Abstract.

It has been reported that Rare Earth Elements (REE) can promote plant growth as well as other physiological activities. Since the ion radius of Nd³⁺ is very close to that of Ca²⁺, the interaction of Neodymium(Nd) and Calcium might be one of the important mechanisms to understand such phenomena. Treated with 3*10-6 mol*L-1 Nd(NO3)3 in Ca2+-deficient solution, the effects of Nd on membrane damage in oilseed rape(Brassica napus) were studied. It showed that the symptom of Ca- starvation was relieved and the peroxidization process in rape was deferred. It indicated that added Nd could lower the root relative permeability and MDA content in leave, increase CAT, POD, and SOD activities in rape. Likewise, the added Nd to Hoagland solution showed similar results. The interpretation is offered that the effects are a consequence of the Nd function in substituting some roles of Calcium through interacting with cellular membrane. REE compound fertilizer is extensively applied in China; better understanding of physiological mechanism of REE could be used to direct agricultural operation.

As early as 1879, it was reported that there were trace Rare Earth Elements (REE) in barley, tomato and grape. China has very rich resource of Rare Earth. Since 1972, synthetically studies of interdiciplines have been conducted on agricultural applications of REE in China. Rare Earth compound fertilizers were extensively applied there and increased the crop productivity significantly. However, the mechanism of REE’s biological effects has not been very clear yet. Comparing to La or Ce, there have been few reports on Nd, while the ion radius of which is most similar to Ca²⁺, so it is necessary to do further study to make clear the relationship of Nd³⁺ and Ca²⁺. It was founded that free Nd³⁺ at low concentrations can specially bind to the high-affinity Ca²⁺ binding sites on the Ca-ATPase. In this work we investigated the effect of Nd on some physiological activities in oilseed rape, especially the influence on cell membrane.

Materials and methods

Plant material. After sterilization and water treatment, seeds of oilseed rape (Brassica napus L.) CV Zheyou You 2 were set on filter paper moistened with deionized water, and germinated in the dark at 25℃ for 2 days. Then, Seedlings were moved into and cultured on silica in growth chamber for 7 days. Five uniform seedlings were placed in a polyethylene foam float, provided with Hoagland’s solution for 2 weeks for different treatments. There were four treatments in this experiment: control (Hoagland, CK), control added in Nd(NO₃)₃ (CK + Nd), Ca deficient solution (-Ca) and Ca deficient solution added in Nd(NO₃)₃ (-Ca + Nd). The concentration of Nd(NO₃)₃ used in this experiment is 3*10-6 mol*L^-1 . 20 seedlings were cultured for each treatment. Samples were taken at 10^th day for following experiments.

Relative permeability of root membrane. The root of rape were excised and rinsed thoroughly with deionized water. After incubation in 9 cm culture dish with 30ml deionized water at 30℃ for 15 min, the root were washed several times to remove the inorganic ion outside of the membrane. Then, the samples were transferred into tubes with 10 ml deionized water to measure the conducting value(E₀); conducting value (E₁) was measured after incubating the tubes at 30℃ for 30min; at last, tubes were placed in boiling water for 10min to make electrolyte in tissues completely leak out, and the conducting value (E₂) of solution was measured after the tubes had gotten to be cold. The relative permeability of root membrane was given according to the following formula:

Cell permeability (%) = (E₁-E₀) / (E₂-E₀)×100

SOD activity. Washed 2 g leafs were ground with 0.05 mol⋅L^-1 phosphate buffer at pH 7.8 in ice bath and centrifuged at 13000g for 20 min. Supernatant was collected to measure enzyme activity using NBT reduce method.

MDA content. The procedures of enzyme extraction were the same as above. MDA content was measured by TBA method: 3 ml extract and 5 ml 0.5% TBA were incubated for 10-15 min in boiling-water bath, then centrifuged at 1800g for 10 min after it had been cold down. Supernatant was collected to measure the absorbance at 532 and 600 nm, respectively.

POD activity measurement. 1 g leafs were ground with 0.02 mol⋅L^-1 KH₂PO₄ in ice bath and centrifuged at 13000 g for 15min. Supernatant was collected to measure the absorbance at 470 nm.

CAT activity measurement. 1 g leafs were ground with CaCO₃ and H₂O, and transferred to 100ml bottle and added water to the mark. Supernatant was then used to measure the enzyme activity using Na₂S₂O₃ titration method.

Results

Effect of Nd on membrane permeability of rape root during Ca-starvation

Comparing to the CK treatment, the membrane permeability lowered a little for the treatment of CK+Nd. For the –Ca treatment, the permeability rose significantly as much as 120.6%, which was 97.9% of control for –Ca+Nd treatment (fig1). It indicated that Nd could reverse the membrane damage induced by Ca deficiency.

