Molecular Mechanism Of FIP1 Gene Regulating Nitrate Signaling In Arabidopsis

Nitrate?NO₃^-?is a major form of nitrogen absorbed by most of terricolous plants.NO₃^-is not only a nutrient substance,composed of bio-macromolecule,but also as a signal molecule regulating growth and development of many plants.Revealing the molecular mechanism of nitrate sensing,transport and mentalism are key step to improve nitrogen utility efficiency and reduce environmental pollution.In model plant Arabidopsis,there are four gene families responsible for nitrate transport and allocation,including NRT1/PTR(nitrate transporter 1/peptide transporter family,53members,NRT2?7 members?,CLC?7 memberes?and SLAC1/SLAH?5 members?.The CHL1of NRT1 gene family,is not only a dual-affinity nitrate transporter,but also as a sensor regulating many intracellular genes expression.The last decade,more and more researchers focus on screening and identification of nitrate-regulatory genes involving in nitrate response.Many labs screened these nitrate-regulatory genes used different strategies,such as forward and reverse genetics,systems biology,Ch IP-chip/sequenceing.Several nitrate-regulatory genes in genome scale were identified,included transcription factors?ANR1,NLP7,TGA1?TGA4,SPL9,LBD37/38/39 and TCP20?,protein kinases?CIPK8 and CIPK23?,microRNA?miR393/AFB3?and so on.However the molecular network of NO₃^-response is comprehensive and refined,the regulatory genes mentioned above maybe the tip of the iceberg in this network.Our lab constructed a nitrate-response promoter?a fragment form NiR promoter and NIA1 enhancer fused YFP?and then transform into Col-0.The root of homozygous transgenic seeding included this promoter?SS204-9?showed yellow fluorescence in presence of NO₃^-.M₂ population after treated with EMS was screened,many important nitrate-regulatory genes?NRT1.1,LP7,NRG2 and CPSF30?were identified.CPSF30,a major component of cleavage and polyadenylation specificity factor?CPSF?,contained three CCCH zinc figures in N-terminal.The third zinc figure performed endonuclease could interact with FIP1,which was a central protein of CPSF.Moreover,the interaction between FIP1 and CPSF30 affected endonuclease activity of CPSF30.Recently,CPSF30 was identified as a nitrate-regulatory gene.The expression of nitrate response gene NIA1,NiR and NRT2.1 were decreased when point mutation was present in the third zinc figure caused by EMS treatment.Fluorescence idensity of double mutant suggested that CPSF30 and NRT1.1 were in the same pathway to regulate nitrate signaling.In addition,CPSF30 affected nitrate accumulation by regulation expression of nitrate transporter genes?NRT1.1,NRT1.5,NRT1.8?and assimilation genes?NIA1,NiR?.In this paper,the interaction between N-terminal of FIP1 protein and CPSF30 were determined by Yeast two hybrid.This interaction abolished when the third zinc figure of CPSF30 was mutation.The seedlings cultured on?NH₄?₂Suc for 7d and then treated with10mM KNO₃ for 2h,the expression of nitrate response genes NIA1,NiR and NRT2.1significantly decreased in fip1 mutant,compare with WT.The fluorescence idensity was reduced when FIP1 mutation in SS204-9 in presence of nitrate.These results indicated that FIP1 was a nitrate-regulatory gene.The expression of FIP1 in different tissues after 10mM KNO₃ treatment at various time were determined,it suggested that this genes is constitutive expression and not induced by nitrate.FIP1 was mainly expressed in vascular tissuse,stoma and stele,basal lateral root in adult and young seedling,respectively.FIP1 could afftect NO₃^-allocation between shoot and root.The NO₃^-concentration was significantly decreased in seedling and shoot of fip1 mutant under 1/2 MS culture,but that of increased in root,compared with WT.The expression of nitrate transporter and assimilation genes were determine,they showed that NRT1.5 and NRT1.8 decreasd in shoot,in contrast to NRT1.5 increased in root of fip1 mutant,comparte with WT.The expression of NiR both in shoot and root and NIA1 only in shoot remarkably increased in fip1 mutant.These results might be the reason for FIP1 affecting nitrate allocation between shoot and root.The expression of several nitrate-regulatory genes in various mutants was determined to reveal the relationship between FIP1 and these genes.These results showed that the expression of ANR1,CIPK8,CIPK23 in fip1 mutant were significant higher than that of WT.Further research found that nitrate concentration was lower in CIPK23 overexpression line,compared with WT.It suggested that FIP1 might affecte nitrate concentration by improving the expression of CIPK23.Geneticis analysis of double mutants showed that fluorescence idensity of fip1cpsf30 and fip1nlp7 were similar with fip1 mutant,and the expression of ANR1,CIPK8 and CIPK23 in fip1nrt1.1 was between fip1 and nrt1.1 single mutant.These results suggested that FIP1,CPSF30 and FIP1,NLP7 were in the same pathway to regulating nitrate signaling,FIP1 and NRT2.1 were in the different pathway.The long-term inflluence of FIP1 regulating nitrate signaling was affecting lateral root development.The expression of GATA23,a gene participated in improving primordium initiation,was increased because the expression of its upstream inhibitor IAA14 was decreased in fip1 mutant.The expression of ANR1 and IAA transporter genes PIN6,PIN7involved in elongation were increased and decreased,respectively.However,FIP1 likely not affected meristem activation mediated by NRT2.1 under high C/N ratio.These results suggested that FIP1 affected lateral root intensity was a overlay result of above four steps of lateral root development.In this paper,the function of nitrate-regulatory gene FIP1 was identified.The result suggested that CPSF components participated in nitrate response.FIP1 could affect gene expression of nitrate transport,assimilation and regulation.Several nitrate-related phenotype such as seedling growth,nitrate concentration and lateral root development were observed in fip1 mutant.These result improve illumination of NO₃^-response network,metabolism and lateral root development in Arabidopsis.Moreover,this research provides the basis for nitrogen utility efficiency in plant.