A novel transcription factor, ERD15 (Early Responsive to Dehydration 15), connects endoplasmic reticulum stress with an osmotic stress-induced cell death signal

Abstract

As in all other eukaryotic organisms, endoplasmic reticulum (ER) stress triggers the evolutionarily conserved unfolded protein response in soybean, but it also communicates with other adaptive signaling responses, such as osmotic stress-induced and ER stress-induced programmed cell death. These two signaling pathways converge at the level of gene transcription to activate an integrated cascade that is mediated by N-rich proteins (NRPs). Here, we describe a novel transcription factor, GmERD15 (Glycine max Early Responsive to Dehydration 15), which is induced by ER stress and osmotic stress to activate the expression of NRP genes. GmERD15 was isolated because of its capacity to stably associate with the NRP-B promoter in yeast. It specifically binds to a 187-bp fragment of the NRP-B promoter in vitro and activates the transcription of a reporter gene in yeast. Furthermore, GmERD15 was found in both the cytoplasm and the nucleus, and a ChIP assay revealed that it binds to the NRP-B promoter in vivo. Expression of GmERD15 in soybean protoplasts activated the NRP-B promoter and induced expression of the NRP-B gene. Collectively, these results support the interpretation that GmERD15 functions as an upstream component of stress-induced NRP-B-mediated signaling to connect stress in the ER to an osmotic stress-induced cell death signal.

FIGURE 1.

A tunicamycin- and PEG-induced ERD15 soybean homolog associates stably with the NRP-B promoter in yeast. A, isolation of GmERD15 by one-hybrid screening in yeast. An S. cerevisiae strain carrying the pNRPB-HIS3 reporter was transformed with the GAL4 activation domain-encoded vector or GAL4AD vector containing the cDNA of GmERD15. After growth for 12 h, the yeast culture was diluted, as indicated in the figure, and plated on medium lacking histidine but supplemented with 100 mm 3-aminotriazole and incubated for 3 days. B, LacZ reporter expression. β-Galactosidase activity was determined from total protein extracts of yeast strains carrying the indicated combinations of the empty expression vector and AD-GmERD15. As a control, a pBip_350bp-LacZ integrated strain was transformed with AD-GmERD15. Thin bars indicate the standard deviation of three independent experiments. C, kinetics of GmERD15 and GmNRP-B induction by PEG-induced osmotic stress in soybean-cultured cells. Levels of RNA were examined at various times after PEG treatment by quantitative RT-PCR. Expression values were calculated using the 2 − ΔC_t method with helicase used as an endogenous control. cDNA was prepared from three biological replicates. Thin bars indicate the standard deviation. D, induction of GmERD15 and NRP-B by tunicamycin. Soybean-cultured cells were treated with tunicamycin for the indicated times. Levels of RNA were examined by quantitative RT-PCR, as described in C. E, GmERD15 associates to a discrete region of NRP-B promoter in yeast. The schematic representation of the promoter constructs indicates the 5′-flanking sequences of NRP-B fused to LacZ that were integrated into the W303 strain and transformed with AD-GmERD15 or the empty vector. The activity of the β-galactosidase was expressed in units as described under “Experimental Procedures.” Thin bars indicate the standard deviation of three independent experiments.

FIGURE 2.

GmERD15 is a DNA-binding protein that exhibits transactivation activity in yeast. A, gel shift assays. A 187-bp biotin-labeled DNA fragment of the GmNRP-B 5′-flanking sequences (positions −330 to −517; lane 1) was incubated with purified, His-tagged GmERD15 in the absence (lane 2) and presence of 4 pmol of unlabeled DNA probe (lane 3) for 20 min at RT. The products were separated by electrophoresis in a 5% polyacrylamide gel in TB buffer. Lane 4 indicates probe plus anti-GmERD15 antisera, and lane 5 indicates probe plus anti-GmERD15 antisera plus recombinant GmERD15 protein. The solid arrow indicates the free DNA, and the open arrow designates the DNA-protein complexes. B, transactivation activity of the GmERD15 protein in yeast. pBD-GmERD15 and the empty vector (pDEST32) were introduced separately into yeast strain AH109. Overnight growth cultures were diluted as indicated in the figure and plated on medium lacking histidine but supplemented with 100 mm 3-aminotriazole and incubated for 3 days. C, GmERD15 transactivates expression of a lacZ reporter gene. β-Galactosidase activity was determined from total protein extracts of overnight growth yeast strains carrying the indicated DNA construct. Thin bars indicate the standard deviation of the three independent experiments.

FIGURE 3.

