Structural And Functional Analysis Of Arabidopsis ThalianaNRAMPs In Metal Transport

Iron(Fe)is a vital micronutrient for the survival of all organisms.In plants,Fe plays important role in cellular respiration,photosynthetic electron transport,stabilization of chlorophyll,and various other metabolic functions.Absence of Fe will affect the growth and development of plants.Natural resistanceassociated macrophage proteins(NRAMPs)are responsible for the absorption and transport of metal ions.However,the detailed functions of many members are still unclear.In this study,we identified and characterized the function of NRAMP6(AtNRAMP6)in Arabidopsis thaliana.We demonstrated that AtNRAMP6 plays an important role in the growth of the lateral root.The main results are summarized as follows:Experiments of qRT-PCR and GUS-staining revealed that AtNRAMP6 is highly expressed in the lateral roots and young leaves of Arabidopsis.Mn,Fe,Zn or Cu treatment did not significantly affect AtNRAMP6 expression in either the roots or the shoots.When transiently expressed in Arabidopsis mesophyll protoplasts or tobacco leaves,AtNRAMP6 localized to the Golgi/trans-Golgi network.Knockout of AtNRAMP6 impaired lateral root growth without any effects on the principal root growth,while overexpression of AtNRAMP6 improved plant growth under Fe-deficient conditions.However,Fe concentrations in shoots and roots had no significant differences between wild-type,mutant,and overexpression lines.AtNRAMP6 expression could not complement Fe uptake in yeast mutant △fet3fet4,but exacerbated the sensitivity of △cccl mutant to excessive Fe.These results indicated that AtNRAMP6 might not involved in the absorption of external Fe,but involved in intracellular utilization of Fe by releasing Fe from the Golgi/TGN vesicles to the cytosol under Fe-deficient conditions,which might be important for the growth of the lateral root.In many studies,NRAMPs showed low ion selectivity.However,the molecular mechanism of ions binding and selection in NRAMPs is not clear.In this study,in order to reveal the structure-function relationship of AtNRAMP3,we used site-directed mutagenesis to identify the key amino acid residues which are responsible for ion binding and selection in AtNRAMP3.Based on the crystal structure,the ion-binding site of NRAMPs was formed by three residues(Asp,Asn and Met)which are strongly conserved from bacteria to humans.In AtNRAMP3,these corresponded to D72,N75 and M248.We found that the D72A and N75A mutations resulted in a significant reduction in Fe and Mn transport activity.These results suggested that in AtNRAMP3,D72 and N75 are essential for the transport activity.To better understand the function of M248 in AtNRAMP3,this residue was extensively and variably substituted with Ala,Ile,Cys,Ser or Asp,respectively.M248A showed a significant decrease in Cd transport activity.M248S abolished Fe and Mn transport but remained Cd transport.Substitution of Met248 with Cys or Asp resulted in reduced Fe and Cd transport activity but had no effects on Mn transport.M248I showed reduced Fe,Mn and Cd transport.These results indicated that M248 was involved in ion selectivity in AtNRAMP3.Two residues,E194 and R262,may play an important role in stabilizing outward-facing conformation,which is essential for transport activity.G171 mutation disrupted Fe transport activity but had no effects on Mn and Cd transport,suggesting that G171 was also involved in ion selectivity.Deletion mutant results indicated that the N-terminus,but not C-terminus,is necessary for the function of AtNRAMP3.