Studies Of The Hyperaccumulation Ability Of Pronephrium Simplex And Pronephrium Triphyllum To Rare Earth Elements And Their Binding Peptides

With the widely application of rare earth elements(REEs),the potential environmental and health risks attract more and more attention.Especially,the acceptable daily intake and the agricultural safety evaluation of REEs become hot research topics.High dose of REEs is thought to be poisonous not only for the growth of plants but also for the health of bones, brain,viscera and endocrine system.On the other hand,compared with the traditional chemical and physical remediation techniques of the environment,phytoremediation,a strategy using plant hyperaccumulator to remove contaminants from soil and water,becomes more important as it is environmental friendly and cost-effective.Finding out the hyperaccumulators is the premises of phytoremediation.Although the plant hyperaccumulators of heavy metals were intensively studied,less attention was paid to REE-hyperaccumulator.Currently,most research emphasizes on REEs fractionations in plants,but few molecular mechanism studies of REEs hyperaccumulation have been reported so far.In this dissertation,a REE-hyperaccumulator fern,Pronephrium simplex,was investigated for its hyperaccumulation ability in natural environment.It was cultured under Nd³⁺ stress to study the tolerance and enrichment abilities as well as the fractionation of REEs during uptake and migration processes.Furthermore,the REE binding peptide was separated and characterized,which plays a very important role in hyperaccumulation,to discuss the potential hyperaccumulation mechanism.In addition,a newly discovered REE-hyperaccumulator,Pronephrium triphyllum,was introduced.First of all,P.simplex was sampled from Nanjing natural reservation of semitropical rain-forest for investigating its REE-hyperaccumulation behavior.The content of REEs in the lamina of P.simplex was up to 1249.37μg g^-1,which was respectively 51.8 and 650.7 times of those in its host soil and bioavailable fractions of its host soil.Ce content was the highest in the lamina of natural P.simplex followed by La and Nd.The REE binding peptide separated by SEC and purified by RPLC from P.simplex was 5072Da characterized by MALDI-TOF-MS and ESI-IT-MS.Moreover,the P.simplex was cultivated under different concentrations of Nd³⁺ stress to investigate its accumulation mechanisms.Results showed that P.simplex could survive even at a concentration as high as 500μg mL^-1,confirming that P.simplex has not only hyperaccumulation ability but also strong tolerance for REEs.With increasing of the Nd³⁺ concentration in the solution,the content of Nd in the lamina increased while the Ce content decreased.Nd³⁺ could be absorbed into the cell to promote the expression of the binding peptides.ICPMS results indicated that Nd content in the binding peptide was respectively 94.1 and 57.9 times higher than La and Ce.Thus,the main binding peptide was characterized as Nd binding peptide.There were only 4Da difference between the Ce and Nd binding peptides,which were 5072 and 5076 Da respectively,suggesting that Nd replaced Ce in the peptide.A 632 Da fragment(5076 - 4444 = 632 Da) potentially contained the Nd binding site was found after demetalization of Nd peptide.V8 protease(Endoproteinase Glu-C) hydrolysis results showed there were four fragments(-E-T-M-E-,-P-N-L-L-G-E-, -F-V-I-Y-E-,-F-V-I-A-) in the peptide.Finally,a new REE-hyperaccumulator,Pronephrium triphyllum,was discovered in Nanjing natural reservation of semitropical rain-forest,Fujian,China.The REEs fractionation and storage were studied.It could accumulate 1026.53μg g^-1 REEs in the lamina,which was respectively 42.6 and 557.9 times of those in its host soil and bioavailable fraction of the host soil.The REEs were increasingly hyperaccumulated in vivo by absorption from the bioavailable fraction of the soil by root,migration from root to stem and translocation from petiole to lamina.Based on these findings,P.triphyllum was discovered for the first time as a REE-hyperaccumulator.The REE binding peptide in this fern was 5070.97 Da identified by MALDI-TOF-MS,and the molecular weight was just 1 Da different from the peptide in P. simplex,maybe because that P.simplex and P.triphyllum are intergeneric plants.Further investigations are undergoing.