Effects Of Rare Earth Element Activating The Endocytosis Of Arabidopsis Leaf Cells

It is indisputable that rare earth elements（REEs） have been accumulating in the environment and entering organisms through food chain. Thus, the biosecurity of REEs has been attracting more and more attention of global researchers. However, the mechanisms of the biological effects of REEs are still unknown. Our previous study showed that after the treatment with REEs on plant leaves, REEs were first anchored in/on the plasma membrane, then they activated the endocytosis of plant leaf cells, which is naturally inactive. It was the first observed cellular response after the treatment with REEs, therefore, it is of great significance for studying the cytobiological effects of REEs on plant. However, the mechanism underlying the activation, especially the binding targets of REEs activating the endocytosis is still not clear. Therefore, in this thesis, the effects of La（III） and Ce（III）（representatives of REEs） activating endocytosis in Arabidopsis（a model plant） leaf cells were investigated by optimized combinating methods. The effects of REEs on the endocytosis-related downstream signal transduction were also preminilarily discussed. The results of this thesis would provide scientific basis and references for elucidating the cytobiological effects of REEs as well as the safty evaluation. The main results of this thesis were as follows:（1） The anchoring targets through which REEs activate the endocytosis of plant cells are in/on plasma membrane, and the endocytosis activated by REEs in low and high concentrations were in different significances for the growth and development of plants. By using electron microscopic autoradiography, we discovered that after the treatment with REEs in low concentration(30 μmol·L^-1), they were first anchored in/on plasma membrane. Meanwhile, by using confocal laser scanning microscopy, we observed that the endocytosis of Arabidopsis leaf cells was activated. Moreover, the membrane permeability and the MDA content in Arabidopsis leaves were not significantly increased, indicating that the plasma membrane was not damaged. Therefore, the activated endocytosis might be for promoting nutrient uptake. After the treatment with REEs in high concentration(80 μmol·L^-1), more REEs were anchored in/on the plasam membrane. Meanwhile, the activated endocytosis was in higher activity. In addition, the membrane permeability and MDA content in Arabidopsis leaves were significantly increased, indicating that the plasma membrane was damaged. Therefore, the activated endocytosis might be for internalizing the anchored REEs to reduce their toxic effects. After the treatment with REEs in a higher concentration(160 μmol·L^-1), the activity of the endocytosis was obviously decreased. Meanwhile, the membrane permeability and MDA content in Arabidopsis leaves were further increased, indicating that the plasma membrane was serverely damaged. Thus the normal function of plasma membrane was destoried, so that it could not trigger endocytosis.（2） Vitronectin-like protein（VN） is an anchoring target of REEs in/on plasma membrane of plant cells, and the binding of REEs to VN may activate the endocytosis of plant leaf cells. The results of electrochemical analysis showed that the treatment with REEs could increase the VN content at the surface of Arabidopsis leaf cells. The VN content was increased with the increasing REEs concentration, which in accord with the change in endocytosis activity. By using immune-Au combined with transmission electron microscope, it was observed that the intracellular VN expression was increased with the increasing treatment concentration of REEs. Meanwhile, the intracellular VN migrated onto the plasma membrane. It was worth noting that the distribution areas of VN in/on the plasma membrane partly overlapped those of REEs, indicating that VN might be the anchoring target of REEs. By using energy dispersive spectroscopy, it was showed that VN and REEs could locate at same sites, indicating that VN was anchoring site of REEs. The results of homologous modeling, molecular dynamics simulation and quantum chemical calculation showed that REEs could form Lewis acid-base complexes with VN through coordinatively binding to the O in VN. Meanwhile, the hydrogen bonds would stabilize the complexes. REEs could change the molecular structure and charge distribution of VN, which would further trigger a series of cellular responses. Because VN can trigger the integrin-mediated endocytosis, it may be anchoring target of REEs activating the endocytosis of Arabidopsis leaf cells.（3） Arabinogalactan proteins（AGPs） are the main anchoring targets of REEs activating the endocytosis of plant leaf cells. After the incubation with the antagonist of AGPs, it was observed that the activity of the endocytosis of Arabidopsis leaf cells activated by REEs was obviously decreased, indicating that AGPs play a key role in REEs activating the endocytosis of plant leaf cells. The results of electrochemical analysis showed that the treatment with REEs could increase the AGPs content at the surface of Arabidopsis leaf cells. The AGPs content was increased with the increasing REEs concentration, which in accord with the change in endocytosis activity. By using immune-Au combined with transmission electron microscope, it was observed that the intracellular VN expression was increased with the increasing treatment concentration of REEs. Meanwhile, the intracellular AGPs migrated onto the plasma membrane. It was worth noting that the distribution areas of AGPs in/on the plasma membrane partly overlapped those of REEs, indicating that AGPs might be the anchoring target of REEs. By using energy dispersive spectroscopy, it was showed that AGPs and REEs could locate at same sites, indicating that AGPs was anchoring site of REEs activating the endocytosis of Arabidopsis leaf cells. The results of homologous modeling, molecular dynamics simulation and quantum chemical calculation showed that REEs could form Lewis acid-base complexes with AGPs through coordinatively binding to the O in AGPs. Meanwhile, the hydrogen bonds would stabilize the complexes. REEs could change the molecular structure and charge distribution of AGPs, which would further trigger a series of cellular responses.（4） REEs could change the gene expression and activity of Rho-related GTPase from Plants 2（ROP2）, and may influence the downstream Ca²⁺ signal pathway through interacting with calmodulin（CaM）. After the treatment with La（III） in low concentration on the wildtype of Arabidopsis and its four ROP2 mutants, the decrease in ROP2 gene expression and activity would increase root length. After the treatment of La（III） in high concentration, there was no obvious relationship between the gene expression and activity of ROP2 and root length. Meanwhile, it was discovered that the increase in the gene expression of ROP2 could increase the survival ratio of the Arabidopsis under the stress of La（III） in high concentration. The effects of La（III） was investigated by using real-time polymerase chain reaction, and the results showed that La（III） in low concentration could increase the gene expression of ROP2, which further increase the survival ratio of the Arabidopsis, while La（III） in high concentration presented an opposite effect. The effects of La（III） on ROP2 activity was investigated by using western boltting, and the results showed that the increasing concentration of La（III） could increase the gene expression and activity, which is in accord with the change in the endocytosis activity. In addition, the interaction betwwen REEs and CaM was investigated by using homologous modeling, molecular dynamics simulation and quantum chemical calculation. The results showed that REEs could form Lewis acid-base complexes with CaM by forming coordination bond with O in CaM. Meanwhile, the hydrogen bonds in the complexes would make the complexes more stable. Thus, REEs changed the molecular structure and charge distribution of CaM, which would further change its cellular functions. Therefore, REEs could influence the downstream Ca²⁺ signals.