**3. The ER-stress-induced NRP-mediated cell death response**

NRP-mediated cell death signaling is a distinct, plant-specific branch of the ER stress path‐ way that has been uncovered in soybean and has been shown to integrate the ER and osmot‐ ic stress signals into a full response. This integrative pathway was first identified through genome-wide approaches and expression profiling, which revealed the existence of a mod‐ est overlap of the ER and osmotic stress-induced transcriptomes in soybean seedlings treat‐ ed with PEG (an inducer of osmotic stress) or tunicamycin and AZC (potent inducers of ER stress; Irsigler et al., 2007). The co-regulated genes were first considered to be downstream targets of the integrated pathway based on similar induction kinetics and a synergistic re‐ sponse to the combination of osmotic and ER stress-inducing treatments. Based on these cri‐

teria, the selected downstream components of this ER and osmotic stress responseintegrating pathway encode proteins with diverse roles, such as plant-specific development and cell death (DCD) domain-containing proteins (NRP-A and NRP-B), an ubiquitin-associ‐ ated (UBA) protein homolog and NAC domain-containing proteins (GmNAC6). Among them, NRP-A and NRP-B were the first ones to be characterized and to show to induce a cell death response when ectopically expressed in tobacco leaves or soybean protoplasts (Costa et al., 2008). As a consequence, the ER and osmotic stress response-integrating pathway has been designated as the NRP-mediated cell death response.

An upstream component of the NRP-mediated cell death response, GmERD15 (*Glycine max* Early Responsive to Dehydration 15), has been recently identified using one-hybrid screen‐ ing that targeted the NRP-B promoter in yeast (Alves et al., 2011). GmERD15 is induced by ER and osmotic stress to activate the expression of *NRP* genes (NRP-A and NRP-B). Up-reg‐ ulation of NRP-B leads to the induction of an NAC domain-containing protein, GmNAC6, which is a critical mediator of stress-induced cell death in plants (Faria et al., 2011). These components of the ER stress-induced NRP-mediated cell death signaling pathway, GmERD15, NRPs and GmNAC6, have been further characterized.

## **3.1. GmERD15 is a ssDNA binding transcriptional activator**

The Early Responsive Dehydration (ERD) genes are rapidly induced in response to water deficit and form a family comprised by ERD1 to ERD16 representatives. The ERD encoded proteins exhibit diverse and heterogeneous biochemical functions and fall into different classes of proteins, such as chloroplast ATP-dependent protease (ERD1), cytosolic HSP70 (ERD2), glutationa-S-transferases (ERD9,ERD11, ERD13) among others (Soitano et al., 2008; Kiyosue et al., 1994; Kiyosue et al., 1993). ERD15 was first identified in Arabidopsis as a hy‐ drophilic protein that possesses a PAM2 domain that interacts with polyA-binding proteins (PABP11; Kiyosue et al., 1994; Kariola et al., 2006)., ERD15 has been shown to function as a negative regulator of the abcisic acid (ABA)-mediated response (Kariola et al., 2006). Over‐ expression of ERD15 reduces the ABA sensitivity of Arabidopsis, whereas silencing of ERD15 by RNAi promotes hypersensitivity to the hormone. The negative effect of ERD15 on ABA signaling enhances salicylic acid-dependent defense because overexpression of ERD15 was associated with increased resistance to the bacterial necrotroph Erwiniacarotovora, and the enhanced induction of marker genes for systemic acquired resistance. These results are consistent with the observed antagonistic effect of ABA on salicylic acid-mediated defense and may implicate ERD15 as a shared component of these responses.

The soybean GmERD15 homolog has been described as a new ER stress- and osmotic stress-in‐ duced transcription factor that binds to the promoter and induces the expression of the NRP-B gene. In fact, GmERD15 was isolated by its capacity to associate stably with the promoter of NRP-B in yeast cells using the one-hybrid system (Alves et al., 2011). The GmERD15 binding site in the NRP-B promoter was mapped to a 12-bp palindromic sequence (511 AG‐ CAnnnnTGCT -500) that resembles binding sites for ssDNA binding proteins, such as NF1C and PBF2 that recognize the sequences -TTGGCnnnnnGCCAA-3' and 5- TGACAnnnnTGT‐ CA-3', respectively (Wang and Kiledjian., 2000). Furthermore, GmERD15 is located in the nu‐ cleus, and chromatin immunoprecipitation (ChIP) assays revealed that it binds to the NRP-B promoter in vivo (Alves et al., 2011). The ectopic expression of GmERD15 in soybean cells acti‐ vates the NRP-B promoter and induces NRP-B expression. Collectively, these results indicate that GmERD15 functions as an upstream component of the NRP-mediated cell death signal‐ ing pathway that is induced by ER stress and osmotic stress

