Any condition that disrupts the ER homeostasis activates a cytoprotective signaling cascade, designated as the unfolded protein response (UPR), which is transduced in plant cells by a bipartite signaling module. Activation of IRE1/bZIP60 and bZIP28/bZIP17, which represent the bipartite signaling arms and serve as ER stress sensors and transducers, results in the upregulation of ER protein processing machinery-related genes to recover from stress. However, if the ER stress persists and the cell is unable to restore ER homeostasis, programmed cell death signaling pathways are activated for survival. Here, we describe an ER stress-induced plant-specific cell death program, which is a shared response to multiple stress signals. This signaling pathway was first identified through genome-wide expression profile of differentially expressed genes in response to combined ER stress and osmotic stress. Among them, the development and cell death domain-containing N-rich proteins (DCD/NRPs), NRP-A and NRP-B, and the transcriptional factor GmNAC81 were selected as mediators of cell death in plants. These genes were used as targets to identify additional components of the cell death pathway, which is described here as a regulatory circuit that integrates a stress-induced cell death program with leaf senescence via the NRP-A/NRP-B/GmNAC81:GmNAC30/VPE signaling module.
- osmotic stress
The onset of leaf senescence is a highly regulated developmental program that is controlled by both genetics and the environment. Multiple stresses in plants induce programmed cell death, and the underlying regulatory mechanisms are often associated with molecular links of developmentally programmed senescence. The transcriptome changes induced by different environmental stressors are not entirely overlapping, but functional analysis of genes commonly induced as shared responses can give clues on signaling integration. This approach has been used to select for overlapping genes as candidate regulatory components that integrate the ER stress and osmotic stress responses, which were shown later to participate also in natural leaf senescence. Among genes identified as components of the ER and osmotic stress shared response, the developmental and cell death (DCD) domain-containing asparagine-rich proteins (NRP-A and NRP-B) were the first ones to be characterized as cell death-promoting proteins, and hence this multiple stress-integrating signaling was designated as stress-induced DCD/NRP-mediated cell death response. Further characterization of the cell death pathway implicated in the discovery of the signaling module ERD15/NRPs/GmNAC81:GmNAC30/VPE that also has been shown to operate in developmentally programmed leaf senescence. This plant-specific cell death signaling module, which operates in both stress-induced and natural leaf senescence, constitutes the primary focus of this chapter.
2. Modest overlapping of ER stress and osmotic stress response identifies NRPs and NACs as cell death-promoting genes
2.1 Osmotic stress responses
Organisms, in general, are continually adapting to internal and external stimuli, which activate sensor proteins to subsequently transmit the signal to downstream effectors responsible for the assembly of adaptive cellular responses . Abiotic stresses consist of a set of adverse environmental conditions that limits plant development. Cold, high temperature, salinity, water availability (drought or overflow), radiation, pollution, and chemical exposure are the most common examples of types of abiotic stresses .
Generally, a signaling sensor network connects internal and external stimuli to adaptive responses leading to molecular modifications that allow physiological adjustments, which ultimately cause susceptibility or tolerance to the exposed conditions. Molecular responses to abiotic stress conditions in plants are crucial for survival and productivity as these stresses often limit yield. Among abiotic stresses, drought and excess salinity conditions induce sophisticated adaptive responses in plants to cope with or acclimate to these adverse environmental conditions [3, 4]. Some types of abiotic stress responses are better understood than others. In plants, for example, the molecular mechanisms of perception and responses to drought, high salinity, and endoplasmic reticulum stress are well characterized, and many stress-related cell signaling pathways are completely elucidated, revealing some convergence points between them.
The osmotic stress in plants, caused by water deprivation or high salinity, for example, undergoes a set of characteristic morphological, molecular, and physiological changes. One of the most notorious symptoms in plants under low water availability is the ABA-mediated stomatal closure . This hormone-mediated morphological change affects plant physiology. The stomatal closure prevents the evapotranspiration, optimizing the cell water use, but it also compromises carbon dioxide uptake, causing imbalances on photosynthetic apparatus, which culminates on reactive oxygen species (ROS) production [6, 7]. The ROS accumulation acts as a signal to the cell, which triggers mechanisms of ROS-associated detoxification, including upregulation of antioxidant enzymes, osmolyte, and electron-carrier synthesis . There is evidence that osmotic stress and temperature changes are capable of generating lipid-derived signal transducers, including the phosphatidic acid, phosphoinositides, sphingolipids, lysophospholipids, oxylipins, N-acylethanolamines, and others. Water deprivation causes a collapse on the organization of membrane lipids, disrupting its permeability and some significant molecular interactions between lipids and proteins, which act as a cell signal to stress-mediated physiological changes. The mechanisms of how stress responses are connected with membrane lipid transducer generation are still unclear, but lipid messengers can alter protein and enzymatic functions .
