Genome-wide characterization of the laccase gene family in Setaria viridis reveals members potentially involved in lignification

  • 143 Accesses


Main conclusion

Five laccase genes are potentially involved in developmental lignification in the model C4 grass Setaria viridis and their different tissue specificities suggest subfunctionalization events.


Plant laccases are copper-containing glycoproteins involved in monolignol oxidation and, therefore, their activity is essential for lignin polymerization. Although these enzymes belong to large multigene families with highly redundant members, not all of them are thought to be involved in lignin metabolism. Here, we report on the genome-wide characterization of the laccase gene family in the model C4 grass Setaria viridis and further identification of the members potentially involved in monolignol oxidation. A total of 52 genes encoding laccases (SvLAC1 to SvLAC52) were found in the genome of S. viridis, and phylogenetic analyses showed that these genes were heterogeneously distributed among the characteristic six subclades of the family and are under relaxed selective constraints. The observed expansion in the total number of genes in this species was mainly caused by tandem duplications within subclade V, which accounts for 68% of the whole family. Comparative phylogenetic analyses showed that the expansion of subclade V is specifically observed for the Paniceae tribe within the Panicoideae subfamily in grasses. Five SvLAC genes (SvLAC9, SvLAC13, SvLAC15, SvLAC50, and SvLAC52) fulfilled the criteria established to identify lignin-related candidates: (1) phylogenetic proximity to previously characterized lignin-related laccases from other species, (2) similar expression pattern to that observed for lignin biosynthetic genes in the S. viridis elongating internode, and (3) high expression in S. viridis tissues undergoing active lignification. In addition, in situ hybridization experiments not only confirmed that these selected SvLAC genes were expressed in lignifying cells, but also that their expression showed different tissue specificities, suggesting subfunctionalization events within the family. These five laccase genes are strong candidates to be involved in lignin polymerization in S. viridis and might be good targets for lignin bioengineering strategies.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5



Secondary cell wall


  1. Bao W, O’Malley DM, Whetten R, Sederoff RR (1993) A laccase associated with lignification in loblolly pine xylem. Science 260:672–674.

  2. Barceló AR, Ros LVG, Carrasco AE (2007) Looking for syringyl peroxidases. Trends Plant Sci 12:486–491.

  3. Barros J, Serk H, Granlund I, Pesquet E (2015) The cell biology of lignification in higher plants. Ann Bot 115:1053–1074.

  4. Berthet S, Demont-Caulet N, Pollet B et al (2011) Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems. Plant Cell 23:1124–1137.

  5. Brutnell TP, Bennetzen JL, Vogel JP (2015) Brachypodium distachyon and Setaria viridis: model genetic systems for the grasses. Annu Rev Plant Biol 66:465–485.

  6. Bryan AC, Jawdy SS, Gunter L et al (2016) Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release. Plant Biotechnol J 14:2010–2020.

  7. Cannon SB, Mitra A, Baumgarten A et al (2004) The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol 4:10.

  8. Carrió-Seguí À, Ruiz-Rivero O, Villamayor-Belinchón L et al (2019) The altered expression of microRNA408 influences the Arabidopsis response to iron deficiency. Front Plant Sci 10:324.

  9. Cesarino I (2019) Structural features and regulation of lignin deposited upon biotic and abiotic stresses. Curr Opin Biotechnol 56:209–214.

  10. Cesarino I, Araujo P, Sampaio Mayer JL et al (2013) Expression of SofLAC, a new laccase in sugarcane, restores lignin content but not S:G ratio of Arabidopsis lac17 mutant. J Exp Bot 64:1769–1781.

  11. Cesarino I, Simões MS, dos Brito M, et al (2016) Building the wall: recent advances in understanding lignin metabolism in grasses. Acta Physiol Plant 38:269.

  12. Chou EY, Schuetz M, Hoffmann N et al (2018) Distribution, mobility, and anchoring of lignin-related oxidative enzymes in Arabidopsis secondary cell walls. J Exp Bot 69:1849–1859.

