Abstract
In this study, a novel laccase gene, EuLAC1, was cloned from Eucommia ulmoides Oliver (E. ulmoides). An overexpression vector harboring the EuLAC1 was constructed and introduced into the tobacco (Nicotiana tabacum cv. Xanthi). The laccase activity, resistance to Botrytis cinerea (B. cinerea) and lignin level in wild-type and transgenic plants were thereafter investigated. Interestingly, the transgenic tobacco displayed a significantly higher laccase activity and resistance to gray mold as compared to the wild-type tobacco. Additionally, the lignin contents in the leaves and stems of the transgenic tobacco were significantly higher in comparison to the wild-type tobacco. Scanning electron microscopy was used to observe the cross sections of wild-type and transgenic tobacco stems and it was noted that the cell wall near the xylem catheter of the transgenic tobacco was substantially thicker and the outline clearer than that of the wild-type. Thus, the EuLAC1 gene can significantly increase laccase activity and lignin content in tobacco, leading to an increase in the physical defenses, thereby increasing tobacco resistance to gray mold.
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The data that support the findings of this study are available from the corresponding author on reasonable request.
References
Balasubramanian VK, Rai KM, Thu SW, Hii MM, Mendu V (2016) Genome-wide identification of multifunctional laccase gene family in cotton (Gossypium spp.); expression and biochemical analysis during fiber development. Sci Rep 6:4132–4136. https://doi.org/10.1038/srep34309
Barros J, Serk H, Granlund I, Pesquet E (2015) The cell biology of lignification in higher plants. Ann Bot 115:1053–1074. https://doi.org/10.1093/aob/mcv046
Berthet S, Demont-Caulet N, Pollet B, Bidzinski P, Cézard L (2011) Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems. Plant Cell 23:1124–1137. https://doi.org/10.1105/tpc.110.082792
Bonello P, Blodgett JT (2003) Pinus nigra - Sphaeropsis sapinea as a model pathosystem to investigate local and systemic effects of fungal infection of pines. Physiol Mol Plant Pathol 63:249–261. https://doi.org/10.1016/j.pmpp.2004.02.002
Cesarino I, Araujo P, Sampaio Mayer JL, Vicentini R, Berthet S, Demedts B, Vanholme B, Boerjan W, Mazzafera P (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. https://doi.org/10.1093/jxb/ert045
Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16:664–670. https://doi.org/10.1006/abio.1994.1170
Chou EY, Schuetz M, Hoffmann N, Watanabe Y, Sibout R, Samuels AL (2018) Distribution, mobility, and anchoring of lignin-related oxidative enzymes in Arabidopsis secondary cell walls. J Exp Bot 69:1849–1859. https://doi.org/10.1093/jxb/ery067
Coego A, Ramirez V, Gil MJ, Flors V, Mauch-Mani B, Vera P (2005) An Arabidopsis homeodomain transcription factor, overexpressor of cationic peroxidase 3, mediates resistance to infection by necrotrophic pathogens. Plant Cell 17:2123–2137. https://doi.org/10.1105/tpc.105.032375
Dixon RA, Achnine L, Kota P, Liu C, Reddy MSS, Wang L (2002) The phenylpropanoid pathway and plant defence-a genomics perspective. Mol Plant Pathol 3:371–390. https://doi.org/10.1046/j.1364-3703.2002.00131.x
Dong X, Zhao Y, Ran X, Guo L, Zhao D (2017) Overexpression of a new chitinase gene EuCHIT2 enhances resistance to Erysiphe cichoracearum DC. in tobacco plants. Int J Mol Sci 18:2361. https://doi.org/10.3390/ijms18112361
Fadden HGMC, Chapple R, Feyter RD, Dennis E (2001) Expression of pathogenesis-related genes in cotton stems in response to infection by Verticillium dahliae. Physiol Mol Plant Pathol 58:119–131. https://doi.org/10.1006/pmpp.2001.0320
Gayoso C, Pomar F, Novo-Uzal E, Merino F, de Ilárduya ÓM (2010) The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H2O2, peroxidase and lignins and drives PAL gene expression. BMC Plant Biol 10:232. https://doi.org/10.1186/1471-2229-10-232