2.2 ER stress responses
The endoplasmic reticulum is one of the most dynamics organelles in cell machinery. It is the gateway for the synthesis of secretory proteins and contains the necessary apparatus to ensure quality protein synthesis, protein maturation, and secretion in eukaryotic cells . Furthermore, the ER can modulate some chronic stress-related pathways, promoting oxidative stress, autophagy, and apoptotic cell death in mammals and plant cells [11, 12, 13].
Several adverse environmental conditions can affect the ER quality control machinery, causing unfolded/misfolded protein accumulation in the ER lumen. The secretory proteins are synthesized in ER membrane-bound polysomes, and, as soon as they enter the organelle, they are processed by the ER processing machinery. Under normal conditions, there is a perfect balance between the rate of protein synthesis and ER processing capacity. Any conditions that disrupt this balance promote unfolded/misfolded protein accumulation in the ER lumen. As a consequence, the perturbation on ER function triggers a sophisticated and coordinated signal cascade, perceived by ER membrane-associated sensors, which activate the expression of ER-resident chaperones, foldases, and components of the ER quality control machinery. Collectively, these cytoprotective mechanisms are known as the unfolded protein response pathway (UPR, Figure 1) .
The detection of ER stress is mediated by membrane-associated sensors, identified both in mammals and plants. In mammals, there are three of these sensors: kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and protein kinase RNA-like ER kinase (PERK) , which are regulated by the ER-resident molecular chaperone BiP (binding protein). The ER sensors initiate the UPR to restore ER homeostasis under stress condition. If the adverse physiological status is prolonged, they can initiate some alternative routes leading to cell death.
Under normal conditions, BiP is bound to the luminal domain of these receptors, keeping them inactive. With the stress progression and consequent misfolded protein accumulation, the BiP molecular chaperone function is required to prevent aggregation of the unfolded proteins. Therefore, under these stress conditions, BiP is released from the ER receptors, which leads to their activation. The three ER signal transducers act in different ways, but in convergent stress-responsive pathways. IRE1 (IRE1a and IRE1b) displays a dual biochemical activity. It harbors a ribonuclease and kinase activity at the C-terminus, responsible for the unconventional spliceosome-independent splicing of X-box binding protein 1 (XBP1) mRNA. Stress-mediated BiP release from the IRE1 N-terminus promotes IRE1 homodimerization, which sequentially activates its kinase via autophosphorylation and endoribonuclease activity, culminating on spliceosome-independent splicing of XBP1, a bZIP transcriptional factor. Under normal conditions, the XBP1u (unspliced form) is constitutively translated into a low-functional transcription factor, which is rapidly degraded by the proteasome and does not effectively activate UPR. The IRE1-mediated mRNA splicing removes an unconventional intron of 26 nucleotides, which causes a shifting frame in XBP1 mRNA translation, generating a protein of 376 amino acids instead of 261 amino acids when unprocessed. This unconventional splicing seems to prevent the degradation of XBP1s (spliced form) product by the proteasome and increase its transactivation activity, causing activation of UPR-related genes [16, 17]. Thus, the XBP1s is a soluble and functional transcription factor, which is reallocated to the cell nucleus to activate genes involved in cytoprotective pathways, such as some members of ER quality control or programmed cell death-related genes, including the apoptotic signaling kinase 1 (ASK1) and Jun-N-terminal kinase (JNK) [16, 17, 18, 19].
The ER signal transducer ATF6 is anchored to the ER membrane and harbors an N-terminal sensor domain facing the ER lumen and a C-terminal bZIP domain facing the cytosolic side. Under normal conditions, ATF6 is inactivated by BiP binding to the ER stress sensor domain. ER stress conditions promote the BiP disassociation and reallocation of ATF6 to the Golgi apparatus, where it is specifically processed by SP1 and SP2 proteolytic enzymes. The limited proteolysis of ATF6 transmembrane domain allows that the bZIP domain of ATF6 be directed to the nucleus, where it acts in concert with XBP1 to induce genes involved in ER protein processing, ER quality control, and ER-associated protein degradation (ERAD) pathway. Finally, the PERK activation upon BiP release by stress conditions promotes global translation suppression through the phosphorylation of the translation initiation factor IF2α . PERK also activates the transcription factor CHOP, involved in the regulation of apoptosis-related genes [10, 21].
