Accumulation Of Lanthanum In Horseradish Root Cells Under Acid Rain Stress And Action Mechanism To Potassium Channel

The extensive use of rare earth elements（REEs） has increased their environmental accumulation. Acid rain is a serious environmental issue around the world. Therefore, acid rain exists simultaneously with REE accumulation in many agricultural regions. Plant roots absorb water and nutrients from soil, which puts them in direct contact with REEs in the soil.However, little has been reported about the effect on plant roots of REEs under acid rain stress. In particular, no results have been reported regarding the effects on the potassium channel（K⁺ channel） of REEs under acid rain stress in plant roots. Lanthanum（La） is one of most abundant REEs in the environment. Horseradish（Armoracia rusticana） is an important economic crop. Therefore, in this thesis, accumulation of La（Ⅲ） in horseradish root cells under acid rain and action mechanism to potassium channel was investigated with the optimizing combination among the cytobiological, biochemical, physicochemical, molecular biological and computer simulation methods. Meanwhile, horseradish was selected as the experimental material. The main results from our experiments were shown as follows:（1） Acid rain promoted accumulation of La in horseradish root cells: inductively coupled plasma-mass spectrometry（ICP-MS） and scanning electron microscope-energy dispersive spectrometer（SEM-EDS） were employed for determining the La content in horseradish root cells, and the results showed that the La content in roots and intracellular La content of horseradish treated with only La（Ⅲ） increased compared to the control. In addition, the size of the increase got larger as the La（Ⅲ） concentration rose. The La content in horseradish roots is determined by the La（Ⅲ） level in external environment, and the intracellular La content is determined by the extracellular La level in roots. Acid rain promoted the increase of accumulation of La（Ⅲ） in horseradish roots and root cells（except for the treatment of 20mg·L-1 La（Ⅲ） under pH 4.5 acid rain stress had no significant change）, the size of the increase got larger as the La（Ⅲ） concentration and acid rain strength rose. It was observed by using confocal laser scanning microscopy that La was mainly distributed in cell wall and plasma membrane, a part of La（Ⅲ） could enter the cells by endocytosis. Acid rain promot La（Ⅲ） to enter the cells by endocytosis and then accumulate in cells.（2） La（Ⅲ） could influence the functions of K⁺ channel in horseradish root cells under acid rain stress: the K channel current of horseradish root cell was determined by patch clamp,and the K content in horseradish root and root cells was determined by ICP-MS and SEM-EDS, respectively. The results showed that acid rain enhanced the inhibition effects of La（Ⅲ） on the inward current of K⁺ channel. The inhibition effects were enhanced with increasing La（Ⅲ） concentration and acid rain strength. The results of correlation analysis showed that under the stress of acid rain, the accumulation of La（Ⅲ） in horseradish root cells could significantly influence the intracellular contents of K, Zn and Fe. The content of La（Ⅲ）in horseradish root cells positively correlated with the intracellular contents of Fe and Zn,while it significantly correlated with the intracellular content of K in negative way. When it was treated with only La（Ⅲ）, La（Ⅲ） in low concentration（20 mg·L-1） promoted the uptaking K of horseradish roots, while La（Ⅲ） in high concentration（100 and 300 mg×L-1） presented anopposite effect. Under the stress of acid rain at pH 4.5, La（Ⅲ） in 20 and 100 mg·L-1 promoted the uptaking K of horseradish roots, while La（Ⅲ） in 300 mg·L-1 presented no obvious effect on the uptaking K of horseradish roots; under the stress of acid rain at pH 3.0, all the treatments of La（Ⅲ）（20, 100 and 300 mg·L-1） inhibited the uptaking K of horseradish roots.