuations in the ratio between the lipids of these classes were observed within three days of experiment. These fluctuations were apparently caused by the beginning of flowering of plants. The stage of regeneration of reproductive organs is usually accompanied by reorganization of metabolic processes in plants (Good-
Table 2. Content of total protein, lipids, and chlorophyll in P. perfoliatus leaves (mg per g dry weight) Day of exposure C(NO3)2, µM 0* 1 10 100 1000
1
2
3
Protein
Chlorophyll
Lipids
Protein
Chlorophyll
Lipids
Protein
Chlorophyll
Lipids
20.3 44.4 30.8 21.5 26.0
13.3 31.1 18.7 19.5 19.3
82.0 187.5 117.5 100.9 208.1
23.2 45.6 28.4 33.6 32.5
17.2 21.7 18.6 26.3 24.5
86.6 100 97.2 102.3 –
27.5 23.9 25.5 34.3 54.5
18.4 10.3 17.5 13.2 16.2
110.7 118.9 155.2 88.1 81.2
* Mean values of two control experiments (K1 and K2). BIOLOGY BULLETIN
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100 80 60 40 20 0 100 80 60 40 20 0
Content of lipids, % (a) 100 80 60 40 20 0 0 1 2 (b)
1 2 3 3 day
(c)
(e)
(d)
0
1
70 60 50 40 30 20 10 0
2
3
0
1
2
3
Fig. 1. Changes in the content of lipids of different classes in P. perfoliatus leaves under exposure to cadmium: (1) polar lipids, (2) glycolipids, and (3) phospholipids. Abscissa: the incubation time (days); ordinate: the content of lipids (% of total content). Designations: (a), control; (b), 1 µM Cd(NO3)2; (c), 10 µM Cd(NO3)2; (d), 100 µM Cd(NO3)2; and (e), 1000 µM Cd(NO3)2.
win and Merser, 1986). However, an increase in LNs and a decrease in PLs and GLs were observed on the first day of exposure of plants to low doses of Cd(NO3)2 (1 and 10 µM). On the second and third day of exposure, the level of PLs remained unchanged, whereas the ratio between MLs and GLs varied. As a result, the level of NLs restored to the control level or increased or decreased with the level of GLs. High concentrations of cadmium in water (100 and 1000 µM) changed the ratio between the main classes of lipids, decreasing the content of PLs and GLs and increasing the content of NLs. It is known that, in plants, NLs are represented by reserve fats and structural components contained in the cuticular layer. Separation of NLs to individual components by TLC showed that exposure of plants to increased concentrations of cadmium on the second and third day was accompanied by an increase in the proportion of the compounds representing the group of waxes, such as sterols, sterol esters, alcoholic and acidic components, and carbohydrates (data not shown). These compounds are the main components of the cuticle (Goodwin and Merser, 1986). Therefore, the increase in the content of NLs in leaves of P. perfoliatus was apparently caused by the thickening of the cuticular layer in order to create a barrier preventing cadmium penetration across cell walls. Polar lipids (GLs and PLs) are the structural components of membranes; patterns of their distribution in the BIOLOGY BULLETIN
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70 60 50 40 30 20 10 0 70 60 50 40 30 20 10 0
0
1
191
Content of glycolipids, % (a) 1 2 3
0
(b)
3 day (c)
(d)
(e)
2
1
3
2
0
1
2
3
Fig. 2. Changes in the content of glycolipids (% of total content) in P. perfoliatus leaves under exposure to cadmium: (1) monogalactosyldiacylglycerol, (2) digalactosyldiacylglycerol, and (3) sulfoquinovosyldiacylglycerol. For designations, see Fig. 1.
