Evolutionary history and expression analysis of the RWP-RK gene family and its potential regulatory network in root nodules

The RWP-RK protein family is divided into two subfamilies: NODULE INCEPTION (NIN) -like proteins (NLPs) and RWP-RK domain proteins (RKDs), which are involved in key biological processes including nitrate response, symbiotic nitrogen fixation, and embryonic development. We investigated the evolutionary history and functional divergence of these two subfamilies in green plants through phylogenetic analysis, motif analysis, expression profiling, and regulatory network construction. Both NLPs and RKDs originated from the early green algae ancestor, with multiple duplications during the seed plant period driving their lineage-specific expansion. Conserved motifs are more abundant among NLP proteins, whereas the number of conserved motifs among RKDs is relatively smaller. Expression analysis in various samples showed that GmNLP2a/b in soybean exhibit expression patterns analogous to those of the four NIN genes, while GmRKD4/13 also display abnormally high expression in root nodules. Therefore, there are at least eight RWP-RK genes that are specifically expressed or highly expressed in root nodules. Co-expression and functional enrichment analyses of transcriptome data further revealed that the expression patterns of eight nodule-specific/highly expressed genes of NLPs and RKDs in soybean can be divided into those associated with early development and late maturation. Integrating ATAC-seq data, we further constructed a potential regulatory network of eight nodule-specific/highly expressed genes and their co-expressed transcription factors. In summary, our study elucidates the evolutionary expansion and expression divergence of NLPs and RKDs across plants, providing insights into dissecting the transcriptional regulatory network underlying soybean root nodule development and adaptive evolution of plant gene families.