The Soybean Laccase Gene Family: Evolution and Possible Roles in Plant Defense and Stem Strength Selection

Abstract

Laccase is a widely used industrial oxidase for food processing, dye synthesis, paper making, and pollution remediation. At present, laccases used by industries come mainly from fungi. Plants contain numerous genes encoding laccase enzymes that show properties which are distinct from that of the fungal laccases. These plant-specific laccases may have better potential for industrial purposes. The aim of this work was to conduct a genome-wide search for the soybean laccase genes and analyze their characteristics and specific functions. A total of 93 putative laccase genes (GmLac) were identified from the soybean genome. All 93 GmLac enzymes contain three typical Cu-oxidase domains, and they were classified into five groups based on phylogenetic analysis. Although adjacent members on the tree showed highly similar exon/intron organization and motif composition, there were differences among the members within a class for both conserved and differentiated functions. Based on the expression patterns, some members of laccase were expressed in specific tissues/organs, while some exhibited a constitutive expression pattern. Analysis of the transcriptome revealed that some laccase genes might be involved in providing resistance to oomycetes. Analysis of the selective pressures acting on the laccase gene family in the process of soybean domestication revealed that 10 genes could have been under artificial selection during the domestication process. Four of these genes may have contributed to the transition of the soft and thin stem of wild soybean species into strong, thick, and erect stems of the cultivated soybean species. Our study provides a foundation for future functional studies of the soybean laccase gene family.

Keywords: Cu-oxidase; GmLac; laccase; resistance; selection; soybean; stem strength.

Figure 1

Physical map of the soybean laccase genes. Chromosome size is indicated by its relative length. The scale bar is shown on the left, and the numbers represent the physical position of the genes on the chromosomes. Tandemly duplicated genes are represented by boxes with red outlines.

Figure 2

The phylogenetic tree of the Arabidopsis and soybean laccase proteins. In total, 93 soybean and 17 Arabidopsis laccase proteins were aligned using the ClustalW program. The neighbor-joining tree was constructed using MEGA7.0. The bootstrap value was set as 1000 replicates. The length of each branch is shown next to the branches.

Figure 3

Collinearity of the laccase gene (GmLac) gene pairs. The outermost scale represents the chromosomes. Genes are listed in the inner circle according to their chromosomal location. Synteny relationships between gene pairs are marked with a black line.

Figure 4

Phylogenetic relationships, gene structure, and motif compositions of the soybean laccase gene family. (A) A neighbor-joining tree of 93 GmLac protein sequences constructed using MEGA v7.0. (B) The structure of the 93 GmLac genes. Yellow squares correspond to exons and linking black lines indicate introns, while the blue squares refer to the 5′ untranslated region (UTR) and 3′ UTR sequences. (C) Schematic motif composition of 93 GmLac proteins. The colored boxes represent the different motifs, indicated in the top right-hand corner. The scales at the bottom of the image indicate the estimated exon/intron length in kb and motif length in numbers of amino acids (aa).

Figure 5

Expression profiles of the 93 soybean laccase genes following Phytophthora sojae infection. (A) Transcriptome heatmap. The hierarchical cluster color code: the highest induction values vs. controls are displayed in red (hot), while the values displayed in blue (cool) indicate the most reduced expression levels vs. controls; intermediate expression levels are represented by lighter intermediate colors. The color scale represents △FPKM normalized to log2-transformed vales. The genes marked by the black brackets on the right in (A) are those obviously mis-regulated genes. (B) Expression levels of 21 GmLacs after P7076 infection. Bars represent average values of three replicates ± standard deviation (SD). All expression levels of the GmLac genes were normalized to the expression levels of GmPDF. ***, **, and * indicate significant differences compared to the control (0 h) at p < 0.001, p < 0.01, and p < 0.05, respectively.

Figure 6

Selective pressure analysis of laccase gene family. The outermost scale represents chromosomes, and the green bar chart in the middle represents π_G.max/π_G.soja values per chromosome. The top 5% selected laccase genes are listed according to their physical location.

Figure 7

Phylogenetic tree of 302 accessions. SNPs of the 10 selected genes among resequenced genomes of the 302 soybean accessions were used to construct the neighbor-joining (N-J) phylogenetic tree. The bootstrap value was set as 1000 replicates. Corresponding specie idnetifiers (IDs) of the accessions are available from Reference [57].