biological trace element research

Volume 93 • Nos. 1–3 (Three Issues) Summer 2003

EDITOR-IN-CHIEF

BIOLOGICAL TRACE ELEMENT RESEARCH

GERHARD N. SCHRAUZER EDITORS

PETER SCHRAMEL YASUSHI KODAMA ALAIN FAVIER

ISSN: 0163–4984

HUMANA PRESS

© Copyright 2003 by Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/03/93(1–3)–0257 $20.00

Effects of Europium Ions (Eu3+) on the Distribution and Related Biological Activities of Elements in Lathyrus sativus L. Roots HONG ER TIAN, YONG SHENG GAO, FENG MIN LI, AND FULI ZENG* State Key Laboratory of Arid Agroecology, School of Life Science, Lanzhou University, Lanzhou, 730000, China Received July 31, 2002; Accepted August 25, 2002

ABSTRACT Scanning electron microscopic and energy-dispersive X-ray analyses were used to study the distributions of different types of elements in the epidermis, exodermis, endodermis, and vascular cylinder of the fracture face in the Lathyrus sativus L. roots in the presence or absence of Eu3+. Some index of the biological activity related to the elements binding with protein were determined also. The results showed that the tissular distributions of elements in the fracture face are different in the presence and absence of Eu3+. The atomic percentages of P, S, Ca, and Mn were influenced more than those of other elements. Eu3+ promoted the biological activities of various kinds of element. The one possible mechanism changing the biological activities was +e that the reaction of Eu3+ → Eu2+ would influence the electron capture or transport in elements of binding protein. Another mechanism was that CaM–Ca2+ becoming CaM–Eu3+ through Eu3+ instead of Ca2+ would affect the biological activity of elements by regulating the Ca2+ level in the plant cell. Index Entries: Europium ion (Eu3+); Lathyrus sativus L.; root; elements; biological activities.

INTRODUCTION As a exotic species, Lathyrus sativus L. has been known since 1960. Its habitat is the arid and semiarid region in the west of China. Influenced by *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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the arid environment, the vegetal body form of L. sativus has changed due to ecobiomorphism. Its stem is creeping and dwarf, its leaf is small, its horny layer of leaf is thick, its root system is developed, and its metabolism is adapted to drought. All of these changes affect drought resistance and drought tolerance significantly. Recent studies showed that the abundant protein content could be used for animal forage (1). Thus, some researchers attempted to determine the mechanism producing toxicity in L. sativus and the reports related to toxicity are increasing (2–5). On the basis of the reported biochemical properties, we determined the distribution of elements and their related physiological activities (6,7). The purpose of all of the above research work is to develop and safely utilize L. sativus as forage. As in our previous studies, we used Eu3+ as a probe to study the affect of Eu3+ in treated L. sativus roots (8).

MATERIALS AND METHODS Lathyrus Sativus L. Cultivation Selected seeds of L. sativus L. were carefully sterilized with 0.1% HgCl2 for 8 min and washed with distilled water. After soaking in water for 24 h at 18 ± 0.2°C, the seeds were germinated on filter paper in the dark at 25 ± 0.2°C. Buds were planted in sterilized quartz sand (38 µmol/m2/s light, 25 ± 0.2°C during the day, 18 ± 0.2°C at night, a 15/9-h day/night period). After being cultured for 30 d, the seedlings were divided into two groups: one group was treated with 0.5 mmol/L Eu3+ for 3 d and then grown for 7 d continuously; the other was grown concurrently in distilled water. Identical positions of roots in the two groups were used to measure the contents of all the elements.

Methods of Measurement Scanning electron microscopic and energy-dispersive X-ray analyses were used to determine the tissular percentages of all element in the fracture face of Eu3+-treated L. sativus roots and the control. The activity of root system in the fracture face of treated L. sativus roots was determined as described by Stenponkus and Lanphear (9). The content of soluble protein in the identical position of the fracture face in treated L. sativus roots was measured as described by Bradford (10). Concurrently, the specific activity of protein hydrolase in treated L. sativus roots was measured as described by Zeng (11). An Hitichi 835-50-type high-rate amino acid analyzer was used to measure the contents of threonine (Thr), serine (Ser), and tyrosine (Tyr) in the identical position of the fracture face in treated L. sativus roots as described by Talley and Mary (12). The contents of free radicals in the identical position of the fracture face in treated L. sativus roots were determined as described by Wang and Biological Trace Element Research

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Luo (13), Steiner and Babbs (14), and Brennan and Frenkel (15). The content of polyamine in the identical position of fracture face in treated L. sativus roots was determined as described by Flores and Galson (16).

