**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 [1]. 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 [2].

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 [5]. 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 [8]. 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,

**63**

*A Regulatory Circuit Integrating Stress-Induced with Natural Leaf Senescence*

messengers can alter protein and enzymatic functions [9].

unfolded protein response pathway (UPR, **Figure 1**) [14].

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

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 [10]. Furthermore, the ER can modulate some chronic stress-related pathways, promoting oxidative stress, autophagy, and apoptotic cell

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

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) [15], 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

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.

*DOI: http://dx.doi.org/10.5772/intechopen.89498*

death in mammals and plant cells [11–13].

**2.2 ER stress responses**

leading to cell death.

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 [9].

### **2.2 ER stress responses**

*Plant Science - Structure, Anatomy and Physiology in Plants Cultured in Vivo and in Vitro*

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) domaincontaining asparagine-rich proteins (NRP-A and NRP-B) were the first ones to be characterized as cell death-promoting proteins, and hence this multiple stressintegrating 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 stressinduced and natural leaf senescence, constitutes the primary focus of this chapter.

**2. Modest overlapping of ER stress and osmotic stress response identifies** 

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 [1]. 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

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

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 [5]. 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 [8]. 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,

**NRPs and NACs as cell death-promoting genes**

**2.1 Osmotic stress responses**

types of abiotic stresses [2].

convergence points between them.

**62**

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 [10]. Furthermore, the ER can modulate some chronic stress-related pathways, promoting oxidative stress, autophagy, and apoptotic cell death in mammals and plant cells [11–13].

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**) [14].

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) [15], 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.

#### **Figure 1.**

*The endoplasmic reticulum stress response in* Arabidopsis*. The secretory proteins are synthesized in ER-bound polysomes (1) attached to the ER membrane through the interaction of signal recognition particle (SRP) and membrane receptor. As soon as they enter the lumen of the organelle, they are bound to a series of molecular chaperones, including BiP, to assist correct folding (2). Upon ER stress, the accumulation of unfolded protein (UP) activates a protective signaling cascade, designated as unfolded protein response, which allows communication of ER with the nucleus via a bipartite signaling module: the bZIP28/bZIP17 and IRE1a/IRE1bbZiP60 signaling modules. Under normal conditions, BiP is bound to bZIP28/17, keeping the transducer in an inactive configuration (4). Upon ER stress, UP causes the dissociation of BiP from bZIP28/bZIP17, which is, then, translocated to the Golgi (5), where it is proteolytically cleaved to release the bZIP28/bZIP17 domain from the membrane that, in turn, is translocated to the nucleus (6). UP accumulation also causes the oligomerization of IRE1a/IRE1b, subsequent activation of its kinase domain by phosphorylation, and the endonuclease activity (6). The activated IRE1a/IRE1b endonuclease domain promotes unconventional splicing of bZIP60 mRNA to remove a transmembrane motif-encoding fragment, generating bZIP60 spliced mRNA that is translated into a soluble bZIP60 protein (bZIP60s) (7), which otherwise would be translated into the membrane-associated bZIP60us as it occurs under normal conditions. bZIP60s is, then, translocated to the nucleus (8), where it cooperates with bZIP28/bZIP17 to upregulate UPR genes and ERAD-related genes, increasing the ER protein processing capacity under ER stress to promote recovery (9). However, if the stress persists, and ER homeostasis cannot be restored, cell death signaling pathways are activated. among them, the DCD/NRP-mediated cell death signaling is initiated with activation of AtNRP1 (10) that leads to the induction of AtNRP2 and activation of a signaling cascade that culminates with the induction of ANAC36 that binds to the* VPE *promoter (11) and induces the expression of* VPE*, the executioner of the cell death program via collapse of the vacuole. These ER stress signaling pathways are conserved in other plant species.*

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) [16–19].

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.

**65**

from stress.

