Figure 1.
Up-regulation of NAC089 by ER stress is directly controlled by bZIP28 and bZIP60.
(A–B) Up-regulation of NAC089 in the wild-type (wt) plants by ER-stress inducers tunicamycin (TM, 5 µg/ml) and dithiothreitol (DTT, 2 mM) (A) and in the wt, bZIP28 single mutant (zip28), bZIP60 single mutant (zip60) or double mutant (zip28zip60) by TM (5 µg/ml) treatment (B). The expression of NAC089 is normalized to the expression of the internal control actin. (C–D) Transactivation of NAC089 promoter in the dual-luciferase leaf protoplast assays. Activations of the NAC089 promoter by ER stress treatments (C) and by co-expression of either activated bZIP28 (bZIP28D) or activated bZIP60 (bZIP60S) (D). Relative reporter activity is the firefly luciferase activity normalized to the renilla luciferase activity. Bars depict SE (n = 3) in A–D. The empty vector (EV) was used as a negative control. ** P<0.01, * P<0.05. (E–F) EMSA experiments to detect the protein-DNA binding. Either the purified His-bZIP28D (E) or Trx-His-bZIP60T (F) was incubated with the biotin-labeled pNAC089 DNA. Lane 3, 50× un-labeled pNAC089; lane 4, 200× un-labeled pNAC089; lane 5, 200× un-labeled mutated form pNAC089M1. Arrows and arrow heads point to the positions of shifted bands and free probes, respectively.
Figure 2.
NAC089 relocates from the ER membrane to the nucleus during ER stress responses.
(A) Topology of NAC089 protein. (B) Activation of MYC-NAC089 in response to ER stress. Plant seedlings were treated with H2O (control), tunicaymin (TM, 5 µg/ml) or DTT (2 mM) for 6 hr. Star and asterisk represent the precursor and the processed form, respectively. The beta-estradiol (BE) induced truncated form NAC089D-MYC was used as the migration marker. Coomassie blue staining of RbcS serves as the loading control. (C–D) Nuclear relocation of mGFP-NAC089 in response to ER stresses in Arabidopsis leaf protoplast (C) and root cells (D). TM = 5 µg/ml in C and TM = 10 µg/ml in D. Dashed lines highlight the cell boundary. Bar = 50 µm.
Figure 3.
Down-regulation of NAC089 confers ER stress tolerance.
(A–B) Sensitivity of NAC089 RNAi plants to ER stress. Photos were taken with 10-day-old Arabidopsis seedlings grown on 1/2 MS medium supplied without or with different concentrations of tunicamycin (TM) (A) and percentage of green-big (G-B), green-small (G-S) and yellow-small (Y-S) plants was calculated (B). Bars depict SE (n = 3). The difference between the wild-type (wt) and each NAC089 RNAi line is significant (P<0.05).
Figure 4.
NAC089 promotes programmed cell death in plants.
(A–C) Photos of Arabidopsis NAC089D-MYC transgenic lines (XVE089D) and wild-type (wt) control plants transferred to 1/2 MS medium supplied without (A) or with (B) beta-estradiol (BE) for additional 5 days with the quantitative data in (C). (D–E) Caspase 3/7-like activity in protein extracts from NAC089D-MYC overexpression plants treated with BE (D) or in the wt and NAC089 RNAi plants treated with TM (0.5 µg/ml) (E). The caspase-like activity was normalized to the activity of the wt control under normal condition. Bars depict SE (n = 3) in C–E. ** P<0.01, * P<0.05. (F–I) Esterase activities (F–G) and H2O2 accumulation (H–I) in the XVE089D-13 roots as revealed by FDA and DAB staining without (F, H) or with (G, I) BE treatment for 3 days. (J–N) Membrane rigidity and nucleus morphology in the XVE089D-13 roots as reflected by PI (J–K) and DAPI (L–N) staining without (J, L) or with (K, M–N) BE treatment for 5 days. (O–Q) DNA breakage in the XVE089D-13 roots as detected by TUNEL assay without (O) or with (P–Q) BE treatment for 5 days. Bar = 10 mm in A–B and bar = 10 µm in F–Q.
Figure 5.
NAC089 regulates many downstream genes.
Expression of down-stream genes identified through microarray analysis was examined with qRT-PCR in two lines of NAC089D-MYC plants. Fold change is the gene expression level of plants treated with beta-estradiol (BE) for 16 hr divided by that of plants treated with DMSO (solvent control), both of which were normalized to the expression of actin. Bars depict SE (n = 3). The fold change for each gene is significant (P<0.01) in line XVE089D-13.
Figure 6.
Up-regulation of NAC089D-MYC-regulated genes by ER stress is impaired in the NAC089 RNAi plants.
The expressions of NAC089D-MYC downstream genes (A) and other UPR markers (B) were quantified with qRT-PCR in the wild-type control (wt) and NAC089 knock-down plants (line RNAi089-25). Fold change is the gene expression value in TM (5 µg/ml) -treated plants divided by the value in non-treated plants, both of which were normalized to the expression of actin. Bars depict SE (n = 3). Values for the gene locus with # are log2 values. Fold change of BiP1 (AT5g28540, highlighted with §) also includes that of BiP2 (AT5G42020). ** P<0.01, * P<0.05.
Figure 7.
NAC089 binds to the promoter regions of target genes.
Enrichment is the DNA level of each fragment in beta-estradiol (BE)-treated sample divided by that in DMSO-treated sample, both of which were normalized to the level of TA3. Anti-MYC antibody was used to precipitate NAC089D-MYC and anti-GST was used as a negative antibody control. Bars depict SE (n = 3). ** P<0.01, * P<0.05 except for AT1G79330 (p = 0.057).
Figure 8.
Hypothetical working model of cell survival and cell death pathways in plant UPR.
In response to ER stress, the ER-localized bZIP28P is activated through S1P/S2P sequential cleavage. The precursor bZIP60P is also ER membrane-associated under normal growth condition; ER stress activates the ER-localized IRE1, which splices the bZIP60 mRNA to produce a shorter bZIP60 mRNA encoding the activated form bZIP60S. Both the activated bZIP28D and bZIP60S enter the nucleus and up-regulate downstream genes such as molecular chaperones and ERAD components to ensure cell survival. The activated bZIP60S also induces its own transcription and another transcription factor NAC103 to amplify the cell survival signal. IRE1 could also protect cells through RIDD to reduce the entry of secretory proteins into the ER lumen. On the other hand, the ER membrane-localized NAC089P is activated to produce the nuclear form NAC089D when the ER stress is severe. NAC089D activates several downstream PCD-related genes to promote cell death. Both bZIP28D and bZIP60S control the up-regulation of NAC089 under ER stress condition and may also control the expression of negative regulators for NAC089 activation. Thus, both pro-survival and pro-death signals are controlled by bZIP28 and bZIP60, and the balance between their outputs probably decides the life-or-death cell fate in Arabidopsis plants under ER stress conditions.