Evolutionary divergence of function and expression of laccase genes in plants

Laccases (LACs) are versatile enzymes that catalyze oxidation of a wide range of substrates, thereby functioning in regulation of plant developmental processes and stress responses. However, with a few exceptions, the function of most LACs remains unclear in plants. In this study, we newly identified 4, 12, 22, 26, 27, 28 and 49 LAC genes for Physcomitrella patens, Amborella trichopoda, Zea mays, Ricinus communis, Vitis vinifera, Triticum aestivum and Glycine max, on the basis of exhaustive homologous sequence searches. In these plants, LACs differ greatly in sequence length and physical properties, such as molecular weight and theoretical isoelectric point (pI), but majority of them contain a signal peptide at their N-terminus. The originality of LACs could be traced back to as early as the emergence of moss. Plant LACs are clearly divided into seven distinct classes, where six ancient LACs should be present prior to the divergence of gymnosperms and angiosperms. Functional divergence analysis reveal that functional differentiation should occur among different groups of LACs because of altered selective constraints working on some critical amino acid sites (CAASs) within conserved laccase domains during evolution. Soybean and maize LACs have significantly different exon frequency (6.08 vs 4.82), and they are unevenly distributed and tend to form gene clusters on some chromosomes. Further analysis shows that the expansion of LAC gene family would be due to extensive tandem and chromosomal segmental duplications in the two plant species. Interestingly, *81.6% and 36.4% of soybean and maize LACs are potential targets of miRNAs, such as miR397a/b, miR408d, or miR528a/b etc. Both soybean and maize LACs are tissuespecifically and developmental-specifically expressed, and are in response to different external abiotic and biotic stressors. These results suggest a diversity of functions of plant LAC genes, which will broaden our understanding and lay solid foundation for further investigating their biological functions in plants.