Preliminary Study On The Role Of OsMCO1 In Ferrous Tolerance Of Rice

Ferrous(Fe2+)toxicity is one of the major limiting factors in the rice yield in tropical and subtropical low-lying areas on earth.Rice yield loss due to Fe2+ toxicity is severe in China,especially in the southern rice growing area.Fe2+toxicity is the major limiting factor for the rice growing in cold and soaked fields.It accounts for 80%of the reducing substances.There will be a lot of Fe2+ accumulated in the soil constantly soaked in water for a long period,making it a strong reducing agent.The reducing soil has a toxic effect on rice,which is the main reason for low yield in cold and soaked field.This study focuses on investigating the function of one of the genes induced by high Fe2+ level based on microarray,LOC_Os01g03630,which encodes a multi-copper oxidase,OsMCO1,by using bioinformatics and molecular biology techniques.To begin with,a polygenetic tree was built from multi-copper oxidase(MCO)gene families identified in Arabidopsis and rice.In total,51 MCO members were found in rice.The analysis on OsMCO1 and its subfamily with bioinformatics methods showed that OsMCO1 encodes a multi-copper oxidase,and shows the highest expression level in root during vegetable growth period,slight expression in root and anther during reproductive growth.There are five members in rice MCO subfamily,all of them being localized to peroxisome and having multiple glycosylation sites.The analysis on the amino acid sequences of these genes showed that they all have copper binding sites that are conserved in all multi-copper oxidases.Meanwhile,the expression pattern of OsMCO1 in rice root was analyzed by real-time quantitative PCR.The results showed that OsMCO1 in rice MCO subfamily is induced by high Fe2+ levels,and shows maximum expression level at 10 ?M Fe2+.Moreover,its expression increases as treatment time increases after 12 hours' 10?M Fe2+ treatment.The expression level of OsMCO1 in Fe2+ treatment is 460,820,400,959 times higher than in znic,copper,aluminum,cadmium treatment,indicating that OsMCO1 induction is Fe2+ specific.Transient expression of OsMCO1 in onion epidermal cells demonstrated that both OsMCO1-GFP and GFP-OsMCO1 are scattered in the cell,and localize to organelles.It is inferred that OsMCO1 is likely to be localized to peroxisome after combining bioinformatic prediction.In order to further investigate the biological function of OsMCO1,OsMCO1 T-DNA insertion mutant,osmco1,was treated with different concentrations of Fe2+.Compared to wild type Dongjing,osmcol root growth is inhibitted and has shorter root under high Fe2+treatment.Moreover,the relative root elongation of osmcol is significantly lower than the wild type under 10?M and 100?M Fe2+ treatment,while there is no significant difference between relative root elongation of osmcol and wild type under znic,copper,aluminum,cadmium treatment.Root dry weight of osmcol is also significantly lower than that of wild type under 10 ?M and 100 ?M Fe2+ treatment.osmcol shows darker color at the root tip compared to the wild type Dongjing after being stained by prussian blue.The analysis on Fe2+ levels in both wild type Dongjing and osmcol under Fe2+ gradient treatments revealed that there is no significant difference between their Fe2+ levels in the root.However,Fe2+level in osmcol shoot under 1 ?M and 10 ?M Fe2+ treatment is significantly higher than than in the wild type.Overall,the results indicate that OsMCO1 may play an important role in relieving Fe2+ toxicity in rice,and reducing excess Fe2+ uptake by root.