Guo LX, Dong X, Zhao DG (2016) Transgenic tomato plants expressing a Eucommia ulmoides chitinase gene EuCHIT1 and their resistance to Botrytis cinerea. Chin J Plant Physiol 52:703–714 ((in Chinese))
Huang RH, Zhang Y, Wang DC (2010) Primary structural determination of N-terminally blocked peptides from the bark of Eucommia ulmoides Oliv by mass spectrometric analysis. Rapid Commun Mass Spectrom 17:903–908. https://doi.org/10.1002/rcm.999
Hung J, Yang C, Tsai Y, Huang H, Huang M (2008) Antiproliferative activity of aucubin is through cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells. Clin Exp Pharmacol Physiol 35:995–1001. https://doi.org/10.1111/j.1440-1681.2008.04935.x
Igor C, Pedro A, Lischka SMJ, Renato V, Serge B, Brecht D, Bartel V, Wout B, Paulo M (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. https://doi.org/10.1093/jxb/ert045
Kiefer-Meyer MC, Gomord V, Connell O, A, Halpin C, Faye LC, (1996) Cloning and sequence analysis of laccase-encoding cDNA clones from tobacco. Gene 178:205–207. https://doi.org/10.1016/0378-1119(96)00381-2
Kwan C, Zhang W, Deyama T, Nishibe S (2004) Endothelium-dependent vascular relaxation induced by Eucommia ulmoides Oliv. bark extract is mediated by NO and EDHF in small vessels. Naunyn-Schmiedeberg’s Arch Pharmacol 369:206–211. https://doi.org/10.1007/s00210-003-0822-4
Li L, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215:239–247. https://doi.org/10.1007/s00425-002-0750-4
Liang M, Haroldsen V, Cai X, Wu Y (2006) Expression of a putative laccase gene, ZmLAC1, in maize primary roots under stress. Plant Cell Environ 29:746–753. https://doi.org/10.1111/j.1365-3040.2005.01435.x
Liu J, Zhuang Y, Huang X, Zhao D, Zhao Y (2020) Overexpression of cotton laccase gene LAC1 enhances resistance to Botrytis cinereal in tomato plants. Int J Agric Biol 24:221–228. https://doi.org/10.17957/IJAB/15.1428
Liu Q, Luo L, Wang X, Shen Z, Zheng L (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. https://doi.org/10.3390/ijms18020209
Liu Z, Luan Y, Li J, Yin Y (2016) Expression of a tomato MYB gene in transgenic tobacco increases resistance to Fusarium oxysporum and Botrytis cinerea. Eur J Plant Pathol 144:607–617. https://doi.org/10.1007/s10658-015-0799-0
Ma QH, Zhu HH, Qiao MY (2018) Contribution of both lignin content and sinapyl monomer to disease resistance in tobacco. Plant Pathol 67:642–650. https://doi.org/10.1111/ppa.12767
Morrison IM (1972) A semi-micro method for the determination of lignin and its use in predicting the digestibility of forage crops. J Sci Food Agric 23:791. https://doi.org/10.1002/jsfa.2740230405
Naoumkina MA, Zhao Q, Gallego Giraldo L, Dai X, Zhao PX, Dixon RA (2010) Genome-wide analysis of phenylpropanoid defence pathways. Mol Plant Pathol 11:829–846. https://doi.org/10.1111/j.1364-3703.2010.00648.x
Nie Q, Gao G, Fan Q, Qiao G, Wen X, Liu T, Peng Z, Cai Y (2015) Isolation and characterization of a catalase gene “HuCAT3” from pitaya (Hylocereus undatus) and its expression under abiotic stress. Gene 563:63–71. https://doi.org/10.1016/j.gene.2015.03.007
Pomar F, Novo M, Bernal MA, Merino F, Barceló AR (2004) Changes in stem lignins (monomer composition and crosslinking) and peroxidase are related with the maintenance of leaf photosynthetic integrity during Verticillium wilt in Capsicum annuum. New Phytol 163:111–123. https://doi.org/10.1111/j.1469-8137.2004.01092.x
Powell ALT, Kan JV, Have AT, Visser J, Labavitch JM (2000) Transgenic expression of pear PGIP in tomato limits fungal colonization. Mol Plant Microbe Interact 13:942–950. https://doi.org/10.1094/MPMI.2000.13.9.942
Ranocha P, McDougall G, Hawkins S, Sterjiades R, Borderies G, Stewart D, Cabanes-Macheteau M, Boudet AM, Goffner D (1999) Biochemical characterization, molecular cloning and expression of laccases - a divergent gene family - in poplar. Eur J Biochem 259:485–495. https://doi.org/10.1046/j.1432-1327.1999.00061.x
Sato Y, Wuli B, Sederoff R, Whetten R (2001) Molecular cloning and expression of eight laccase cDNAs in loblolly pine (Pinus taeda)*. J Plant Res 114:147–155. https://doi.org/10.1007/PL00013978