  13. Cosio C, Dunand C (2009) Specific functions of individual class III peroxidase genes. J Exp Bot 60:391–408.

  14. De Meester B, de Vries L, Özparpucu M et al (2018) Vessel-specific reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in dwarfed ccr1 mutants restores vessel and xylary fiber integrity and increases biomass. Plant Physiol 176:611–633.

  15. Demarco D (2017) Histochemical analysis of plant secretory structures. In: Pellicciari C, Biggiogera M (eds) Histochemistry of single molecules. Humana, New York, pp 313–330

  16. Duarte KE, de Souza WR, Santiago TR et al (2019) Identification and characterization of core abscisic acid (ABA) signaling components and their gene expression profile in response to abiotic stresses in Setaria viridis. Sci Rep 9:1–16.

  17. Fernández-Pérez F, Pomar F, Pedreño MA, Novo-Uzal E (2015) Suppression of Arabidopsis peroxidase 72 alters cell wall and phenylpropanoid metabolism. Plant Sci 239:192–199.

  18. Ferreira SS, Simões MS, Carvalho GG et al (2019) The lignin toolbox of the model grass Setaria viridis. Plant Mol Biol 101:235–255.

  19. Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

  20. Halpin C (2019) Lignin engineering to improve saccharification and digestibility in grasses. Curr Opin Biotechnol 56:223–229.

  21. Hatfield R, Ralph J, Grabber JH (2008) A potential role for sinapyl p-coumarate as a radical transfer mechanism in grass lignin formation. Planta 228:919–928.

  22. Hellemans J, Mortier G, De Paepe A et al (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19.

  23. Hu Q, Min L, Yang X et al (2018) Laccase GhLac1 modulates broad-spectrum biotic stress tolerance via manipulating phenylpropanoid pathway and jasmonic acid synthesis. Plant Physiol 176:1808–1823.

  24. Koutaniemi S, Toikka MM, Kärkönen A et al (2005) Characterization of basic p-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming Picea abies suspension culture. Plant Mol Biol 58:141–157.

  25. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874.

  26. Le Bris P, Wang Y, Barbereau C et al (2019) Inactivation of LACCASE8 and LACCASE5 genes in Brachypodium distachyon leads to severe decrease in lignin content and high increase in saccharification yield without impacting plant integrity. Biotechnol Biofuels 12:181.

  27. Li P, Brutnell TP (2011) Setaria viridis and Setaria italica, model genetic systems for the Panicoid grasses. J Exp Bot 62:3031–3037.

  28. Li Q, Feng J, Chen L et al (2019) Genome-wide identification and characterization of Salvia miltiorrhiza laccases reveal potential targets for salvianolic acid B biosynthesis. Front Plant Sci 10:435.

  29. Liu C-J (2012) Deciphering the enigma of lignification: precursor transport, oxidation, and the topochemistry of lignin assembly. Mol Plant 5:304–317.

  30. Liu Q, Luo L, Wang X et al (2017) Comprehensive analysis of rice laccase gene (OsLAC) family and ectopic expression of OsLAC10 enhances tolerance to copper stress in Arabidopsis. Int J Mol Sci 18:209.

  31. Mahon EL, Mansfield SD (2019) Tailor-made trees: engineering lignin for ease of processing and tomorrow’s bioeconomy. Curr Opin Biotechnol 56:147–155.

  32. Marita JM, Hatfield RD, Rancour DM, Frost KE (2014) Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L. Plant J 78:850–864.

  33. Marriott PE, Gómez LD, McQueen-Mason SJ (2016) Unlocking the potential of lignocellulosic biomass through plant science. New Phytol 209:1366–1381.

  34. Martin AP, Palmer WM, Brown C et al (2016) A developing Setaria viridis internode: an experimental system for the study of biomass generation in a C4 model species. Biotechnol Biofuels 9:45.

  35. Martins PK, Ribeiro AP, Cunha BADB et al (2015) A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis. Biotechnol Rep 6:41–44.

