The endoplasmic reticulum binding protein BiP displays dual function in modulating cell death events

Abstract

The binding protein (BiP) has been demonstrated to participate in innate immunity and attenuate endoplasmic reticulum- and osmotic stress-induced cell death. Here, we employed transgenic plants with manipulated levels of BiP to assess whether BiP also controlled developmental and hypersensitive programmed cell death (PCD). Under normal conditions, the BiP-induced transcriptome revealed a robust down-regulation of developmental PCD genes and an up-regulation of the genes involved in hypersensitive PCD triggered by nonhost-pathogen interactions. Accordingly, the BiP-overexpressing line displayed delayed leaf senescence under normal conditions and accelerated hypersensitive response triggered by Pseudomonas syringae pv tomato in soybean (Glycine max) and tobacco (Nicotiana tabacum), as monitored by measuring hallmarks of PCD in plants. The BiP-mediated delay of leaf senescence correlated with the attenuation of N-rich protein (NRP)-mediated cell death signaling and the inhibition of the senescence-associated activation of the unfolded protein response (UPR). By contrast, under biological activation of salicylic acid (SA) signaling and hypersensitive PCD, BiP overexpression further induced NRP-mediated cell death signaling and antagonistically inhibited the UPR. Thus, the SA-mediated induction of NRP cell death signaling occurs via a pathway distinct from UPR. Our data indicate that during the hypersensitive PCD, BiP positively regulates the NRP cell death signaling through a yet undefined mechanism that is activated by SA signaling and related to ER functioning. By contrast, BiP's negative regulation of leaf senescence may be linked to its capacity to attenuate the UPR activation and NRP cell death signaling. Therefore, BiP can function either as a negative or positive modulator of PCD events.

Figure 1.

Global variation of gene expression induced by soyBiPD overexpression. A, The pie chart illustrates the distribution of the differentially expressed genes across functional categories defined by the Gene Ontology Biological process. The numbers represent the frequency of genes in each category. B, The relative quantification of gene expression in transgenic plants compared with the wild type in a log2 scale (± sd, n = three biological replicates) determined by real-time PCR. The identities of each gene are presented in Supplemental Table S3.

Figure 2.

Slightly delayed leaf senescence in 35S::BiP4 lines. A to C, Developmental performance of soybean plants after flowering. Wild-type (WT) and 35S::BiP4 soybean lines were grown under greenhouse conditions and were photographed at 51 DAG (A), at 72 DAG (B), and at 93 DAG (C). D to F, Chlorophyll loss during the progression of leaf senescence. The content of chlorophyll a (D), chlorophyll b (E), and total chlorophyll (F) was determined over time in the leaves of wild-type and 35S::BiP4 lines during leaf senescence. The arrow indicates the start of flowering. G, The carotenoids content during leaf senescence in wild-type and 35S::BiP4 leaves. H, CO₂ assimilation during leaf senescence in wild-type and 35S::BiP4 leaves. I, Lipid peroxidation induced by leaf senescence in wild-type and 35S::BiP4 leaves. The leaf lipid peroxidation was monitored by determining the level of thiobarbituric acid-reactive compounds and expressed as the malondialdehyde content. The bars indicate the confidence intervals, and the asterisks indicate significant differences of P ≤ 0.05 when comparing the transgenic and nontransgenic lines on the same observation day.

Figure 3.

Gene expression analysis of senescence and cell death-associated genes in 35S::BiP4 transgenic plants under normal developmental conditions. Total RNA was isolated from wild-type (WT) and 35S::BiP4 leaves at 37, 72, and 93 DAG, and the induction of GmNAC1 (A), Cys protease, GmCystP (B), soyBiPD (C), CNX (D), PDI (E), and the IRE1 homolog Glyma11g09240 (F) was monitored by qRT-PCR. The bars indicate the confidence interval (P < 0.05, n = 3), and the asterisks indicate significant differences between wild-type and transgenic plants.

Figure 4.

BiP overexpression attenuates mediators of the ER and osmotic stress-induced cell death response during development. Total RNA was isolated from wild-type (WT) and 35S::BiP4 leaves at 37, 72, and 93 DAG, and the induction of the indicated genes was monitored by qRT-PCR, as follows: NRP-A (A), NRP-B (B), GmNAC81 (C), VPE (Glyma01g05135; D), VPE (Glyma04g05250; E), and VPE (Glyma14g10620; F). The expression values were obtained using the comparative cycle threshold (2^−ΔCT) method and the endogenous control helicase. The bars indicate a confidence interval (P < 0.05, n = 3), and the asterisks indicate significant differences between wild-type and transgenic plants.

Figure 5.

