GmNAC30 and GmNAC81 integrate the endoplasmic reticulum stress- and osmotic stress-induced cell death responses through a vacuolar processing enzyme

Abstract

Prolonged endoplasmic reticulum and osmotic stress synergistically activate the stress-induced N-rich protein-mediated signaling that transduces a cell death signal by inducing GmNAC81 (GmNAC6) in soybean. To identify novel regulators of the stress-induced programmed cell death (PCD) response, we screened a two-hybrid library for partners of GmNAC81. We discovered another member of the NAC (NAM-ATAF1,2-CUC2) family, GmNAC30, which binds to GmNAC81 in the nucleus of plant cells to coordinately regulate common target promoters that harbor the core cis-regulatory element TGTG[TGC]. We found that GmNAC81 and GmNAC30 can function either as transcriptional repressors or activators and cooperate to enhance the transcriptional regulation of common target promoters, suggesting that heterodimerization may be required for the full regulation of gene expression. Accordingly, GmNAC81 and GmNAC30 display overlapping expression profiles in response to multiple environmental and developmental stimuli. Consistent with a role in PCD, GmNAC81 and GmNAC30 bind in vivo to and transactivate hydrolytic enzyme promoters in soybean protoplasts. A GmNAC81/GmNAC30 binding site is located in the promoter of the caspase-1-like vacuolar processing enzyme (VPE) gene, which is involved in PCD in plants. We demonstrated that the expression of GmNAC81 and GmNAC30 fully transactivates the VPE gene in soybean protoplasts and that this transactivation was associated with an increase in caspase-1-like activity. Collectively, our results indicate that the stress-induced GmNAC30 cooperates with GmNAC81 to activate PCD through the induction of the cell death executioner VPE.

Keywords: ER stress; NAC transcriptional factors; abiotic stresses; cell death signaling.

Fig. 1.

GmNAC30 binds to GmNAC81 in the nucleus of plant cells. (A) The in vivo interaction between GmNAC81 and GmNAC30 by BiFC analysis. The fluorescence (YFP) images were taken from the soybean leaf protoplasts that coexpressed 35S:GmNAC30-nYFP + 35S:GmNAC81-cYFP and 35S:GmNAC30-cYFP + 35S:GmNAC81-nYFP fusion proteins 36 h after the electroporation of the protoplasts with the indicated DNA constructs. (Scale bars, 20 µm.) (B) Homo- and heterodimerization between GmNAC30 and GmNAC81. The immunoprecipitation of transiently expressed proteins was performed using anti-GFP matrix, and coimmunoprecipitated proteins were detected using anti-HA antibody. (Top) Immunoblots of coimmunoprecipitated GmNAC30-HA and GmNAC81-HA (Co-IP); (Middle) immunoprecipitated GmNAC30-GFP and GmNAC81-GFP (IP); (Bottom) total protein extracts (input HA) from transiently expressed proteins in N. benthamiana leaves. The expression levels of the input HA-fused proteins were assessed by anti-HA of crude extracts. These results were replicated twice from independent experiments.

Fig. 2.

GmNAC30 may be a downstream component of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling pathway. (A–C) GmNAC30 is induced by osmotic stress, ER stress, and cell death inducers. The soybean seedlings were treated with the osmotic stress-inducer PEG for 10 h (A), the ER stress-inducer tunicamycin for 16 h (B), or the cell death-inducer cycloheximide (C) for 6 h and 12 h, and the expression of the indicated genes was monitored by qRT-PCR. The gene expression was calculated using the 2^−ΔCT method and the RNA helicase as an endogenous control. GmSMP is a seed-maturation protein that was used as a control for the PEG treatment. GmCNX (calnexin) and GmBiPD (binding protein) are ER markers. GmNAC81 and GmNRP-B are components of the ER stress- and osmotic stress-integrating cell death pathway. The values are given as the mean ± confidence interval of three independent experiments. The asterisks indicate significant differences from the controls by the t test at P ≤ 0.05. (D) GmNRP-A and GmNRP-B induce the expression of the GmNAC30 gene. The plasmids containing the GmNRP-A and GmNRP-B expression cassettes or the empty vector were electroporated into the soybean protoplasts, and the gene expression of the GmNAC genes was monitored by qRT-PCR as in A–C. (E) DNA fragmentation was promoted by GmNAC30 expression. The cells were sampled 36 h postelectroporation of the soybean protoplasts with the empty vector and GmNAC30 expression cassettes, and submitted to TUNEL labeling. As a positive control, the untransfected cells were also treated with DNase. The nuclei were stained with DAPI. The magnification was 1.3-fold higher than in Fig. 1A.