GmERD15 localizes to the nucleus of tobacco leaf cells and binds to the NRP-B promoter in vivo. A, confocal fluorescence image of transiently expressed GmERD15-YFP in epidermal cells of tobacco leaves. Tobacco leaves were infiltrated with Agrobacterium tumefaciens carrying ³⁵S::GFP-NSP or ³⁵S::GmERD15-YFP DNA constructs and observed by confocal microscopy 3 days after agroinoculation. The white arrows show the nuclear positions of the nuclear viral protein NSPs (left panel) and GmERD15 (right panel). The black arrows in the bottom right panel show the co-localization signal of GmERD15 with the nuclear marker NSP. B, immunoblotting of nuclear fractions of tobacco leaves expressing YFP-GmERD15. Soluble (SF) and nuclear fractions (NF) of tobacco leaves expressing YFP-GmERD15 were immunoblotted with anti-GmERD15 antibodies. The open arrow indicates the position of purified His-GmERD15, and the black arrow indicates the position of YFP-GmERD15. M is molecular mass. C, ChIP with tunicamycin-treated soybean suspension cells using anti-GmERD15 antibodies. The 187-bp DNA fragment of NRP-B 5′-flanking sequences was detected by PCR amplification when immunoprecipitation was mediated by anti-GmERD15 antibodies (lane 2) but not by unrelated anti-polymerase (Pol) II antibodies (lane 4) or IgG (lane 3). Lane 1 represents the amplification from input total DNA, and M represents the molecular mass standards.

FIGURE 4.

GmERD15 activates the NRP-B promoter and induces NRP-B expression. A, transient expression of GmERD15 in soybean protoplast activates a NRP-B promoter::β-glucuronidase gene. Soybean protoplasts were co-electroporated with plasmids carrying −1000pNRP-B::β-glucuronidase (Gus) gene and either ³⁵S::GmERD15 DNA constructs (light gray bars) or empty vector (dark gray bars). After 48 h, β-glucuronidase activity (nmol/min/mg protein) was measured from the total protein extracts of transfected soybean cells. The thin bars indicate the standard deviation of 15 biological replicates. The asterisks indicate the mean values statistically different from the control treatment according to Student's t test with the two-tailed p value 0.000846. B, an internal deletion in the promoter sequence of NRP-B abolishes ERD15 transactivation of NRP-B promoter in soybean cells. Soybean protoplasts were co-electroporated with plasmids carrying Δ(−666/−403)pNRP-B::β-glucuronidase gene and either ³⁵S::GmERD15 DNA constructs (light gray bars) or empty vector (dark gray bars). The mean values do not differ from each other according to Student's t test with the two-tailed p value 0.083458. C, transient expression of GmERD15 in soybean protoplasts. Plasmid containing a GmERD15 expression cassette was electroporated into soybean protoplasts, and gene expression was monitored by quantitative RT-PCR. Expression values were calculated using the 2^−ΔCt method and helicase as an endogenous control. The thin bars indicate the standard deviation of five biological replicates. The asterisks denote mean values statistically different from the control treatment according to Student's t test with the two-tailed p value 0.0000527. D, transient expression of GmERD15 in soybean protoplasts induces GmNRP-B expression but not soyBiPD expression. GmNRP-B (light gray bars) and soyBiPD (dark gray bars) transcript levels were determined by quantitative RT-PCR in protoplasts transformed with empty vector pK7WG2 (PK7) or a GmERD15 expression cassette (PK7 GmERD-15). Expression values were calculated using the 2^−ΔC_t method and helicase as an endogenous control. The thin bars indicate the standard deviations of five biological replicates. The asterisks denote the mean values statistically different from the control treatment according to Student's t test with the two-tailed p value 0.00563.

FIGURE 5.

ERD15 binds specifically to an inverted repeat sequence to activate NRP-B promoter. A, GmERD15 binds to a 50-bp sequence limited by positions −550 to −500. Sequential 50- and 60-bp biotin-labeled DNA fragments of the NRP-B promoter spanning from positions −550 to −330 (as indicated in the figure) were incubated with purified, His-tagged GmERD15 for 20 min at room temperature. The products were separated by electrophoresis in a 5% polyacrylamide gel in TB buffer. The solid arrow indicates the free DNA, and the open arrow designates the DNA-protein complexes. B, the 50-bp fragment (−550/−500) is sufficient to promote GmERD15-mediated transactivation of the minimal promoter of the yeast CYC1 gene. The 50-bp fragment of NRP-B promoter, positions −550 to −500, was cloned into pLACZi upstream to the minimal promoter of CYC1 and to the LacZ gene. β-Galactosidase activity was determined from total protein extracts of overnight growth yeast strains carrying the indicated DNA construct. C and D, GmERD15 binds to the −12-bp ⁻⁵¹¹AGCAnnnnTGCT⁻⁵⁰⁰ palindromic sequence but not to a −25-bp palindromic sequence (⁻⁴²⁴ACGATTCTAnnnnnnnnTAGAACGT⁻³⁹⁹) of the NRP-B promoter. The 12-bp (C) and the 25-bp (D) palindromic sequences were biotin-labeled and incubated with purified, His-tagged GmERD15 in the absence (lanes 1, 3, and 5) and presence of 4 pmol of unlabeled DNA probe as double-stranded form (lanes 2), noncoding strand (lanes 4), and coding strand (lanes 6) for 20 min at room temperature. The products were separated by electrophoresis in a 5% polyacrylamide gel in TB buffer. The open arrows designate the DNA-protein complexes. DS, double-stranded DNA; SSC, single-stranded coding sequence; SSNC, single-stranded noncoding sequence.