Roles Of A Chloroplastic Manganese Transporter CMT1 In Arabidopsis Thaliana

Manganese(Mn)is one of the essential microelements in the life cycle of plants.As the cofactor or activator of numerous enzymes,Mn plays the key role in diverse metabolic pathways,including the antioxidant defense,DNA synthesis,sugar metabolism and protein modification.In photosyntesis,Mn is the central atom of oxygen evolving complex that catalyzes water-splitting in PSII.Therefore,Mn deficiency is detrimental to photosynthesis thus leading to the decrease of crop yield and quality.Chloroplast is a semi-autonomous organelle with a dual-membrane system.The transport of nutrient ions through the outer envelope membrane is generally viewed as non-selective.In contrast,specific transporters are required for ions transport through the inner envelope membrane.Mn homeostasis in chloroplast is vital for plant growth and development.However,little is known about the mechanism of Mn uptake into chloroplast.In this study,we identified a new type of Mn2+transporter CMT1(Chloroplastic Manganese Transporter 1)by the reverse genetics.The molecular mechanism of CMT1 in vivo was analyzed by various methods,including molecular genetics,cell biology,proteomics and ionomics.The main conclusions are as follows:(1)CMT1 is essential for plant growth and developmentTo explore chloroplastic transporters,we collected putative multi-transmembrane proteins localized in the inner envelope membrane based on proteomics analysis and established a T-DNA mutants' library corresponding to these candidate proteins.In screening the library,we found a mutant with a T-DNA insertion in the intron of At4g13590 showed a chlorosis,dwarf and stunted growth.To demonstrate that the phenotype was attributable to the mutation of At4g13590,complementation experiment was conducted with the cmt1 mutant as background by its genomic fragment,and the phenotypic defects in cmt1 could be complemented.At4g13590 ecncoded a protein of 359 amino acids with 5 putative transmembrane helices and a chloroplast transit peptide in its N-terminal.At4g13590 belonged to a well conserved family,UPF0016(Uncharacterized protein family 0016),and was named as CMT1 based on its properties according to our research described in this thesis.(2)CMT1 is localized in the inner envelope membrane of chloroplastTo investigate the subcellular localization of CMT1,the CMT1-GFP fusion construct was introduced into Arabidopsis mesophyll protoplasts by transient expression.The GFP signals were specifically located at the periphery of chloroplasts only,which was similar to PIC1,a well known inner envelope membrane protein.To further verify the inner envelope membrane localization of CMT1,PIC1-RFP and CMT1-GFP were co-transformed into Arabidopsis mesophyll protoplasts.GFP signals produced by CMT1-GFP were well overlapped with RFP signals generated by PIC1-RFP.Taken together,these results confirmed that CMT1 was a inner envelope protein.To analyze the expression pattern of CMT1,we performed the histochemical staining and qRT-PCR assay.The results revealed that CMT1 was expressed in various tissues,predominantly in aerial part.(3)CMT1 is crucial for photosynthesisThe chlorosis and stunted growth suggested a possible defect in photosynthesis of cmt1 mutant.Firstly,we analyzed the the ultrastructure of chloroplast by transmission electron microscopy.The results showed that few stroma lamellas were observed in cmt1,whereas grana stacks appeared to be more thick than wild type.Then we conducted the BN-PAGE followed by SDS-PAGE to investigate the thylakoid membrane protein complexes.The results exhibited that the PS? supercomplexes(PS? SC)and other supercomplexes were significantly reduced in the mutant.By contrast,the LHC? trimer and LHC? monomer showed a relative increase in abundance.To assess the levels of thylakoid membrane protein quantitatively,the immunoblotting was performed.In analysis of the nuclear-encoded subunits of the oxygen evolving complex(OEC)in PS? SC,we noticed that level of PsbO was not altered in cmt1,however,contents of PsbP and PsbQ were reduced to only 25%compared with wild type.The chlorophyll a fluorescence parameters and oxygen evolution rate were analyzed respectively.Fv/Fm and ?PS? in cmt1 were significantly decreased compared with wild type,and the oxygen evolution activity in cmt1 is only 32%of that in wild type.These results indicated that the defect of PS? is probably due to the loss function of OEC.(4)CMT1 functions in Mn2+ transport in the chloroplastMembers of UPF0016 family have been linked to the Ca2+/H+ homeostasis in yeast and human.So we speculated that CMT1 might alter the ion levels in the chloroplast.To assess the effect of the cmt1 mutation on metal homeostasis,we measured the ion contents of leaves and intact chloroplasts by inductively coupled plasma-mass spectrometry(ICP-MS).Even though total Mn levels in leaves were comparable between wild type and mutant,the content of Mn was significantly reduced in chloroplast of cmtl compared with wild type.To examine the transport property in vitro,CMT1 was introduced into the ?smf1 yeast strain which is defective in Mn uptake.The plasma membrane localized ?N65CMT1(without chloroplast transit peptide)could complement the phenotype of ?smf1 in Mn limited condition.These results demonstrated that CMT1 was directly involved in Mn2+ transport.In conclusion,we identified a new Mn2+ transporter localized in the inner envelope membrane of chloroplast,which mediating Mn2+ uptake into stroma from cytoplasm.Mutation of CMT1 results in Mn deficiency in chloroplasts,affecting the OEC activity,thus leading to the suppression of plant growth and development.The thesis provides useful theoretical guidances and experimental evideces for molecular breeding to improve Mn2+utilization efficiency of crops.