Fig. 3.

GmNAC81 and GmNAC30 determine the full activation or repression of target promoters and bind specifically to the core sequence TGTGTT in vitro. (A) GmNAC30 and GmNAC81 repress an ACC synthase promoter. The soybean protoplasts were coelectroporated with plasmids carrying the 1000pACC_pro:β-GUS gene and either the 35S:GmNAC81 or 35S:GmNAC30 DNA constructs or a combination of both DNA constructs. After 48 h, the β-GUS activity (nmol⋅min⋅mg protein) was measured from the total protein extracts of the transfected soybean cells. An unrelated MyB transfactor, 35S:At5g05800, was used as a negative control. The error bars represent the confidence interval (α = 0.05) of three biological replicates. (B) The full activation of the carboxypeptidase (ACPase) promoter requires both GmNAC81 and GmNAC30. The soybean protoplasts were coelectroporated with plasmids carrying the 2000pACPase_pro:β-GUS gene and the same combinations of DNA constructs, as described in A, and the samples were processed for β-GUS activity, as in A. (C) Quantitation of the GUS reporter gene expression by qRT-PCR. The soybean protoplasts were electroporated with the same DNA constructs as described in A and B, and the GUS transcript levels were monitored by qRT-PCR. The relative quantitation as demonstrated by a log₂ scale of gene expression was calculated using the 2^−ΔΔCt method and helicase as an endogenous control. The values are relative to the control treatment (empty vector), and the error bars represent the confidence interval (α = 0.05) of three biological replicates. An unrelated MyB transfactor, 35S::At5g05800, was used as a negative control. (D) The specific binding of GmNAC81 and GmNAC30 to their core DNA binding sites. An 18-bp biotin-labeled fragment harboring the directly repeated core sequence TGTGTT was incubated with an E. coli-produced and purified GST, GST-tagged GmNAC30 (N30), GST-GmNAC81 (N81), and both NAC proteins (N30+N81) for 20 min at room temperature in the absence and presence of a 100-fold molar excess of unlabeled probe, as indicated in the figure. The products were separated by electrophoresis in a 4% (wt/vol) polyacrylamide gel in TB buffer. The arrow indicates the DNA:protein complexes.

Fig. 4.

GmNAC81 and GmNAC30 bind to VPE promoter in vivo, transactivate VPE expression and induce caspase-1–like activity. (A) GmanC81 and GmNAC30 bind to the VPE promoter in vivo. ChIP assay was performed with GmNAC30-GFP–, GmNAC81-GFP–, or GFP-expressing leaves using anti-GFP antibodies. The 103-bp fragment of VPE 5′ flanking region was detected by PCR amplification when DNA was immunoprecipitated from GmNAC30-GFP– or GmNAC81-GFP–expressing leaves but not from GFP-expressing leaves. “C” represents amplification of control plasmid. (Right) Amplifications from input total DNA. (B) GmNAC30 and GmNAC81 transactivate the VPE promoter. Tobacco leaves were agroinfiltrated with a construct harboring the 2000pVPE::Luciferase gene alone or in combination with 35S::GmNAC30 or 35S::GmNAC81. Luciferase activity was determined 36 h postagroinoculation. (C) GmNAC81 and GmNAC30 induce VPE expression in the soybean protoplasts. The transcript levels of VPE were quantified by qRT-PCR 36 h postelectroporation of the soybean protoplasts with GmNAC81, GmNAC30, or the empty vector. The gene expression was calculated using the 2^−ΔCT method and RNA helicase as an endogenous control. The error bars represent the confidence interval (α = 0.05) of three biological replicates. (D) GmNAC30 and GmNAC81 induce caspase-1–like activity in the soybean protoplasts. Caspase-1–like activity was determined from GmNAC81- and GmNAC30-expressing protoplasts in the absence and presence of a specific competitor. The asterisks indicate significant differences by the t test at P ≤ 0.05 (n = 3).