Functional Study Of High-affinity Nitrate Transporter Genes SsNRT2.1 And SsNRT2.5 In Suaeda Salsa

Nitrate（NO₃^-）is an essential nutrient for plant growth,and it is one of the major sources of nitrogen which is closely correlated to the salt tolerance of plants.Suaeda salsa L.is a salt-tolerant halophytic herb with succulent leaves,which can be used in developing saline agriculture.S.salsa adapt to low NO₃^-and high salt habitat,and the species can grow normally and complete its life cycle,indicating that S.salsa may have an efficient NO₃^-uptake capacity.In this study,we cloned the NO₃^-transporter genes SsNRT2.1 and SsNRT2.5 from S.salsa and provided a reference for understanding the physiological mechanisms related to NO₃^-uptake in euhalophytes.The main results of this study are as follows:（1）Analysis of RNA sequencing（RNA-seq）in S.salsa under high salinity and low NO₃^-conditionsSeedlings of S.salsa were treated with low NO₃^-and high salinity.The results of RNA-seq showed that NRT2.1,2.2,2.4,2.5 and NRT3.1 were up-regulated at 200 mM NaCl.These genes may play an important role in NO₃^-uptake under low NO₃^-condition in the studied species.It is regarded that both NRT1 and NRT2 families are proton-coupled symporters that rely on the activity of PM-ATPase.In S.salsa,several genes（AHA1,4,8）related to PM H⁺-ATPase were up-regulated at 200 mM NaCl.This indicates that the high ability to absorb NO₃^-under salinity in S.salsa may be related to the up-regulation of PM H⁺-ATPase genes.The correlation between the data of RNA-seq and q RT-PCR was consistent,indicating that the results of NRT2.1and NRT2.5 in the transcriptome were reliable,and the two genes can be used for further study.（2）Cloning SsNRT2.1,SsNRT2.5 and SsNRT2.5 promoterThe putative c DNA sequence encoding SsNRT2.1 and SsNRT2.5 was isolated from S.salsa roots by RACE PCR.The sequence of SsNRT2.1 contained a complete open reading frame（ORF）of 1653 bp,which encodes 550 amino acids.The prediction of transmembrane domains was performed using the TMHMM predictor,and it was found that there were 12 transmembrane domains.The SsNRT2.1 protein contained two conserved MFS and NNP domains,indicating that SsNRT2.1 may play a role in absorbing NO₃^-.It was found that SsNRT2.1 had the highest homology and the closest genetic relationship with physcomitrella patens when the phylogenetic analysis with MEGA software was done.The sequence of SsNRT2.5 contained a complete ORF of 1524 bp,which encodes 507amino acids with 12 transmembrane domains.The SsNRT2.5 protein also contained two conserved MFS and NNP domains.It was found that SsNRT2.5 had the highest homology and the closest genetic relationship with Chenopodium quinoa and Beta vulgaris.Under low NO3^-conditions,SsNRT2.1 and SsNRT2.5 were up-regulated under salinity in S.salsa.Under salinity,the up-regulation of SsNRT2.5 was continuous and was more significant compared with SsNRT2.1,indicating that SsNRT2.5 may play an important role in NO₃^-uptake under high salinity and low NO₃^-condition.Therefore,the SsNRT2.5 promoter was cloned and its function was verified in this experiment.The SsNRT2.5 promoter sequence of 2774 bp was isolated from S.salsa roots by TAIL-PCR.The sequence contained several cis acting elements,such as NaCl-responsive elements GT-1,DREB and ABRE,and transcription factors binding sites,which can be predicted that SsNRT2.5 is a salt-responsive promoter.（3）The positive effect of salinity on nitrate uptake in S.salsaSalinity of 200 mM NaCl significantly reduced the root length,surface area,volume and number of root tips,while the expression of SsNRT2.1 and SsNRT2.5 in the roots were up-regulated at 200 mM NaCl,and the shoot biomass increased compared to that at 0 mM NaCl under 0.5 mM NO₃^-for 7 d;Seedlings of S.salsa were treated with 0,200 and 500 mM NaCl under 0.5 mM NO₃^-with or without Na₃VO₄（the inhibitor of plasma membrane H⁺-ATPase）for24 h.Salinity of 200 mM NaCl up-regulated the gene expression of SsNRT2.1 and SsNRT2.5 in the roots,increased the root net influx of H⁺and NO₃^-,and ¹⁵NO₃^-accumulation in the leaves and roots.The ¹⁵NO₃^-accumulation at 200 mM NaCl with Na₃VO₄ treatment was lower than that without supplying Na₃VO₄ in the leaves and roots.Supplying Na₃VO₄ had no significant effect on the net H⁺flux,but induced a net NO₃^-efflux in the roots at 200 mM NaCl.In conclusion,salinity may directly activate the expression of SsNRT2.1 and SsNRT2.5 when the root absorption area was reduced,and promote NO₃^-uptake via the increment of pumping H⁺by PM H⁺-ATPase in S.salsa.