RESULTS AND ANALYSIS Comparison of Microstructure of the Fracture Face in Roots of Eu3+-Treated L. sativus and Control Under Scanning Electron Microscope Figure 1 shows that the structure of L. sativus roots are composed of, from outside to inside, epidermis, exodermis, endodermis, and vascular cylinder. The figures and structures of cells in the fracture face of L. sativus roots have no differences significantly. The structures of the formed tissues and organs include primary phloem, vessel (triarch), secondary xylem vessel, cambium layer, phloem sieve, and so forth.

Atomic Percentages of Elements Distributed in the Fracture Face of L. sativus Roots in the Presence and Absence of Eu3+ Figure 2 shows that the amount of silicon (Si) in the epidermis of Eu3+treated L. sativus roots was greater than in the control. Therefore, the contents of Si in the exodermis, endodermis, and vascular cylinder of Eu3+-treated L. sativus roots were less than that in the control. The possible reason was that the absorbed element Si was transported to the other tissues and organs. As a necessary element, Si promotes the roots system development and growth, enhances the ability of stress resistance in plants, reduces the harm caused by heavy metal 3, and protects plants against pathogens (17,18). The contents of phosphorus (P) and sulfur (S) in various tissues of the epidermis, exodermis, endodermis, and vascular cylinder of Eu3+-treated L. sativus roots were less than that in the control. Phosphorus is the necessary macroelement in plant growth. It is absorbed as monovalent and/or bivalent phosphoryl by plants. The light energy absorbed by the chloroplast is transformed to chemical energy and stored in the high-energy phosphate bond of ATP (adenosine triphosphate). The energy is released through the reaction of ATP → ADP + Pi, when plants need it for growth. The reaction of ATP → ADP + Pi is regulated and controlled by Na+, K+ATPase. The energy generated by oxidation phosphorylation and phostosynthesis phosphorylation in plant cells is stored in ATP. Sulfur combines with protein by forming sulfur-containing amino acids (cysteine and methionine) in plant cells. Being the acceptor of electron, the -HS bond activates and restrains the activity of the related enzyme and regulates the metabolism reaction related to sulfur. Biological Trace Element Research

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Fig. 1. (A) Photograph of various tissues in the fracture face of water-treated L. sativus roots (the control) during X-ray microanalysis. (B) Photograph of various tissues in fracture face of Eu3+-treated L. sativus roots during X-ray microanalysis.

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Fig. 2. Comparison of the atomic percentages of various elements distributed in fracture faces of L. sativus roots treated with Eu3+ solution and with water (the control). The content of each element (%) is the atomic percentage of various elements in the fracture faces of L. sativus roots which water (the control) taken from Eu3+-treated.


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Iron (Fe) combining with sulfur and phosphorus participate in synthesizing iron–sulfur protein (FeSX, FeSA, FeSB) and changing the oxidation–reduction potential. Ferritin combining with cytochrome-b6 and cytochrome-f form the cytochrome-b6/f complex. The electrons are transFe3+ + e. ported through the reversible reaction of Fe2+ – The content of chlorine (Cl ) in the epidermis, endodermis, and vascular cylinder of Eu3+-treated L. sativus roots is less than that in the control. However, the content of chlorine in the Eu3+-treated exodermis is greater than that in the identical position of the control. The content of chlorine in the plant body is very low. Chlorine absorbed by roots is transported to leaves to participate in photosynthesis. As an electron acceptor in the reaction of photolysis of water, chlorine can stabilize water photolyase through → Cl– (19). Chlorine can also promote the growth and the reaction of Cl +e development of roots. The content of sodium (Na+) in the epidermis of Eu3+-treated L. sativus roots is more than in the control, whereas that in the exodermis, endodermis, and vascular cylinder of Eu3+-treated L. sativus roots is almost equal to that in the control. However, the content of potassium (K+) in various tissues of Eu3+-treated L. sativus roots are all more greater than in the control. Sodium and potassium belonging to the same race in the periodic table of elements are mobile and reusable elements. They are generally similar in chemical properties. Potassium is the necessary element in the synthesis of protein. Sodium is the necessary element in salt-tolerant plants but not the necessary element in salt-sensitive plants. The Na+/K+ ratio is nearly constant in plant cells (20). No evidence shows that there is a specific sodium channel. The absorption of sodium is competitive with potassium. K+,Na+-ATPase might regulate the store and release of energy of ATP in plants. The content of magnesium (Mg2+) in the epidermis of Eu3+-treated L. sativus roots is greater than that of the control, whereas that in the exodermis, endodermis, and vascular cylinder of Eu3+-treated L. sativus roots is equal to or less than in the control. Magnesium is the core element in the molecular structure of chlorophyll. ATP and Mg2+ are necessary when aspartic acid is combined with NH4+. As a activator of the enzyme, Mg2+ catalyzes the reaction of synthesizing ATP. Mg2+ absorbed by roots is quickly transported to the shoot and leaves. The contents of aluminum (Al) in the exodermis, endodermis, and vascular cylinder of Eu3+-treated L. sativus roots are less than that in the control, whereas the content of aluminum in the Eu3+-treated epidermis is more than that of control. The variable valence of Al, which is influenced by the pH value of the microenvironment in the plant, would affect the physiological activities and stimulate some plant growing. There are few specific reports on Al. Calcium (Ca2+) is a necessary element of plants. A low Ca2+ concentration can maintain the normal architecture and selective permeability of plant cell membranes. The content of Ca2+ in various types of cell is reguBiological Trace Element Research

