A wound-induced small polypeptide gene family is upregulated in soybean nodules

Small peptides function as key signals in processes, such as plant cell differentiation, organ development and defenses to biotic stresses. A large number of small peptide precursor genes have been predicted from the analysis of the soybean (Glycine max) whole genome DNA sequence. However, most of these genes have unknown characteristics and functions. In this report, we systemically searched for the gene families of small peptide precursors that are up-regulated in soybean nitrogen-fixing root nodules. We found 212 genes (encoding peptides shorter than 150 amino acids) that were up-regulated, and among them, 79 genes belong to 38 multiple-gene families, but the other 133 genes are unique. Twenty-eight of 38 families are conserved in Arabidopsis, but the other 10 only exist in legumes. We also identified 16 out of the 38 members of the wound-induced polypeptide (WIP) gene family to be upregulated in nitrogen-fixing nodules. We further analyzed homologs of WIP genes in Medicago, Lotus, Arabidopsis and Oryza species and found that a few homologous genes from Medicago truncatula and Lotus japonicus were also upregulated in their nodules and some WIP genes were induced by specific fungal pathogens on soybean and rice. Structure prediction indicated that all WIP prepropeptides contain a conserved DUF3774 domain (including two hydrophobic regions) and most of them have an N-terminal signal sequence. Fluorescence microscopy analysis of two WIP prepropeptides fused to GFP revealed that these proteins are located on the plasma membrane of tobacco leaf cells. Interestingly, 34 soybean WIP genes are clustered onto three soybean chromosomes, different from known peptide gene families (such as CLE). Among them, 11 highly identical genes are aligned on the 6th chromosome, 12 on the 12th, and 11 on the 13th chromosomes. Most of WIP genes from the 12th chromosome share the highest identities with their homologs on the 13th chromosome, suggesting that ancestral WIP genes could have originated from the 13th chromosome, then spread onto the 12th chromosome by chromosome homologous recombination; the new WIP genes could have existed in multiple copies by gene duplication which then spread onto the 6th chromosome. In Arabidopsis and Oryza species, half of the WIP genes are also aligned on one chromosome and showed higher identity with those from the soybean 12th and 13th chromosomes, suggesting that WIP genes originated from one common ancestor.