**signaling pathway**

*A Regulatory Circuit Integrating Stress-Induced with Natural Leaf Senescence*

Finally, the PERK activation upon BiP release by stress conditions promotes global translation suppression through the phosphorylation of the translation initiation factor IF2α [20]. PERK also activates the transcription factor CHOP, involved in the

In plants, the UPR pathway has, at least, two arms (**Figure 1**). The first one activates IRE1 (IRE1a–AT2G17520 and IRE1b–AT5G24360, in *Arabidopsis thaliana*), and the other is transduced through bZIP membrane-associated transcription factors (bZIP17–AT2G40950 and bZIP28–AT3G10800, in *Arabidopsis thaliana*) [22, 23]. In the first arm of plant UPR, like in mammals, the accumulation of misfolded proteins leads to the activation of IRE1, which promotes unconventional cytosolic splicing of bZIP60 mRNA [24]. The unspliced bZIP60 mRNA, called bZIP60us, is translated into an ER membrane-associated transcription factor and does not exhibit transcriptional activity. Upon IRE1 activation by UPR, the spliced bZIP60 mRNA, called bZIP60s, does not display the transmembrane domain coding region, and its translation generates an active transcription factor, which is reallocated to the nucleus to activate UPR and cytoprotective genes, such as *BiP3*, *CNX* (calnexin), *CRT* (calreticulin), etc. [24–26]. This mechanism is conserved among plants, as the rice (*Oryza sativa*) bZIP60 orthologs, OsbZIP74 or OsbZIP50, display similar IRE-mediated mRNA splicing to render the activation of ER stressinducible promoters [27, 28]. Likewise, in maize (*Zea mays*), ZmbZIP60 mRNA splicing leads to the activation of ER stress-inducible promoters [29], and, in soybean (*Glycine max*), the ZIP60 ortholog GmbZIP68 harbors a canonical site for IRE1 endonuclease activity and is efficiently spliced under ER stress conditions to

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 [31]. 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 [31–34]. 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

**2.3 Convergence of ER stress and osmotic stress responses into a cell death** 

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**) [12]. This cell death pathway was

*DOI: http://dx.doi.org/10.5772/intechopen.89498*

regulation of apoptosis-related genes [10, 21].

activate UPR genes [30].

*A Regulatory Circuit Integrating Stress-Induced with Natural Leaf Senescence DOI: http://dx.doi.org/10.5772/intechopen.89498*

Finally, the PERK activation upon BiP release by stress conditions promotes global translation suppression through the phosphorylation of the translation initiation factor IF2α [20]. 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 *Arabidopsis thaliana*), and the other is transduced through bZIP membrane-associated transcription factors (bZIP17–AT2G40950 and bZIP28–AT3G10800, in *Arabidopsis thaliana*) [22, 23]. In the first arm of plant UPR, like in mammals, the accumulation of misfolded proteins leads to the activation of IRE1, which promotes unconventional cytosolic splicing of bZIP60 mRNA [24]. The unspliced bZIP60 mRNA, called bZIP60us, is translated into an ER membrane-associated transcription factor and does not exhibit transcriptional activity. Upon IRE1 activation by UPR, the spliced bZIP60 mRNA, called bZIP60s, does not display the transmembrane domain coding region, and its translation generates an active transcription factor, which is reallocated to the nucleus to activate UPR and cytoprotective genes, such as *BiP3*, *CNX* (calnexin), *CRT* (calreticulin), etc. [24–26]. This mechanism is conserved among plants, as the rice (*Oryza sativa*) bZIP60 orthologs, OsbZIP74 or OsbZIP50, display similar IRE-mediated mRNA splicing to render the activation of ER stressinducible promoters [27, 28]. Likewise, in maize (*Zea mays*), ZmbZIP60 mRNA splicing leads to the activation of ER stress-inducible promoters [29], and, in soybean (*Glycine max*), the ZIP60 ortholog GmbZIP68 harbors a canonical site for IRE1 endonuclease activity and is efficiently spliced under ER stress conditions to activate UPR genes [30].

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 [31]. 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 [31–34]. 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**) [12]. This cell death pathway was

*Plant Science - Structure, Anatomy and Physiology in Plants Cultured in Vivo and in Vitro*

