Functional Analysis And Validation Of NAC Transcription Factors PwNAC30/PwNAC31 In Picea Wilsonii

Picea(P.)wilsonii,a unique coniferous tree species,is one of the most important afforestation and landscape trees widely distributed in northern China.Due to its strong adaptability,especially its strong tolerance for cold stress,P.wilsonii provides abundant resources of candidate genes responding to stress.Despite the ability of P.wilsonii to tolerate cold,it is more sensitive to drought stress than other Picea species.Due to global warming and frequent drought,in recent years,the distribution scope of P.wilsonii has continuously shrunk,while the molecular mechanism of P.wilsonii in response to drought stress is not elucidated.NAC(NAM,ATAF1/ATAF2 and CUC2)transcription factor is one of the largest transcription factor families in plant,which is widely reported to participate in the multiple life processes of plant,especially plays vital roles in plant response to abiotic stresses.To improve the drought tolerance of P.wilsonii,it is important to illuminate its tolerance mechanisms and explore key regulating genes during the process.In the previous study,we identified two NAC transcription factors which were evidently induced by drought stress through RNA-seq data and q RT-PCR analysis,named as PwNAC30 and PwNAC31.In the present study,we further elucidate the biology function of PwNAC30 and PwNAC31 by heterologous expression in Arabidopsis,the detailed results are as follows:1): The results show that PwNAC30 and PwNAC31 are mainly localized in the nucleus by transient expression in N.benthamiana and can function as a nuclear localization transcription factor.2): The promoter of PwNAC30 is analyzed by β-glucuronidase(GUS)staining.By transient expression in N.benthamiana,we found that the promoter of PwNAC30 is responsive to ABA,GA,Me JA,drought,salt and cold stress,and Dual luciferase reporter assays(LUC)further quantify this result.By heterologous expression in Arabidopsis,the results show that the expression pattern of PwNAC30 throughout the plant development process,which is expressed in most of the plant tissues except stamens and petals.3): By transactivation assay,we detect that as for PwNAC30,the transcriptional activity is only presented in the C-terminal instead of its full-length,and the PwNAC30 can form homologous dimer by itself.For PwNAC31,the full-length of it has transcriptional activity,and the transactivation domain is located in the C terminal,and it can not form homologous dimer by itself.4): By overexpression of PwNAC30 in Arabidopsis,we find that overexpression of PwNAC30 significantly represses seedlings’ and mature plants’ tolerance to both drought and salt stresses,while itdoes not affect flowering time and pod development.It is further found that the accumulation of reactive oxygen species(ROS)is significantly increased in transgenic plants,while the expression of stress-responsive genes is obviously inhibited in PwNAC30 overexpression lines.5): The homologous gene of PwNAC31 in Arabidopsis is At NAC72,by overexpression of PwNAC31 in atnac72 mutant,we find that transgenic plants could restore the drought defects of atnac72 mutant.