plasmalemma, endoplasmic reticulum, and organoids are different (Curr and Harwood, 1991; Harwood, 1998). Important components of photosynthetic membranes are galactolipids—monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). MGDG and DGDG are contained in the membranes of chloroplasts, grains, and thylakoids. These two lipids stabilize photosynthetic membranes related to functioning of PS I and PS II; however, MGDG also plays a key role in stabilization of deflected regions of thylakoid membranes and is involved in the electron transfer (Gounaris et al., 1986; Gennis, 1997). The MGDG-to-DGDG ratio lower than 2 is indicative of disturbances in the structure of thylacoid membranes, detachment of grains, and degradation of the PS II-containing photosynthetic domains (Murphy, 1986). These changes are observed in different types of stress in plants, including exposure to heavy metals (Stefanov et al., 1992; Rama Deli and Prasad, 1999). SQDG is located only in thylakoid membranes and is believed to ensure such a configuration of membranes that is optimal for active electron transport in PS II (Benning, 1993). Figure 2 shows the time course of the content of MGDG, DGDG, and SQDG in P. perfoliatus leaves during three days. The content of glycolipids in the control varied and accounted for 55–65% for MGDG,
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1
40
2 3
30
4
20
5
10 0 60
0
1
2
3 (c)
(b)
50 40 30 20 10 0 60 50 40 30 20 10 0
(e)
(d)
0
1
2
3
0
1
2
3
Fig. 3. Changes in the content of phospholipids (% of total content) in P. perfoliatus leaves under exposure to cadmium: (1) phosphatidic acid, (2) phosphatidylinositol, (3) phosphatidylcholine, (4) phosphatidylethanolamine, and (5) phosphatidylglycerol. For designations, see Fig. 1.
25–27% for DGDG, and 8–20% for SQDG. These fluctuations are indicative of lability of membrane structures and correlated changes in the concentrations of MGDG and SQDG in plants under normal conditions. The ratio between galacto- and sulfolipids, as well as the MGDG-to-DGDG ratio, which varied from 2.2 to 2.4, corresponded to the values characteristic of photosynthetic tissues of higher terrestrial plants (Gurr and Harwood, 1991). Under exposure to low concentrations of cadmium (1 and 10 µM), the curves that reflect the content of MGDG and SQDG in P. perfoliatus leaves during three days had an oscillatory mode, suggesting that the content of these compounds changed every day in opposite directions. Note that the level of DGDG practically did not change. The MGDG-to-DGDG ratio varied due to changes in MGDM content: it decreased to 1.6 and 1.3 on the first day of exposure to 1 and 10 µM Cd(NO3)2, respectively, and was then restored to 2.0 on the next day. At high concentrations of cadmium (100 and 1000 µM), the dependence had a linear mode, suggesting a decrease in MGDG content and an
increase in SQDG content. As a result, the proportion of these compounds became equal on the third and second day of exposure. The level of DGDG at high concentrations of cadmium also changed only slightly (as in the case of low concentrations). The MGDG-to-DGDG ratio decreased to 1.5 and 1.3 due to a decrease in the MGDG content. These results indicate that the lability of glycolipids is related predominantly to cadmium sensitivity of MGDG and SQDG. The level of these compounds in leaves varies in an oscillatory manner. Note that only one third of the total MGDG content varied, being first decreased and then restored to the initial level, whereas two thirds of MGDG content did not undergo changes, being retained even at the highest concentrations of cadmium. Figure 3 shows the daily time course of the ratio between the most important phospholipids in dependence on the concentration of cadmium. In the control, the ratio between phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) varied within 44–54, 22–24, and 14–18%, respectively. BIOLOGY BULLETIN
