Pedro A. B. Reis

The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco.

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought.

Cryptochromes Interact Directly with PIFs to Control Plant Growth in Limiting Blue Light

Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome wide and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions.

Expression profiling on soybean leaves reveals integration of ER- and osmotic-stress pathways.

BackgroundDespite the potential of the endoplasmic reticulum (ER) stress response to accommodate adaptive pathways, its integration with other environmental-induced responses is poorly understood in plants. We have previously demonstrated that the ER-stress sensor binding protein (BiP) from soybean exhibits an unusual response to drought. The members of the soybean BiP gene family are differentially regulated by osmotic stress and soybean BiP confers tolerance to drought. While these results may reflect crosstalk between the osmotic and ER-stress signaling pathways, the lack of mutants, transcriptional response profiles to stresses and genome sequence information of this relevant crop has limited our attempts to identify integrated networks between osmotic and ER stress-induced adaptive responses. As a fundamental step towards this goal, we performed global expression profiling on soybean leaves exposed to polyethylene glycol treatment (osmotic stress) or to ER stress inducers.ResultsThe up-regulated stress-specific changes unmasked the major branches of the ER-stress response, which include enhancing protein folding and degradation in the ER, as well as specific osmotically regulated changes linked to cellular responses induced by dehydration. However, a small proportion (5.5%) of total up-regulated genes represented a shared response that seemed to integrate the two signaling pathways. These co-regulated genes were considered downstream targets based on similar induction kinetics and a synergistic response to the combination of osmotic- and ER-stress-inducing treatments. Genes in this integrated pathway with the strongest synergistic induction encoded proteins with diverse roles, such as plant-specific development and cell death (DCD) domain-containing proteins, an ubiquitin-associated (UBA) protein homolog and NAC domain-containing proteins. This integrated pathway diverged further from characterized specific branches of ER-stress as downstream targets were inversely regulated by osmotic stress.ConclusionThe present ER-stress- and osmotic-stress-induced transcriptional studies demonstrate a clear predominance of stimulus-specific positive changes over shared responses on soybean leaves. This scenario indicates that polyethylene glycol (PEG)-induced cellular dehydration and ER stress elicited very different up-regulated responses within a 10-h stress treatment regime. In addition to identifying ER-stress and osmotic-stress-specific responses in soybean (Glycine max), our global expression-profiling analyses provided a list of candidate regulatory components, which may integrate the osmotic-stress and ER-stress signaling pathways in plants.

A New Branch of Endoplasmic Reticulum Stress Signaling and the Osmotic Signal Converge on Plant-specific Asparagine-rich Proteins to Promote Cell Death

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

NIK1-mediated translation suppression functions as a plant antiviral immunity mechanism

Plants and plant pathogens are subject to continuous co-evolutionary pressure for dominance, and the outcomes of these interactions can substantially impact agriculture and food security. In virus–plant interactions, one of the major mechanisms for plant antiviral immunity relies on RNA silencing, which is often suppressed by co-evolving virus suppressors, thus enhancing viral pathogenicity in susceptible hosts. In addition, plants use the nucleotide-binding and leucine-rich repeat (NB-LRR) domain-containing resistance proteins, which recognize viral effectors to activate effector-triggered immunity in a defence mechanism similar to that employed in non-viral infections. Unlike most eukaryotic organisms, plants are not known to activate mechanisms of host global translation suppression to fight viruses. Here we demonstrate in Arabidopsis that the constitutive activation of NIK1, a leucine-rich repeat receptor-like kinase (LRR-RLK) identified as a virulence target of the begomovirus nuclear shuttle protein (NSP), leads to global translation suppression and translocation of the downstream component RPL10 to the nucleus, where it interacts with a newly identified MYB-like protein, L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB), to downregulate translational machinery genes fully. LIMYB overexpression represses ribosomal protein genes at the transcriptional level, resulting in protein synthesis inhibition, decreased viral messenger RNA association with polysome fractions and enhanced tolerance to begomovirus. By contrast, the loss of LIMYB function releases the repression of translation-related genes and increases susceptibility to virus infection. Therefore, LIMYB links immune receptor LRR-RLK activation to global translation suppression as an antiviral immunity strategy in plants.