Schuetz M, Benske A, Smith RA, Watanabe Y, Tobimatsu Y, Ralph J, Demura T, Ellis B, Samuels AL (2014) Laccases direct lignification in the discrete secondary cell wall domains of protoxylem. Plant Physiol 166:798–807. https://doi.org/10.1104/pp.114.245597
Sun AP, Choi MS, Kim MJ, Jung UJ, Kim HJ, Park KK, Noh HJ, Park HM, Yong BP, Lee JS (2006) Hypoglycemic and hypolipidemic action of Du-zhong (Eucommia ulmoides Oliver) leaves water extract in C57BL/KsJ-db/db mice. J Ethnopharmacol 107:412–417. https://doi.org/10.1016/j.jep.2006.03.034
Tabei Y, Kitade S, Nishizawa Y, Kikuchi N, Kayano T, Hibi T, Akutsu K (1998) Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea). Plant Cell Rep 17:159–164. https://doi.org/10.1007/s002990050371
Tobimatsu Y, Schuetz M (2019) Lignin polymerization: how do plants manage the chemistry so well? Curr Opin Biotechnol 56:75–81. https://doi.org/10.1016/j.copbio.2018.10.001
Vanholme R, Demedts BB, Morreel K, Ralph J, Boerjan W (2010) Lignin biosynthesis and structure. Plant Physiol 153:895–905. https://doi.org/10.1104/pp.110.155119
Wang GF, Shi LP, Ren YD, Liu QF, Liu HF, Zhang RJ, Li Z, Zhu FH, He PL, Tang W (2009) Anti-hepatitis B virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro. Antiviral Res 83:186–190. https://doi.org/10.1016/j.antiviral.2009.05.002
Wang J, Zhu ML, Wei ZM (2008) Expression of cotton laccase gene in transgenic Populus alba var. pyramidalis and its effect on lignin synthesis. J Mol Cell Biol 1:11–18 ((in Chinese))
Yang C, Liang Y, Qiu D, Zeng H, Yuan J, Yang X (2018) Lignin metabolism involves Botrytis cinerea BcGs1- induced defense response in tomato. BMC Plant Biol 18:103. https://doi.org/10.1186/s12870-018-1319-0
Yang Q, He Y, Kabahuma M, Chaya T, Kelly A, Borrego E, Bian Y, El Kasmi F, Yang L, Teixeira P, Kolkman J, Nelson R, Kolomiets M, Dangl JL, Wisser R, Caplan J, Li X, Lauter N, Balint-Kurti P (2017) A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens. Nature Genet 49:1364. https://doi.org/10.1038/ng.3919
Yen GC, Hsieh CL (2000) Reactive oxygen species scavenging activity of Du-zhong (Eucommia ulmoides oliv.) and its active compounds. J Agric Food Chem 48:3431–3436. https://doi.org/10.1021/jf000150t
Zhang Y, Wang X, Rong W, Yang J, Li Z, Wu L, Zhang G, Ma Z (2017) Histochemical analyses reveal that stronger intrinsic defenses in Gossypium barbadense than in G. hirsutum are associated with resistance to Verticillium dahliae. Mol Plant-Microbe Interact 30:984. https://doi.org/10.1094/MPMI-03-17-0067-R
Zhang Y, Wu L, Wang X, Chen B, Zhao J, Cui J, Li Z, Yang J, Wu L, Wu J, Zhang G, Ma Z (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. https://doi.org/10.1111/mpp.12755
Zhao Q, Nakashima J, Chen F, Yin Y, Fu C, Yun J, Shao H, Wang X, Wang ZY, Dixon RA (2013) Laccase is necessary and nonredundant with peroxidase for lignin polymerization during vascular development in Arabidopsis. Plant Cell 25:3976–3987. https://doi.org/10.1105/tpc.113.117770
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31870285, 31660076), the Construction Program of Biology First-class Discipline in Guizhou (GNYL [2017] 009), the Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region Ministry of Education (MOELP-201803), and Guizhou Province High-level Innovative Talent Training Program Project ([2016]4003).
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YCZ and DGZ conceived and designed the experiments; YCZ, YQL, XD and JJL performed the experiments and analyzed the data; YQL wrote the paper.
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Zhao, Y., Liu, Y., Dong, X. et al. Identification of a novel laccase gene EuLAC1 and its potential resistance against Botrytis cinerea. Transgenic Res 31, 215–225 (2022). https://doi.org/10.1007/s11248-022-00297-8
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DOI: https://doi.org/10.1007/s11248-022-00297-8