Functional Analysis Of A Putative High-affinity Ni²⁺/Co²⁺ Transporter （AtHAN） Of Arabidopsis Thaliana

Nickel is an essential micronutrient in plants. It is the metal cofactor of urease. Urease mediates hydrolysis of urea, which has been identified in bacteria, fungi, algae and plants. In addition, nickel is an essential compoment of Ni-Fe hydrogenase, carbon monoxide dehydrogenase, acetyl-coenzyme A decarbonylase/synthase, superoxide dismutase SodN and methyl-coenzyme M reductase in microorganisms. Cobalt has been found in all kinds of plants despite not been defined as essential nutrient. Many studies have shown that adding a small amount of cobalt has a positive effect on crop yield. Cobalt has been defined as a beneficial element.The metal ion transporters mediate in uptake and transport of metal ions between the cells, tissues and organs. Metal ion transporter family mainly includes ZIP, YSL and NRAMP. High-affinity nickel and cobalt transporter were found in microorganisms. The high-affinity nickel and cobalt uptake is mainly mediated by ATP-binding cassette (ABC) systems and by secondary transporters. Mechanism of nickel and cobalt uptake in plants is unknown. Nickel and cobalt transporter have not been reported in plants. In the present study, a gene (At4g35080) was cloned from Arabidopsis thaliana, which encodes a putative high-affinity Ni2+/Co2+transporter (AtHAN for short). The open reading frame (ORF) of AtHAN gene was1098bp, which encoded a protein of365amino acids. The prediction of functional domains shows that amino acid(140-364)participate in high-affinity transport of nickel and cobalt. AtHAN is characterised by5transmembrane (TM) helices predicted by computer algorithms.pREP3-AtHAN recombinant plasmid and pREP3vector was transformed into yeast strain Aniclp, which is deficient in nickel uptake.The Ni2+/Co2+concentrations in the yeast transformants were determined with an atomic absorption spectrophotometer.The transgenic yeast showed higher total Ni2+/Co2+than control.But there is no significant difference between the growth of transgenic yeast and control under the treatment of nickel.The growth of transgenic yeast were weaker than control when cultured in medium containing0.6mM Co2+. It is speculated that yeast cells requires nickel ions with low content (nM) and nickel content in the medium is sufficient to ensure the normal growth of the yeast. Excess nickel is toxic and therefore no significant difference was detected in the growth of both yeast strains. GFP fusion proteins of AtHAN localized in endoplasmic reticulum in yeast cells.The relative expression levels of AtHAN in different tissues and in the presence of Ni2+and Co2+were studied by qRT-PCR. The results showed that the expression levels of AtHAN are higher in flower and in root.The expression levels of the AtHAN genes were effected by the concentration of nickel and cobalt ions in enrironment. AtHAN gene was cloned into pBI121 expression vector.The pBI121-AtHAN recombinant plasmid was introduced into Arabidopsis thaliana. The over-expression lines of AtHAN gene were confirmed. The growth of over-expression plants were weaker than wide-type plants in the presence of Co2+. There is no difference between the growth of over-expression plants and the wide-type plants in the presence of Ni2+. The Co2+accumulation in the shoot of over-expression plants were higher than wide-type plants and it was the same in the root under40μM,150μM Co2+treatment. There is no difference between the Ni2+accumulation of over-expression plants and the wide-type plants in the presence of Ni2+.It is speculated that AtHAN involved in cobalt ion transport and the accumulation of cobalt is excessive in the shoot of over-expression plants, thereby affecting the growth of plants.