The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress-and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiPoverexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress-and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP-and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.

BackgroundThe endoplasmic reticulum (ER) is a major signaling organelle, which integrates a variety of responses against physiological stresses. In plants, one such stress-integrating response is the N-rich protein (NRP)-mediated cell death signaling pathway, which is synergistically activated by combined ER stress and osmotic stress signals. Despite the potential of this integrated signaling to protect plant cells against different stress conditions, mechanistic knowledge of the pathway is lacking, and downstream components have yet to be identified.ResultsIn the present investigation, we discovered an NAC domain-containing protein from soybean, GmNAC6 (Glycine max NAC6), to be a downstream component of the integrated pathway. Similar to NRP-A and NRP-B, GmNAC6 is induced by ER stress and osmotic stress individually, but requires both signals for full activation. Transient expression of GmNAC6 promoted cell death and hypersensitive-like responses in planta. GmNAC6 and NRPs also share overlapping responses to biotic signals, but the induction of NRPs peaked before the increased accumulation of GmNAC6 transcripts. Consistent with the delayed kinetics of GmNAC6 induction, increased levels of NRP-A and NRP-B transcripts induced promoter activation and the expression of the GmNAC6 gene.ConclusionsCollectively, our results biochemically link GmNAC6 to the ER stress- and osmotic stress-integrating cell death response and show that GmNAC6 may act downstream of the NRPs.

The onset of leaf senescence is a highly regulated developmental change that is controlled by both genetics and the environment. Senescence is triggered by massive transcriptional reprogramming, but functional information about its underlying regulatory mechanisms is limited. In the current investigation, we performed a functional analysis of the soybean (Glycine max) osmotic stress- and endoplasmic reticulum (ER) stress-induced NAC transcription factor GmNAC81 during natural leaf senescence using overexpression studies and reverse genetics. GmNAC81-overexpressing lines displayed accelerated flowering and leaf senescence but otherwise developed normally. The precocious leaf senescence of GmNAC81-overexpressing lines was associated with greater Chl loss, faster photosynthetic decay and higher expression of hydrolytic enzyme-encoding GmNAC81 target genes, including the vacuolar processing enzyme (VPE), an executioner of vacuole-triggered programmed cell death (PCD). Conversely, virus-induced gene silencing-mediated silencing of GmNAC81 delayed leaf senescence and was associated with reductions in Chl loss, lipid peroxidation and the expression of GmNAC81 direct targets. Promoter-reporter studies revealed that the expression pattern of GmNAC81 was associated with senescence in soybean leaves. Our data indicate that GmNAC81 is a positive regulator of age-dependent senescence and may integrate osmotic stress- and ER stress-induced PCD responses with natural leaf senescence through the GmNAC81/VPE regulatory circuit.