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

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 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 transgenic lines [52].

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

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

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

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

*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.*

cell death in plants [36–38].

**66**

**Figure 2.**

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 singlestranded and double-stranded configurations [53].