  36. Martins PK, Mafra V, de Souza WR et al (2016) Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis. Sci Rep 6:28348.

  37. McCaig BC, Meagher RB, Dean JFD (2005) Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in Arabidopsis thaliana. Planta 221:619–636.

  38. Méchin V, Baumberger S, Pollet B, Lapierre C (2007) Peroxidase activity can dictate the in vitro lignin dehydrogenative polymer structure. Phytochemistry 68:571–579.

  39. Muro-Villanueva F, Mao X, Chapple C (2019) Linking phenylpropanoid metabolism, lignin deposition, and plant growth inhibition. Curr Opin Biotechnol 56:202–208.

  40. Novo-Uzal E, Fernández-Pérez F, Herrero J et al (2013) From Zinnia to Arabidopsis: approaching the involvement of peroxidases in lignification. J Exp Bot 64:3499–3518.

  41. Petrik DL, Karlen SD, Cass CL et al (2014) p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon. Plant J 77:713–726.

  42. Pourcel L, Routaboul J-M, Kerhoas L et al (2005) TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat. Plant Cell 17:2966–2980.

  43. Ralph J, Lapierre C, Boerjan W (2019) Lignin structure and its engineering. Curr Opin Biotechnol 56:240–249.

  44. Ranocha P, Chabannes M, Chamayou S et al (2002) Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar. Plant Physiol 129:145–155.

  45. Saha P, Sade N, Arzani A et al (2016) Effects of abiotic stress on physiological plasticity and water use of Setaria viridis (L.). Plant Sci 251:128–138.

  46. Shigeto J, Tsutsumi Y (2016) Diverse functions and reactions of class III peroxidases. New Phytol 209:1395–1402.

  47. Shigeto J, Kiyonaga Y, Fujita K et al (2013) Putative cationic cell-wall-bound peroxidase homologues in Arabidopsis, AtPrx2, AtPrx25, and AtPrx71, are involved in lignification. J Agric Food Chem 61:3781–3788.

  48. Sterjiades R, Dean JFD, Gamble G et al (1993) Extracellular laccases and peroxidases from sycamore maple (Acer pseudoplatanus) cell-suspension cultures. Planta 190:75–87.

  49. Tobimatsu Y, Schuetz M (2019) Lignin polymerization: how do plants manage the chemistry so well? Curr Opin Biotechnol 56:75–81.

  50. Turlapati PV, Kim K-W, Davin LB, Lewis NG (2011) The laccase multigene family in Arabidopsis thaliana: towards addressing the mystery of their gene function(s). Planta 233:439–470.

  51. van der Weijde T, Kamei CLA, Torres AF et al (2013) The potential of C4 grasses for cellulosic biofuel production. Front Plant Sci 4:107.

  52. Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(research0034):1.

  53. Vanholme R, De Meester B, Ralph J, Boerjan W (2019) Lignin biosynthesis and its integration into metabolism. Curr Opin Biotechnol 56:230–239.

  54. Vargas L, Cesarino I, Vanholme R et al (2016) Improving total saccharification yield of Arabidopsis plants by vessel-specific complementation of caffeoyl shikimate esterase (cse) mutants. Biotechnol Biofuels 9:139.

  55. Wang Y, Wang X, Paterson AH (2012) Genome and gene duplications and gene expression divergence: a view from plants. Ann N Y Acad Sci 1256:1–14.

  56. Wang J, Feng J, Jia W et al (2015a) Lignin engineering through laccase modification: a promising field for energy plant improvement. Biotechnol Biofuels 8:145.

  57. Wang Y, Bouchabke-Coussa O, Lebris P et al (2015b) LACCASE5 is required for lignification of the Brachypodium distachyon culm. Plant Physiol 168:192–204.

  58. Wang J, Feng J, Jia W et al (2017) Genome-wide identification of Sorghum bicolor laccases reveals potential targets for lignin modification. Front Plant Sci 8:714.