In plants, the UPR pathway has, at least, two arms (Figure 1). The first one activates IRE1 (IRE1a–AT2G17520 and IRE1b–AT5G24360, in
The second arm of plant UPR pathway is mediated by posttranslational modification of bZIP17 and bZIP28 transcription factors, the functional analogs of ATF6. Both bZIP17 and bZIP28 display a canonical SP1 site in their C-terminal domain, facing the ER lumen . Upon stress conditions, BIP is released from the bZIP28 and bZIP17 ER sensor domain, and the transcription factors are reallocated from the ER to the Golgi apparatus, where they are processed by SP1 and SP2 proteases. These proteases remove the transmembrane domain of bZIP17 and bZIP28, exposing their cytosolic regions, which will activate UPR-related genes in the nucleus [31, 32, 33, 34]. Like the IRE1/bZIP60 signaling module of plant UPR, the bZIP28/bZIP17 arm triggers the evolutionarily conservative UPR but also accommodates cross-talk with several other adaptive signaling responses [24, 30, 31]. In summary, upon ER stress, bZIP60s and bZIP28 use a different mechanism to be translocated to the nucleus where they act in concert to induce the expression of UPR genes and ERAD-related genes to increase the ER protein processing capacity for recovery from stress.
2.3 Convergence of ER stress and osmotic stress responses into a cell death signaling pathway
At a physiological level, the UPR encompasses three protective mechanisms: (i) global translation suppression by PERK-mediated IF2α phosphorylation; (ii) upregulation of ER-resident molecular chaperones, and (iii) proteasome-mediated protein degradation by ERAD pathway. However, if the stress conditions are sustained and the UPR pathway fails to restore ER homeostasis, apoptotic pathways are triggered as an ultimate attempt to survive. In plants, there is a specific branch of ER stress that integrates the osmotic stress and leads to programmed cell death (PCD), the development and cell death domain-containing N-rich protein (DCD/NRP)-mediated cell death signaling (Figure 1) . This cell death pathway was first identified via genome-wide and expression profiling approaches, which revealed a modest overlapping between ER and osmotic stress-induced transcriptomes of soybean seedlings treated with PEG (an osmotic stress inducer) and tunicamycin and AZC (ER stress inducers). Several genes displayed similar kinetics and a synergistic induction under combined ER and osmotic stresses, indicating that the ER stress response integrates the osmotic signal to potentiate transcription of shared target genes. Among them, two plant-specific DCD/N-rich proteins, NRP-A and NRP-B, an ubiquitin-associated protein homolog (UBA), and a NAC domain-containing protein, GmNAC81, displayed the most robust synergistic upregulation by the combination of both stresses . Transient expression of NRPs or GmNAC81 in soybean protoplasts and
The NRP-A and NRP-B display a highly conserved DCD domain at their C-terminal protein region and a high number of asparagine residues at their more divergent N-terminus (Figure 2) . Consistent with the presence of a DCD domain, overexpression of
Similar to NRPs, GmNAC81 (
3. Early dehydration responsive gene 15, ERD15-like, controls NRP expression
The early dehydration responsive (
Consistent with the multiple stress-responsive expression profiles, the soybean
4. The stress-induced NRP/NAC081/VPE module transduces a cell death signal
As components of the DCD/NPR-mediated cell death signaling, NRPs and GmNAC81 are critical mediators of cell death derived from ER stress and osmotic stress signals. More recent progress toward deciphering this branch of stress-induced cell death signaling includes the identification of two additional downstream components, the NAC transcriptional factor (GmNAC30) and the vacuolar processing enzyme (VPE) .
GmNAC30 was identified as a nuclear partner of GmNAC81 via two-hybrid screening using GmNAC81 as a bait.
