A transcription factor, PbWRKY24, contributes to russet skin formation in pear fruits by modulating lignin accumulation

Abstract

Skin color is one of the major traits of fruit appearance quality in pear (Pyrus) that affects the fruit commodity value. Russet skin protects pear fruits from environmental stresses and its formation process is closely linked to lignin accumulation. However, the molecular regulatory networks underlying russet skin formation in pear fruits involve complex secondary metabolic pathways and remain elusive. Here, we explored the regulatory mechanisms underlying lignin accumulation in pear skin based on transcriptome sequencing, co-expression network analysis, and gene expression profiling. We identified a WRKY transcription factor gene, PbWRKY24, that regulates russet skin formation in pear fruits. The relative expression of PbWRKY24 in russet pear skin was significantly correlated with lignin content. We then verified the function of PbWRKY24 in lignin accumulation via genetic transformation. DNA affinity purification sequencing revealed that PbWRKY24 directly binds to the promoter of a lignin biosynthesis gene, PbPRX4. This binding was confirmed by yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays. Overexpression of PbPRX4 in pear skin stimulated lignin accumulation and consequently promoted russet skin formation. This study provides a glimpse into the intricate lignin biosynthesis mechanisms during russet skin formation in pear fruits, which is of practical significance to pear breeding for fruit quality.

Acknowledgements

This research was supported by the Funds for the Shandong Provincial Natural Science Foundation, China (ZR2021MC186 and ZR2020QC146) and the Modern Agricultural Industry Technology System in Shandong Province, China (SDAIT-06-06).

Author contributions

C.M. conceived and supervised the research. J.W., D.W., and R.Z. conducted most of the experiments and data analysis. M.Y., X.Z., Z.S., X.L., Y.T., and C.W. participated in data collection, analysis, and preservation. C.M. and J.W. drafted and revised the manuscript. All authors contributed to writing and editing the final manuscript.

Data availability

All data supporting the findings are available within the paper or from the corresponding author upon request. The RNA sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) SRA database under accession number PRJNA948064.

Conflict of interest statement

The authors declare that they have no conflicts of interest associated with this work.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Zhang J, Zhang Y, Zhang P. et al. An integrated metabolic and transcriptomic analysis reveals the mechanism through which fruit bagging alleviates exocarp semi-russeting in pear fruit. Tree Physiol. 2021;41:1306-18

[2] Yamamoto T, Terakami S, Takada N. et al. Identification of QTLs controlling harvest time and fruit skin color in Japanese pear (Pyrus pyrifolia Nakai). Breed Sci. 2014;64:351-61

[3] Wang Y, Dai M, Cai D. et al. Proteome and transcriptome profile analysis reveals regulatory and stress-responsive networks in the russet fruit skin of sand pear. Hortic Res. 2020;7:16

[4] Schuetz M, Benske A, Smith RA. et al. Laccases direct lignification in the discrete secondary cell wall domains of protoxylem. Plant Physiol. 2014;166:798-807

[5] He J, Liu Y, Yuan D. et al. An R2R3 MYB transcription factor confers brown planthopper resistance by regulating the pheny-lalanine ammonia-lyase pathway in rice. Proc Natl Acad Sci USA. 2020;117:271-7

[6] Heng W, Liu L, Wang M. et al. Differentially expressed genes related to the formation of russet fruit skin in a mutant of ‘Dangshansuli’ pear (Pyrus bretchnederi Rehd.) determined by suppression subtractive hybridization. Euphytica. 2014;196:285-97

[7] Shi C, Qi B, Wang X. et al. Proteomic analysis of the key mecha-nism of exocarp russet pigmentation of semi-russet pear under rainwater condition. Sci Hortic. 2019;254:178-86

[8] Xue C, Yao J, Xue Y. et al. PbrMYB 169 positively regulates lignification of stone cells in pear fruit. JExp Bot. 2019;70:1801-14

[9] Lam PY, Tobimatsu Y, Takeda Y. et al. Disrupting flavone syn-thase II alters lignin and improves biomass digestibility. Plant Physiol. 2017;174:972-85

[10] Wang Y, Dai M, Zhang S. et al. Exploring candidate genes for pericarp russet pigmentation of sand pear (Pyrus pyrifolia)via RNA-Seq data in two genotypes contrasting for pericarp color. PLoS One. 2014;9:e83675

[11] Zhu X, Jiang L, Cai Y. et al. Functional analysis of four Class III peroxidases from Chinese pear fruit: a critical role in lignin polymerization. Physiol Mol Biol Plants. 2021;27:515-22

[12] Zhang J, Liu Y, Li C. et al. PtomtAPX is an autonomous lig-nification peroxidase during the earliest stage of secondary wall formation in Populus tomentosa Carr. Nat Plants. 2022;8:828-39

[13] Yeh SY, Huang FC, Hoffmann T. et al. FaPOD 27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.). Front Plant Sci. 2014;5:518

[14] Zhao X, Wang Q, Wang D. et al. PagERF 81 regulates lignin biosyn-thesis and xylem cell differentiation in poplar. J Integr Plant Biol. 2023;65:1134-46

[15] Wu X, Wang Q, Wang Y. et al. Multi-omics analysis of green-and russet skin pear cultivars identify key regulators of skin russeting. Sci Hortic. 2023;318:112116

[16] Viana VE, Busanello C, da Maia LC. et al. Activation of rice WRKY transcription factors: an army of stress fighting soldiers? Curr Opin Plant Biol. 2018;45:268-75

[17] Mahiwal S, Pahuja S, Pandey GK. Review: structural-functional relationship of WRKY transcription factors: unfolding the role of WRKY in plants. Int J Biol Macromol. 2024;257:128769

