Research On Mechanism Of The Excessive Accumulation Of Cadmium In Arabidopsis Thaliana Mutant Of Ferroxidase Gene,lpr1lpr2

Cadmium(Cd)is a toxic element that is ubiquitous in soils and has been classified as a Group I carcinogen by the International Agency for Research on Cancer(IARC).Recently,the farmland soil in some areas of our country has been polluted by Cd due to the rapid industrialization,the discharge of industrial wastes and the improper application of chemical fertilizers and pesticides containing Cd.Cd is easily taken up by plants and can harm hunman health,causing kidney damage,osteoporosis and other diseases through the food chain.Therefore,it is necessary to develop environmentally friendly and sustainable remediation technology for Cd contaminated soil.Phytoremediation is a potential method to deal with soil Cd pollution.With the development of molecular biology,it is promising to improve plant Cd accumulation and remediation through biotechnology.The molecular mechanism of plant Cd accumulation is the basis for improving plant Cd remediation through biotechnology.In this study,we found the excessive accumulation of Cd in shoots of Arabidopsis thaliana ferroxidase gene mutants lpr1lpr2 through ionomics analysis and further analyzed the phenotype,the expression and regulation mode of LPR1 and LPR2 gens and the subcellular localization and enzymatic properties of their proteins.We also studied the molecular mechanism that control the excessive accumulation of Cd in lpr1lpr2 and explored its application prospect in the phytoremediation of Cd contaminated soil.The main results are summarized as follows:(1)Arabidopsis thaliana mutants lpr1,lpr2,lpr1lpr2 and the wild-type Col-0 were cultured in soil,their growth phenotypes were surveyed and the elements concentration were analyzed by ionomics.The results showed that the growth of lpr1lpr2 was inhibited and the Fe,Mn,Zn and Cd concentration in shoots were significantly higher than Col-0,lpr1 and lpr2,especially for Cd.Further studies showed that the growth inhibition of lpr1lpr2 was caused by the excessive accumulation of Fe.The Cd concentration in shoots,roots and seeds of lpr1lpr2 was much higher than that of wild type when exposed to Cd,suggesting that these two genes have potential application in plant remediation.(2)The RT-q PCR analysis showed that the expression level of LPR1 gene was similar in roots and shoots,while the LPR2 expression was much higher in shoots than that in roots and also much higher than LPR1.The expression of LPR1 and LPR2 in both shoots and roots were not affected by Fe concentration.GUS staining showed that LPR1 and LPR2 were mainly expressed in vascular tissue of leaves.The complement lines were obtained by expressing the GFP fused LPR1 or LPR2 driven by their own promoter,respectively.The fluorescence signal of GFP in the petiole of the complement lines was observed,and it was shown that the GFP fused proteins of LPR1 or LPR2 was mainly located on the cell wall of xylem vessels.In addition,LPR2 was proved to have ferroxidase activity as well as the LPR1 by heterologously expressed in tobacco leaves.(3)The expressions of Fe-deficiency induced Fe metabolism genes,such as IRT1,FRO2,BHLH39,FEP1,FEP2 and FEP3 were observed to be significantly higher in roots of lpr1lpr2 than Col-0 under normal Fe(20 μM)condition,which indicated that there is still Fe deficiency signal in roots of lpr1lpr2 even Fe is overaccumulated.The distribution of Fe in shoots of lpr1lpr2 was analyzed by chemical staining and synchrotron-based X-Ray fluorescence microscopy.Fe was found to deposite at the apoplast of the xylem vessels and the protective cell of vascular tissues in the petioles and leaves.The Fe concentration in the phloem sap of lpr1lpr2 was found to be significantly lower than that of Col-0.These results indicated that the mutation of LPR1 and LPR2 genes lead to the deposition of Fe at the apoplast of the xylem vessel of petiole and main leaf vein of lpr1lpr2,reducing the transfer of Fe to the phloem.The reduced Fe concentration in the phloem will induce the Fe deficiency signal and the expression of IRT1 and FRO2,causing the excessive accumulation of Cd.We also found that the growth inhibition of lpr1lpr2 was significantly alleviated when grown in neutral soil and the Cd concentration in shoots of lpr1lpr2 was further increased,which showing the potential for application in the phytoremediation.In conclusion,this study is firstly found a new phenotype of ferroxidase gene mutant lpr1lpr2,which is characterized by the growth inhibition and excessive accumulation of Fe,Mn,Zn and Cd.We also found that the lost function of LPR1 and LPR2 at the apoplast of main leaf veins lead to Fe deposition there and reduced Fe translocation to the phloem,resulting in the decrease of Fe concentration in the phloem sap,which inducing the Fe deficiency response and the expression of IRT1.The continuous high expression of IRT1 eventually increased the uptake and accumulation of Cd in lpr1lpr2,thus reveals the molecular mechanism of excessive accumulation of Cd in lpr1lpr2.In addition,we also investigated the potential of the application of lpr1lpr2 in the phytoremediation of Cdcontaminated soils and found it has an application prospect in phytoremediation of neutral Cd-contaminated soils.