Functional Analysis Of Arabidopsis Molybdate Transporter MOT1;1 And Screening For Rescued Mutant

Molybdenum(Mo)is an essential micronutrient for normal growth and development of plants.Plants uptake molybdenum in the form of molybdate from soil.Molybdenum itself is not biologically active and need to combine with organic pterinate to form molybdenum cofactor(Moco).Moco combines with molybdenase in animals and plants to complete the intracellular redox reactions.The main molybdenum enzymes in plant cells include nitrate reductase,xanthine dehydrogenase,aldehyde oxidase and sulfite oxidase.They are involved in key biological process,such as nitrogen assimilation,purine degradation,plant hormone synthesis and sulfite detoxification in plants.Molybdenum also plays an irreplaceable role in the biogeochemical cycle of carbon and nitrogen and the assimilation of sulfur.In agricultural production,plants grown in acid soil often appear defective phenotypes due to molybdenum deficiency,affecting plants growth and crop yield.Therefore,improving plant tolerance to molybdenum deficiency and promoting plant uptake of molybdenum under low molybdenum conditions have great significance on promoting plant growth and ensuring crop yields and varieties.Previous studies showed that the Arabidopsis molybdate transporter MOT1;1 control shoot molybdenum content,but the specific mechanism is still unclear.We studied the function of MOT1;1 in Arabidopsis molybdenum uptake and transport by analyzing the phenotype of the mot1;:1 mutant.Besides,we explored the subcellular localization of MOT1;1,to elucidate its possible action mechanism on affecting plants uptake of molybdenum.The research results will help to further study the molecular mechanism of plant molybdenum absorption,improve the plant's ability to absorb molybdenum in acid soils,enhance the plant tolerance to molybdenum stress,thereby promote plant growth and development and improve crop yield and quality.In addition,based on the low molybdenumsensitive phenotype of the mot1;1 mutants we screened for the rescued mutants which can recover the low molybdenum-sensitive phenotype to the wild-type level,and mapped the mutant gene on the chromosome 2 by bulked segregant analysis(BSA).It will benefit to clone the mutant gene and clarify the molecular mechanism of MOT1;1 involved in Arabidopsis thaliana absorption of molybdenum.The specific research content and main results are as follows:(1)First,we studied the function of molybdate transporter MOT1;1 in the process of Arabidopsis molybdenum absorption under low molybdenum conditions.Compared to the wild type,mot1;1 mutant is more sensitive to low-pH environment,both biomass and shoot molybdenum content being significantly lower than the wild type.The molybdenum bioavailability was significantly reduced in low-pH media.The growth of mot1;1 mutant was more severely inhibited than the wild type on the low-pH medium,and this defect phenotype could be restored to wild type level by adding appropriate amount of molybdenum to the medium,indicating that MOT1;1 plays an important role in Arabidopsis molybdenum absorption and transport.(2)The Arabidopsis sulfate transporters have high sequence identity with MOT1;1 and may be involved in the molybdate absorption.The short-term molybdenum uptake experiments show that MOT1;1 was mainly involved in molybdate uptake under low molybdenum conditions.Under sulfur deficiency conditions,Arabidopsis uptakes molybdenum from the environment mainly through the sulfate transporters SULTR1;1 and SULTR1;2.We studied the effects of different molybdenum,sulfur,and iron concentration on the uptake of molybdenum.Both molybdenum and iron deficiency inhibited molybdenum uptake in mot1;1 mutant.Molybdenum and sulfur compete for the same transporter,and the Arabidopsis molybdenum content increased significantly under sulfur deficiency conditions,and high sulfur concentrations inhibited the molybdenum uptake.(3)We studied the characteristics of the low-molybdenum phenotype and molybdenum uptake of the transgenic lines overexpressing MOT1;1 in the mot1;1 mutants.Under acidic growth conditions,compared with the mot1;1 mutant,the transgenic plant lines overexpressing GFP fusion MOT1;1 driven by the 35S promoter or MOT1;1 native promoter can restore its low pH sensitive phenotype to wild-type levels,and molybdenum content levels also increased significantly.We observed the GFP fluorescence of the transgenic lines through fluorescence confocal microscope and studied the subcellular localization of MOT1;1.The results indicated that MOT1;1 is localized to the mitochondria.The above results indicated that the mitochondria localized MOT1;1 is functional.(4)By separation and purification of the mitochondria of wild-type and mot1;1 mutants and measuring the molybdenum content in the isolated and purified mitochondria,we found that,compared to the wild-type,the mot1;1 mutant's mitochondrial molybdenum content was significantly reduced.These results indicate that MOT1;1 may be responsible for transporting molybdenum from the cytoplasm to the mitochondria and maintaining the homeostasis of molybdenum in the cytoplasm.(5)We perform EMS mutagenesis on the mot1;1 mutants to construct a mutant library.Taking the advantage of the low pH-sensitive phenotype of the mot1;1 mutant,we screened for rescued mutants 28-3 with enhanced tolerance to acidic stress and identified a mutant named 28-3.Under acidic environment,the biomass and molybdenum content of the rescued mutant 28-3 was significantly higher than the mot1;1 mutant,which was close to the wild type.F2 mapping population was constructed by crossing 28-3 with mot1;1.Under low-pH conditions,the F2 generation appeared trait segregation,and the ratio of the number of sensitive plants to the number of tolerant plants was about 3:1,indicating that 28-3 is a recessive mutant.By extracting DNA of extreme individuals and performing BSA,the mutated gene were roughly mapped in in an 1Mb genome region on chromosome 2.In summary,the Arabidopsis molybdate transporter MOT1;1 is involved in the root molybdenum uptake,especially in the process of uptake molybdenum under low molybdenum conditions.MOT1;1 is localized to mitochondria and may be responsible for transporting molybdenum from the cytoplasm to the mitochondria.It plays an important role in maintaining the molybdenum homeostasisin cytoplasm.The research results in this article are helpful to further analyze the molecular mechanism of plant molybdenum uptake,and lay a theoretical foundation for improving crops through genetic engineering methods,cultivating low pH-tolerant plant varieties,and increasing plant molybdenum absorption capacity.