The Structural Basis For Rare Earth Elements To Initiate Leaf Endocytosis

Rare earth elements(REEs)are widely used in many fields,which results its accumulation in the environment.It is urgent to establish the standard of REE limit concentration due to that REEs can be absorbed by plants and then enter human bodies through food chain,which affects human health.The primary issue for establishing the standard is revealing the molecular and cellular biological mechanisms of REEs acting on plants.Our previous study discovered that REEs can break the endocytic inertia of leaf cells,which is a result of long-term evolution,and initiate the endocytosis in leaf cells.The initiation of endocytosis is the primary response to REEs,which opened a window for investigating the molecular and cytobiological mechanism of REEs acting on plants.However,it is still not clear that what is the target through which REEs activated endocytosis?And what is the structural basis of the endocytosis initiation.In this paper,Arabidopsis was chosen as the model plant,La(?),Ce(?)and Tb(?)were chosen as the representatives of REEs.By using a multi-disciplinary strategy involved microscope,confocal laser scanning microscope,nanometre immune colloidal gold technique,ultraviolet-visible spectroscopy,fluorescence spectrometry,circular dichroism,and computational simulation,the structural mechanism of REEs initiating the endocytosis of leaf cells was investigated.The main results are as follows:(1)It was confirmed that Arabinogalactan proteins(AGPs)are the targets REEs initiating the endocytosis of Arabidopsis leaf cells.Without the treatment of AGPs inhibitor,La(?),Ce(?)and Tb(?)can initiate the endocytosis of Arabidopsis leaf cells;After the treatment of AGPs inhibitor,the endocytosis initiated by REEs was greatly weakened,but did not completely disappear.As the endocytosis initiated by the treatments of La(?),Ce(?)and Tb(?),AGPs was recruited onto the plasma membrane of leaf cells.And the AGPs were increasingly recruited with the increasing concentrations of La(?),Ce(?)and Tb(?).(2)The the structural basis of REEs-AGPs initiating activating the endocytosis of Arabidopsis leaf cells was revealed.Under the condition of pH=6.5,La(?),Ce(?),Tb(?)in low concentrations interacted with AGPs through weak interactions(such as electrostatic interaction,van der waals' force,hydrogen bond,intermolecular force,hydrophobic interaction,etc.),which further caused a micro-perturbation on the molecular structures of AGPs.Although it made the molecular structure more orderly,it finally made the molecules much more looser due the increase of beta angle;the La(?),Ce(?),Tb(?)in high concentrations can serve as Lewis acids to coordinately bind to the O in the carboxyl or carbonyl groups of AGPs,which are Lewis bases,to form stable REEs-AGPs complexes,which increased the randomness of AGP molecular structures.In conclusion,after the interaction with La(?),Ce(?)and Tb(?),the structure of AGPs have been changed,which endowed AGPs a new function—initiating the endocytosis of leaf cells.This is the structural basis of REEs-AGPs initiating the endocytosis of Arabidopsis leaf cells.(3)It was found that the interactions between REEs and AGPs depend on the pH value.Under the conditions of different pH values,the surface of AGPs contain the negatively charged regions,which could bind to the positively charged La(?),Ce(?)and Tb(?).Under the condition of pH=6.0,La(?),Ce(?)and Tb(?),especially they in high concentrations showed the strongest binding to AGPs which resulted the greatest influences on the molecular structure of AGPs.The effects of pH value on the interactions between La(?),Ce(?),Tb(?)and AGPs depends on the kind of REEs:Ce(?)>La(?)>Tb(?),and a possible reason is that Tb(?)has the best binding ability.Considering the principle of rare earth chemistry,the interaction modes between La(?),Ce(?),Tb(?)(especially in high concentration)and AGPs are not only coordinative binding bonding,but also van der waals' force,hydrogen bond,intermolecular force,hydrophobic interaction,etc.(4)Vitronectin-like protein(VN)is an anchoring target of REEs on the plasma membrane of plant leaf cells.After the treatment with low-concentrations of La(?)and Ce(?),it was found that almost all of La(?)and Ce(?)were anchored in the cell wall-plasma membrane area,which was precisely the distribution area of VN in cells;After the treatment with high-concentrations La(?)and Ce(?),more La(?)and Ce(?)were anchored in the cell wall-plasma membrane area,and it increasingly overlapped the distribution area of VN.Therefore,VN is a target of REEs acting on leaf cells.Low-concentration La(?)and Ce(?)could interact with VN through weak interactions,whereas high-concentrations of La(?)and Ce(?)could coordinately bind to the O in the carboxyl or carbonyl group of VN to form stable Ln-VN complexes.Theses interactions changed the molecular structure of VN,making it become another target of REEs initiating the endocytosis of Arabidopsis leaf cells except AGPs.This study can provide a theoretical basis for revealing the molecular and cellular biological mechanism of REEs acting on plants and establishing the standard of REEs limit concentrations in plants.