Cloning And Functional Analysis Of MDG1 And MDG2 That Are Required For Manganese Utilization In Arabidopsis Thaliana

Manganese,as an essential microelement to plant growth and development,plays important role in many metabolism processes,such as photosynthesis,synthesis of protein and lipid,regulation of many enzyme activities and oxidative stress etc.Manganese,which has active chemical property,owns many valence states in the environment,in which Mn2+is the most effective form for plant obsorption and utilization.Soil pH is the major factor influencing the content of effective Mn2+ in soil.Manganese deficiency is becoming an important factor limiting crop yield in calcareous soil.Therefore,exploring transport,obsorption and homeostasis mechanism of manganese in plants,especially the research about adaptation mechanism and manganese nutrition efficiency under Mn-deficient condition,is of great significance to improve plant nutrition efficiency for manganese.So far,molecular mechanism of manganese absorption,transport and inner balance reported less,mainly including the NRAMP family(natural resistance associated macrophage protein),CDF family(cation diffusion facilitator/metal tolerance protein),ZIP family(zinc regulated transpoter/iron-regulated transpoter[ZRT/IRT1]-related protein),YSL family(yellow stripe-like),etc.In Arabidopsis,NRAMP 1 was reported to be a high affinity manganese transporter,involved in the absorption of manganese under Mn-deficient condition.In order to better explore the mechanism of manganese absorption,transport and inner balance,the study built mutagenesis library in the nramp1 background through the EMS chemical method,screened manganese-defective growth mutant at low manganese medium,in order to clone more unknown genes,which involved in manganese transport and inner balance.In this study,two mutants were isolated,and characterized as Mn-dependent short root mutant mdg1nramp1 and Mn-dependent yellow leaf mutant mdg2nramp1.The research mainly focused on phenotypic,physiological and genetic analysis,gene cloning and function analysis.The main results were as follows:(1)Through physiological analysis,results showed that mdglnrampl was specifically response to Mn,but not to other divalent metal ions,such as iron,zinc and cadmium.Under Mn-deficient condition,cell length of root epidermis cell and root hair of mdglnrarp1 were significantly inhibited,and the short root phenotype was independent of auxin and the SHR/SCR regulation pathway.Compared with WT and nramp1,H2O2 accumulated more on the root tip of mdg1nramp1 by ROS staining under Mn-deficient condition,and the root inhibition phenotype of mdg1nramp1 could be partially rescued by using H2O2 scavenger KI under Mn-deficient medium.So we suggested that H2O2 accumulation in the root tip of mdg1nramp1 might be the reason why mdg1nramp1 showed short root phenotype under Mn-deficient condition.By whole genome sequencing and gene mapping,and phenotype of transgenic complemented line,MDG1 gene was cloned finally.MDG1 encodes NRAMP2 member of NRAMP metal transport family.nramp2 single mutant was specifically response to Mn,but not iron and zinc,could be significantly inhibited under Mn-deficient condition.Metal content analysis showed that,Mn content decreased significantly in young leaf of nramp2,and accumulated in root,suggested that NRAMP2 involved in Mn transport from root to shoot.Tissue localization results showed that NRAMP2 mainly expressed in vascular tissue of root and shoot,especially higher in young leaf than mature leaf,and expression level was not induced by Mn-deficiency.Subcellular localization showed that NRAMP2 was localized in TGN of endomembrane system.Metal uptake assay in yeast indicated that NRAMP2 not only had transport activity for Mn,but also for Fe and Zn ions.-So it could be concluded that NRAMP2 was involved in Mn utilization in cell under Mn-deficient condition.Compared with Col-0,overexpression of NRAMP2 showed better growth,,and more tolerant to Mn-deficiency condition.(2)Through the physiological analysis,results showed that mdg2nramp1 was specifically response to Mn.Under Mn-deficient condition,abnormal chloroplast structure was observed,chlorophyll content and maximum photochemical quantum yield Fv/Fm decreased significantly,suggesting that photosynthetic electron transport might be damaged.Metal content analysis showed that,compared with nramp1,whether in young leaf,mature leaf,flower or root,Mn content showed no significant differences in mdg2nramp1 mutant,besides manganese,other metal ions,such as iron,zinc also showed no difference,so we suggested that the yellow leaf phenotype was not caused by reduced metal absorption under Mn-deficient condition.By whole genome sequencing and gene mapping,and phenotype of transgenic complemented line,MDG2 gene was cloned finally.MDG2 encodes a chaperon protein Cpn60? in Arabidopsis,which involved in Rubisco folding.Study showed that Cpn60a localized in chloroplast stroma,involved in early embryo and chloroplast development,and homogyzous cpn60? showed lethal,while mdg2 mutation in our study was a weak allele,which was caused by single-base substitution.Under Mn-deficient condition,protein complexes of photosystem II,especially D1 and D2 protein were decreased significantly in the young leaf of mdg2nramp1 mutant.So we suggested that Cpn60? was involved in the process how Mn transport into PSII complex in chloroplast.In conclusion,we cloned NRAMP2 and Cpn60? gene by forward genetics.Under Mn-deficient condition,NRAMP2 is important for plant growth,which involved in Mn utilization.Molecular chaperon Cpn60? protein was involved in Mn utilization in chloroplast.