Stem lodging Resistance-1 controls stem strength by positively regulating the biosynthesis of cell wall components in Capsicum annuum L.

Abstract

Lodging presents a significant challenge in cultivating high-yield crops with extensive above-ground biomass, yet the molecular mechanisms underlying this phenomenon in the Solanaceae family remain largely unexplored. In this study, we identified a gene, CaSLR1 (Capsicum annuum Stem Lodging Resistance 1 ), which encodes a MYELOBLASTOSIS (MYB) family transcription factor, from a lodging- affected C. annuum EMS mutant. The suppression of CaSLR1 expression in pepper led to notable stem lodging, reduced thickness of the secondary cell wall, and decreased stem strength. A similar phenotype was observed in tomato with the knockdown of SlMYB61, the orthologous gene to CaSLR1. Further investigations demonstrated that CaNAC6, a gene involved in secondary cell wall (SCW) formation, is co-expressed with CaSLR1 and acts as a positive regulator of its expression, as confirmed through yeast one-hybrid, dual-luciferase reporter assays, and electrophoretic mobility shift assays. These findings elucidate the Ca NAC6- Ca SLR1 module that contributes to lodging resistance, emphasizing the critical role of CaSLR1 in the lodging resistance regulatory network.

Acknowledgements

We thank Dr Xingyao Xiong and Dr. Feng Liu for their helpful discussions on this manuscript. This research was supported by the National Natural Science Foundation of China (32172584), the Natural Science Foundation of Hunan Province (2021JJ30339), the Hunan Provincial Innovation Foundation for Postgraduate (CX20200655), and the National Natural Science Foundation of China (32002040).

Author contributions

Q.L. performed experiments and analysed data; C.F., H.H., and Q.X. performed RNA-seq and RT-qPCR; B.Y. constructed F₂ population; H.Y. analysed Mutmap sequencing data; W.M., R.L., W.C., and Z.Z. performed field management; Q.L. and B.H. wrote the paper; Z.Z. and L.O. directed the project.

Data availability

BSA-seq and RNA-seq data generated in this study are available at the NCBI. BSA-seq data bioproject accession: PRJNA1115904; VIGS RNA-seq data bioproject accession: PRJNA1115904; WT and slr1 RNA-seq data bioproject accession: PRJNA1113712.

Conflict of interest statement

The authors declare that they have no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Li Q, Fu C, Liang C. et al. Crop lodging and the roles of lignin, cellulose, and hemicellulose in lodging resistance. Agronomy. 2022; 12 :1795

[2] Liu S, Huang Y, Xu H. et al. Genetic enhancement of lodging resistance in rice due to the key cell wall polymer lignin, which affects stem characteristics. Breed Sci. 2018; 68 :508-15

[3] Coen E, Cosgrove DJ. The mechanics of plant morphogenesis. Science. 2023; 379 :eade8055

[4] Zhang Y, Yu J, Wang X. et al. Molecular insights into the com- plex mechanics of plant epidermal cell walls. Science. 2021; 372 : 706-11

[5] Fan CF, Li Y, Hu Z. et al. Ectopic expression of a novel OsExtensin- like gene consistently enhances plant lodging resistance by reg- ulating cell elongation and cell wall thickening in rice. Plant Biotechnol J. 2018; 16 :254-63

[6] Li CH, Luo YL, Jin M. et al. Response of lignin metabolism to light quality in wheat population. Front Plant Sci. 2021; 12

[7] Nakano Y, Yamaguchi M, Endo H. et al. NAC-MYB-based tran- scriptional regulation of secondary cell wall biosynthesis in land plants. Front Plant Sci. 2015; 6 :288

[8] Zhong R, Lee C, McCarthy RL. et al. Transcriptional acti- vation of secondary wall biosynthesis by rice and maize NAC and MYB transcription factors. Plant Cell Physiol. 2011; 52 : 1856-71

[9] Fang S, Shang X, Yao Y. et al. NST-and SND-subgroup NAC pro- teins coordinately act to regulate secondary cell wall formation in cotton. Plant Sci. 2020; 301 :110657

[10] Zhong R, Ye ZH. The Arabidopsis NAC transcription factor NST2 functions together with SND1 and NST1 to regulate secondary wall biosynthesis in fibers of inflorescence stems. Plant Signal Behav. 2015; 10 :e989746

[11] Zhong R, Richardson EA, Ye ZH. The MYB46 transcription factor is a direct target of SND1 and regulates secondary wall biosyn- thesis in Arabidopsis. Plant Cell. 2007; 19 :2776-92

[12] Negi S, Tak H, Ganapathi T. Native vascular related NAC tran- scription factors are efficient regulator of multiple classes of secondary wall associated genes in banana. Plant Sci. 2017; 265 : 70-86

[13] Zhong R, Kandasamy MK, Ye ZH. XND1 regulates secondary wall deposition in xylem vessels through the inhibition of VND functions. Plant Cell Physiol. 2021; 62 :53-65

[14] Zhong R, Lee C, Haghighat M. et al. Xylem vessel-specific SND5 and its homologs regulate secondary wall biosynthesis through activating secondary wall NAC binding elements. New Phytol. 2021; 231 :1496-509

