Study On The Molecular Mechanisms Of Camellis Sinensis Metal Tolerance Protein 8(Csmtp8)in Transporting Manganese Ion

Tea plant[Camellia sinensis(L.)0.Kuntze]is an important economic crop,which is one of the most popular non-alcoholic beverages worldwide.Manganese(Mn)is one of micronutrient elements required for C.sinensis growth and development,but it can be toxic if accumulated at high concentration.Due to the excessive use of fertilizers in tea gardens,the phenomenon of Mn toxicity caused by soil acidification is becoming more and more serious.However,the study on the absorption and transportion of Mn element and the functional characterization of Mn tolerance genes is unavailable in C.sinensis.Research on the physiological law of the absorption,transportion,and accumulation of Mn in tea leaves is helpful to clarify the molecular mechanisms of C.sinensis accumulation and tolerance of Mn,and provide a new idea for the efficient cultivation of tea plants.In this study,C.sinensis cultivars ’Yingshuang’ and ’Longjing-changye’ were used as experimental materials.Photosynthesis and chlorophyll fluorescence,as well as secondary metabolite contents,were investigated under different Mn concentrations.Proteomic analysis of the leaves revealed that C.sinensis Metal Tolerance Protein 8(CsMTP8)accumulated in response to Mn toxicity in cultivar ’Longjing-changye’.In addition,the biological functions of CsMTP8 were investigated using yeast and Arabidopsis thaliana.The main contents and conclusions are summarized as follows:1.To analyze the tolerance of tea plant(Camellia sinensis)to manganese(Mn)stress,concentrations of 5,100,250 and 1 000 μM Mn were used to treat plants of’Longjing-changye’ cultivar.Photosynthesis and chlorophyll fluorescence,as well as secondary metabolite contents,were investigated.The results show that the photosynthesis of C.sinensis was inhibited when Mn concentration was>250 μM;both antioxidation and plant protection systems were disrupted.We also discovered that the activities of superoxide dismutase(SOD)and peroxidase(POD)were decreased.As for accumulation of secondary metabolites after Mn stress treatment,total amino acid content increased while tea polyphenol content decreased,which can be attributed to stimulation of polyphenol oxidase(PPO)activity.However,Mn at low concentration(5 μM)promoted photosynthesis and C.sinensis growth.Presumably,C.sinensis growth inhibition under Mn stress was due to photosystem damage.Our results will provide a theoretical basis for further studies on C.sinensis tolerance to Mn and will be helpful for manganese fertilizing applications in various tea plantations.2.Through the high Mn treatment of C.sinensis cultivar ’Yingshuang’ and’Longjing-changye’,it was found that the ’Longjing-changye’ had higher Mn tolerance than cultivar ’Yingshuang’.Mn tolerance of C.sinensis cultivar ’Longjing-changye’ were analyzed using two-dimensional electrophoresis(2-DE)combined with matrix-assisted laser desorption ionization(MALDI)-time of flight(TOF)/TOF mass spectrometry(MS)analysis.A total of more than 700 protein spots were detected,and 20 protein spots were differentially expressed,in which 9 protein spots were down-regulated and 11 up-regulated.Further analysis of protein spots E20 indicated that the protein is related to metal tolerance.The C.sinensis metal tolerance protein CsMTP8 was obtained by rapid-amplification of cDNA ends(RACE)and reverse transcription PCR(RT-PCR).The ORF of CsMTP8 is 1218 bp,encoding 405 amino acids with a molecular mass of 44.5 kDa.Evolutionary relationship analysis showed that CsMTP8 had high homology with metal tolerance protein from other plant species,such as Arabidopsis thaliana,rice and Stylosanthes hamate.Subcellular localization results showed that CsMTP8 localized to the plasma membrane.Quantitative real-time PCR(qRT-PCR)results showed that the expression of CsMTP8 in leaves was higher than that in roots,stems and flowers under basal Mn concentration,while excess Mn significantly up-regulated the expression of CsMTP8 in C.sinensis leaves(P<0.05).3.The molecular biological functions of CsMTP8 were studied using the eukaryotic model organisms yeast(Saccharomyces cerevisiae)and Arabidopsis thaliana.Heterologous expression of CsMTP8 in yeast cells resulted in enhanced tolerance to high concentrations of Mn by an efflux of Mn2+ to the external medium.Moreover,overexpression of CsMTP8 in A.thaliana increased plant tolerance and reduced Mn accumulation in plant tissues under excess Mn conditions.Taken together,the results suggest that CsMTP8 is a Mn-specifc transporter,which is localized in the plasma membrane,and transports excess Mn out of plant cells.The results also suggest that it is needed for Mn tolerance in shoots.