Functional Study Of Two NAC Transcription Factor Genes In Brassica Napus And Arabidopsis Thaliana

NAC transcription factor（TF）genes constitute a plant-specific family and play diverse roles in plant growth and development,as well as in responses to abiotic and biotic stresses.Among NAC TFs,a few members are special since they have a transmembrane domain and therefore are associated with membrane system,which are called NTL（NTM-like）.It is proposed that NTLs may plany an essential role in fine-tunning signaling upon stress challenge.However,how NTLs regulate ROS（Reactive oxygen species）to promote leaf senescence in oilseed rape（Brassica napus L.）at the transcriptional level and how NTLs regulate the expression of heat shock protein（HSP）genes to improve heat tolerance in Arabidopsis remain largely unknown.In this paper,through the reverse genetic approach,the functions and regulatory mechanisms of Bna NTLx in oilseed rape and AtNTLa in Arabidopsis were explored in terms of leaf senescence and heat stress tolerance.The Bna NTLx encoded a protein that has a single transmembrane domain at the carboxy terminus.Using the green fluorescent protein（GFP）reporter gene,the subcellular localization analysis revealed that the full-length form of Bna NTLx was localized at the endoplasmic reticulum（ER）,and its truncated form without transmembrane domain is localized in the nucleus.Both the transcriptional activity assay in yeast and the dual luciferase reporter assay（Dual LUC）showed that Bna NTLx-ΔTM has transcriptional activation activity.When GFP-Bna NTLx was expressed in protoplasts of oilseed rape,it was translocated into the nucleus after treatment with an ER stress inducer such as tunicamycin（TM）,dithiothreitol（DTT）and oxidative stress inducer hydrogen peroxide（H₂O₂）.q RT-PCR analysis showed that Bna NTLx was up-regulated only in the late stage of leaf senescence.Bna NTLx and Bna NTLx-ΔTM were constitutively overexpressed in Arabidopsis using the Ca MV35S promoter.The natural aging phenotypic assay revealed that Bna NTLx-ΔTM overexpression lines showed accelerated leaf senescence process and promoted the accumulation of ROS.Furthermore,q RT-PCR results showed that Bna NTLx-ΔTM can activate expression of programmed cell death（PCD）-related genes AtBFN1,AtCEP1,AtαVPE,AtβVPE,AtγVPE,ROS-related gene AtRboh D,and senescence-associated genes AtNYE1,AtSAG13 and AtSEN4 under natural aging conditions.The results of the dual luciferase reporter assay showed that PCD-related genes of AtBFN1,AtγVPE and AtδVPE were up-regulated,and the expression of JA-related genes AtOPCL1,AtAOC3,as well as Bna ZAT12,Bna BFN1,BnaαVPE,BnaβVPE,BnaγVPE could also be activated.Electrophoretic mobility shift assay（EMSA）in vitro showed that Bna NTLx-ΔTM binds to promoter regions of AtBFN1,AtγVPE,AtδVPE,AtOPCL1,AtAOC3,Bna ZAT12,Bna BFN1,BnaαVPE,BnaβVPE and BnaγVPE.Atthe same time,Ch IP-q PCR in vivo further confirmed that AtOPCL1,AtAOC3,AtBFN1,AtγVPE and AtδVPE could function as downstream targets of Bna NTLx.Secondly,according to preliminary results,the basic mechanism of a membrane-bound NAC transcription factor gene AtNTLa was also explored,which may regulate heat tolerance in Arabidopsis.The GFP-AtNTLa fusion plasmid was transfected into Arabidopsis protoplasts and treated with TM,DTT,H₂O₂ and 37°C heat stress,respectively.It can be observed that GFP-AtNTLa relocated from the plasma membrane to the nucleus.Subsequently,it was found that the active form of AtNTLa-ΔTM can activate the LUC expression driven by promoters of heat shock-related genes such as AtHSP90.1,AtHSP101,AtHSP70,AtHSP70-2,AtHSP21,AtHSP17.6II and AtHsf B2A by a Dual LUC assay.A series of homozygous T-DNA insertion mutants such as Atntla,AtHSP90.1,AtHSP101 and AtHSP70 were identified by PCR and RT-PCR,which will be used in subsequent heat stress related assays and genetic analysis.In conclusion,Bna NTLx acts as a transmembrane transcription factor that positively regulates ROS accumulation and natual senescence of leaves.In addition,AtNTLa may regulate heat stress tolerance by regulating the expression of heat shock-related genes.