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Fig. 3. Activity of the root system in the Eu3+-treated L. sativus roots.

lated by the activity of Ca2+-ATPase and the absorption of Ca2+ is enhanced by Eu3+. Second-messenger hypothesis of Ca2+ was reported by Rasmussen in 1981. It has been determined that Ca2+ plays an important role in the second signal system (21,22). Eu3+ is a rare-earth-element ion with chemical properties similar to Ca2+ and it is an antagonist of Ca2+. As a result, Eu3+ was used to study the function of Ca2+ in the signal-transferring system. A low Eu3+ concentration is able to replace Ca2+ as a signal (8). The content of manganese (Mn) is very low in the plant cell. Influenced by Eu3+, the content of Mn in the epidermis, exodermis, endodermis, and vascular cylinder of Eu3+-treated L. sativus roots is less than that in the control. The results showed that Eu3+ was able to promote the process of absorbing and transporting Mn. Mn has the role of stabilizing the normal structure of membrane system of the chloroplast and activating the fatty acid synthetase, the related enzymes synthesizing deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and the respiratory enzyme in TCA (tricarboxylic acid cycle). Mn also participates in the reaction of hydrolysis and releasing oxygen (2H2O → O2 + 4H+) in photosynthesis. The alterable valence of Mn has the function of a electron carrier.

Effects of Eu3+ on the Activity of the Root System of Eu3+-Treated L. sativus The activity of root system was measured by the standard curve of TTC method. The results (see Fig. 3) showed that the activity of the root system in Eu3+-treated L. sativus is nearly 6% higher than that of control. The Eu3+ affects the oxidation–reduction reaction of substrate in the activity of root systems through the change of the S valence. Being an electron acceptor, S participates in the reaction of TTC (2,3,5-triphenyltetrazolium chloride) + 2H → TTCH (triphenyl formazan). Cl has the function of promoting the activity of the root system. Eu3+ plays the important role of enhancing the absorption of nutrient ion and the capability of anabolism and, further, promotes the plant growth. Biological Trace Element Research

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Fig. 4. Content of Protein in the Eu3+-treated L. sativus roots.

Fig. 5. Specific activity of proteolytic enzyme in Eu3+-treated L. sativus root cells.

Effect of Eu3+ on the Content of Proteins and the Related Enzymes Activity in Eu3+-Treated L. sativus Roots The content of protein synthesized in Eu3+-treated L. sativus roots increased by 410.0% (see Fig. 4). S forms the -HS bond of sulfur-containing amino acid in the protein. K plays an important role in the synthesis of protein and enhances drought tolerance. Adapting to the arid environment, the Eu3+-treated L. sativus is able to increase the specific activity of the proteolytic enzyme and then increase the concentration of amino acid in the root cells (see Fig. 5). A higher amino acid concentration lowers the osmotic potential in order to imbibe more water and stabilize the content of water in the plant cell in an arid environment.

Effect of Eu3+ on the Content of Three Kinds of Amino Acid In Eu3+-treated L. sativus, the content of serine (Ser) was equal to the control; however, the content of threonine (Tyr) increased by 50.0% and the content of tyrosine (Thr) decreased by 125.0% compared with those of control (see Fig. 6). The changes of the contents of three kinds of amino acid (Ser, Thr, Tyr) were relevant to the function of related metabolic enzymes. Ser participated in the synthesis of the related protein; however, Tyr and Thr played a role in signal transferring in L. sativus roots. The possible mechanism is that Eu3+ instead of Ca2+ increases the calmodulin content (23). Biological Trace Element Research

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Fig. 6. Contents of three kinds of amino acid in Eu3+-treated L. sativus roots.

