Genome-wide identification, molecular structures and functional exploration of the membrane attack complex/perforin domain-containing proteins and validation of GmmiRNA169o-GmMACPF-9 module in soybean cold stress

Soybean (Glycine max L.) is a widely cultivated legume and an enriched source of edible oil and protein. Membrane Attack Complex/Perforin Domain-Containing Proteins (MACPFs) played significant roles in plant growth and biotic and abiotic stresses. Here, we performed a genome-wide scanning for the putative GmMACPFs and a total of 20 GmMACPFs were identified in soybean. The conserved domain and phylogenetic analysis of MACPF showed the evolutionary diversification of MACPF genes family among legumes. The GmMACPFs protein characteristics were predicted, including theoretical molecular weight (38.06 to 68.38 kDa), protein isoelectric point (6.61 to 9.41), subcellular localization (nucleus, cytoplasm or mitochondria) and various TFs binding sites. GmMACPFs are located on 12 chromosomes out of a total 20 chromosomes in soybean. The 2 kb promoter analysis revealed that GmMACPFs contains a large number of phytohormones and stress responsive cis-acting elements (CAE) and transcription factor binding sites. We explored the 3D protein structure of GmMACPFs, their posttranscriptional regulators especially abiotic stress responsive miRNAs, and the evolutionary relationship among different legumes and Arabidopsis. GmMACPFs expression pattern was retrieved from RNA-seq libraries for tissue specific and under various abiotic stresses in soybean. According to the results, GmMACPF genes exhibited spatiotemporal, tissue, development stage and abiotic stress specific expression patterns in soybean. Our RT-qPCR analysis indicates the antagonistic expression pattern between GmmiRNA-GmMACPF modules under cold, salt, drought and heat stresses. Transient over expression of GmmiRNA169o-GmMACPF-9 module in soybean hairy root and biochemical analysis showed that GmmiRNA169o-OE plants accumulated less reactive oxygen species (ROS), malondialdehyde (MDA) and accumulated higher superoxide dismutase (SOD) and peroxidase (POD) to enhance cold stress tolerance. These results enriched our knowledge of the functions of GmmiRNA169o and GmMACPF-9 and provided candidate miRNAs-GmMACPFs modules especially GmmiRNA169o for environmental stress resilient soybean breeding through genetic engineering.