The Functional Analysis Of Arabidopsis Thaliana Magnesium Transporter 4

Magnesium is one of the essential macro-elements for plant growth and development,participated in photosynthesis and various metabolic processes.At MGT family in Arabidopsis is homologous with the bacterial CorA family.The Mg²⁺-transport abilities of the AtMGT?Magnesium Transporter?were identified in bacteria or yeast mutant system.In recent years,functions of some members of AtMGT family in Arabidopsis were elucidated,but functions of AtMGT4 in Arabidopsis were still unknown.In this project,studies on AtMGT4 would deepen our understanding of biological functions of At MRS2/AtMGT gene family and Mg²⁺transport mechanisms in Arabidopsis.By using At MGT4 transposon insertion mutant,functions of AtMGT4 during Arabidopsis development were investigated with the methods of molecular and cellular biological technology.The main results were as follows:Firstly,the AtMGT4 transposon insertion mutant?Ler.ecotype?was obtained,named mgt4-1.No homozygote was obtained from progenies of self-crossed+/mgt4-1.About 50%pollen grains of+/mgt4-1 harbored no nuclei with the staining of Hoechst 33342 fluorescent dye.Reciprocal cross tests between+/mgt4-1 and wild type?Ler.?plants were performed,the segregation ratio between Ler.and+/mgt4-1 genotypes closed to 1:1in the progenies of+/mgt4-1???×Ler.???,while all were wild type genotype in the progenies of Ler.???×+/mgt4-1???.Meanwhile,AtMGT4 RNAi driven by At MGT4 promoter in the background of Col.also caused pollen abotion,furthermore,transgenic plants expressing AtMGT4 in the mgt4-1 background could recover the pollen fertility to the wild type.These results demonstrated that the disruption of AtMGT4in Arabidopsis could result in a defect of pollen development.Secondly,analysis of the stage at which visible pollen abortion appeared was performed.With the staining of Hoechst 33342 dye,nearly50%bicellular pollens nuclei were degraded and showed markedly abnormal shape.Then anthers from wild type and+/mgt4-1 lines were sliced with transverse paraffin sections.The development and degradation of tapetum were natural.At stage 11 of anther development,a ring of vacuole appeared in some of the pollens of+/mgt4-1 mutant,which showed light color when stained with hematine.These results indicated that the visible pollen abortion appeared at bicellular pollen stage in+/mgt4-1.Thirdly,tissues expression pattern analysis based on GUS reporter showed AtMGT4 mainly expressed in root tips,the sites of lateral root elongation,root-stem transition region,shoot apical meristems and vascular tissues of the vegetative organs.In the reproductive organs,AtMGT4 mainly expressed in tapetum tissue,bicellular pollen,tricellular pollen and mature pollen grains.Fourthly,the subcellular localization of AtMGT4 was observed.Arabidopsis mesophyll protoplasts were co-transfected with ER fluorescence marker Bip-RFP and AtMGT4-EGFP,EGFP signal overlapped well with the RFP signal under confocal microscopy,indicating that AtMGT4 was localized to the ER in Arabidopsis cells.Furthermore,the chimeric mgt4 mutant was constructed,with normal expression of At MGT4 in pollens but knockout of AtMGT4 in vegetative organs by expressing AtMGT4 in the background of+/mgt4-1driven by anther-specific AtMGT5 promoter,which was named as mgt4-1for short.Under 3 mM external Mg²⁺level,growth of mgt4-1 was nearly consistent with the wild type.While under 0.01 mM external Mg²⁺level,on the 4^th day,the difference between mgt4-1 and the wild type was remarkable,cotyledon of mgt4-1 was yellow and smaller,compared with the wild type,hypocotyl of mgt4-1 was also shorter than the wild type.Following sustainable growth and development,mgt4-1 showed dwarf and rare later root phenotype.Palisade cells size and number of mgt4-1 were measured with the whole mount clearing technique.Under 3 mM external Mg²⁺level,palisade cells size of mgt4-1 were smaller than the wild type,but cells number of mgt4-1 was the same as the wild type.While under 0.01 mM external Mg²⁺level,both of cells size and number of mgt4-1 were lower than the wild type,indicating it were smaller size and fewer number of palisade cells of mgt4-1 compared with the wild type.that caused dwarf phenotype of mgt4-1.Mg²⁺contens of mgt4-1 under various external Mg²⁺level were measured with ICP-OES technology.Under 3 mM external Mg²⁺level,Mg²⁺content of the shoot of mgt4-1 was lower than the wild type.Under0.01 mM external Mg²⁺level,both the shoot and root Mg²⁺contents of mgt4-1 were lower than the wild type respectively.Otherwise,the ratio of shoot/root Mg²⁺content of Ler.was 1.6:1 under 3 mM external Mg²⁺level,and decreased to 1:1 under 0.01 mM Mg²⁺.But the ratio of mgt4-1always closed to 1:1 under either 3 mM Mg²⁺or 0.01 mM Mg²⁺level,indicating mgt4-1 failed to redistribute the Mg²⁺contents between the shoot and root in response to diverse external Mg²⁺contents.q RT-PCR showed the expression levels of ER-stress related gene AtBip1,AtBip2,AtCNX1,AtCRT1a and At CRT1b in mgt4-1 could be up-regulated by the inducer of tunicamycin,but the up-regulation of AtCRT1b in mgt4-1 was greater than in Ler.In addition,under 0.01 mM Mg²⁺level the expression level of AtCRT3?involved in the BR signal pathway?in mgt4-1 was higher than that in Ler..In conclusion,AtMGT4 was essential for pollen development in Arabidopsis,and also important for the adaption of vegetative organs to low external Mg²⁺level.Mg²⁺concentration in the endoplasmic reticulum might impact the expression level of AtCRT3,and then controlled the cell division,cell growth and male fertility through coordination with BR signal pathway.