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，我们获得了番茄根中金属结合蛋白LeGlp 1功能的新发现。(1)金属亲和层析纯化重组LeGlp 1，作为抗原培养抗体。抗体对LeGlp 1具有很强的特异性，可以定量检测到LeGlp 1。(2) LeGlp 1以单体和六聚体形式与金属结合。(3)在转基因烟草LeGlp 1的叶片中，其草酸氧化酶活性较弱，表达形式与番茄根中的原生LeGlp 1相同。(4)GUS和GFP定位于转基因烟草的根中，这些烟草被构建来表达任意一种报告蛋白，其n端与LeGlp 1的靶序列融合。显微镜观察发现，LeGlp 1可能靶向于根的皮层质膜或内胚层细胞，而不分泌到外质体中。(5)将新鉴定的LeGlp 1 n端金属结合位点上的Cys残基突变为Ser，突变后的LeGlp 1无法在转基因烟叶中构建六聚体天然形式的LeGlp 1。(6)转基因烟叶蛋白片段对大肠杆菌生长有抑制作用。由于金属结合的Cys残基取代了LeGlp 1的Ser，降低了LeGlp 1的活性，因此LeGlp 1可能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紊乱。综上所述，LeGlp 1在皮层或内胚层细胞的质膜上作为金属离子通道，参与了微量营养物质从外质体向木质部输送的过程。

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.