### **3.2. NRPs: Molecular and functional characterization**

teria, the selected downstream components of this ER and osmotic stress responseintegrating pathway encode proteins with diverse roles, such as plant-specific development and cell death (DCD) domain-containing proteins (NRP-A and NRP-B), an ubiquitin-associ‐ ated (UBA) protein homolog and NAC domain-containing proteins (GmNAC6). Among them, NRP-A and NRP-B were the first ones to be characterized and to show to induce a cell death response when ectopically expressed in tobacco leaves or soybean protoplasts (Costa et al., 2008). As a consequence, the ER and osmotic stress response-integrating pathway has

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

An upstream component of the NRP-mediated cell death response, GmERD15 (*Glycine max* Early Responsive to Dehydration 15), has been recently identified using one-hybrid screen‐ ing that targeted the NRP-B promoter in yeast (Alves et al., 2011). GmERD15 is induced by ER and osmotic stress to activate the expression of *NRP* genes (NRP-A and NRP-B). Up-reg‐ ulation of NRP-B leads to the induction of an NAC domain-containing protein, GmNAC6, which is a critical mediator of stress-induced cell death in plants (Faria et al., 2011). These components of the ER stress-induced NRP-mediated cell death signaling pathway,

The Early Responsive Dehydration (ERD) genes are rapidly induced in response to water deficit and form a family comprised by ERD1 to ERD16 representatives. The ERD encoded proteins exhibit diverse and heterogeneous biochemical functions and fall into different classes of proteins, such as chloroplast ATP-dependent protease (ERD1), cytosolic HSP70 (ERD2), glutationa-S-transferases (ERD9,ERD11, ERD13) among others (Soitano et al., 2008; Kiyosue et al., 1994; Kiyosue et al., 1993). ERD15 was first identified in Arabidopsis as a hy‐ drophilic protein that possesses a PAM2 domain that interacts with polyA-binding proteins (PABP11; Kiyosue et al., 1994; Kariola et al., 2006)., ERD15 has been shown to function as a negative regulator of the abcisic acid (ABA)-mediated response (Kariola et al., 2006). Over‐ expression of ERD15 reduces the ABA sensitivity of Arabidopsis, whereas silencing of ERD15 by RNAi promotes hypersensitivity to the hormone. The negative effect of ERD15 on ABA signaling enhances salicylic acid-dependent defense because overexpression of ERD15 was associated with increased resistance to the bacterial necrotroph Erwiniacarotovora, and the enhanced induction of marker genes for systemic acquired resistance. These results are consistent with the observed antagonistic effect of ABA on salicylic acid-mediated defense

The soybean GmERD15 homolog has been described as a new ER stress- and osmotic stress-in‐ duced transcription factor that binds to the promoter and induces the expression of the NRP-B gene. In fact, GmERD15 was isolated by its capacity to associate stably with the promoter of NRP-B in yeast cells using the one-hybrid system (Alves et al., 2011). The GmERD15 binding site in the NRP-B promoter was mapped to a 12-bp palindromic sequence (511 AG‐ CAnnnnTGCT -500) that resembles binding sites for ssDNA binding proteins, such as NF1C and PBF2 that recognize the sequences -TTGGCnnnnnGCCAA-3' and 5- TGACAnnnnTGT‐ CA-3', respectively (Wang and Kiledjian., 2000). Furthermore, GmERD15 is located in the nu‐

been designated as the NRP-mediated cell death response.

Relationships

264

GmERD15, NRPs and GmNAC6, have been further characterized.

and may implicate ERD15 as a shared component of these responses.

**3.1. GmERD15 is a ssDNA binding transcriptional activator**

The N Rich Protein (NRP) gene was first identified by its rapid induction in response to pathogen incompatible interactions in soybean (Ludwig and Tenhaken, 2001). The NRP des‐ ignation was derived from its high content of asparagine residue, about 25 %. NRP is repre‐ sented in the soybean genome by a small family of three genes: NRP-A, NRP-B and NRP-C. The encoded proteins share a highly conserved development and cell death (DCD) domain at the C-terminal portion in addition to a high content of asparagine residues at their more divergent N termini. The asparagine rich domain is not well characterized but harbors puta‐ tive glycosylation and myristoylation sites that may be relevant for function. The DCD do‐ main is found exclusively in plant proteins and it is composed of about 130 amino acid residues, organized into several conserved motifs: FGLP and LFL in the N-treminal region of the domain, PAQV and PLxE at its C-terminus (Tenhaken et al; 2005). DCD domain-con‐ taining proteins may be subdivided into four groups, according to the localization of the DCD domain in the primary structure. NRPs belong to the the subgroup I of DCD domaincontaining family of proteins, as their domains are located at the C-terminal portion of the protein (Tenhaken et al; 2005).