[15] Liu F, Yu H, Deng Y. et al. PepperHub, an informatics hub for the chili pepper research community. Mol Plant. 2017; 10 :1129-32

[16] Liu H, Ding YD, Zhou YQ. et al. CRISPR-P 2.0: an improved CRISPR-Cas 9 tool for genome editing in plants. Mol Plant. 2017; 10 :530-2

[17] Zou X, Ma Y, Dai X. et al. Spread and industry development of pepper in China. Acta Horticul Sin. 2020; 47 :1715-26

[18] Yang B, Zhou S, Ou L. et al. Construction of mutant population and analysis of dwarf mutants in "6421" (Capsicum annuum L.) through EMS mutagenesis. Agric Sci Technol. 2016; 17 :1322

[19] Arisha MH, Shah SN, Gong ZH. et al. Ethyl methane sulfonate induced mutations in M2 generation and physiological varia- tions in M1 generation of peppers (Capsicum annuum L.). Front Plant Sci. 2015; 6 :399

[20] Liu F, Zhao JT, Sun HH. et al. Genomes of cultivated and wild capsicum species provide insights into pepper domestication and population differentiation. Nat Commun. 2023; 14 :5487

[21] Takagi H, Tamiru M, Abe A. et al. MutMap accelerates breeding of a salt-tolerant rice cultivar. Nat Biotechnol. 2015; 33 :445-9

[22] Zong Y, Song QN, Li C. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat Biotechnol. 2018; 36 :950-3

[23] Chen S, Zhou Y, Chen Y. et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018; 34 :i884-90

[24] Li H, Durbin R. Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics. 2009; 25 :1754-60

[25] McKenna A, Hanna M, Banks E. et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20 :1297-303

[26] Chen X, Schulz-Trieglaff O, Shaw R. et al. Manta: rapid detec- tion of structural variants and indels for germline and cancer sequencing applications. Bioinformatics. 2016; 32 :1220-2

[27] Danecek P, McCarthy SA. BCFtools/csq: haplotype-aware variant consequences. Bioinformatics. 2017; 33 :2037-9

[28] Wang J, Shan Q, Yi T. et al. Fine mapping and candidate gene analysis of CaFCD1 affecting cuticle biosynthesis in Capsicum annuum L. Theor Appl Genet. 2023; 136 :46

[29] Wang W, Li Y, Cai C. et al. Auxin response factors fine-tune lignin biosynthesis in response to mechanical bending in bamboo. New Phytol. 2023; 241 :1161-76

[30] Rozewicki J, Li S, Amada KM. et al. MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Res. 2019; 47 :W5-10

[31] Nguyen LT, Schmidt HA, Von Haeseler A. et al. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum- likelihood phylogenies. Mol Biol Evol. 2015; 32 :268-74

[32] Liu ZY, Hou SG, Rodrigues O. et al. Phytocytokine signalling reopens stomata in plant immunity and water loss. Nature. 2022; 605 :332-9

[33] Zhang JW, Wang Y, Shu X. et al. Magnetic chitosan hydro- gel induces neuronal differentiation of neural stem cells by activating ras-dependent signal cascade. Carbohydr Polym. 2023; 314 :120918

[34] Zhang P, Yan Y, Gu S. et al. Lodging resistance in maize: a function of root-shoot interactions. Eur J Agron. 2022; 132 :126393

[35] Zhang Z, Liu Y, Yuan Q. et al. The bHLH1-DTX35/DFR module regulates pollen fertility by promoting flavonoid biosynthesis in Capsicum annuum L. Hortic Res. 2022; 9 :uhac172

[36] Song JL, Sun BM, Chen CM. et al. An R-R-type MYB transcrip- tion factor promotes non-climacteric pepper fruit carotenoid pigment biosynthesis. Plant J. 2023; 115 :724-41

[37] Sun B, Zhou X, Chen C. et al. Coexpression network analysis reveals an MYB transcriptional activator involved in capsaici- noid biosynthesis in hot peppers. Hortic Res. 2020; 7 :162

[38] Ouyang B, Chen Y, Li H. et al. Transformation of tomatoes with osmotin and chitinase genes and their resistance to fusarium wilt. J Hortic Sci Biotechnol. 2005; 80 :517-22

[39] Modi A, Vai S, Caramelli D. et al. The Illumina Sequencing Protocol and the NovaSeq Mengoni A, Bacci G, Fondi M,eds. Bacterial Pangenomics. Methods in Molecular Biology. Vol. 2242. Humana: New York, 2021;15-42

[40] Saremi B, Gusmag F, Distl O. et al. A comparison of strategies for generating artificial replicates in RNA-seq experiments. Sci Rep. 2022; 12 :7170

[41] Qin C, Yu C, Shen Y. et al. Whole-genome sequencing of culti- vated and wild peppers provides insights into capsicum domesti- cation and specialization. Proc Natl Acad Sci. 2014; 111 :5135-40

[42] Pertea M, Kim D, Pertea GM. et al. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and ballgown. Nat Protoc. 2016; 11 :1650-67

