Cloning And Functional Analysis Of Three Stress-Related NAC Transcription Factor Genes From Ammopiptanthus Mongolicus

The NAC(NAM,ATAF,and CUC)proteins are an important class of plant-specific transcription factors(TFs),which play important roles in growth and development of plants as well as in the resistance of plants to adverse environments.Ammopiptanthus mongolicus,the unique evergreen broadleaf plant in the northwest desert of China,has special uses in the studies on stress-resistant mechanisms and the mining of stress-resistant genes in plants.In our previous study,eight drought-and cold-induced genes coding for NAC TFs were identified in the transcriptome of this species.In the present study,we cloned three of them,namely AmNAC3,AmNAC4 and AmNAC5,and analyzed the structural characteristics,subcellular localization and transcriptional activation activities of the proteins encoded by these genes.We also performed functional analyses of these genes in response and tolerance to different abiotic stresses.The main results are as follows:(1)The complete cDNA and gDNA coding regions of AmNAC3,AmNAC4 and AmNACS were cloned by PCR.The cDNA coding regions consist of 846,879 and 891 bp,and their predicted proteins contain 281,292 and 296 amino acid residues,respectively.The corresponding gDNA coding regions consist of 1213,1231 and 1729 bp,respectively,and each of them contains three exons and two introns in its coding region.(2)Sequence alignments and conserved domain analyses showed that the AmNAC3,AmNAC4 and AmNAC5 sequences contain a typical NAC domain at their N-terminal regions,respectively.Each NAC domain includes A to E five subdomains,and both the C and D subdomains contain a nuclear localization signal.The AmNAC3,AmNAC4 and AmNAC5 proteins were separately confirmed to be located in the nucleus by using their GFP fusion proteins and have transcriptional activation capacity in a yeast self-activation system.Furthermore,each of the AmNAC proteins contains transcriptional activation domain at its C-terminal region.(3)Expression patterns of three genes were analyzed by RT-qPCR.In the shoots and/or roots of laboratory-cultured A.mongolicus seedlings,the transcription levels of these genes were differentially up-regulated under drought,salt,cold and heat stresses and ABA treatment.By comparison,AmNAC3 was induced more rapidly and obviously by the drought,salt and ABA treatments;AmNAC4 was induced more rapidly and/or greatly by all the four stresses,and AmNAC5 was mainly induced by the drought,salt,heat and ABA treatments.In young leaves of A.mongolicus shrubs naturally growing in the wild,both AmNAC3 and AmNAC5 were abundantly transcribed in November and December compared to other months,while AmNAC4 had much higher transcript levels in January,November and December compared to those in other months within the years of sampling.In the shrubs growing in the wild in spring,AmNAC3 was highly transcribed in lateral roots and twigs but weakly transcribed in young leaves,flower buds and immature pods;AmNAC4 had much higher transcript level in young leaves rather than other organs,and AmNAC5 was mainly transcribed in lateral roots and young leaves.(4)Transgenic Arabidopsis lines constitutively expressing AmNAC3,AmNAC4 or AmNAC5 were constructed,and the physiological roles of these genes in plant stress tolerance were evaluated through the phenotypic and physiological alterations of the transgenic lines under different stress and ABA treatments.AmNAC3 could significantly enhance salt tolerance but reduce heat tolerance and water holding capacity of the leaves.AmNAC4 played significant roles in the improvement of the tolerances to drought,high salinity and freezing.AmNAC5 could also improve the tolerances to drought and high salinity but reduce the sensitivity to exogenous ABA.Moreover,all the three genes conferred their transgenic lines the abilities to endure oxidative damage and to maintain the stability of cellular membranes and chlorophylls during the stress treatments.(5)Three AmNAC genes could increase the transcription levels of multiple stress-related genes in their transgenic lines.AmNAC3 mainly increased the transcription of RD29A,RD29B,POD and NHX1.AmNAC4 could enhance the transcription of RD29A,RD29B,POD,ALDH7B4,P5CS1 and CORI5a.The genes up-regulated by AmNAC5 were RD29A,RD29B,POD,CAT and ALDH7B4.(6)The promoter sequence of AmNAC4 contains multiple cis-acting elements such as those responding to low temperature,drought,light and ABA,as well as the binding site for MYB TFs.In transgenic Arabidopsis,the promoter mainly plays a role in aerial parts of the seedlings and flowers of the adult plants.Moreover,the promoter's activity in the seedlings could be mainly induced by cold,salt and heat stresses.The above results laid a foundation for further investigating and utilizing these AmNAC genes with respect to their stress resistance function,as well as for the analysis of stress resistance mechanisms of A.mongolicus.