NRPs are critical mediators of ER and osmotic stress-induced cell death in soybeans (Costa et al., 2008). The cell death response mediated by NRPs resembles a programmed cell death event. The overexpression of NRPs in soybean protoplasts induces caspase-3-like activity and promotes extensive DNA fragmentation. Furthermore, the transient expression of NRPs in plants causes leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolu‐ tion and the induction of senescence marker genes, which are hallmarks of leaf senescence.

NRPs are up-regulated by ER or osmotic stress but need both stress signals for full induction (Isrigler et al., 2007). This synergistic interaction of both signals upon NRP induction indi‐ cates that the ER stress and osmotic stress responses converge at the level of gene expression to potentiate a NRP-mediated cell death response (Costa et al., 2008). NRPs are also upregulated by other abiotic and biotic signals, such as salt stress, oxidative stress and patho‐ gens. Because the NRP-mediated cell death signaling pathway represents a shared response to multiple stress signals in plants, it might permit coordinate adaptive cellular responses under a large array of stress conditions

#### **3.3. GmNAC6 as a downstream component of the NRP-mediated cell death response**

NAC domain-containing proteins are plant-specific transcriptional factors that are ex‐ pressed in several tissues and developmental stages. The NAC transfactors are organized in‐ to a general structure that consists of a highly conserved N-terminal domain involved in

DNA binding (called NAC domain) and a C-terminal region highly divergent in sequence and length that functions as the activation domain. The NAC domain was derived from comparison of consensus sequences among NAM from Petunia, ATAF1/2 and CUC2 from Arabidopsis (Souer et al., 1996.). It comprises nearly 160 amino acid residues, divided into five subdomains (A–E) exhibiting a negative net charge and a nuclear localization signal (Xie et al., 1999; Seoet al., 2008). The subdomains A, C and D are conserved among plant species whereas B and E subdomains are variable (Ooka et al., 2003). The C- terminus har‐ bors a protein-protein interaction domain in some NAC-containing proteins while a trans‐ membrane domain is present in other transcriptional factors (Seo et al., 2008). Therefore, the NAC family is comprised by both soluble, nuclear transactivators and membrane proteins.

The members of the NAC gene family are involved in a variety of developmental events and defense responses, such as shoot apical meristem formation and maintenance (SAM; Aida et al., 1997; Souer et al.,1996; Weir et al., 2004), hormone signaling (Fujita et al., 2004; Xie et al., 2000), response to pathogen infection (Ren et al., 2000; Selth et al., 2005; Xie et al., 1999), leaf senescence (John et al., 1997) and response to different abiotic stresses (Hegedus et al., 2003; Tran et al.,2004).

The soybean NAC family is comprised by 180 putative sequences of NAC domain-contain‐ ing proteins, which display different expression profiles in response to distinct environmen‐ tal stress conditions and developmental signals (Mochida et al.,2009; Mochida et al., 2010; Wang et al., 2010). Frequently, the stress-induced expression profile of the soybean NAC genes reflects the functional profile of the encoded protein (Pinheiro et al., 2009). GmNAC6 was identified by its synergistic induction in response to a combined treatment of inducers of osmotic stress (polyethylene glycol) and ER stress (tunicamycin) and was functionally linked to the NRP-mediated cell death response (Faria et al., 2011). Transient expression of GmNAC6 promotes cell death and hypersensitive-like responses *in planta*. GmNAC6 and NRPs also share overlapping responses to biotic signals, but the induction of NRPs peaks before the increased accumulation of GmNAC6 transcripts. Consistent with the delayed ki‐ netics of GmNAC6 induction, increased levels of NRP-A and NRP-B transcripts induce pro‐ moter activation and the expression of the GmNAC6 gene. Therefore, GmNAC6 is biochemical and functionally linked to the ER stress- and osmotic stress-integrating cell death response, in which it acts downstream of the NRPs.

GmNAC6 encodes a 33kDa protein that belongs to the TERN (Tobacco elicitor-responsive gene encoding NAC domain protein) group of the NAC family, which is induced by elici‐ tors of the pathogen response (Ooka et al.,2003). Likewise, GmNAC6 is induced by the pathogenic bacteria *Pseudomonas syringaepatovar tomato* which elicits an incompatible interac‐ tion in soybean and by cell wall-degrading enzymes, which mimic bacterial pathogen attack. Like GmNAC6, the other components of the ER- and osmotic-stress induced cell death sig‐ naling pathway, GmERD15 and NRPs, are also induced by other biotic and abiotic signals, such drought and pathogen incompatible interactions. Therefore, the activation of the NRPmediated senescence-like response is not specific to ER stress or osmotic stress but is, rather, a shared branch of general environmental adaptive pathways.
