The Functional Analysis Of OsMTP11,a Member Of The Cation Diffusion Facilitator Family In Rice

Manganese(Mn)is an essential micronutrient for plant growth?In plants,Mn plays roles in oxygen generation in a domain of photosystem ? and in decomposing superoxide in mitochondria by Mn containing proteins.Further more,Mn2+ is a cofactor that activates 35 different enzymes.Too less or too much Mn2+accumulation will block the grow of plants.In Mn deficient plants,dry matter production,net photosynthesis and chlorophyll content decline rapidly.In this case,to study the cellular mechanism of rice plants absorbing and accumulating heavy metals and consequently helps cultivate green rice with high yield and good quality is of great significance.Here we show that an Rice Cation diffusion facilitator,OsMTP11,mediates Mn transport and is essential for metal homeostasis and tolerance?To investigate the cellular function of OsMTP11,the rice protein was expressed in S.cerevisiae yeast mutants.The yeast mutants were transformed with OsMTP11 expressed from pYES2 through the Lithium acetate transformation method.We find that OsMTP11 has apreference for Mn2+as the transported ion,but not in other heavy metals.To determine the growth rates and Mn2+accumulation in ?pmr1 cells that transformed with OsMTP11 or empty vector and S.cerevisiaestrainwas transformed by the pYES2 empty vector.We find that OsMTP11 protein confer Mn2+tolerance to ?pmr1 via a Mn2+efflux mechanism.To investigate the tissue specific expression pattern determined using quantitative real-time RT-PCR,We find that the expression of OsMTP11 was investigated in different tissuesat different growth stages.At seedling and tilling stages,OsMTP11 wasmainly expressed in the roots,but at the reproductive growth stage,the expression of OsMTP11 was increased especially in the shoots.A dose response experiment with different Mn2+concentrations showed that compared with plants grown under control conditions,root and shoots OsMTP11 transcript levels were increased in response to Mn2+ deficiency and shoots OsMTP11 transcript levels increased upon Mn2+excess(3-fold increase),root OsMTP11 transcript levels 1.5-fold increase.To investigate whether the expression of OsMTP11 is affected by a deficiency of essential metals,including Zn,Fe,or Cu,we exposed the seedlings to a solution that lacked each of these metals.However,the expression of OsMTP11 was only increased in roots and shoots by a Mn deficiency and was unaffected by Fe Zn and Cu deficiency.These findings suggest that the expression of OsMTP11 is specific and regulated by manganese.To determine subcellular localization and the function of OsMTP11,we transiently expressed an enhanced green fluorescent protein(EGFP)-OsMTP11 fusion gene in protoplasts prepared from rice leaves and tobacco leaves was transfected by infiltration using an tumefaciens method.We find that OsMTP11-EGFP and mRFP-SYP61 as a marker that is present in the Trans Golgi network were coexpressed,the two fluorescence signals overlapped considerably.Overall,these transient expression analyses provide evidence that OsMTP11 is localized in the Trans Golgi network.When tobacco leaves transiently expressing OsMTP11-EGFP were infiltrated with perfusion media containing excessive Mn2+,a new OsMTP11-EGFP signal emerged overlapping with the signal from the PM marker PIP2A-RFP.These results reveal that OsMTP11 sequestered Mn2+into vesicles that traffic to the plasma membrane to release the metal from the cell via exocytosis.We generated transgenic lines with suppressed expression by using the LH-FAD2-1390 rice interference vector and obtained a T-DNA insertion line from the Rice Mutant Database.When the plants were subjected to deficiency Mn2+ concentrations,Compared with wild-type plants,Mn2+accumulation and the dry weight in roots and shoots of the mutant was slightly increased,Shoot growth and root elongation were increased.Similar results were obtained with RvAi lines.At excessive Mn2+treatment,the dry weight of the shoots and roots in the mutant and OsMTP11 RNAi lines were reduced,the mutant and RNAi lines exhibited symptoms of Mn toxicity,such as brown spots and chlorosis.Mn2+accumulation in shoots and roots were increased about 20%.Urlike Mn2+,there were no large differences in the accumulation of other microelements(Zn,Cu,and Fe).In conclusion,OsMTP11 are suggestive of asecretory pathway mediated mechanism of heavy metal detoxification in plants.Transcriptome sequencing revealed that when the mutant rice under the stress of Mn2+,there were a large number of transporters of Fe up regulated,and Pi transports down regulated.What's more,jasmonic acid signaling was involved in alleviating the toxic effects of riceIn conclusion,the present set of data including phenotypic characterization of rice transgenic lines,yeast complementation,tissue expression,and subcellular localization.OsMTP11 are suggestive of a secretory pathway-mediated mechanism of heavy metal detoxification in plants through sequestered Mn2+into vesicles that traffic to the plasma membrane to release the metal from the cell via exocytosis.