Physiological And Molecular Biological Analysis On Osnaat1, The Lost Function Of Fe(III) Uptake Mutant In Rice

A rice mutant was isolated from a ethyl methane sulfonate(EMS)mutagenized rice(cultivar Nipponbare)mutant library.The mutant could not grow with citrate-Fe(â…¢)as the sole iron source.Using map-based cloning method,we identified a point mutation in a gene encoding nicotianamine aminotransferease, OsNAAT1(LOC_Os02g20360).The Osnaat1 mutant failed to produce deoxymugineic acid(DMA),the phytosiderophore,and defects in absorption of Fe(â…¢).Nicotianamine(NA),the substrate for NAAT1,accumulated markedly in roots and shoots of the mutant.Osnaat1 mutant is able to grow normally under supplement of Fe(â…¡)-EDTA in the cultural solution,and in water-logged padding field which Fe(â…¡)is abundant. Under the condition of supplement of Fe(â…¡)or grown in water-logged field,the mutant accumulated more iron,zinc and other divalent metals in roots and shoots than wild-type.For iron,higher Fe concentration in polished grains was also found. Microarray analysis showed that the expression of a number of the genes involved in Fe(â…¡)acquisition and transportation were upregulated in Osnaat1 mutant.The results demonstrate that disruption of DMA biosynthesis can stimulate Fe(â…¡)acquisition and increase iron accumulation in rice.In addition,when Fe(â…¡)was supplied to the rice young seedlings,expression of genes involved in Fe(â…¢)uptake and transportation were up-regulated in the root, whatever for the Osnaatl or wild type.The amount of DMA was also secreted more than that in the wild type even though the seedlings had no phenotype of iron deficiency under supplement of Fe(â…¡).This implicated that Fe(â…¢)possibly has special physiological function and it maybe help to product some oxygen through the Fenton reaction,relieving the damage brought by low O₂ pressure in the paddy soils. So,Fe(â…¢)uptake in rice seedling is necessary under the anaerobic conditions.When Osnaat1 was supplied with Fe(â…¢)and the phosphorus was removed from cultural solution,the mutant also can be rescued from the abnormal growth.The ICP-MS analysis results showed that Fe,Zn concentration markedly increased in the shoot and root under this condition compared to Fe(â…¢)supplied with phosphorus.The similar result was also found in wild type.But microarry analysis showed the expression of genes involved Fe(â…¢)and Fe(â…¡)uptake were both repressed in rice, implicating a different Fe(â…¢)uptake mechanism may exist under Pi-starvation.The mutation of OsNAAT1 also results in an increase in ethylene in roots,but decrease in shoots when Osnaat1 cultured in the solution under Fe(â…¢).When AVG or AgNO₃,the ethylene inhibitor,was added to this cultural solution,the phenotype of Osnaat1 was alleviated.In the analysis of microarray,we found OsACO1 and some genes related to hypoxia stress were up-regulated.The results showed Fe deficiency in rice could disrupt the ethylene balance and this disruption possibly was caused by hypoxia stress.On the contrary,the better aerobic condition can make rice seedlings escape from this harm.