Molecular Mechanisms And Functions Of RtNAC100 And RtNAC055 In Reaumuria Trigyna Regulating Plant Response To Salt And Drought Stresses

NAC(NAM,ATAF1/2,CUC1/2)is one of the largest transcription factor families of plant,which is involved in plant development and response to abiotic/biotic stresses.In past decade,a lot of researches about the regulation networks of the NAC transcription factor(TFs)were reported,but few studies focused on the abiotic stresses function of NAC055 and NAC100 TFs.Reaumuria trigyna,an endangered recretohalophyte,is a small archaic wild shrub endemic to arid and semiarid plateau regions of Inner Mongolia,China.R.trigyna has evolved distinct morphological and physiological features,which has stronger ability to adapt drought and salinization desert environment.The studies about R.trigyna were confined to the morphological and physiological ecology and little reports about the regulatory mechanism of transcription factor in R.trigyna.Based on salt-related transcriptomic data,we analyzed the NAC family genes and identified 2 NAC TFs(RtNAC100 and RtNAC055).To verify the function of these two NAC TFs regulating plant response to abiotic stress,they were translated into Arabidopsis thaliana and Populus davidiana X P.bolleana.We employed qRT-PCR,Yeast one-hybrid,Dual-luciferase assay to seek the regulation networks.The results are as follows:1.Fifty-six NAC genes were annotated from the R.trigyna transcriptome data,35 had complete ORFs(contain a complete NAM domain)and were divided into four groups by phylogenetic comparison with the A.thaliana NAC family.Bioinformatics analysis showed that the NAC family had a highly conserved NAM domain and divided into subdomains A to E.RtNAC100 was induced by salt,Me JA and ABA,but RtNAC055 was induced by salt and drought via qRT-PCR analysis.2.RtNAC055/100 located in nucleus,had transcription activity and bounded to the NACRS.These 2 NAC gene's promoters were translated into A.thaliana to detect the NAC genes expression pattens.Gus staining showed the promoter of RtNAC100 was mainly induced by Me JA and expressed in lateral root,leaf,flower and silique.The promoter of RtNAC055 mainly expressed in root and stomatal.3.Salt and Me JA could induce the leaves senescence of R.trigyna,qRT-PCR results showed that the expressions of senescence-associated gene(Rt SAG12),ROS production related gene(Rt Rboh E)and RtNAC100were induced.After drought treatment to the seedlings of R.trigyna,RtNAC055,proline synthesis related gene(Rt P5CS1),ROS production related gene(Rt Rboh E)and dehydration responsive gene(Rt DREB1.1)were induced.These co-expression genes maybe the target gene of RtNAC100/RtNAC055.The promoters of these target genes were isolated and contained multiple NACRS.Yeast one-hybrid and Dual-luciferase assay certified RtNAC055 could bound to the promoter of Rt P5CS1,Rt Rboh E,Rt DREB1.1 and enhanced plant drought tolerance.Meanwhile,RtNAC100 may direct regulate Rt SAG12,Rt Rboh E expression to accelerate plant PCD.4.To certify the gene function,we obtained the transgenic plants of RtNAC100.The over-expression lines exhibited sensitive to hypersaline conditions,and the root length,gemination rate,plant height,fresh weight,chlorophyll content was lower compared with wild type.Histochemistry staining assay displayed that the over-expression lines accumulated more H₂O₂and presented higher levels of cell death than wild type.The RtNAC100 OE lines exhibited more wilting than those of WT plants.Under salt treatment,the RtNAC100 OE lines accumulated more Na⁺,Ca²⁺,and had higher level of Na⁺/K⁺,moreover,the POD,CAT activities of OE lines were lower than wild type,accompanied by higher contain of H₂O₂,MDA,O₂.^-.Salt treatment caused PCD related gene(At PDCD5,At AEP1/3),senescence-associated gene(Rt SAG12),ROS production related gene(Rt Rboh E)higher expression,and ion transport protein lower expression.These results indicated the balance of ion transport system and ROS level were broken,indicating RtNAC100 accelerated salt-induced plant PCD.5.To deep dig the gene's abiotic function,the RtNAC055 was translated into atnac055 mutant and Populus davidiana X P.bolleana.Under drought treatment,the complementary lines exhibited higher leaf temperature and higher survival rate compared with atnac055 mutant,and also improved the root length of atnac055 mutant,indicating RtNAC055enhanced drought tolerance of atnac055 mutant.The transgenic poplar over-expressing had a lower stomatal conductance and transpiration rate,but higher WUE than wild type under normal condition.Under drought treatment,the transgenic poplar had a better physiological and biochemical indexes,including Pro,chlorophyll,MDA,POD,RWC,Fv/Fm and water loss rate compared with wild type.The OE line's stomatal would accumulate more ROS(H₂O₂)than wild type,which accelerated stomatal closure.Drought treatment caused proline synthesis related gene(Pt P5CS2),ROS production related gene(Pt Rboh D/F)and dehydration responsive gene(Pt DREB2.2/2.6)up-expression.These results indicated RtNAC055 regulated stomatal closure,reduced transpiration rate and improved plants drought resistance via maintaining the balance between antioxidant system and ROS level.