Possible participation of metal-binding germin like protein of tomato roots in the acquisition of soil mineral nutrients

在我们的研究项目中，获得了以下新的发现的金属结合蛋白，LeGlp 1，在番茄根的功能。（1）重组LeGlp 1经金属亲和层析纯化后作为抗原制备抗体。抗体对LeGlp 1非常特异，这使得能够定量检测LeGlp 1。（2）LeGlp 1以单体和六聚体形式与金属离子结合。(3)In LeGlp 1转基因烟草的叶子以与番茄根中天然LeGlp 1相同的形式表达具有弱草酸氧化酶活性的LeGlp 1。（4）GUS和GFP在转基因烟草的根中定位，所述转基因烟草已经被构建为表达与LeGlp 1的靶序列融合的报告蛋白中的任一种。显微镜下观察LeGlp 1可能定位于根皮层或内胚层细胞的质膜上，而不分泌到质外体。(5)By将LeGlp 1 N端新发现的金属结合位点的Cys残基定点突变为Ser，突变的LeGlp 1在转基因烟草叶片中未能构建LeGlp 1的六聚体天然形式。（6）转LeGlp 1基因烟草叶片的蛋白组分抑制大肠杆菌的生长。由于LeGlp 1的半胱氨酸残基被丝氨酸残基取代后，LeGlp 1的这种活性降低，因此LeGlp 1被认为是大肠杆菌质膜上的金属离子通道，导致细菌细胞内金属稳态的紊乱。LeGlp 1在皮层或内胚层细胞的质膜上作为金属离子通道参与了质外体向木质部管的微量营养素吸收。

In our research project following novel findings were obtained on the function of a metal-binding protein, LeGlp 1, in tomato root.(1)Recombinant LeGlp 1 was purified by metal affinity chromatography and used as antigen to raise antibodies. Antibodies were very specific to LeGlp 1 which enables to quantitatively detect LeGlp 1.(2)Metal binding of LeGlp 1 was shown for its monomeric and hexameric forms.(3)In the leaves of LeGlp 1 transgenic tobacco LeGlp 1 was expressed with a weak oxalate oxidase activity in a form that is identical with the native LeGlp 1 in tomato roots.(4)GUS and GFP were localized in the roots of transgenic tobacco that had been constructed to express either of the reporter proteins fused their N-terminal with the target sequence of LeGlp 1. According to microscopic observation LeGlp 1 may be targeted to plasma membrane of cortex or endoderm cell in roots and may not be secreted to apoplast.(5)By a site-directed mutagenesis of Cys residues to Ser in the region of newly identified metal binding site at the N-terminal of LeGlp 1, mutated LeGlp 1 was shown to fail in the construction of hexameric natural form of LeGlp 1 in transgenic tobacco leaves.(6)Protein fraction of LeGlp 1 transgenic tobacco leaves suppressed growth of E.coli. Since this activity of LeGlp 1 was lowered by the substitution of metal-binding Cys residues to Ser of LeGlp 1, LeGlp 1 was suggested to be a metal ion channel at the plasma membrane of E.coli which resulted in the disorder of metal homeostasis in the bacterial cells.In conclusion, LeGlp 1 is shown to function at the plasma membrane of cortex or endoderm cells as a metal ion channel in the rout of micronutrient uptake from apoplast to xylem tube.