**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 stressinduced cell death signaling includes the identification of two additional downstream components, the NAC transcriptional factor (GmNAC30) and the vacuolar processing enzyme (VPE) [55].

GmNAC30 was identified as a nuclear partner of GmNAC81 via two-hybrid screening using GmNAC81 as a bait. *GmNAC30* and *GmNAC81* exhibit similar expression profiles and cell death activity. They are upregulated by ER stress, osmotic stress, and by the cell death-inducer cycloheximide. Consistently, GmNAC30 promotes cell death when transiently expressed in soybean protoplasts and, as a downstream component of the cell death signaling, is induced by expression of NRP-A and NRP-B.

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*) gene, which is involved in PCD in plants [55]. Consistent with their transcriptional function as a heterodimer, *GmNAC81* and *GmNAC30* display overlapping and coordinate expression profiles in response to multiple environmental and developmental stimuli. Therefore, the stress-induced *GmNAC30* cooperates with *GmNAC81* to activate PCD through the upregulation of the cell death executioner VPE.

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 [56]. It is synthesized as an inactive preprotein precursor, which is selfcatalytically converted into the active mature form, under a processing step that resembles the activation of caspase 1 (**Figure 2**). It has been associated with *Tobacco mosaic virus*-induced hypersensitive cell death and developmental PCD [57, 58]. As an executioner of a cell death program, VPE is self-activated by hydrolytic cleavage and, in turn, mediates the initial activation of vacuolar enzymes, which degrade the vacuolar membrane and initiate the proteolytic cascade leading to PCD. Therefore, VPE activation may result in vacuolar collapse-mediated cell death, a type of plantspecific programmed cell death.

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

**69**

**Figure 3.**

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

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

The DCD/NRP-mediated programmed cell death pathway is conserved and operates with similar regulatory mechanisms in plants [40]. Soybean prototypes of each component of the cell death pathway were used to search for orthologs in the *Arabidopsis* genome (**Figure 3**) [30]. Arabidopsis AtNRP1 is most closely related to GmNRP-A and GmNRP-B, whereas a third homolog GmNRP-C was

*Integration of developmental signal and stress signals into the DCD/NRP-mediated cell death response. Leaf senescence, ER stress, and osmotic stress induce the expression of ERD15-regulated NRP-A that in turn upregulates NRP-B to initiate a signaling cascade that culminates with the induction of* GmNAC30 *and*  GmNAC81 *expression. The NAC transcription factors form a heterodimer to fully induce the activation of* VPE *promoter, which leads to* VPE *upregulation and subsequent execution of a cell death program. The ER-resident molecular chaperone BiP acts as a negative regulator of cell death by modulating the expression and activity of the cell death pathway components. The DCD/NRP-mediated cell death signaling is conserved in other plant* 

*species, and the* Arabidopsis *orthologs are shown on the right.*

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

signaling components.

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

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 [40]. Soybean prototypes of each component of the cell death pathway were used to search for orthologs in the *Arabidopsis* genome (**Figure 3**) [30]. Arabidopsis AtNRP1 is most closely related to GmNRP-A and GmNRP-B, whereas a third homolog GmNRP-C was

#### **Figure 3.**

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

stranded and double-stranded configurations [53].

**signal**

processing enzyme (VPE) [55].

sion of NRP-A and NRP-B.

death executioner VPE.

specific programmed cell death.

was identified using one hybrid screening that targeted the NRP-B promoter in yeast. As an upstream member of the NRP-mediated cell death response, GmERD15 binds the *NRP-B* promoter region in vivo and in vitro and induces the *NRP-B* expression [53]. Despite its role as a transcription factor, GmERD15 does not harbor a typical DNA-binding motif, but instead, it contains a conserved sequence of 13 amino acids at positions 71–83 (DEDEKERKEgKEv), which is a part of a tripartite motif domain derived from ssDNA-binding transcriptional regulators [54]. Accordingly, the GmERD15 binding site was mapped to a 12-bp palindromic sequence <sup>−</sup>511AGCAnnnnnTGCT−500 on the *NRP-B* promotor in both single-