  59. Wilkerson CG, Mansfield SD, Lu F et al (2014) Monolignol ferulate transferase introduces chemically labile linkages into the lignin backbone. Science 344:90–93.

  60. Yang Z (2007) PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24:1586–1591.

  61. Yu Y, Li Q-F, Zhang J-P et al (2017) Laccase-13 regulates seed setting rate by affecting hydrogen peroxide dynamics and mitochondrial integrity in rice. Front Plant Sci 8:1324.

  62. Zhang J (2003) Evolution by gene duplication: an update. Trends Ecol Evol 18:292–298.

  63. Zhang Y-C, Yu Y, Wang C-Y et al (2013) Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nat Biotechnol 31:848–852.

  64. Zhang Y, Wu L, Wang X et al (2019) The cotton laccase gene GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and lignin components in the cell walls of plants. Mol Plant Pathol 20:309–322.

  65. Zhao Q, Nakashima J, Chen F et al (2013) LACCASE is necessary and nonredundant with PEROXIDASE for lignin polymerization during vascular development in Arabidopsis. Plant Cell 25:3976–3987.

  66. Zhao Y, Lin S, Qiu Z et al (2015) MicroRNA857 is involved in the regulation of secondary growth of vascular tissues in Arabidopsis. Plant Physiol 169:2539–2552.

Download references


This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) via the BIOEN Young Investigators Awards research grant (Processo FAPESP no. 2015/02527-1). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. IC thanks FAPESP for the SPRINT Project Grant number 2016/50189-0. IC is indebted to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the research fellowship 302927/2018-2. MSS was funded by CAPES for a predoctoral fellowship. GGC was funded by CNPq-PIBIC for a scientific initiation fellowship. SSF was funded by FAPESP for a postdoctoral fellowship (Processo FAPESP no. 2016/06917-1).

Author information

Correspondence to Igor Cesarino.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Peptide sequences of all 52 laccases of S. viridis in FASTA format (TXT 31 kb)

Dataset of laccase peptide sequences from Arabidopsis thaliana + grasses in FASTA format (TXT 319 kb)

Dataset of laccase peptide sequences from Arabidopsis thaliana + eudicots in FASTA format (TXT 157 kb)

Dataset of laccase peptide sequences from Arabidopsis thaliana + other monocots + Amborella trichopoda in FASTA format (TXT 54 kb)

Exon-intron structure and conserved domains distribution of SvLAC genes/proteins (PDF 357 kb)

Maximum likelihood tree built with laccase proteins from Arabidopsis thaliana and grasses. Numbers at nodes indicate branch support performed by the SH-aLRT test and the ultrafast bootstrap test, respectively (PDF 89 kb)

Maximum likelihood tree built with laccase proteins from Arabidopsis thaliana and eudicots. Numbers at nodes indicate branch support performed by the SH-aLRT test and the ultrafast bootstrap test, respectively (PDF 45 kb)

Maximum likelihood tree built with laccase proteins from Arabidopsis thaliana and other monocots + Amborella trichopoda. Numbers at nodes indicate branch support performed by the SH-aLRT test and the ultrafast bootstrap test, respectively (PDF 29 kb)

List of primers used in this study (XLSX 21 kb)

In silico prediction of some physico-chemical properties of SvLAC proteins (XLSX 14 kb)

RNAseq expression data (FPKM) of all laccase genes in S. viridis retrieved from the work of Martin et al. 2016. Data for different subclades are shown in different sheets (XLSX 42 kb)

Analysis of the presence of stress-related cis-elements in the promoter of SvLAC genes (XLSX 21 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Simões, M.S., Carvalho, G.G., Ferreira, S.S. et al. Genome-wide characterization of the laccase gene family in Setaria viridis reveals members potentially involved in lignification. Planta 251, 46 (2020) doi:10.1007/s00425-020-03337-x

Download citation


  • Gene expression
  • Grasses
  • In situ hybridization
  • Laccases
  • Lignin
  • Monolignol polymerization
  • Paniceae