The NAC domain-containing protein, GmNAC6, is a downstream component of the ER stress- and osmotic stress-induced NRP-mediated cell-death signaling pathway

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.

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

Significance An endoplasmic reticulum stress- and osmotic stress-induced cell death pathway has emerged as a relevant adaptive response of plant cells to multiple environmental stimuli. We identified a unique component of this integrated circuit of stress-induced cell death, the GmNAC30 transcriptional factor, which binds to GmNAC81 in the nucleus of plant cells to coordinately regulate the expression of the vacuolar processing enzyme, a plant-specific executioner of cell death. In addition to describing a plant-specific endoplasmic reticulum stress cell death response that communicates with other environmental stimuli, the study deciphered the regulation of vacuolar processing enzyme expression that has been shown to be involved in several events of cell death in plants. 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.

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

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.

N-rich protein (NRP)-mediated cell death signaling: a new branch of the ER stress response with implications for plant biotechnology.

Upon disruption of ER homeostasis, plant cells activate at least two branches of the unfolded protein response (UPR) through IRE1-like and ATAF6-like transducers, resulting in the upregulation of ER-resident molecular chaperones and the activation of the ER-associated degradation protein system. Here, we discuss a new ER stress response pathway in plants that is associated with an osmotic stress response in transducing a cell death signal. Both ER and osmotic stress induce the expression of the novel transcription factor GmERD15, which binds and activates N-rich protein (NRP) promoters to induce NRP expression and cause the upregulation of GmNAC6, an effector of the cell death response. In contrast to this activation mechanism, the ER-resident molecular chaperone binding protein (BiP) attenuates the propagation of the cell death signal by modulating the expression and activity of components of the ER and osmotic stress-induced NRP-mediated cell death signaling. This interaction attenuates dehydration-induced cell death and promotes a better adaptation of BiP-overexpressing transgenic lines to drought.

Comprehensive analysis of the endoplasmic reticulum stress response in the soybean genome: conserved and plant-specific features

BackgroundDespite the relevance of the eukaryotic endoplasmic reticulum (ER)-stress response as an integrator of multiple stress signals into an adaptive response, knowledge about these ER-mediated cytoprotective pathways in soybean (Glycine max) is lacking. Here, we searched for genes involved in the highly conserved unfolded protein response (UPR) and ER stress-induced plant-specific cell death signaling pathways in the soybean genome.MethodsPreviously characterized Arabidopsis UPR genes were used as prototypes for the identification of the soybean orthologs and the in silico assembly of the UPR in soybean, using eggNOG v4.0 software. Functional studies were also conducted by analyzing the transcriptional activity of soybean UPR transducers.ResultsAs a result of this search, we have provided a complete profile of soybean UPR genes with significant predicted protein similarities to A. thaliana UPR-associated proteins. Both arms of the plant UPR were further examined functionally, and evidence is presented that the soybean counterparts are true orthologs of previously characterized UPR transducers in Arabidopsis. The bZIP17/bZI28 orthologs (GmbZIP37 and GmbZIP38) and ZIP60 ortholog (GmbZIP68) from soybean have similar structural organizations as their Arabidopsis counterparts, were induced by ER stress and activated an ERSE- and UPRE-containing BiP promoter. Furthermore, the transcript of the putative substrate of GmIREs, GmbZIP68, harbors a canonical site for IRE1 endonuclease activity and was efficiently spliced under ER stress conditions. In a reverse approach, we also examined the Arabidopsis genome for components of a previously characterized ER stress-induced cell death signaling response in soybean. With the exception of GmERD15, which apparently does not possess an Arabidopsis ortholog, the Arabidopsis genome harbors conserved GmNRP, GmNAC81, GmNAC30 and GmVPE sequences that share significant structural and sequence similarities with their soybean counterparts. These results suggest that the NRP/GmNAC81 + GmNAC30/VPE regulatory circuit may transduce cell death signals in plant species other than soybean.ConclusionsOur in silico analyses, along with current and previous functional data, permitted generation of a comprehensive overview of the ER stress response in soybean as a framework for functional prediction of ER stress signaling components and their possible connections with multiple stress responses.