[18] Hu Q, Xiao S, Wang X. et al. GhWRKY1-like enhances cotton resistance to Verticillium dahliae via an increase in defense-induced lignification and S monolignol content. Plant Sci. 2021;305:110833

[19] Fan H, Shen X, Ding Y. et al. DkWRKY transcription factors enhance persimmon resistance to Colletotrichum horii by promot-ing lignin accumulation through DkCAD 1 promotor interaction. Stress Biol. 2024;4:17

[20] Li D, Li X, Wang Z. et al. Transcription factors RhbZIP17 and RhWRKY30 enhance resistance to Botrytis cinerea by increasing lignin content in rose petals. JExp Bot. 2024;75:1633-46

[21] Hou Y, Yu X, Chen W. et al. MdWRKY75e enhances resistance to Alternaria alternata in Malus domestica. Hortic Res. 2021;8:225

[22] Wang Z, Liu S, Huo W. et al. Transcriptome and metabolome analyses reveal phenotype formation differences between rus-set and non-russet apples. Front Plant Sci. 2022;13:1057226

[23] Zhang J, Liu Z, Zhang Y. et al. PpyMYB 144 transcriptionally regulates pear fruit skin russeting by activating the cytochrome P450 gene PpyCYP86B1. Planta. 2023;257:69

[24] Legay S, Guerriero G, Deleruelle A. et al. Apple russeting as seen through the RNA-seq lens: strong alterations in the exocarp cell wall. Plant Mol Biol. 2015;88:21-40

[25] Wang Y, Zhang S, Dai M. et al. Pigmentation in sand pear (Pyrus pyrifolia) fruit: biochemical characterization, gene discovery and expression analysis with exocarp pigmentation mutant. Plant Mol Biol. 2014;85:123-34

[26] Quan M, Du Q, Xiao L. et al. Genetic architecture underlying the lignin biosynthesis pathway involves noncoding RNAs and transcription factors for growth and wood properties in Populus. Plant Biotechnol J. 2019;17:302-15

[27] Wang H, Chen W, Xu Z. et al. Functions of WRKYs in plant growth and development. Trends Plant Sci. 2023;28:630-45

[28] Xian P, Yang Y, Xiong C. et al. Overexpression of GmWRKY 172 enhances cadmium tolerance in plants and reduces cad-mium accumulation in soybean seeds. Front Plant Sci. 2023;14:1133892

[29] Davin LB, Jourdes M, Patten AM. et al. Dissection of lignin macromolecular configuration and assembly: comparison to related biochemical processes in allyl/propenyl phenol and lig-nan biosynthesis. Nat Prod Rep. 2008;25:1015-90

[30] Fernández-Pérez F, Vivar T, Pomar F. et al. Peroxidase 4 is involved in syringyl lignin formation in Arabidopsis thaliana. J Plant Physiol. 2015;175:86-94

[31] Ma C, Wang X, Yu M. et al. PpMYB 36 encodes a MYB-type tran-scription factor that is involved in russet skin coloration in pear (Pyrus pyrifolia). Front Plant Sci. 2021;12:776816

[32] Mortazavi A, Williams BA, McCue K. et al. Mapping and quan-tifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008;5:621-8

[33] Ma C, Jing C, Chang B. et al. The effect of promoter methylation on MdMYB 1 expression determines the level of anthocyanin accumulation in skins of two non-red apple cultivars. BMC Plant Biol. 2018;18:108

[34] Li R, Yu C, Li Y. et al. SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics. 2009;25:1966-7

[35] Trapnell C, Roberts A, Goff L. et al. Differential gene and tran-script expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562-78

[36] Kim D, Paggi JM, Park C. et al. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37:907-15

[37] Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B. 1995;57:289-300

[38] Huang da W, Sherman BT, Lempicki RA. Systematic and inte-grative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4:44-57

[39] Kanehisa M, Araki M, Goto S. et al. KEGG for linking genomes to life and the environment. Nucleic Acids Res. 2008;36:D480-4

[40] Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9:559

[41] Ren L, Zhu B, Zhang Y. et al. The research of applying primer premier 5.0 to design PCR primer. J Jinzhou Med Coll. 2004;25:43-6

[42] Alves Oliveira D, Tang JD, Warburton ML. Reference gene selec-tion for RT-qPCR analysis in maize kernels inoculated with Aspergillus flavus. Toxins (Basel). 2021;13:386

[43] Wang C, Bian C, Li J. et al. Melatonin promotes Al3+ compartmen-talization via H⁺ transport and ion gradients in Malus hupehensis. Plant Physiol. 2023;193:821-39

[44] Jia D, Wu P, Shen F. et al. Genetic variation in the promoter of an R2R3-MYB transcription factor determines fruit malate content in apple (Malus domestica Borkh.). Plant Physiol. 2021;186:549-68

[45] Langmead B, Salzberg SL.Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357-9

[46] Zhang Y, Liu T, Meyer CA. et al.Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008;9:R137

[47] Wang H, Zhang S, Fu Q. et al. Transcriptomic and metabolomic analysis reveals a protein module involved in preharvest apple peel browning. Plant Physiol. 2023;192:2102-22

[48] Dang Q, Sha H, Nie J. et al. An apple (Malus domestica) AP2/ERF transcription factor modulates carotenoid accumulation. Hortic Res. 2021;8:223

[49] Yao W, Zhang D, Zhou B. et al. Over-expression of poplar NAC 15 gene enhances wood formation in transgenic tobacco. BMC Plant Biol. 2020;20:12

[50] Li M, Cheng C, Zhang X. et al. PpNAC 187 enhances lignin syn-thesis in ‘Whangkeumbae’ pear (Pyrus pyrifolia) ‘hard-end’fruit. Molecules. 2019;24:4338