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Phosphatidylinositol (PI) and phosphatidic acid (PA) were present in low quantities (5–8 and 3–8%, respectively). These results correspond to the published data on the ratio between membrane phospholipids in photosynthesizing tissues in higher terrestrial plants (Harwood, 1998) and the data obtained by us earlier for aquatic macrophytes (Rozentsvet et al., 2000). Under exposure to cadmium, the ratio between individual phospholipids differs from the control, as follows from the change in curve pattern. Low and high concentrations of cadmium caused both an increase and decrease in the content of PC in leaves. Under exposure to low cadmium concentrations, changes were expressed only on the second and third day of exposure, whereas high concentrations of cadmium induced changes as early as on the first day. Note that the content of PC varied within the range of 20–35% of the initial level, with the major part of PC being retained on the second and third day of exposure even to the highest concentration of cadmium. The content of PG increased as the concentration of cadmium in medium increased (on the third day in the case of low concentrations of cadmium and on the first and second day of exposure to high concentrations). At high cadmium concentrations, almost a twofold increase in the content of PG was observed. In contrast to PG, the content of PE decreased two or three times at all cadmium concentrations as early as on the first day of exposure. At lower concentrations of cadmium, the level of PE was restored on the second and third day of exposure; however, at higher concentrations of cadmium, it decreased to trace levels. The content of minor components, PI and PA, varied to different extent within three days of exposure to 1–100 µM Cd(NO3)2. The highest concentration of Cd(NO3)2 (1000 µM) had no effect on their content. Thus, with respect to resistance to increased concentrations of cadmium, especially labile components (PC, PI, and PA), whose content varied (increased or decreased) and the components whose content either increased (PG) or decreased (PE) can be distinguished. If phospholipids are considered from the standpoint of their location and function in plant cells, PC is the major structural component of the plasmalemma and prevails over other phospholipids in mitochondria (45 and 41% of the total content of phospholipids, respectively). In addition to PC, mitochondria also contain lower quantities of PE and PI; the content of these phospholipids in chloroplasts is two- or threefold lower (Goodwin and Merser, 1986). These phospholipids are located predominantly on the outer side of the membrane. The main function of PC, as well as other phospholipids, comes to maintaining the general bilayer structure of membranes (Gurr and Harwood, 1991; Gennis, 1997). It is also known that PC is involved in the formation of the cuticle (Goodwin and Merser, 1986). Changes in the content of PC, PE, and PI observed in the presence of cadmium allow indirect assessing the state of outer membrane structures of the cell (predominantly, plasmalemma and mitochondria BIOLOGY BULLETIN
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and, in part, chloroplasts). Our results suggest that, under exposure to high concentrations of cadmium, one fourth or one third of the outer membrane structures in P. perfoliatus leaves underwent changes, because the content of PC and PI varied by 20–35% as the content of PE decreased. It should be noted that PE easily undergoes chemical modifications (Gennis, 1997). It was reported that, in different types of stress, PE undergoes oxidation and is substituted in membranes with PC, which is resistant to oxidation. It was shown that such rearrangement represents an adaptive response to oxidative stress induced by heavy metals in some lichens (Kotlova, 2000). In our experiments, fluctuations in PC concentrations observed simultaneously with a decrease in PE content are indicative of such possibility under exposure to low concentrations of cadmium. The effect of cadmium on the inner membranes can be assessed by a change in the content of PG, which accounts for up to 60% of the total phospholipids of chloroplasts in higher plants (Gurr and Harwood, 1991). It was assumed that, in metal-induced oxidative stress, an increase in PG content is due to additional synthesis of this phospholipid during formation of new complexes of PS II (Kotlova, 2000). Possibly, the increase in the content of PG in P. perfoliatus leaves under exposure to high concentrations of cadmium is also related to additional synthesis of it, which undoubtedly plays an adaptive role. To conclude, it should be noted that we should differentiate the effect of low (1 and 10 µM) and high (100 and 1000 µM) concentrations