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

*The endoplasmic reticulum stress response in* Arabidopsis*. The secretory proteins are synthesized in ER-bound polysomes (1) attached to the ER membrane through the interaction of signal recognition particle (SRP) and membrane receptor. As soon as they enter the lumen of the organelle, they are bound to a series of molecular chaperones, including BiP, to assist correct folding (2). Upon ER stress, the accumulation of unfolded protein (UP) activates a protective signaling cascade, designated as unfolded protein response, which allows communication of ER with the nucleus via a bipartite signaling module: the bZIP28/bZIP17 and IRE1a/IRE1bbZiP60 signaling modules. Under normal conditions, BiP is bound to bZIP28/17, keeping the transducer in an inactive configuration (4). Upon ER stress, UP causes the dissociation of BiP from bZIP28/bZIP17, which is, then, translocated to the Golgi (5), where it is proteolytically cleaved to release the bZIP28/bZIP17 domain from the membrane that, in turn, is translocated to the nucleus (6). UP accumulation also causes the oligomerization of IRE1a/IRE1b, subsequent activation of its kinase domain by phosphorylation, and the endonuclease activity (6). The activated IRE1a/IRE1b endonuclease domain promotes unconventional splicing of bZIP60 mRNA to remove a transmembrane motif-encoding fragment, generating bZIP60 spliced mRNA that is translated into a soluble bZIP60 protein (bZIP60s) (7), which otherwise would be translated into the membrane-associated bZIP60us as it occurs under normal conditions. bZIP60s is, then, translocated to the nucleus (8), where it cooperates with bZIP28/bZIP17 to upregulate UPR genes and ERAD-related genes, increasing the ER protein processing capacity under ER stress to promote recovery (9). However, if the stress persists, and ER homeostasis cannot be restored, cell death signaling pathways are activated. among them, the DCD/NRP-mediated cell death signaling is initiated with activation of AtNRP1 (10) that leads to the induction of AtNRP2 and activation of a signaling cascade that culminates with the induction of ANAC36 that binds to the* VPE *promoter (11) and induces the expression of* VPE*, the executioner of the cell death program via collapse of the* 

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.

(ASK1) and Jun-N-terminal kinase (JNK) [16–19].

*vacuole. These ER stress signaling pathways are conserved in other plant species.*

**64**

**Figure 1.**

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 [35]. Transient expression of NRPs or GmNAC81 in soybean protoplasts and *Nicotiana benthamiana* leaves demonstrated that they are critical mediators of ER stress- and osmotic stress-induced cell death in plants [36–38].

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**) [39]. Consistent with the presence of a DCD domain, overexpression of *NRPs* in soybean protoplasts induces caspase-3-like activity and promotes extensive DNA fragmentation. Furthermore, transient

#### **Figure 2.**

*Schematic representation of the cell death pathway components. The predicted domains of each protein are highlighted. The indicated domains are delimited by the amino acid positions in the primary structure shown by the numbers. For ERD-15, PAM2 is a PABP-interacting motif, PAE2 is PAM2-associated element 1 motif, DEDEKERKEGKEV is a conserved sequence representing a putative motif of ssDNA-binding transcriptional regulators, and QPR is a highly conserved C-terminal QPR motif. As for GmNAC81, GmNAC30, and ANAC36, the N-terminal NAC domain is subdivided into five conserved motifs (A to E) as indicated. In the AtNRP1, AtNRP2, NRP-A, and NRP-B schemes, DCD is development and cell death domain.*

**67**

*A Regulatory Circuit Integrating Stress-Induced with Natural Leaf Senescence*

referred to as the DCD/NRP-mediated cell death response.

the DCD/NRP-mediated cell death signaling [40].

expression of NRPs *in planta* causes leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker genes, which are hallmarks of leaf senescence and cell death [36, 38, 40]. The cell death response mediated by NRPs resembles a programmed cell death event. Because NRPs were the first components of the ER stress and osmotic stress-integrating cell death response to be characterized, this signaling pathway is commonly

Similar to NRPs, GmNAC81 (*Glycine max* NAC81, formerly designated as GmNAC6) is another target of the ER stress- and osmotic stress-integrating pathway that induces a senescence-like response *in planta* and cell death in soybean protoplasts [37, 41]. GmNAC81 belongs to the plant-specific transcriptional factor superfamily of domain-containing proteins, represented by 111 members in *Arabidopsis*, 151 in rice, 152 in maize, and 180 in soybean [42, 43]. Members of this family function in development and stress response. The NAC transcriptional factors display a highly conserved N-terminal domain, called NAC domain, responsible for recognition of cis-regulatory elements on target promoters and DNA binding (**Figure 2**). The C-terminal domain is more divergent in sequence but is undoubtedly responsible for transcriptional activity [44, 45]. In addition, a subset of NAC proteins, which also exhibits protein binding activity, harbors an additional transmembrane domain present in the membrane-tethered NAC