（4）Functional study of SsNRT2.1 in ArabidopsisThe subcellular localization of SsNRT2.1 revealed that SsNRT2.1 was expressed in the plasma membrane.Treated with 0.5 mM NO₃^-nutrient solution,the two mutant Arabidopsis lines nrt2.1-2 and nrt2.1-3showed a lower NO₃^-content compared to that in WT at 0 and 50 mM NaCl.The Arabidopsis overexpression lines showed a higher NO₃^-content compared to WT.A similar trend was observed in the root length.This indicates that SsNRT2.1 may be a high-affinity nitrate transporter in S.salsa,which is involved in NO₃^-uptake at low external NO₃^-environments.（5）Functional study of SsNRT2.5 in ArabidopsisSsNRT2.5 was also expressed in the plasma membrane.Treated with 0.5 mM NO₃^-nutrient solution,At NRT2.5 was expressed at extremely low levels in the two mutant Arabidopsis lines M2 and M22,but the expression of At NRT2.1 was increased.Meanwhile,At NRT2.1 and At NRT2.5 gene were down regulation in the SsNRT2.5 overexpression lines.The SsNRT2.5overexpression Arabidopsis lines showed a higher NO₃^-content compared to WT at 0,50 and100 mM NaCl.Meanwhile,more NO₃^-was accumulated in the seeds of overexpression lines compared to WT.This indicates that SsNRT2.5 may contribute to the efficient NO₃^-uptake in S.salsa under high salt and low NO₃^-conditions.（6）Function study of SsNRT2.5 promoterThe 35S promoter was replaced with SsNRT2.5 promoter in the p CAMBIA1300-35S-s GFP-SsNRT2.5 expression vector.Treated with 0.5 mM NO₃^-nutrient solution,the transgenic lines showed a higher fresh weight,root length and NO₃^-content compared with WT at 50 mM NaCl.In 1/2 MS medium,the root length and fresh weight of transgenic lines increased significantly when they were treated with 100 mM NaCl in the seedling growth stage.With 150 mM NaCl treatment,WT could not grow,but the growth of transgenic lines was much better than WT.At100 and 150 mM NaCl,the NO₃^-content increased,the DAB and NBT staining was shallow,SOD,CAT and POD activity increased,the expression of corresponding gene of these enzymes increased,the Na⁺content decreased,the expression of At SOS1 and At HKT1 genes increased,the MDA content decreased and proline content increased in transgenic lines compared with WT.This study revealed that the transgenic lines had higher salt tolerance compared with WT.SsNRT2.5 may affect the expression of key genes related to ROS scavenging,antioxidant enzymes and ion transport under salt stress,thus increasing salt tolerance.In transgenic Arabidopsis lines,GUS staining analysis of SsNRT2.5 promoter showed that SsNRT2.5 was expressed in flowers,pods and seeds at the reproductive stage.It can be speculated that it is related to the NO₃^-accumulation in seeds.The 5’deletion method was used to analyze the function of the promoter,and GUS activity was observed by transient transformation in S.salsa and Nicotiana benthamiana.In S.salsa,the GUS staining was enhanced at 100 mM NaCl in the Q1 and Q2 segment,while decreased in the Q3 segment.The same trend was shown in N.tabacum,indicating that the promoter of Q1 and Q2 segment can be induced by NaCl.In transgenic Arabidopsis,GUS staining was consistent with the transient transformation results.It was found that the GUS staining of Q1 and Q2 became darker after salt treatment,indicating that salt induced the expression of GUS gene,and the expression of GUS genes showed the same trend.The results showed that the SsNRT2.5 promoter was a salt-inducible promoter.In the Q1 and Q2 segment of the promoter,the gene expression was increased under salinity due to the salt-related cis-acting elements.In conclusion,200 mM NaCl up-regulated the gene expression of SsNRT2.1 and SsNRT2.5in S.salsa.SsNRT2.1 and SsNRT2.5 overexpressed in Arabidopsis may contribute to the efficient NO₃^-uptake in low NO₃^-conditions.SsNRT2.5 transgenic Arabidopsis lines had higher salt tolerance.The results showed that the SsNRT2.5 promoter was a salt-inducible promoter.NRT2.5may play a more important role in the efficient uptake of NO₃^-than NRT2.1 under low NO₃^-and high salt conditions in S.salsa.