Molecular Mechanisms Of Enhancement Of Iron And Zinc Uptake And Utilization Through Repression Of Fe Uptake Strategy Ⅱ In Rice

An EMS (ethyl methane sulfonate) induced Fe-uptake strategy II defective (std) mutant was isolated from a japonica rice, cv. Nipponbare. The phenotypic and physiological characterization of the mutant was conducted under the circumstances of different iron supply. The positional cloning was used to identify the mutated gene. The gene cloned is for nicotianamine aminotransferase (NAAT) which is involved in the mugeinic acids synthesis in rice. The concentrations of nicotianamine (NA) and deoxymugineic acid (DMA) were measured in the mutant under different Fe supplied conditions. Microarray analysis was used to examine gene expression profile in the mutant under different growth conditions. The results are summarized as follows:1.In contrast to the wide type (WT), the mutant showed small and chlorosis with short adventitious and lateral roots. Shortened lateral roots formed densely and emerged on the root apical region in the citrate-Fe solution. These mutant traits could be restored when the mutant plants were grown in the solution supplied with EDTA-Fe. The element analysis showed that the Fe concentrations of both shoots and roots in the mutant were remarkably decreased compared to the WT with citrate-Fe supply, while increased with EDTA-Fe supply. While the concentrations of another metal ion Zn was increased in the mutant under both Fe conditions, and even when Fe was withdrawn from the solution.2.An F₂ population was developed from a cross between the mutant and an indica rice cv. Kasalath. The segregation of mutated phenotypic traits among the F₂ population indicated that the mutant possesses a recessive mutation at a single locus. The mutated gene was mapped on chromosome 2 flanked by the markers RM13046 and RM13051 based on a map-based strategy using 2900 F₂ mutants. In the region, one gene which encodes nicotianamine aminotransferase (EC 2.6.1.80) was found by BLAST searches. With the sequencing analysis of this gene, a substitution in the alternative splicing site was occurred, which resulted in the fourth exon of 60bp in the gene was deleted.3.Measurement of root DMA secretion showed that plenty of DMA from the WT was induced under Fe-deficiency but not from the naatl mutant. The precursor of DMA, NA is considerably increased in the naatl mutant compared to the WT, under the three iron conditions.4.Microarray analysis was performed to investigate the gene expression profile in the naatl mutant under different iron conditions compared to the WT. Under citrate-Fe supplied condition, the genes involved in the systemâ…¡, such as these for NAS,NAAT1 and DMAS, the iron and Zn transporters and some transcriptional factors were upregulated in shoots or roots or both of the mutant. One gene for plant acid phosphase was upregulated in roots. These indicated that the mutant was suffered by iron deficiency. Under EDTA-Fe supplied condition, most of the above genes were still upregulated in the mutant roots. One ferritin gene was repressed in root of the mutant. The results support that the impact of iron metabolism by function loss of NAAT1 in the mutant strongly induced the expression of genes involved in iron and zinc uptake.The results demonstrated that repression of Fe uptake strategyâ…¡would enhance iron and zinc uptake and utilization in rice. This would be helpful to molecular design breeding on microelement uptake.