Functional Characterization Of A Transcription Factor NAC103 In The ER Stress Response In Arabidopsis

Protein folding plays important roles in the synthesis of protein, and the processing of the protein folding is very fastidious in response to adverse environmental conditions. Under these conditions, much misfolded or unfolded proteins accumulate in the ER, triggering the ER stress. The ERQC system upregulate many genes encoding components of protein folding to assistant protein folding which is called UPR, or encoding some ER-associated degradation(ERAD) genes for degradation misfolded or unfolded proteins. In the model plant arabidopsis, two arms of UPR signaling pathway have been described, including bZIP28-S1P/S2P and IRE1-bZIP60. However, the underlying mechanism of many genes in the UPR pathway is largely unknown. Here we studied the biology function of NAC103 and how the NAC103 involved in the UPR signaling pathway by means of methods in genetics,cell biology, biology chemical and molecular biology.NAC103 belongs to NAC family which is special for plants. NAC 103 protein is typically conserved with a conserved DNA binding domain in the N-terminus and a transcriptional active domain in the C-terminus. NAC 103 form homodimer in the nuclei of the yeast and Nicotiana benthamiana cells. NAC103-YFP fluorescence was observed in the nuclei of roots after the seedlings were incubated with ER stress-inducing agent tunicamycin (TM) or the potent proteasome inhibitor MG132 for 4 hours. All these data indicate that NAC103 is a transcription factor of the NAC family. Overexpressing of 35S::NAC103-YFP seedlings were smaller than wild-type (wt) plants with retarded growth, an increased number of rosette leaves and leaf marginal serration. On the 1/2 MS medium with TM, the number of seedlings with true leaves in the overexpressing 35S::NAC103-YFP plants is more than that in the wt plants, and the number of seedlings with true leaves in the NAC 103 knockdown plants is less than that in the wt plants, indicating that NAC 103 plays important roles in response to ER stress.The expression of NAC103 is upregulated in the wt treated with TM/DTT, and its inducing expression is dependent on the bZIP60 because the TM-induced NAC 103 expression was completely blocked in the bZIP60 null T-DNA mutant bzip60, as well as in the bZIP28 and bZIP60 double mutant bzip28 bzip60. A new ER stress-responsive cis-element UPRE-III (TCATCG) on the NAC 103 promoter was identified in a dual-luciferase reporter assay system. It was confirmed that bZIP60 activated the UPRE-Ⅲ in both yeast cells and Arabidopsis protoplasts and the direct binding of bZIP60 to UPRE-Ⅲ-containing DNA was also demonstrated in an EMS A. Of 53 genes dependent on bZIP60, ten genes have at least one copy of UPRE-Ⅲ in the 500 bp upstream promoter, and 19 genes have at least one copy of UPRE-Ⅲ in the 1000 bp upstream promoter, indicating that UPRE-Ⅲ may be responsible for upregulation of these UPR genes.Compared with the wild-type, overexpression of NAC103-YFP upregulated expression of several UPR downstream genes in Arabidopsis under normal growth conditions. The activation of UPR gene promoters by NAC103D was also demonstrated in effector/reporter protoplast assays.Thus, our study demonstrates that NAC103 is a trancription factor of NAC family and overexpression of NAC 103 has pleiotropic effects on the plant growth. Under the ER stress conditions, NAC 103 relays ER stress signals from bZIP60 to UPR downstream genes through a newly identified ER stress cis-element UPRE-Ⅲ (TCATCG) and transcriptional activation activity of its encoded protein NAC 103 in the nuclei.