Isolation And Expression Analysis Of A Putative Nitrate Transporter Gene OsNPF4.5 In Rice

China is the largest agricultural country in the world.Over the years,as the demand for high crop yields,excessive and inappropriate application of fertilizers(especially N and P fertilizers)becomes normal during agricultural production,which has caused severe evironmental and ecological issues,such as soil acidification,eutrophication,and ground water pollution.Therefore,increasing plant nutrient uptake efficiency,and reducing the loss of N fertilizers,is of great significance for the development of sustainable agriculture and for the ecological environment protection.Nitrogen(N)is one of the three most essential macronutrients required by plants,and is also the most critical limiting factor for crop production.Nitrate(NO3-)and ammonium(NH4+)are the most important inorganic N forms absorbed by plants.In natural ecosystems,as a result of root absorption,chemical fixation,leaching losses,nitrification and denitrification,etc,the availability of N drastically fluctuates in both space and time,which makes N-limiting conditions a general rule in many soils.Plants have evolved several strategies to improve nutrient acquisition from nutrient-poor soils,including activation of high-affinity nutrient transport systems and formation of symbiotic associations with arbuscular mycorrhizal(AM)fungi in soils.Several AM-induced plant transporters responsible for transporting NH4+,across the symbiotic interface have been identified.However,no genes involved in symbiotc NO3-transfer have been verified so far.In our previous work,we have identified an AM-induced gene,OsNPF4.5,from the rice NPF gene family.Since multiple homologous genes in the plant NPF family have been demonstrated to have the ability of transporting NO3-across the plasma membrane,it is tempting to speculate that OsNPF4.5 may be involved in the N transfer across the symbiotic interface.To testify this hypothesis,in this study,a series of investigations,such as quantitative expression analysis,tissue-specific expression paterns and heterogeneous expression analysis were carried out.The main results are as follows:1.The full-length cDNA sequence of OsNPF4.5 was obtained through RACE(Rapid amplification of cDNA ends)assay.Sequence analysis revealed that this gene harbors seven exons and six introns in its coding region,and contains a 1830 bp open reading frame(ORF)encoding a predicted protein of 609 amino acids.Phylogenetic analysis showed that OsNPF4.5 could be grouped into the IV subfamliy of the plant NPF gene family,and shows substantial homologies to the known NPF genes from Arabidopsis and rice,such as AtNPF4.1,AtNPF4.2,AtNPF4.3 and OsNPF4.1.2.The expression patterns of the OsNPF4.5 in response to AM fungal colonization were investigated.The transcripts of OsNPF4.5 could be highly induced in the roots colonized by the AM fungus(Rhizophagus irregularis),whereas its transcripts in the leaves of the colonized rice plants,or in the roots and leaves of the uncolonized rice plants were barely detectable.None of the other paralogues in the IV subfamily of rice NPF family showed significant upregulation in response to AM symbiosis.3.The expression profiles of the OsNPF4.5 in response to different N forms and different phytohormone applications were determined.It was shown that OsNPF4.5 were observably induced upon NO3-application,and its transcripts could be downregulated in response to ABA and SA applications.However,application of IAA,JA or GA had no significant effect on the transcript abudances of OsNPF4.5.4.Histochemical staining demonstrated that a 2258-bp and a 844-bp OsNPF4.5 promoter fragments were sufficient to drive the GUS expression strongly in AM fungal colonized tobacco and soybean roots.It was observed that the GUS staining was predominantly confined in distinct cortical cells containing arbuscules.However,the GUS staining could not be detected in the uncolonized plant roots.5.Subcellular localization analysis revealed that the protein encoded by OsNPF4.5 is a plasma membrane-localized protein.Heterogeneous expression of OsNPF4.5 in Xenopuslaevis oocytes showed a low-affinity transport activity for nitrate.6.Trangenic rice plants expressing a CRISPR/Cas9 construct designed to specifically knock out the OsNPF4.5 were generated.Several homozygous mutant lines have been obtained,which will facilitate the dissection of the physiological roles of OsNPF4.5 in symbiotic N uptake and AM symbiosis.