Isolation And Characterization Of A Mutant Defective In Manganese Utilization In Arabidopsis Thaliana

Manganese(Mn)is an essential nutrient element for plant growth and development.It acts as a component in photosynthesis and involves in protein and lipid metabolism as a direct cofactor of a variety of enzymes.Mn has very active chemical properties,but in the alkaline soil(pH>7.5),the chemical activity of Mn decreases dramatically.Hence,Mn deficiency is becoming a severe problem second to iron deficiency on the global calcareous soil.Compared with other metal ions,the molecular mechanism for Mn transport is poorly understood,with a few protein families been reported,including the NRAMP family,the ZIP family,the CDF family,P-type ATPases and cation/H+ reverse transporters.NRAMP 1 is a high affinity transporter that plays an important role in Mn uptake under manganese deficiency condition in Arabidopsis thaliana.For discovering more genes that take part in Mn absorption and utilization in plants,we establish a mutant library on the background of nr amp1,and screen mutants with yellow leaves phenotype under Mn deficiency condition for further study.The main results were as following:(1)We established a nrampl-EMS mutagenesis library,screening chlorosis seedlings defected in Mn uptake and utilization under Mn deficiency condition,and observe whether they will be recovered in Mn-replete condition.After phenotypic verification of M3 generation,five mutants with yellow newer leaf phenotype;myl2/+nramp1-myl6nrampl were confirmed.Yellow leaves of myl3nrampl and myl4nrampl couldn't be recovered completely by adding replete Mn,while myl2/+nramp1,myl5nrampl and myl6nrampl could.(2)myl2/+nra,mpl showed three different phenotypes:albino,chlorisis in young leaves,normal on vertical medium lacking Mn.The plant with yellow leaves could be recovered by added 2 ?M MnSO4 into the medium,but the albino couldn't.Cultivated Col,nr ampl,myl2/+nrampl in nutrient solution with or without Mn,the phenotype was consistent with that on vertical medium.There was no difference in Mn,Fe,Zn uptake between Col,nrampl,myl2/+nrampl by determine the concentrations of Mn,Fe,Zn in newer leaves,older leaves and roots respectively.Measuring SPAD value of these three materials' newer leaves,we found that the SPAD value of myl2/-+nrampl newer leaves was only half of the other two materials.And there was no difference in Mn-replete culture solution.By collecting younger leaves of Col,nr amp 1,myl2/+nrampl grown on Mn-deficiency and Mn-replete medium,we observed their chloroplast structure with transmission electron microscopy.Results showed that chloroplast of myl2/+nrampl was severely defected under Mn-deficiency conditions.(3)Some experiments were conducted to study the influence of other divalent metal ions on phenotype of myl2/+nrampl.There was no difference between Col,nr ampl,and myl2/+nrampl when added 10-40 ?M Ferrozine into the medium.There was no difference in Mn,Fe,Zn uptake under Fe-replete and Fe-deficiency conditions.Zn-deficiency and different concentrations of Cd treatment had also no difference.(4)Under Mn-deficient medium,myl2/+nrampl showed three phenotype(albino,chlorisis,normal)and the separation ratio accorded with 1:2:1,indicating that the mutation is controlled by an incomplete dominant gene.According to the whole genome sequencing results,we developed dCAPS markers to clone the responsive gene.Results showed that responsive gene was mapped on long arm of chromosome 5.MYL2 is a member of metallo-?-lactamase family,which plays a key role in chloroplast development.In conclusion,MYL2 was not involved in absorption of manganese and other divalent cations,the mutated gene specifically influenced Mn utilization in chloroplast.MYL2 encodes a member of metallo-beta-lactamase family which is located in the chloroplast.How MYL2 involved in Mn utilization in the chloroplast needs further study.