Effect of Eu3+ on the Contents of Free Radicals and Polyamine The effect of Eu3+ on antioxidant capacity embodied in the changing of the contents of O.–2, ·OH, and H2O2. The results in Fig. 7 show that the contents of O–2, ·OH, and H2O2 all increased in Eu3+-treated L. sativus roots compared with those in the control. The possible reason for the increase of the contents of all three radicals was that Eu3+ inhibited the production of Fe2+ and then the Fenton-type Haber–Weiss reaction produced more free radicals (24). Figure 8 shows that Eu3+ helped the L. sativus roots produce more polyamine. The contents of putrescine (put), spermidine (spd), and spermine (spm) were all higher than that in the control. However, a larger amount of polyamine was able to stabilize the membrane and nucleic acid and to protect the membrane from injury (24). The previous reports indicated that polyamine perhaps had the function of scavenging the free radical (25). Thus, Eu3+ made the L. sativus roots produce more free radical, but at the same time, it made L. sativus roots produce more polyamine. The above results showed that in L. sativus roots there were complementary metabolic mechanisms that helped the plant adapt to the stress. The theory of genetic screening indicated that long-term adaptation of L. sativus under the stress of a drought environment could lead to the consequences that promote the antioxidant capacity and the absorbing power of water and organic nutrition.

DISCUSSION The effects of Eu3+ on the distribution and related biological activities of all types of element in L. sativus roots were analyzed in this article. On the one hand, Eu3+ affected the contents of mobile and reusable elements (P, S, Cl, Na, K) more than the that of other elements (Si, Al, Ca, Mn, Fe). Biological Trace Element Research

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Fig. 7. Contents of (a) O.–2, (b) ·OH, and (c) H2O2 in Eu3+-treated L. sativus roots.

The valences of the four elements (P, S, Ca, Mn) that play an important role in metabolic regulation are affected substantially by Eu3+. The contents of the above four elements in the fracture face of Eu3+-treated L. sativus roots were less than that in the control (see Fig. 2). P is the core element in forming the high-energy bond of ATP. S is the necessary element in synthesis of sulfur-containing protein and exist in the plant body in the form of a -HS bond and Fe–S protein. The variable valence of S functions as an electron acceptor and assists in transferring the electron. Ca2+ plays an important Biological Trace Element Research

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Fig. 8. Content of polyamine in Eu3+-treated L. sativus roots.

role in transferring signal. The change of its content leads to the change of the contents of other elements in the plant (26). Eu3+ is able to replace Ca2+ in CaM–Ca2+ and transfer the signal. The evidence is that the synthesis of amranthin was promoted in the dark after yellowing seedlings of Amaranthus caudatus were treated with Eu3+ (unpublished data). Mn plays an important role in the reaction of PSII as an activator and electron acceptor. The changes of the contents of all types of element in the epidermis, exodermis, endodermis, and vascular cylinder of the root’s cross-section in the presence or absence of Eu3+ indicated that the transporting tendency of elements was from the epidermis to the vascular cylinder. The effect of Eu3+ made the activity of translocation and absorption of elements in the epidermis, exodermis, endodermis, and vascular cylinder stronger. On the other hand, Eu3+ affects the physiological activity of some kinds of element by affecting the distribution of related elements in treated L. sativus roots. As an electron acceptor, S and Cl activate the activity of the root system (see Fig. 3) and enhance the absorption of organic nutrition. Sulfur-containing amino acids promote the synthesis of protein by affecting the transcription of mRNA in the root (see Fig. 4). Eu3+ activated the proteolytic enzyme to catabolize the protein and produce the necessary amino acids that were transported to the growing points (see Fig. 5). Especially, the effect of Eu3+ on the contents of amino acids related to the signal-transferring process is high. The increase of the content of Tyr and the decrease of the content of Thr were relevant to the presence and absence of Eu3+ (see Fig. 6). The → Fe3+) favored the formation of more decrease of the Fe2+ content (Fe2+ –e free radicals and lowering the antioxidant capacity. However, at the same time, the larger amount of polyamine (put, spd, spm) produced was able to scavenge more free radicals and keep the relative steady state of free radicals and prevent the membrane from injury. The complementary mechanism of producing more free radicals and more polyamine is perhaps a consequence of long-term adaptation and genetic screening under an arid environment. Biological Trace Element Research

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CONCLUSION The effects of Eu3+ on the amounts of all types of element in the fracture face of L. sativus is relevant to the biological activity and location of the elements measured. The mobile and reusable active elements were affected more than the inactive elements. Especially, the four elements (P, S, Ca, Mn) that have the function of depot energy, electron acceptor, and transferring electrons come into being with the regularity variation in the presence of Eu3+. The large amount of polyamine perhaps complement the larger amount of free radicals to prevent the cell membrane from injury in the Eu3+-treated L. sativus roots. The one possible action mechanism of → Eu2+ would affect the biological activiEu3+ is that the reaction of Eu3+ +e ties of the related elements. Another action mechanism was that of Eu3+, which has identical chemical properties as Ca2+, was able to replace the Ca2+ in CaM–Ca2+ (Eu3+ + CaM–Ca2+ → CaM–Eu3+ + Ca2+) and regulate the related physiological reaction through regulate the transferring of the signal.

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