of cadmium. The effect of low concentrations of cadmium is retarded and manifests itself only on the second and third day of exposure, whereas the effect of high concentrations can be detected as early as on the first day of exposure. Low concentrations of cadmium mostly induce fluctuations in the content of all glycolipids and phospholipids. However, high concentrations of cadmium have a directed effect: they decrease the content of MGDG, PE, and PC; increase the content of SQDG and PG; and increase fluctuations in the content of phospholipids PC, PI, and PA. When comparing these results with the data on the effect of cadmium on general metabolism of proteins, lipids, and pigments, it can be concluded that different sensitivity to low (1 and 10 µM) and high (100 and 1000 µM) concentrations of cadmium in water is expressed in P. perfoliatus leaves at the biochemical level. Low concentrations of cadmium activate metabolism of proteins, lipids, and pigments, whereas high concentrations of this cation decrease the content of total lipids (due to a decrease in the content of polar lipids) and pigments but increase the content of proteins. As mentioned above, high concentrations of cadmium do not cause death of plants, and metabolism of total lipids and pigments in this case is suppressed by no more than 30% (as judged by the content of these com-
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pounds in leaves). All these data indicate that P. perfoliatus exhibits resistance to cadmium when accumulating it in leaves. Different mechanisms ensure the resistance to cadmium at the physiological and biochemical level—when cadmium penetrates leaves, when it attacks the inner and outer membranes, and when it enters the cytoplasm. The resistance to cadmium is developed, first of all, due to the creation of a barrier in the cell wall at the stage of water entry into the cytoplasm. The fact that such a barrier is indeed created is confirmed by the morphological changes related to the thickening of the cuticle, increase in the proportion of neutral lipids in the total lipid fraction, and disturbance of the concentration dependence of cadmium accumulation in leaves. When cadmium attacks the inner and outer membranes, resistant polar lipids (PG, DGDG, and SQDG) can be revealed. Despite the lability of the major structural components of PC and MGDG, almost 70–80% of them is retained in P. perfoliatus leaves under exposure to high concentrations of cadmium. This finding, as well as the fact that the content of DGDG is maintained at a constant level whereas the content of PG and SQDG, which ensure the functioning of the chlorophyll–protein complexes of PS II, increases indicate that the exchange of polar lipids in P. perfoliatus in stress is regulated at a high level. The next factor that ensures cadmium resistance is the induction of defensive mechanisms when cadmium penetrates the cytoplasm. This is confirmed by the increase in the total protein content and by the changes observed in the microstructure of the leaf under exposure to cadmium. However, despite the role of the protein component in cadmium detoxication, which apparently takes place in P. perfoliatus, we believe that it is the lipid component (specifically, the polar lipids, which ensure the stability of cell walls, plasmalemma, and chloroplasts) that plays the key role in protecting plants against this metal. REFERENCES Arduini, I., Godbold, D.L., and Onnis, A., Cadmium and Copper Change Root Growth and Morphology of Pinus pinea and Pinus pinaster Seedlings, Physiol. Plant., 1994, vol. 92, pp. 675–680. Barcelo, J. and Poschenrieder, C., Plant Water Relations as Affected by Heavy Metal Stress: A Review, J. Plant Nutr., 1990, vol. 13, pp. 1–37. Benning, C., Beatty, J.T., Prince, R.C., and Somerville, C.R., The Sulfolipid Sulfoquinovosyldiacylglycerol Is Not Required for Photosynthetic Electron Transport in Rhodobacter sphaeroides but Enhances Growth under Phosphate Limitation, Proc. Natl. Acad. Sci. USA, 1993, vol. 90, pp. 1561– 1565. Bligh, E.G. and Dyer, W.J., A Rapid Method for Total Lipid Extraction and Purification, Canad. J. Biochem. Physiol., 1959, vol. 37, pp. 911–919. Breckle, S.W., Growth under Stress: Heavy Metals, in Plant Roots: the Hidden Half, Waisel, Y. Eshel, A., and Kafkafi, U., Eds., New York: Marcel Dekker, 1991, pp. 351–373.
