Study On Bioinformatics Characteristics Of Tobacco NPFs And Interaction Proteins With NtNPF50

Tobacco is highly sensitive to the application of nitrogen and potassium during its growth and development.Inadequate or excessive use of these nutrients can have detrimental effects on tobacco leaves.Therefore,investigating the nitrogen transport function and potassium response function of the NPF gene family in tobacco holds significant implications for nitrogen and potassium fertilizer absorption and utilization.While there is extensive research on the i family in model plants like Arabidopsis and cereal crops such as rice,information on tobacco remains scarce.As a dicotyledonous plant belonging to the Solanaceae family,tobacco is an economically important crop in China.This paper aims to perform bioinformatics analysis on the NPF gene family in tobacco,explore different modes of gene expression,and analyze the potassium response function of the nitrogen transporter NPF50.The key findings of this research are outlined as follows:1.Bioinformatics Characteristics of the Tobacco NPF Gene FamilyIn this study,a total of 143 NPF genes were identified in the tobacco genome.By employing parameter adjustments to reduce thresholds and conducting multiple screenings on the online SMART website,the selection was narrowed down to 80 Nt NPFs members,designated from Nt NPF1 to Nt NPF80.A systematic analysis of the Nt NPFs family revealed several key characteristics.Physicochemical property analysis indicated that the length of tobacco NPF proteins ranged from 466 to 1178 amino acids(aa),with the majority falling around 600 aa.The relative molecular weight varied between 51.63 k D and 131.11 k D.The theoretical isoelectric points ranged from 5.53 to 9.63.Chromosomal location predictions showed an uneven distribution of 46 Nt NPFs across 17 chromosomes.Evolutionary analysis classified the 80 selected tobacco NPF genes and 53 Arabidopsis NPF genes into 8 subfamilies.Gene structure analysis demonstrated that most Nt NPF genes consisted of 2 to 12 exons,with the majority having 3 to 5 exons.Protein secondary structure prediction revealed that tobacco NPF proteins were predominantly irregularly curled and α-helical,primarily located in vacuoles,with limited presence in the cell nucleus,membrane,and chloroplasts.Cis-acting element analysis identified 14 types of cis-acting elements within the tobacco gene family,encompassing jasmonic acid response elements,salicylic acid response elements,hormone response elements,damage-induced response elements,and stress response elements.However,no nitrogen and potassium responsive cis-elements were found.Regarding tobacco species,eight gene pairs exhibited collinearity.Interspecies collinearity analysis between tobacco NPF genes and Arabidopsis NPF genes revealed 11 gene pairs with collinearity,whereas 76 gene pairs displayed collinearity between tobacco NPF genes and tomato NPF genes.GO annotation analysis discovered that the Nt NPFs gene family participates in various biological processes,with most Nt NPFs proteins located on the cell membrane and possessing material transport functions.2.Different Expression Modes of Tobacco NPFsThe expression of NPFs genes is ubiquitous across various tissues,with leaves exhibiting the highest expression levels.Within the NPF VII subfamily,gene expression is predominantly observed in flowers,stems,leaves,and roots.Notably,the relative expression of most NPF genes is highest in the stem tissue.The expression patterns of tobacco NPFs vary depending on the potassium chloride treatments,displaying distinct period-specificity.Specifically,during the maturity period,Nt NPF65,Nt NPF15,Nt NPF23,Nt NPF66,Nt NPF6,Nt NPF53,Nt NPF7,Nt NPF63,Nt NPF50,Nt NPF2,Nt NPF10,Nt NPF61,Nt NPF70,Nt NPF52,Nt NPF37,Nt NPF3,Nt NPF46,and Nt NPF56 exhibit relatively high expression,which significantly diminishes during the budding period and becomes virtually absent during the vigorous growth phase.Conversely,the remaining genes,namely Nt NPF77,Nt NPF78,Nt NPF58,Nt NPF57,Nt NPF45,Nt NPF26,Nt NPF36,Nt NPF60,Nt NPF29,Nt NPF22,and Nt NPF17,display elevated expression during the vigorous growth period,reduced expression during the budding phase,and minimal expression during the maturity period.Upon application of increasing doses of potassium chloride,the expression of relevant functional genes initially rises,followed by either a decline or a continuous increase within a certain range.To investigate this phenomenon,tobacco plants at the rosette stage were subjected to low potassium,high potassium,low nitrogen,high nitrogen,and salt stress treatments.Subsequently,qRT-PCR experiments were conducted,focusing on ten genes belonging to the NPF VII subfamily.The results reveal significant variations in the expression of most Nt NPF genes following different stress treatments,albeit with divergent trends of change.Notably,Nt NPF50 exhibited a 2.5-fold increase under low potassium treatment and a 3-fold increase under high potassium treatment.Furthermore,under low nitrogen conditions,its expression rose by 1.7-fold,while under high nitrogen conditions,it exhibited a 4-fold increase.Conversely,salt stress resulted in a reduction of Nt NPF50 expression to 0.8 times that of the control group.These findings suggest that NPF50 may possess dual characteristics,functioning as both a nitrogen transporter and a responder to potassium signals.3.Tobacco NPF50 Interacting Proteins and Functional AnalysisThrough protein interaction prediction,it has been suggested that NPF50 exhibits interactions with members of the HAKs family,which are potassium transporter proteins,as well as members of the CIPKs gene family involved in the CIPK-CBL signaling pathway.The subcellular localization results indicate that Nt NPF50 is situated on the cell membrane,while Nt HAKS21 is present both on the cell membrane and in the cytoplasm.Yeast two-hybrid experiments further support these findings,revealing interactions between Nt NPF50 and Nt HAKS21,Nt NPF50 and Nt CIPK34,as well as Nt NPF50 and Nt CIPK27 on triple-deficient medium.Moreover,BIFC fluorescent complementation experiments demonstrate that NPF50-CYFP physically interacts with HAKS21-NYFP on the cell membrane,whereas NPF50-CYFP interacts with CIPK37-NYFP in the cell nucleus.Based on these results,it is postulated that Nt NPF50 may respond to potassium transport,potentially through its own potassium transport function.Additionally,it is plausible that Nt NPF50 responds to potassium by interacting with HAKS21 and CIPKs,thus influencing downstream signaling events.