Revisiting the Soybean GmNAC Superfamily

Abstract

The NAC (NAM, ATAF, and CUC) genes encode transcription factors involved with the control of plant morph-physiology and stress responses. The release of the last soybean (Glycine max) genome assembly (Wm82.a2.v1) raised the possibility that new NAC genes would be present in the soybean genome. Here, we interrogated the last version of the soybean genome against a conserved NAC domain structure. Our analysis identified 32 putative novel NAC genes, updating the superfamily to 180 gene members. We also organized the genes in 15 phylogenetic subfamilies, which showed a perfect correlation among sequence conservation, expression profile, and function of orthologous Arabidopsis thaliana genes and NAC soybean genes. To validate our in silico analyses, we monitored the stress-mediated gene expression profiles of eight new NAC-genes by qRT-PCR and monitored the GmNAC senescence-associated genes by RNA-seq. Among ER stress, osmotic stress and salicylic acid treatment, all the novel tested GmNAC genes responded to at least one type of stress, displaying a complex expression profile under different kinetics and extension of the response. Furthermore, we showed that 40% of the GmNACs were differentially regulated by natural leaf senescence, including eight (8) newly identified GmNACs. The developmental and stress-responsive expression profiles of the novel NAC genes fitted perfectly with their phylogenetic subfamily. Finally, we examined two uncharacterized senescence-associated proteins, GmNAC065 and GmNAC085, and a novel, previously unidentified, NAC protein, GmNAC177, and showed that they are nuclear localized, and except for GmNAC065, they display transactivation activity in yeast. Consistent with a role in leaf senescence, transient expression of GmNAC065 and GmNAC085 induces the appearance of hallmarks of leaf senescence, including chlorophyll loss, leaf yellowing, lipid peroxidation and accumulation of H2O2. GmNAC177 was clustered to an uncharacterized subfamily but in close proximity to the TIP subfamily. Accordingly, it was rapidly induced by ER stress and by salicylic acid under late kinetic response and promoted cell death in planta. Collectively, our data further substantiated the notion that the GmNAC genes display functional and expression profiles consistent with their phylogenetic relatedness and established a complete framework of the soybean NAC superfamily as a foundation for future analyses.

A Regulatory Circuit Integrating Stress-Induced with Natural Leaf Senescence

“…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 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].…”

“…In soybean, a transcriptomic analysis of senescing leaves reveals that 44% of the GmNAC genes were differentially expressed at the onset of leaf senescence. The most representative subfamilies of soybean senescence-associated NAC genes were the abiotic stress-induced SNAC-A (ATAF) subfamily, in which 90% of the members were differentially expressed during senescence, followed by the biotic stress-induced TERN subfamily, displaying 80% of the members differentially expressed during leaf senescence [43]. GmNAC30 and GmNAC81, which belong to the SNAC-A and TERN subfamilies, respectively, are among the upregulated genes by leaf senescence [43,59].…”

“…The most representative subfamilies of soybean senescence-associated NAC genes were the abiotic stress-induced SNAC-A (ATAF) subfamily, in which 90% of the members were differentially expressed during senescence, followed by the biotic stress-induced TERN subfamily, displaying 80% of the members differentially expressed during leaf senescence [43]. GmNAC30 and GmNAC81, which belong to the SNAC-A and TERN subfamilies, respectively, are among the upregulated genes by leaf senescence [43,59]. These results raise the hypotheses that the (i) DCD-NRP/NAC/VPE signaling module may integrate stress-induced with natural leaf senescence and (ii) other NAC genes may be involved in integrated circuits between age-and stress-induced cell death pathways.…”

“…Regarding the first hypothesis, several lines of evidence indicate that the regulatory circuit NRPs/GmNAC81:GmNAC30/VPE integrates osmotic stress-and ER stress-induced PCD response with natural leaf senescence. First, not only GmNAC30 and GmNAC81 but also the other cell death pathway components, NRP-A, NRP-B, and VPE, are induced by leaf senescence [43,59,70]. Second, the activity of VPE is also induced during the onset of leaf senescence [59].…”