Ectopic overexpression of a membrane-tethered transcription factor gene NAC60 from oilseed rape positively modulates programmed cell death and age-triggered leaf senescence.

ABSTRACT

Senescence is an integrative final stage of plant development that is governed by internal and external cues. The NAM, ATAF1/2, CUC2 (NAC) transcription factor (TF) family is specific to plants and membrane-tethered NAC TFs (MTTFs) constitute a unique and sophisticated mechanism in stress responses and development. However, the function of MTTFs in oilseed rape (Brassica napus L.) remains unknown. Here, we report that BnaNAC60 is an MTTF associated with the endoplasmic reticulum (ER) membrane. Expression of BnaNAC60 was induced during the progression of leaf senescence. Translocation of BnaNAC60 into nuclei was induced by ER stress and oxidative stress treatments. It binds to the NTLBS motif, rather than the canonical NAC recognition site. Overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, induces significant reactive oxygen species (ROS) accumulation and hypersensitive response-like cell death in both tobacco (Nicotiana benthamiana) and oilseed rape protoplasts. Moreover, ectopic overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, in Arabidopsis also induces precocious leaf senescence. Furthermore, screening and expression profiling identified an array of functional genes that are significantly induced by BnaNAC60 expression. Further it was found that BnaNAC60 can activate the promoter activities of BnaNYC1, BnaRbohD, BnaBFN1, BnaZAT12, and multiple BnaVPEs in a dual-luciferase reporter assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative PCR assays revealed that BnaNAC60 directly binds to the promoter regions of these downstream target genes. To summarize, our data show that BnaNAC60 is an MTTF that modulates cell death, ROS accumulation, and leaf senescence.