（3） The first reason of La（Ⅲ） changing the functions of K⁺ channel in horseradish root cells under acid rain stress is that La（Ⅲ） could directly interact with K⁺ channel protein. The results of molecular dynamics simulation and quantum chemical calculation showed that under acid condition, La（Ⅲ） could bind to K⁺ channel protein, it then changed the molecular structure of protein, and finally influence the functions of K⁺ channel protein; the results of real-time polymerase chain reaction and western blot showed that La（Ⅲ） could influence the synthesis of K⁺ channel protein. Acid rain at pH 4.5 could increase the promoting effects of La（Ⅲ） in 20 mg×L-1 on the transcription and translation of AKT1 in horseradish root cells,while decrease the inhibiting effects of La（Ⅲ） in 100 and 300 mg×L-1 on the transcription and translation of AKT1 in horseradish root cells. Acid rain at pH 3.0 made La（Ⅲ） in low concentration（20 mg×L-1） inhibit the the transcription and translation of AKT1 in horseradish root cells, while it increased the inhibiting effects of La（Ⅲ） in high concentration（100 and300 mg×L-1） on the transcription and translation of AKT1 in horseradish root cells. The results of correlation analysis showed that the transcription and translation levels of AKT1 were positively correlated with the K content in horseradish roots, and under the stress of acid rain,the transcription and translation levels of AKT1 were positively correlated with the La（Ⅲ）content in horseradish root cells.（4） The second reason of La（Ⅲ） changing the functions of K⁺ channel in horseradish root cells under acid rain stress is that the intracellular environment was changed. First, the results of flow cytometry showed that when it was treated with La（Ⅲ）, acid rain could decrease the cytosolic pH value of horseradish root cells, as well as inhibit the inward current of K⁺ channel and activate its outward current. Second, acid rain at pH 4.5 could promote La（Ⅲ） in low concentration（20 mg×L-1） increasing the cytosolic Ca²⁺ level in horseradish root cells, as well as inhibit the inward current of K⁺ channel and promote the synthesis of K⁺channel protein. Under the stress of acid rain at pH 3.0, La（Ⅲ） in 20 mg×L-1 decreased the cytosolic Ca²⁺ level in horseradish root cells and inhibited the synthesis of K⁺ channel protein;while La（Ⅲ） in high concentrations（100 and 300 mg×L-1） greatly decreased the cytosolic Ca²⁺level in horseradish root cells, and the synthesis of K⁺ channel protein was also further inhibited. Third, when it was treated with La（Ⅲ）, acid rain could promote the accumulation of ROS in horseradish root, inhibit the inward current of K⁺ channel, and activate the outward current of K⁺ channel. Fourth, the surface and morphology of horseradish root protoplasms were observed by using SEM and atomic force microscopy, and the results showed that under the stress of acid rain, the treatment of La（Ⅲ） would aggravate plasma membrane peroxidation, and then change the structure of plasma membrane, and influence the functions of K⁺ channel.（5） Under the stress of acid rain, La（Ⅲ） changed the functions of K⁺ channel and the physiological functions of horseradish: the contents of mineral elements, root activity,photosynthesis and biomass accumulation of horseradish root were determined by using ICP-MS, 2,3,5-triphenyl four azole nitrogen chloride mothed and some other normal detetion methods. The results showed that La（Ⅲ） in low concentration（20 mg×L-1） could promote the root activity and mineral element uptaking of horseradish root, as well as accelerate photosynthesis and the biomass accumulation, while La（Ⅲ） in high concentrations（100 and300 mg×L-1） showed opposite effects. The stress of acid rain at pH 4.5 inhibited the above mentioned promoting effects of La（Ⅲ） in 20 mg × L-1; all the above mentioned promoting effects of La（Ⅲ） in 20 mg×L-1 would be changed into inhibiting effects under the stress of acid rain at pH 3.0; the stress of acid rain at pH 4.5 and 3.0 enhanced the inhibiting effects of La（Ⅲ） in high concentrations（100 and 300 mg × L-1） on the physiological activities of horseradish root, and the inhibiting effects were enhanced with increasing strength of acid rain. The results of correlation analysis showed that the index, such as root activity,photosynthesis, biomass, etc., significantly correlated with La（Ⅲ） content in horseradish root cells in negative way, and with the functions of K⁺ channel protein in positive way.（6） The results in recovery period showed that due to the high La content in horseradish root cells under the stress of acid rain, as well as its strong effects on the cellular structure and function of root cells, the recovery was insufficient. Especially, under the stress of acid rain at pH 3.0 and 4.5, the treatment of La（Ⅲ） in high concentrations（100 and 300 mg·L-1） mainly exhibited destructive effects, and more insufficient recovery. Therefore, more attention should be paid to the biological effect of REEs under the stress of acid rain.