Effect of Nd on SOD activity and MDA content of rape leaf during Ca-starvation

Under the condition of Ca deficiency, the SOD activity decreased significantly, declining 23.9% of the control. When 3μmol⋅L^-1 Nd(NO₃)₃ was added into Ca deficient solution, SOD activity rose to 145.0%, while the activity of CK+Nd treatment was 16.3% higher than the control (fig2). It indicated that Nd treatment could stimulate SOD activity, which decreased during Ca starvation.

MDA content is used as an important index reflecting cell peroxidation degree. During Ca deficiency, MDA content of rape leaf increased to 6.19*10-9 mol*g-1 , 37.1% higher than the control, while MDA content increased 21.0% for –Ca+Nd treatment (fig3). It suggested that Nd application could defer the senescence process induced by Ca starvation.

Effect of Nd on POD and CAT activities of rape leaf

For the treatments of –Ca and –Ca+Nd, POD activity of rape leaf is 91.6, 106.0

OD₄₇₀ min^-1.mg^-1 protein, increasing 37.9% and 59.7%, respectively (fig4). The POD activity increased 31.2% when Nd(NO₃)₃ was added into the control, while CAT activity increased to 107.5%. During Ca deficiency, CAT activity declined obviously, only 76.0% of the control (fig5). When the plant was treated with Nd, the enzyme activity increased 4.7%. It indicated that POD and CAT had different reaction to Ca starvation. However, Nd treatment could facilitate the

activities of both enzymes.

Discussions

Senescence is one of the obstacles for high productivity. In the undesirable environment and the process of senescence, oxygen free radical, which damages membrane lipid, is an important factor for plant damage and senescence. There have been a great many reports on the relationship of calcium and senescence. As one of the stabilizing agents for cell membrane, calcium can prevent membrane damage and leakage, maintain structural stability and integrity of cell membrane, strengthen the structure and connection of cell wall. In addition, it also can lower the peroxidation effects of membrane lipid, increase activities of membrane protecting enzyme and keep the balance state of ion transport, to relieve the damage of plant by environmental stresses.

The results of our experiments showed that the rape development was impeded during Ca-starvation: the membrane permeability rose; MDA, product of membrane peroxidation increased; and enzyme activities (SOD, CAT) decreased obviously. It indicated that the membrane integrity was destroyed and the ability to clear free radical of protecting enzyme system deceased. SOD, POD and CAT are clear agents for free radical. They are important components of protecting system for plant to defer senescence.

When Nd(NO3)3 (3*10-6 mol*L-1 ) was added in solution, the physiological indexes of rape increased in varying degrees. The damage induced by Ca-starvation was improved to a certain extent. The membrane permeability lower, MDA content decreased and enzyme activities increased. It suggested that Nd³⁺ had some biological effects similar to Ca²⁺ and could perform some functions of Ca²⁺ under given conditions. The results were consistent with precious reports.

There have many reports on the relationship of Ln³⁺ and cell membrane. Comparing with Ca²⁺, since Ln³⁺ have higher charge density and intrinsic affinity, they can make membrane fuse more efficiently, and then change the physical state of membrane lipid environment of membrane enzymes and effect their activities. The results showed that Nd³⁺ could alleviate the damage induced by Ca-starvation, especially the state of membrane. By changing the permeability and stability of cell membrane, and by improving the protecting function of cell membrane, Ln³⁺ can increase organism’s ability to resist undesirable environment. With cytolocalization, it was found that Ln³⁺ didn’t penetrate cell wall, but amassed around the cell wall and outside of the membrane.

Ln³⁺ can combine with a great number of large biological molecular to form stable compounds, and the combining ability is much higher than Ca²⁺, so that it could substitute some biological functions of Ca²⁺ at certain degree. As second messenger in cell signal transport, Ca²⁺ plays an important role in many key physiological and biochemical reactions. In most cases, such effects are preformed through controlling the Calmodulin content within cell. It has been reported that Ln³⁺ can occupy the four binding-sites of Calmodulin subunits and the combining constant is much higher than Ca²⁺. Further studies need to be done to find out whether the effects of Nd on plant also function through Calmodulin .

Conclusions

1. Ca²⁺ plays an important role in stabilizing membrane structure and maintaining the balance of production and clearance of free radical. Ca-starvation quickens the process of cell senescence.

2. Nd treatment can relieve Ca-starvation symptom of rape, increase the activities of protecting enzyme system, lower the extent of membrane lipid peroxidation, and inhibit membrane peroxidation process.

3. With many charaters similar to Ca²⁺, Nd³⁺ might perform its biological functions as a type of “ Calcium-like element” in plant. Cell membrane might be the place at which Nd exerts its biological effects.

Acknowledgment

This research was supported by National Natural Science Foundation of China.

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