The PAM2 motif is the second of two PABC domain-binding motifs, PAM1 and PAM2, located in the C-terminus of poly(A)-binding proteins (PABPs), primarily involved in RNA metabolism (Albrecht and Lengauer, 2004). Initially identified in the human polyadenylate-binding protein-interacting protein 1 (Paip1) protein, PAM1 and PAM2 motifs are coactivators in translation initiation regulation for 3' polyadenylated mRNAs (Roy, 2002). The PAM2 motif is typically found in proteins associated with mRNA metabolism or involved in the formation of the scaffold for the assembly of the ribonucleoprotein (RNP) complex on the poly-A tail of mRNAs (Albrecht and Lengauer, 2004). Frequently, the PAM2 domain is linked to other accessory domains, including (i) RRMs (RNA recognition motifs), known forn binding single-stranded RNAs (Maris et al., 2005), (ii) LsmAD (Like Sm associated domains), recognized as an RNA Helicase binding motif (Jiménez and Guzmán, 2014), (iii) TPRs (tetratricopeptide repeats) forming scaffolds for protein interactions (D'Andrea and Regan, 2003), and (iv) the endonuclease Smr (Small MutS-related) domain, involved in nucleic acid mismatch repair (Moreira and Philippe, 1999). Despite extensive studies in the animal kingdom, a more comprehensive analysis of the PAM2 superfamily in the plant kingdom is still lacking. Among PAM2 superfamily members in plants, the PAM2 motif-containing protein ERD15 has emerged as an essential modulator of plant cell signaling (Kariola et al., 2006; Alves et al., 2011a, b).
The Arabidopsis ERD15 (Early responsive to dehydration 15) gene (AT2G41430) was initially identified by screening genes rapidly induced by dehydration stress. A 1 h dehydration treatment induced the ERD genes (ERD1 to ERD16), encoding proteins with different biological functions and cellular localization (Kiyosue et al., 1994). Members of the ERD15-like family are small, acidic proteins of 99 to 177 amino acids. This family is exclusive to the plant kingdom, with orthologs in photosynthetic organisms ranging from green algae (Zygnemophyceae) to higher plants (Embryophyta). The ERD15-like subfamily members share specific protein domains, including the adjacent N-terminal PAM2 (PABP-interacting motif 2) and PAE1 (PAM2 associated element 1) motifs, followed by an acidic region and a highly conserved C-terminal QPR motif (Aalto et al., 2012).
Despite being predominantly found in the C-terminal portion of animal and yeast proteins, the PAM2 motif is located in the N-terminal portion of the ERD15-like proteins. All members of the ERD15 subfamily share the highly conserved PAE1 motif, located adjacent to PAM2; however, its function remains unknown. The non-conserved acidic region is present in all family members and might be a remnant of the PAM1 motif found in the human Paip1 and Paip2 proteins (Aalto et al., 2012). The QPR motif, with a conserved amino acid sequence (isoleucine-glutamine/histidine-glutamine-proline-arginine), is present in most ERD15-like subfamily members but absent in two of the six Glycine max homologs, Glyma03G131900 and Glyma19G133800 (Aalto et al., 2012).
In addition to its rapid induction in response to dehydration, AtERD15 (AT2G41430) has been implicated as a negative modulator of drought tolerance. ERD15 overexpression decreases drought tolerance, while the RNAi silencing of ERD15 confers tolerance to drought (Kariola et al., 2006). Recently, the apple Zinc-finger protein (Malus × domestica) MdBBX7 has been shown to bind the promoter and activate the expression of MdERD15 under drought stress (Chen et al., 2022). Under drought stress, ERD15 is considerably induced in different species, including MiERD15 from Morus indica (Saeed and Khurana, 2017), IbERD15 from Ipomoea batatas (Shao et al., 2014), and SpERD15 from Solanum pennellii (Ziaf et al., 2011), implicating ERD15 as a drought stress marker gene. Furthermore, AtERD15 has been described as a negative regulator of the abscisic acid (ABA) response, controlling the mRNAs encoding proteins involved in the ABA core signaling and regulating stomatal closure (Aalto et al., 2012). ERD15 has also been reported to be induced by pathogen elicitors and positively modulates salicylic acid (SA)-dependent defense pathways (Brader et al., 2001; Kariola et al., 2006). The AtERD15-overexpressing lines display enhanced resistance to Erwinia carotovorum, associated with PR2 upregulation, an SA-mediated systemic acquired resistance (SAR) marker gene (Kariola et al., 2006).
