Cloning And Functional Analysis Of A Manganese-utilization Gene MDG4 In Arabidopsis

Manganese(Mn)is an essential trace element in most organisms.In plants,Mn is required for photosynthesis for forming the oxygen-evolving complex on the thylakoid membrane,and Mn deficiency affects the assembly of the photosynthetic System PSII protein complexes and photosynthesis.In order to probing into how plants balance the Mn ustilizing and the development of chloroplast in plant cells,we construct an EMS mutation library of nrampl background to screen the young yellowing mutant of manganese-deficient dependence and obtain many mutants.In this paper,one of the mutant mdg4nrampl of the yellow manganese deficiency new leaf was used as the experimental material,and the gene cloning,physiology and molecular mechanism were further studied.The following test results were obtained:(1)The phenotype of the young leaf yellowing of the mdg4nrampl mutant was dependent on the supply of exogenous Mn,no difference between mdg4nrampl and nrampl in the absence of iron,zinc,copper or in the present of cadmium.The result indicates that the young leaves chlorosis of mdg4nrampl is relatively special.Mn deficiency seriously interferes with chloroplasts and thylakoid membrane structure of young leaves of mdg4nrampl mutants by transmission electron microscopy.Using ICP-MS measure the content of Mn,Mn-deficiency does not affect the absorption of manganese ions in the newborn leaves of mdg4nrampl mutants.(2)The genetic pattern of MDG4 is a single gene recessive heredity controlled by nucleus,and MDG4 encodes a TPR family protein REC1 which located in cytoplasm and nucleus.The results of GUS staining showed that MDG4 was mainly expressed in the photosynthetic tissues,and the expression of MDG4 gene were decreased in mdg4 and Mn deficiency condition by qRT-PCR.(3)Under the condition of water culture,the content of photosynthetic pigments in mdg4 mutants decreased,the content of Mn in chloroplasts decreased slightly.Using BN-PAGE and Western blotting analysis protein complexes and proteins of chloroplasts,the result was showed that the PSII complexes on the photosynthetic membrane of the leaves decreased,the results of Western blotting showed that the abundance of D1,CP43 and PsbP proteins were decreased most significantly in the total protein of leaves.To sum up,the TPR family protein MDG4 plays an important role in the chloroplast manganese utilization and the assembly of PSII complex in Arabidopsis thaliana.