Functional Analysis Of Metal Transporters TpNRAMP3 And TpNRAMP5 Derived From Dwarf Polish Wheat

Cd is a toxic heavy metal for all living organism,even at low concentration.Since Cd is a non-essential element to plants,no specific transporter is responsible for Cd uptake and translocation.Plants intake and distribute Cd shares the pathway(s)for uptake and transport of essential ions,such as Mn.Natural resistance-associated macrophage protein(NRAMP),one of ancient integrated membrane protein families,absorbs and transports metals including Fe,Mn and Cd.To date,several NRAMP genes have been identified from Arabidopsis,rice,soybean,tobacco and other species.However,no information of NRAMP genes in wheat was unknown.Dwarf Polish wheat(DPW,Triticum polonicum L.)could accumulate high concentrations of Cd,Zn and Fe.Four TpNRAMPs were isolated from DPW;while,their functions were unclear.In the present study,qRT-PCR,subcellular localization,yeast functional analysis and transgenic functional analysis were used to identify the functions of TpNRAMP3 and TpNRAMP5.The main results are as follows:(1)TpNRAMP3 located on chromosome 7BL and consisted of 13 exons and 12 introns.At jointing stage,the expression of TpNRAMP3 was the highest in leaf blades,followed by roots and new leaves,but lower in basal stems and leaf sheaths.At booting stage,the expression of TpNRAMP3 was the highest in old leaves,followed by roots,basal stems and flag leaves,but lower in internodes and nodes.Meanwhile,the expression of TpNRAMP3 was the lowest in peduncles.At grain filling stage,the expression of TpNRAMP3 was the highest in first nodes and the lowest in grains.Furthermore,the expression of TpNRAMP3 in the roots and leaves were not affected by deficiency of Fe,Mg,Zn and Cu.Subcellular localization revealed that TpNRAMP3 was a plasma membrane protein.Expression of TpNRAMP3 enhanced the Cd and Co concentrations,and increased the sensitivity to Cd and Co in yeast.Overexpression of TpNRAMP3 promoted the growth when compared with WT and vector line.Under Mn stress,expression of TpNRAMP3 dramatically improved the seeding root lengths.Under Cd stress,expression of TpNRAMP3 caused the Cd-toxicity and reduced the dry weight of per plant in TpNRAMP3-expressing lines when compared with their individual CK.In addition,expression of TpNRAMP3 in Arabidopsis enhanced the concentrations of Cd,Co and Mn,but not Fe and Zn,in roots,shoots and whole plant.The results revealed that TpNRAMP3 is a Cd,Co and Mn transporter,which is involved in the accumulation of Cd,Co and Mn in Arabidopsis.(2)TpNRAMP5,located on the 4AS chromosome,contains 12 exons and 11 introns.Expression pattern analysis revealed that TpNRAMP5 was mainly expressed in roots,followed by basal stems,but lower in leaves.The subcellular localization results showed that TpNRAMP5 was localized at the plasma membrane of Arabidopsis leaf protoplast.Expression of TpNRAMP5 in yeast significantly increased yeast sensitivity to Cd and Co,and enhanced Cd and Co concentrations.The growths of TpNRAMP5-expressing lines were enhanced compared with the WT in the absence of metal stress conditions.At seedling stage,Mn significantly increased root growth,whereas Cd and Co had no effect.Under Cd stress,TpNRAMP5-expressing lines displayed symptoms of Cd toxicity in seedling leaves and significantly reduced the dry weights of adult-stage plants.Meanwhile,expression of TpNRAMP5 in Arabidopsis significantly increased Cd,Co and Mn concentrations in roots,shoots and whole plants.Furthermore,TpNRAMP5 decreased the translocation factor of Mn.These results indicated that TpNRAMP5 is a Mn,Co and Cd transporter responsible for the accumulation of Mn,Co and Cd.