Subcellular Location And Function Research About Zinc Transporter Of ZNE1 In Arabidopsis

The gene of At3g08650 was predicted coding zinc transporter in Arabidopsis,but there was no direct experimental evidence.We named At3g08650 as ZINC NUTRITION ESSENTIAL 1(ZNE1).To determine the subcellular localization of the ZNE1 protein,the ZNE1-GFP(green fluorescent protein)fusion gene was transiently expressed in the tobacco epidermal cells.ZNE1 was found to be located in the Golgi apparatus under the laser scanning confocal microscope by comparing the position of the marker protein in organelle emiting red fluorescence.ZNE1 was further expressed in mammalian cells HEK 293T and immunofluorescence was used to demonstrate that ZNE1 was present in the cytoplasm.To determine the physiological functions of ZNE1 in plants,two Arabidopsis thaliana T-DNA insertion deletion mutants znel were found by homozygous identification.Furthermore,under water culture,comparing the zinc dependent growth phenotypes of wild-type Arabidopsis thaliana Col and znel,zne1 was found to exhibit sensitive phenotype of yellow lamina in the high zinc culture solution.The integration of a complete ZNE1 coding sequence into the zne1 mutant genome allowed zne1 to revert to wild-type in response to a high zinc environment,demonstrating that ZNEI is an essential gene for Arabidopsis adaptation to high zinc environment.In order to explore the distribution of apoplast Zn2+ in living leaves,we use the immersion method of vitro leaf petiole and membrane impermeable Zn2+ specific fluorescent dye FluoZin-3 and found that the concentration of Zn2+ in Arabidopsis znel one or two leaf veins was significantly higher than that of the wild type Arabidopsis leaves in high zinc processing conditions,which proved that ZNE1 is an important gene to regulate the Zn2+ distribution in Arabcdopsis thaliana leaves.