Bradford, M.M., A Rapid and Sensitive Method for Principle of Protein–Dye Binding, Anal. Biochem, 1976, vol. 72, pp. 248–254. Cumming, J.R. and Taylor, C.J., Mechanisms of Metal Tolerance in Plants: Physiological Adaptation for Exclusion of Metal Ions from the Cytoplasm, in Stress Responses in Plants: Adaptation and Accumulation, Allen, N.S., Ed., 1990, New York: Wiley, pp. 328–356. De Knecht, J.A., van Dillen, M., Koevoets, P.L.M., Schat, H., and Verkleij, J.A.C., Phytochelatins in Cadmium-Sensitive and Cadmium-Tolerant Silene vulgaris, Plant Physiol., 1994, vol. 104, pp. 255–261. Demidchik, V.V., Sokolik, A.I., and Yurin, V.M., Toxicity of Excess Copper and Tolerance of Plants to It, Usp. Sovrem. Biol., 2001, vol. 121, no. 5, pp. 511–525. Drazkievicz, M., Tukendorf, A., and Baszynski, T., AgeDependent Response of Maize Leaf Segments to Cadmium Treatment: Effect on Chlorophyll Fluorescence and Phytochelatin Accumulation, J. Plant Physiol., 2003, vol. 160, no. 3, pp. 247–254. Fodor, E., Szabonagy, A., and Erdei, L., The Effect of Cadmium on the Fluidity and H+-ATPase Activity of Plasma Membrane from Sunflower and Wheat Roots J. Plant Physiol., 1995, vol. 147, pp. 87–92. Gavrilenko, E.E. and Zolotukhina, E.Yu., Accumulation and Interaction of Copper, Zink, Manganese, Cadmium, Nickel, and Lead Ions upon Adsorption by Aquatic Macrophites, Gidrobiol. Zh., 1989, vol. 25, no. 5, pp. 54–61. Gennis, R., Biomembrany: molekulyarnaya struktura i funktsii (Biomembranes: Molecular Structure and Function), Moscow: Mir, 1997. Goodwin, T.W. and Merser, E.I., Introduction to Plant Biochemistry, Oxford: Pergamon, 1983, vol. 1. Translated under the title Vvedenie v biokhimiyu rastenii, Moscow: Mir, 1986, vol. 1. Gounaris, K., Barber, J., and Harwood, J.L., The Thylakoid Membranes of Higher Plants Chloroplasts, Biochem. J., 1986, vol. 237, pp. 313–326. Grill, E., Loffler, S., Winnacker, E.L., and Zenk, M.N., Phytochelatins, the Heavy-Metal-Binding Peptides of Plants, Are Synthesized from Glutathione by a Specific γ-Glutamylcysteine Dipeptidyl Transpeptidase (Phytochelatin Synthase), Proc. Natl. Acad. Sci. USA, 1989, vol. 86, pp. 6838– 6843. Guilizzoni, P., The Role of Heavy Metals and Toxic Materials in the Physiological Ecology of Submersed Macrophytes, Aquatic Botany, 1991, vol. 41, no. 1-3, pp. 87–109. Gupta, M., Rai, U.N., Tripathi, R.D., and Chandra, P., LeadInduced Changes in Glutatione and Phytochelatin in Hydrilla verticillata Royle, Chemosphere, 1995, vol. 30, pp. 2011– 2020. Gurr, M.I. and Harwood, J.L., Lipid Biochemistry, London: Chapman–Hall, 1991, pp. 246–387. Guschina, I.A. and Harwood, J.L., Lipids Metabolism in the Moss Rhytidiadelphus squarrosus (Hedw.) Warnst. from Lead-Contaminated and Not Contaminated Populations, J. Exp. Bot.,. 2002, vol. 53, pp. 455-463. Harwood, J.L., What’s So Special about Plant Lipids? In Plant Lipid Biosynthesis. Fundamentals and Agricultural Application, Harwood, J.L., Ed., Cambridge: Univ. Ontario Press, 1998, pp. 305–363. BIOLOGY BULLETIN
Vol. 32
No. 2