*NRPs* and *GmNAC81* are induced by several different abiotic and biotic stresses

in a coordinated manner, but induction of *NRPs* precedes the upregulation of *GmNAC81*. This early induction kinetics of *NRPs* is consistent with its capacity to activate the promoter and induce the expression of *GmNAC81*. These data placed GmNAC81 downstream of NRPs in the ER and osmotic stress-induced cell death pathway [37]. More recently, using reverse genetics in *Arabidopsis*, NRPs were confirmed to be upstream of ANAC36, the *Arabidopsis* ortholog of GmNAC81, in

**3. Early dehydration responsive gene 15, ERD15-like, controls NRP** 

function, designated as PAM2-associated element 1 (PAE1) and QPR.

*ERD15* is a multiple stress-responsive gene that is involved in adaptation to abiotic and biotic stress. Light treatment, cold stress, and high salinity trigger *ERD15* expression [50, 51]. ERD15 functions as a negative regulator of the abscisic acid (ABA)-mediated response and a positive regulator of the salicylic acid (SA) dependent defense pathway. *ERD15*-overexpressing transgenic lines are less sensitive to ABA and display enhanced salicylic acid-dependent defense pathway, which was associated with increased resistance to the bacterial *Erwinia carotovora* of the

Consistent with the multiple stress-responsive expression profiles, the soybean *ERD15* ortholog (*GmERD15*) is also induced by ER and osmotic stress. *GmERD15*

The early dehydration responsive (*ERD*) genes were first identified due to their rapid induction in response to drought stress. The ERD genes (*ERD1* to *ERD16*) encode a set of proteins that differ in biological functions and cell localization [48]. Among them, ERD15 is a small acidic and hydrophilic protein that belongs to the PAM2 domain-containing protein family (**Figure 2**). The PAM2 domain is a wellcharacterized protein–protein interaction domain, which allows ERD15 to interact with polyA-binding proteins (PABP) regulating mRNA stability and protein translation [49]. In addition to PAM2, ERD15 contains two other domains with unknown

*DOI: http://dx.doi.org/10.5772/intechopen.89498*

proteins [43, 46, 47].

**expression**

transgenic lines [52].

#### *A Regulatory Circuit Integrating Stress-Induced with Natural Leaf Senescence DOI: http://dx.doi.org/10.5772/intechopen.89498*

expression of NRPs *in planta* causes leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker genes, which are hallmarks of leaf senescence and cell death [36, 38, 40]. The cell death response mediated by NRPs resembles a programmed cell death event. Because NRPs were the first components of the ER stress and osmotic stress-integrating cell death response to be characterized, this signaling pathway is commonly referred to as the DCD/NRP-mediated cell death response.

Similar to NRPs, GmNAC81 (*Glycine max* NAC81, formerly designated as GmNAC6) is another target of the ER stress- and osmotic stress-integrating pathway that induces a senescence-like response *in planta* and cell death in soybean protoplasts [37, 41]. GmNAC81 belongs to the plant-specific transcriptional factor superfamily of domain-containing proteins, represented by 111 members in *Arabidopsis*, 151 in rice, 152 in maize, and 180 in soybean [42, 43]. Members of this family function in development and stress response. The NAC transcriptional factors display a highly conserved N-terminal domain, called NAC domain, responsible for recognition of cis-regulatory elements on target promoters and DNA binding (**Figure 2**). The C-terminal domain is more divergent in sequence but is undoubtedly responsible for transcriptional activity [44, 45]. In addition, a subset of NAC proteins, which also exhibits protein binding activity, harbors an additional transmembrane domain present in the membrane-tethered NAC proteins [43, 46, 47].

*NRPs* and *GmNAC81* are induced by several different abiotic and biotic stresses in a coordinated manner, but induction of *NRPs* precedes the upregulation of *GmNAC81*. This early induction kinetics of *NRPs* is consistent with its capacity to activate the promoter and induce the expression of *GmNAC81*. These data placed GmNAC81 downstream of NRPs in the ER and osmotic stress-induced cell death pathway [37]. More recently, using reverse genetics in *Arabidopsis*, NRPs were confirmed to be upstream of ANAC36, the *Arabidopsis* ortholog of GmNAC81, in the DCD/NRP-mediated cell death signaling [40].