Caspase1-like (YVADase) activity in soybean leaves. A, BiP overexpression attenuates caspase1-like activity in leaves. Caspase1-like activity was determined from total protein of wild-type (WT) and 35S::BiP4 leaves at 72 DAG in the presence and absence of the specific inhibitor Ac-YVAD-CHO. B, Enhanced caspase1-like activity displayed by 35S::BiP4 and 35S::BiP2 inoculated leaves compared with wild-type leaves. Soybean wild-type, 35S::BiP4, and 35S::BiP2 leaves were inoculated with P. syringae pv tomato, and caspase1-like (YVADase) activity was determined at the postinoculation time as indicated in the figure. The inclusion of the caspase1-specific inhibitor reduced enzyme activity. The bars indicate the confidence interval based on Student’s t test (P < 0.05, n = 3), and the asterisks indicate significant differences between wild-type and transgenic plants.

Figure 6.

HR in soybean leaves induced by a nonhost-pathogen interaction. A, Leaf necrotic lesions induced by HR. Soybean wild-type (WT), 35S::BiP4, and 35S::BiP2 leaves were mock inoculated or inoculated with P. syringae pv tomato (Pst) and observed at intervals of 6, 12, 18, 24, and 36 h postinoculation. B, H₂O₂ production induced by HR. Wild-type, 35S::BiP2, and 35S::BiP4 leaves were stained with DAB 24 h after inoculation. C, Electrolyte leakage during HR. The conductivity from electrolyte leakage of mock- or P. syringae pv tomato-inoculated wild-type, 35S::BiP2, and 35S::BiP4 leaves was measured at the indicated periods postinoculation. D, Exclusion of Evans blue dye. The frequency of dead cells was quantified by the intensity of Evans blue staining of inoculated leaves as HR progresses. The bars represent a confidence interval based on Student’s t test (P < 0,05, n = 5).

Figure 7.

Induction of PR genes and UPR marker genes during HR elicited by P. syringae pv tomato. Total RNA was isolated from P. syringae pv tomato- or mock-inoculated wild-type (WT), 35S::BiP2 and 35S::BiP4 leaves, and the expression of the PR genes PR1 (A), PR5 (B), and Cys protease (GmCystP; C) was monitored by qRT-PCR. The induction of UPR marker genes, such as soyBiPD (D), PDI (E), CNX (F), the IRE1 homologs Glyma09g32970 (G), and Glyma11g09240 (H), was also monitored by qRT-PCR. The expression values were obtained using the 2^−ΔCT method and a control helicase as the endogenous control. The bars indicate the confidence interval based on Student’s t test (P < 0.05, n = 3), and the asterisks indicate significant differences between wild-type and transgenic plants.

Figure 8.

Inoculation of soybean leaves with P. syringae pv tomato triggers NRP-mediated cell death signaling during the establishment of nonhost resistance. The total RNA was isolated from P. syringae pv tomato- or mock-inoculated wild-type (WT), 35S::BiP2, and 35S::BiP4 leaves, and induction of NRP-A (A), NRP-B (B), GmNAC81 (C), VPE (Glyma01g05135; D), VPE (Glyma14g10620; E), and VPE (Glyma17g34900; F) was monitored by qRT-PCR. Expression values were obtained as in Figure 7.

Figure 9.

HR elicited by nonhost-pathogen interactions in tobacco leaves with enhanced (sense) and suppressed (antisense) levels of BiP. A, The lesions on the tobacco leaves of wild-type (WT), sense, and antisense lines after 24 h of inoculation with a control MgCl₂ buffer or with P. syringae pv tomato (Pst). B, DAB staining in wild-type, sense, and antisense leaves 24 h after inoculation with P. syringae pv tomato or a MgCl₂ solution. C, The membrane integrity of leaf cells during the HR. The conductivity from electrolyte leakage of P. syringae pv tomato- and mock-inoculated wild-type, sense, and antisense leaves was measured at the indicated periods after inoculation. D, The exclusion of vital Evans blue dye from tobacco wild-type, sense, and antisense leaves during the HR. The bars represent the confidence interval (P < 0.05, n = 5).

Figure 10.

Time course of induction of PR genes by the nonhost interaction in tobacco leaves. Wild-type (WT), sense, and antisense tobacco leaves were inoculated with P. syringae pv tomato, and the induction of the PR genes, including PR1 and chitinase, was examined by qRT-PCR. The expression values were obtained using the 2^−ΔCT method and actin as the endogenous control. The bars indicate the confidence interval (P < 0.05, n = 3), and the asterisks indicate significant differences between wild-type and transgenic plants.

Figure 11.

BiP may modulate the NRP-mediated cell death signaling pathway by inhibiting UPR and stimulating SA signaling. The scheme illustrates the propagation of a cell death signal derived from prolonged ER, osmotic, and biotic stress through the NRP-mediated PCD signaling pathway. A broken arrow indicates an effect on gene expression, while a solid arrow indicates that the gene is an immediate downstream target. This investigation revealed that overexpression of BiP inhibits the UPR, the expression of NRPs, and GmNAC81 and as consequence attenuates cell death mediated by the NRP signaling as in leaf senescence. In response to a biotic stimulus, BiP positively modulates the SA signaling and activates the NRP-mediated cell death signaling.