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THE ROLE OF MEMBRANE LIPIDS IN THE RESISTANCE Hollenbach, B., Schreiber, L., Hartung, W., and Dietz, K.J., Cadmium Leads to Stimulated Expression of the Lipid Transfer Protein Genes in Barley: Implication for the Involvement of Lipid Transfer Proteins in Wax Assembly, Planta, 1997, vol. 203, pp. 9–19. Il’in, V.B., Tyazhelye Metally v Sisteme Pochva–Rastenie (Heavy Metals in the Soil–Plant System), Novosibirsk: Nauka, 1991. Jarvis, S.C., Jones, L.H.P., and Hopper, M.J., Cadmium Uptake from Solution by Plants and Its Transport from Roots to Shoots, Plant Soil, 1976, vol. 44, pp. 179–191. Kabara, J.I. and Chen, J.S., Microdetermination of Lipid Classes after Thin-Layer Chromatography, Anal. Chem., 1976, vol. 48, pp. 814–819. Kokin, K.A., Ekologiya vysshikh vodnykh rastenii (Ecology of Higher Aquatic Plants), Moscow: Mosk. Gos. Univ., 1982. Kotlova, E.R., Antioxidant Systems of Lichens, Cand. Sci. (Biol.) Dissertation, St. Petersburg: St. Petersburg State University, 2000. Levina, E.N., Obshchaya toksikologiya metallov (General Toxicology of Metals), Leningrad: Meditsyna, 1972. Lukina, L.F. and Smirnova, N.N., Fiziologiya vysshikh vodnykh rastenii (Physiology of Higher Aquatic Plants), Kiev: Naukova Dumka, 1988. Mazen, A.M. and Maghraby, O.M., Accumulation of Cadmium and Strontium, and a Role of Calcium Oxalate in Water Hyacinth Tolerance Biol. Plant., 1997/1998, vol. 40, pp. 411–417. Mel’nichuk, Yu.P., Vliyanie ionov kadmiya na kletochnoe delenie i rost rastenii (Effect of Cadmium Ions on Cell Division and Growth of Plants), Kiev: Naukova Dumka, 1990. Murphy, D.J., The Molecular Organization of the Photosynthetic Membranes of Higher Plants, Biochim. Biophys. Acta, 1986, vol. 864, pp. 33–94. Quariti, O., Boussama, N., Zarrouk, M., Cherif, A., and Ghorbal, M.H., Cadmium and Copper-Induced Changes in
BIOLOGY BULLETIN
Vol. 32
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195
Tomato Membrane Lipids, Phytochemistry, 1997, vol. 45, pp. 1343–1350. Rai, U.N., Tripathi, R.D., Gupta, M., and Chandra, P., Induction of Phytochelatins under Cadmium Stress in Water Lettuce (Pistia stratiotes L.), J. Environ. Sci., 1995, vol. 30, pp. 2007–2026. Rama Deli, S. and Prasad, M.N.V., Membrane Lipid Alteration in Exposed Plants, in Heavy Metal Stress in Plants. From Molecules to Ecosystems, Berlin: Springer, 1999, pp. 99–117. Rozentsvet, O.A., Saksonov, S.V., Kozlov, V.G., and Koneva, N.V., Ecological and Biochemical Approach to Studying Lipids of Higher Aquatic Plants, Izv. Sam. NTs Ross. Akad. Nauk, 2000, vol. 2, no. 2, pp. 358–366. Seregin, I.V. and Ivanov, V.B., Physiological Aspects of Toxic Effect of Cadmium and Lead on Higher Plants, Fiziol. Rast. (Moscow), 2001, vol. 48, no. 4, pp. 606–630. Severin, S.E. and Solov’eva, G.A., Prakticheskie raboty v biokhimii (Practical Works in Biochemistry), Moscow: Mosk. Gos. Univ., 1989, pp. 23–25. Stefanov, K., Kimenov, G., Popova, I., Pancheva, T., Metodiev, M., Andreev, St., and Popov, S., Lipid and Sterol Changes in Plants and Invertebrates Caused by Environmental Pollution, Bulgar. Chem. Com., 1992, vol. 25, pp. 391– 399. Van der Werff, M. and Pruyt, M.J., Long-Term Effects of Heavy Metals on Aquatic Plants Chemosphere, 1982, vol. 11, pp. 727–739. Vaskovsky, V.E. and Latyshev, L.A., Modified Jungnickel’s Reagent for Detecting Phospholipids and Other Phosphorus Compounds on Thin-Layer Chromatography, J. Chromatogr., 1975, vol. 115, pp. 246–249. Vernon, L.P., Spectrophotometric Determination of Chlorophylls and Pheophytins in Plant Extracts, Anal. Chem., 1960, vol. 32, pp. 1144–1150.