**4. The stress-induced NRP/NAC081/VPE module transduces a cell death** 

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 stressinduced cell death signaling includes the identification of two additional downstream components, the NAC transcriptional factor (GmNAC30) and the vacuolar

GmNAC30 was identified as a nuclear partner of GmNAC81 via two-hybrid screening using GmNAC81 as a bait. *GmNAC30* and *GmNAC81* exhibit similar expression profiles and cell death activity. They are upregulated by ER stress, osmotic stress, and by the cell death-inducer cycloheximide. Consistently,

GmNAC30 promotes cell death when transiently expressed in soybean protoplasts and, as a downstream component of the cell death signaling, is induced by expres-

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*) gene, which is involved in PCD in plants [55]. Consistent with their transcriptional function as a heterodimer, *GmNAC81* and *GmNAC30* display overlapping and coordinate expression profiles in response to multiple environmental and developmental stimuli. Therefore, the stress-induced *GmNAC30* cooperates with *GmNAC81* to activate PCD through the upregulation of the cell

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 [56]. It is synthesized as an inactive preprotein precursor, which is selfcatalytically converted into the active mature form, under a processing step that resembles the activation of caspase 1 (**Figure 2**). It has been associated with *Tobacco mosaic virus*-induced hypersensitive cell death and developmental PCD [57, 58]. As an executioner of a cell death program, VPE is self-activated by hydrolytic cleavage and, in turn, mediates the initial activation of vacuolar enzymes, which degrade the vacuolar membrane and initiate the proteolytic cascade leading to PCD. Therefore, VPE activation may result in vacuolar collapse-mediated cell death, a type of plant-

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

**68**

*Integration of developmental signal and stress signals into the DCD/NRP-mediated cell death response. Leaf senescence, ER stress, and osmotic stress induce the expression of ERD15-regulated NRP-A that in turn upregulates NRP-B to initiate a signaling cascade that culminates with the induction of* GmNAC30 *and*  GmNAC81 *expression. The NAC transcription factors form a heterodimer to fully induce the activation of* VPE *promoter, which leads to* VPE *upregulation and subsequent execution of a cell death program. The ER-resident molecular chaperone BiP acts as a negative regulator of cell death by modulating the expression and activity of the cell death pathway components. The DCD/NRP-mediated cell death signaling is conserved in other plant species, and the* Arabidopsis *orthologs are shown on the right.*

related to AtNRP-2. GmNAC81 and its paralog share sequence conservation with the *Arabidopsis* ortholog ANAC36 (At2G17040), whereas the predicted *Arabidopsis* ortholog of soybean VPE was identified as At4G32940/γVPE. Transient expression of the selected *Arabidopsis* orthologs of pathway components (*AtNRP-1*, *AtNRP-2*, *ANAC36*, and *γVPE)* induces cell death in *Nicotiana benthamiana* leaves with the appearance of hallmarks of PCD and leaf senescence, including DNA fragmentation, leaf yellowing, chlorophyll loss, and lipid peroxidation [38]. In addition, knockout lines for each one of pathway genes in *Arabidopsis* display enhanced tolerance to ER stress-mediated cell death induction. Very importantly, the stress induction of *AtNRP2*, *ANAC36*, and *γVPE* was dependent on the AtNRP1 function, confirming the upstream position of AtNRP1 in the cell death pathway. Therefore, in *Arabidopsis*, the execution of the cell death program has been proposed to occur through AtNRP1-mediated induction of the AtNRP2-ANAC36-γVPE signaling module. Nevertheless, functional information about the GmERD15 and GmNAC30 orthologs in *Arabidopsis* is lacking, and these pathway components have not been identified yet in *Arabidopsis*. Both in soybean and *Arabidopsis*, the DCD/NRPmediated cell death pathway is modulated by the ER-resident molecular chaperone BiP, which negatively regulates the gene expression and activity of these cell deathinducing genes [13, 40].