The ERD15-like genes are classified as multiple stress-induced regulators due to their functional plasticity in response to a wide range of stresses. High salinity, cold, wounding, and hypergravity have been shown to stimulate ERD15 expression in Arabidopsis (Park et al., 2009; Kariola et al., 2006; Walley et al., 2007; Yoshioka et al., 2003). AtERD15 (Lsr1) is one of the light stress-responsive (Lsr1–5) genes highly induced by light stress (Dunaeva and Adamska, 2001). SpERD15 is induced by drought (dehydration), salinity, cold, ABA, gibberellic acid, and ethylene (Ziaf et al., 2011). In contrast to the reduced drought tolerance displayed by AtERD15-overexpressing lines, SpERD15 overexpression enhanced tolerance to dehydration, salinity, and cold (Ziaf et al., 2011). VaERD15 has been shown to improve cold tolerance in Chinese wild Vitis amurensis (Yu et al., 2017). More recently, BplERD15 from Betula platyphylla has also been demonstrated to counteract the AtERD15 negative role in drought tolerance and ABA signaling, functioning as a positive regulator of drought responses (Lv et al., 2020). Therefore, ERD15-like members from different plant species have been described as both positive and negative mediators of drought stress tolerance.
The soybean ERD15 ortholog (Glyma02G260800) is induced by endoplasmic reticulum stress (ER) and osmotic stress. It acts as an upstream member of the NRP-mediated cell death response, binding to the NRP-B promoter region in vivo and in vitro (Alves et al., 2011b). Although Glyma02G260800 does not harbor a typical DNA-binding motif, a conserved sequence of 13 amino acids (-71DEDEKERKEgKEv83-) at the acidic region binds to the 12-bp palindromic sequence − 511AGCAnnnnnTGCT − 500 on the NRP-B promotor to activate NRP-B expression (Alves et al., 2011b). NRP-B, in turn, triggers a signaling cascade that leads to the induction of GmNAC81 and GmNAC30 gene expression (Mendes et al., 2013). These transcription factors work together to induce VPE expression, which acts as the executor of the vacuolar collapse-mediated cell death response, a plant-specific programmed cell death (PCD) event. The NRPs/NACs/VPE signaling circuit is also activated by Cd++ and drought (Reis et al., 2011, 2016; Quadros et al., 2021). The modulation of this signaling module by a negative regulator enhances tolerance to Cd++ toxicity and drought (Valente et al., 2009; Carvalho et al., 2014a; Reis et al., 2016; Quadros et al., 2021).
A recent genetic study using 34-salt tolerant accessions of Glycine soja has identified a naturally variant motif consisting of a 7-bp insertion/deletion in the promotor of Glycine soja (Gs)ERD15B. This variant was linked to enhanced salt tolerance of soybeans and upregulation of the GsERD15B gene (Jin et al., 2021). GsERD15B overexpression has also been associated with increased expression of genes involved in ABA signaling, cation transport, proline content, and dehydration-responsive genes, which may account for the salt tolerance of the soybean accessions harboring the naturally variant GsERD15B promoter. In the soybean accession LY01-10 (Hap2), the 7-bp deletion in the GsERD15B promoter footprinted the formation of an 8-bp inverted sequence, creating a possible binding site for autoregulation or for another transcription factor to enhance the expression of GsERD15B in response to salt stress. Therefore, like Glyma02G260800, the salt-tolerant function of GsERD15B may be associated with its transcriptional activity. In addition to transcriptional activity, GsERD15B interacts with PABPs, as expected from a PAM2-like protein.
ERD15-like subfamily members from different species display diverse subcellular localizations and transactivation activity. The Glyma02G260800 exhibits cytoplasm/nuclear localization and transactivation activity, while the Arabidopsis thaliana ERD15 protein does not exhibit DNA binding activity in yeast (Alves et al., 2011a). The mulberry MiERD15 exhibits transactivation activity, mapped to a non-conserved acidic region before the C-terminal QPR motif, also present in Glyma02G260800 (Saeed and Khurana, 2017). SpERD15 is a nuclear protein (Ziaf et al., 2011), whereas GsERD15B fractionates in the nucleus and cytoplasm (Jin et al., 2021).
Despite the functional relevance of the PAM2 motif and ERD15-like proteins in response to multiple stress, a comprehensive analysis of the PAM2 superfamily and ERD15-like subfamily in Arabidopsis, soybean, and rice still needs to be provided. Here, we performed in silico analyses that revealed the expansion of the ERD15-like subfamily of the PAM2 superfamily in soybean. Our functional complementation assay demonstrated that the members of the GmERD15-like subfamily are functionally redundant in response to drought and dark-induced senescence but display differential expression profiles in response to the hormones SA and ABA. Finally, we provide subcellular localization data of the ERD15-like subfamily members in Arabidopsis, soybean, and rice under different stress inducers.