[43] Shumate A, Wong B, Pertea G. et al. Improved transcriptome assembly using a hybrid of long and short reads with StringTie. PLoS Comput Biol. 2022; 18 :e1009730

[44] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15 :1-21

[45] Thawng CN, Smith GB. A transcriptome software comparison for the analyses of treatments expected to give subtle gene expression responses. BMC Genomics. 2022; 23 :452

[46] Xie Y, Liu Y, Ma M. et al. Arabidopsis FHY3 and FAR1 integrate light and strigolactone signaling to regulate branching. Nat Commun. 2020; 11 :1955

[47] Huang D, Wang S, Zhang B. et al. A gibberellin-mediated DELLA- NAC signaling cascade regulates cellulose synthesis in rice. Plant Cell. 2015; 27 :1681-96

[48] Zhang D, Xu Z, Cao S. et al. An uncanonical CCCH-tandem zinc- finger protein represses secondary wall synthesis and controls mechanical strength in rice. Mol Plant. 2018; 11 :163-74

[49] Jan A, Yang GX, Nakamura H. et al. Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice. Plant Physiol. 2004; 136 :3670-81

[50] Ahmad I, Meng XP, Kamran M. et al. Effects of uniconazole with or without micronutrient on the lignin biosynthesis, lodging resistance, and winter wheat production in semiarid regions. J Integr Agric. 2020; 19 :62-77

[51] Jamet E, Dunand C. Plant cell wall proteins and development. Int J Mol Sci. 2020; 21 :2731

[52] Seveso A, Mazurkewich S, Banerjee S. et al. Polysaccharide uti- lization loci from Bacteroidota encode CE15 enzymes with pos- sible roles in cleaving pectin-lignin bonds. Appl Environ Microbiol. 2024; 90 :e01768-23

[53] Zhu Y, Wang Y, Jiang H. et al. Transcriptome analysis reveals that PbMYB61 and PbMYB308 are involved in the regulation of lignin biosynthesis in pear fruit stone cells. Plant J. 2023; 116 :217-33

[54] Romano JM, Dubos C, Prouse MB. et al. AtMYB61 , an R2R3-MYB transcription factor, functions as a pleiotropic regulator via a small gene network. New Phytol. 2012; 195 :774-86

[55] Penfield S, Meissner RC, Shoue DA. et al. MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat. Plant Cell. 2001; 13 :2777-91

[56] Xiong HC, Guo HJ, Fu MY. et al. A large-scale whole-exome sequencing mutant resource for functional genomics in wheat. Plant Biotechnol J. 2023; 21 :2047-56

[57] Lesa GM. Isolation of Caenorhabditis elegans gene knockouts by PCR screening of chemically mutagenized libraries. Nat Protoc. 2006; 1 :2231-40

[58] Krasileva KV, Vasquez-Gross HA, Howell T. et al. Uncovering hidden variation in polyploid wheat. Proc Natl Acad Sci USA. 2017; 114 :E913-21

[59] Zhang Q, Luo F, Zhong Y. et al. Modulation of NAC transcrip- tion factor NST 1 activity by XYLEM NAC DOMAIN1 regulates secondary cell wall formation in Arabidopsis. J Exp Bot. 2020; 71 : 1449-58

[60] Xiao C, Anderson CT. Interconnections between cell wall polymers, wall mechanics, and cortical microtubules: teas- ing out causes and consequences. Plant Signal Behav. 2016; 11 : 234-49

[61] Gao Y, Xu Z, Zhang L. et al. Identification of traits and genes asso- ciated with lodging resistance in maize. Crop J. 2020; 9 :1408-17

[62] Hennet L, Berger A, Trabanco N. et al. Transcriptional regulation of sorghum stem composition: key players identified through co-expression gene network and comparative genomics analy- ses. Front Plant Sci. 2020; 11 :224

[63] Wang Z, Mao Y, Guo Y. et al. MYB transcription factor161 mediates feedback regulation of secondary wall-associated NAC-Domain1 family genes for wood formation. Plant Physiol. 2020; 184 :1389-406

[64] Xiao R, Zhang C, Guo X. et al. MYB transcription factors and its regulation in secondary cell wall formation and lignin biosynthesis during xylem development. Int J Mol Sci. 2021; 22 : 3560

[65] Zhou J, Lee C, Zhong R. et al. MYB58 and MYB63 are tran- scriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell. 2009; 21 : 248-66

[66] Polko JK, Kieber JJ. The regulation of cellulose biosynthesis in plants. Plant Cell. 2019; 31 :282-96

[67] Blaschek L, Murozuka E, Serk H. et al. Different combinations of laccase paralogs nonredundantly control the amount and composition of lignin in specific cell types and cell wall layers in Arabidopsis. Plant Cell. 2023; 35 :889-909

[68] Hu BW, Li DW, Liu X. et al. Engineering non-transgenic Gynoe- cious cucumber using an improved transformation protocol and optimized CRISPR/Cas9 system. Mol Plant. 2017; 10 :1575-8

[69] Favero D. S. 2020. A Chloroplast-Derived Signal Attenuates Growth in Red Light by Acting on the phyB-PIF Pathway. Plant Physiology. 183 :1408-9