Functional Analyses Of NAC Transcription Factors Of Populus Euphratica In Response To Drought And Salt Stresses

Abiotic stresses are major environmental factors that limit plant growth and productivity.Transcription factors(TFs)play important roles by binding to cis-elements in the promoter regions of target genes or other functional modular structures in abiotic responses.The NAC(NAM/ATAF/CUC)superfamily is a large plant-specific TF family.In this study,Populus euphratica,in desert poplar of NorthWest China,was studied to investigate the molecular mechanisms involved in drought resistance and salt tolerance in NAC TF family.A total of 419 genes ecoding NAC were identified in P.euphratica,P.trichocarpa and Arabidopsis thaliana genomes and classified into nine subfamilies.Gene structure and conserved motif analyses of 137 PeNAC genes indicated that they were relatively conserved.In total 71 paralogous pairs were identified,in which 65 pairs were generated by segmental duplication events,and other six were generated by tandem duplication events.Ka/Ks analysis showed that PeNACs underwent purifying selection pressure.Based on transcriptome analysis in P.euphratica under salt or drought stress,the expression levels of some PeNAC genes,belong to SNAC,NTL1,NTL2 and CUC subfamilies,were down-regulated or up-regulated.Co-expression analysis of ANAC genes indicated that few members,belong to SNAC or NTL2 subfamily,may be involved in response to water or salt stress.Several SNAC-B members exhibit different expression patterns under salt and drought stresses in leaves of P.euphratica.Three putative SNAC proteins were identified,PeNAC034 and PeNAC045 were classified into the ATAF subgroup and PeNAC036 into the RD26 subgroup.Three SNAC transcription factors are localised in the nucleus and contain the activation domain in their C-terminal.PeNAC034 was preferentially expressed in the leaves,whereas PeNAC045 and PeNAC036 were highly expressed in the stems.Under drought and salt stresses,PeNAC036 was strongly induced in the whole plant,but PeNAC034 was significantly suppressed in the roots and stems,and PeNAC045 was inhibited in the roots.PeNAC036 overexpression in A rabidopsis wild-type(WT)(OEPeNAC036)and PeNAC036 complementation in mutant anac072(anac072/PeNAC036)lines increased tolerance to salt and drought,whereas PeNAC034 overexpression in WT(OEPeNAC034)and PeNAC034 complementation in mutant atafl(ataf1/PeNAC034)lines improved salt and drought sensitivity.After drought and salt treatments,the expression levels of stress related genes were up-regulated in OEPeNAC036 and anac072/PeNAC036 lines but were down-regulated in OEPeNAC034 and atof1/PeNAC034 plants.PeNAC045 overexpression in poplar significantly decreased the net photosynthesis rate,stomatal conductance and transpiration rate under salinity and drought conditions.Plant miR164 family is highly conserved and miR164 members regulate conserved targets belonging to NAC transcription factors.P.euphratica miR164 family comprises six members that generate two mature products(miR1 64a-e and miRl 64f)and target seven NAC genes.Co-expression in Nicotiana benthamiana and 5’-RACE confirmed that peu-miR164 directs PeNAC070,PeNAC012 and PeNAC028 mRNAs cleavage.Expression profiles of primary peu-miR164 a/b/c/d/e bear similarity to those of peu-miR164a-e,whereas PeNAC070 and PeNAC081 showed inverse expression patterns with peu-miR164a-e under abiotic stresses.Many stress-related cis-acting elements(ABRE,MBs,Box-W1,GC-motif,and W-box)of PeNAC070 pronoter,but only one stress-related(HSE)of peu-MIR164b promoter further confirmed different responses of peu-miR164 and PeNAC070 to abiotic stresses.Histochemical β-glucuronidase(GUS)staining revealed that GUS activities increased when ProPeNAC070::GUS transgenic Arabidopsis plants were exposed to NaCl,mannitol and abscisic acid(ABA),whereas GUS activity of Propeu-MIR164b::GUS plants decreased under ABA treatment.PeNAC070 protein was localized to the nucleus and exhibited transactivation activity at the C-terminal.Overexpression of PeNAC070 in Arabidopsis promoted lateral root development,delayed stem elongation,and increased sensitivity of transgenic plants to drought and salt stresses.The three SNAC genes differently regulate abiotic stress tolerance.The adaptability of P.euphratica to extreme drought and salt environment may be related to tissue-specific expression patterns of miR164-regulated and specific promoter-regulated PeNAC genes.This study showed that NAC TFs involved in drought and salt stress responses play an important regulatory in P.euphratica adaptation to desert environment,these NAC genes provide genetic material for resistance improvement of trees.