GmNAC30 interacts with GmNAC81 in vitro and in vivo, the complex formed binds to common cis-regulatory sequences in target promoters and synergistically regulates hydrolytic enzyme promoters, including the caspase-1-like vacuolar processing enzyme (
VPE is a vacuole-localized cysteine protease that exhibits caspase-1-like activity and hydrolyzes a peptide bond at the C-terminal side of aspartate and asparagine residues . It is synthesized as an inactive preprotein precursor, which is self-catalytically converted into the active mature form, under a processing step that resembles the activation of caspase 1 (Figure 2). It has been associated with
The discovery of VPE as a downstream target of the coordinate action of GmNAC81 and GmNAC30 underlies a mechanism for the execution of the ER and osmotic stress-induced cell death program (Figure 1). This model holds that prolonged ER and osmotic stresses induce the expression of the transcriptional activator GmERD15 to target the NRP promoter. The upregulation of NRPs initiates a transduction signaling that leads to the induction of GmNAC81 and GmNAC30, which cooperate to activate the VPE promoter and expression. Activation of VPE promotes the disintegration of vacuoles, initiating the proteolytic cascade in plant PCD. As vacuole-triggered PCD is unique to plants, the regulatory circuit linking the stress signal to activation of VPE is fundamentally composed of plant-specific signaling components.
The DCD/NRP-mediated programmed cell death pathway is conserved and operates with similar regulatory mechanisms in plants . Soybean prototypes of each component of the cell death pathway were used to search for orthologs in the
5. A negative regulator of the NRP/NAC081/VPE signaling module confers tolerance to drought
Plants can negatively modulate the NRP/DCD-mediated cell death response to suit the cellular balance during the stress conditions. Moreover, this modulation improves the cellular stableness and consequently increases the plant tolerance to stress conditions in an essential process that is required for plant acclimatization and development. The molecular chaperone BiP plays a crucial role as a negative regulator of NRP/DCD-mediated cell death response. BiP belongs to the HSP70 family, which is essential to protect the cells against environmental stresses and to restore the cell homeostasis .
The molecular chaperone BiP has a catalytic site at the amino-terminal region and a substrate-binding site at the carboxy-terminal region . BiP is involved in the regulation of several processes in the endoplasmic reticulum, a critical organelle that is related to responses to abiotic and biotic stress in plants. In the ER, BiP acts as a sensor that responds to quantitative and qualitative changes in the ER by regulating the activity of ER stress transducers . Furthermore, BiP coordinately regulates the cell death signaling, which connects the signals from osmotic and ER stress in a DCD/NRP-dependent manner [35, 36, 38].
BiP attenuates the NRP/DCD-mediated cell death signal propagation by the modulation of expression and activity of the pathway signaling components (Figure 3). BiP overexpression in soybean attenuates ER stress- and osmotic stress-mediated cell death, a phenotype that is linked to a delay in the induction of
In addition to alleviating ER and osmotic stress-mediated cell death, the
6. The stress-induced DCD/NRP-mediated cell death signaling positively regulates leaf senescence
Leaf senescence is a natural process in plant development, which begins with a physiological transition between active photosynthetic leaves to degenerative and nutrient-recycling leaves. The classical age senescence-related symptom is the leaf yellowing caused by generalized chlorophyll loss. The age-induced senescence or naturally programmed leaf senescence, hereafter referred to as leaf senescence, occurs by plant aging and is precisely regulated by senescence-associated genes (SAGs) [66, 67].
Many SAGs are environmental- and stress-responsive genes, integrating a convergent regulatory cascade between natural plant development and stress-induced PCD . At the molecular level, the onset of senescence is accompanied by a massive reprogramming of gene expression, probably controlled by senescence-associated transcription factors. Among these, several NAC transcription factors have been associated with senescence regulation based on high-resolution temporal expression profiles .
In soybean, a transcriptomic analysis of senescing leaves reveals that 44% of the
Regarding the first hypothesis, several lines of evidence indicate that the regulatory circuit NRPs/GmNAC81:GmNAC30/VPE integrates osmotic stress- and ER stress-induced PCD response with natural leaf senescence. First, not only
Since the discovery of the ER stress- and osmotic stress-induced DCD/NRP-mediated cell death response, considerable progress has been achieved toward deciphering the components and regulation of the pathway (Figure 3). We now know that the combination of multiple stresses synergistically activates a plant-specific PCD response that is initiated by induction of the stress-responsive transcription factor GmERD15, which, in turn, binds and activates the DCD/NRP promoter. Induction of the DCD/NRP genes
We thank the Brazilian government agencies, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), for financial support.
Conflict of interest
The authors declare no conflict of interest.