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 CaNAC6-CaSLR1 module that contributes to lodging resistance, emphasizing the critical role of CaSLR1 in the lodging resistance regulatory network.


Introduction
Stems are essential for plant growth and development, as they not only facilitate the transport of water and nutrients but also provide critical structural support.Stem lodging can drastically affect crop production, significantly reducing yield and quality while increasing harvesting costs [1].Consequently, research into stem lodging resistance (SLR) is vital for advancing sustainable agricultural practices.Many studies have shown that lodging resistance is closely related to the thickness of the secondary cell wall (SCW) [1,2].Cellulose, hemicellulose and lignin are the main components of the cell wall, enhancing the mechanical strength of plants [3,4].In the brittle stem mutant of rice, there was a notable decrease in the stem's mechanical strength and its cellulose content [5].Lignin is considered an important factor in lodging resistance across different varieties [6].Enhancing the expression of cell wall structural genes like 4-coumarate: CoA ligase 3 (4CL3) and phenylalanine ammonia-lyase (PAL) within the lignin biosynthesis pathways bolstered cell wall thickening, thus reinforcing mechanical support in rice [2].Therefore, stem cell wall components play critical roles in enhancing mechanical strength and maintaining stability in stems.
Previous studies have shown that secondary wall NACs (SWNs) transcription factors and MYB transcription factors (e.g., MYB46, MYB83) mediated transcription networks played key regulatory roles in plant SCW formation [7,8].Studies on cotton and Arabidopsis thaliana have showed that concurrent disruption of secondary wall-associated NAC domain protein 1 (SND1) and NAC secondary wall thickening promoter 1 (NST1) results in stem droop, indicating functional redundancy among NAC transcription factors in stem development [9,10].Notably, inhibiting AtMYB46 alone can lead to a stem droop phenotype [11], suggesting that these MYB TFs play a crucial role in regulating stalk development.SWNs bind to specific promoter sequences, known as secondary NAC binding elements (SNBEs), in MYB46 and MYB83, thereby regulating SCW biosynthesis [12][13][14].Nonetheless, our understanding of the pivotal role MYB TFs play in regulating plant cell wall development remains limited.
Pepper (Capsicum annuum L.) is an important vegetable crop due to its distinctive spicy components [15][16][17].However, during production, pepper often faces challenges such as f lower and fruit drop after stem lodging, which severely impairs pepper's yield and quality [1].Despite its importance, research on stem lodging resistance in peppers is notably lacking.In this study, we identified a distinct stem lodging mutant slr1 (stem lodging resistance 1) through ethylmethane sulfonate (EMS) mutagenesis.Genetic analysis revealed that theslr1 phenotype is likely governed by a recessive gene located on chromosome 8, encoding MYB61.Knock down of CaSLR1 resulted in stem bending and lodging, a phenotype was also observed in CRISPR/Cas9-mediated knockout of SlMYB61, a homolog of CaSLR1 in tomato.Additionally, knockdown CaSLR1 led to the downregulation of numerous genes associated with cell wall formation, consequently causing thinner secondary cell walls and reduced stem strength.Furthermore, we identified a SCW formation-related gene, CaNAC6, co-expressed with CaSLR1, with CaNAC6 positively regulating the expression of CaSLR1.This suggests that CaSLR1 may serve as a central hub in the secondary cell wall (SCW) regulatory network, enhancing stem lodging resistance in pepper.Our study provides theoretical support for understanding the molecular regulatory mechanisms in stem development.It offers the target genes for molecular designing breeding programs aimed at enhancing lodging resistance in peppers and tomatoes.

Phenotypic characteristics of slr1
To explore the phenotypic characteristics of stem lodging development in slr1, we compared the growth parameters of WT and slr1 mutants.At 45 days, all slr1 mutant plants exhibited evident stem lodging and twisting compared to the WT, whereas this lodging phenomenon was not observable at 25 days of seedling growth (Fig. 1a and b; Fig. S1a, see online supplementary material).Notably, the slr1mutant exhibited abnormal reproductive development with delayed first f lower and fruit set, accompanied by a significant reduction in the number of fruits and germination rate (Fig. S1b-d, see online supplementary material).Furthermore, we compared the growth differences between 45-day-old WT and slr1 mutants.Compared to WT, the aboveground, root, total fresh weights, and first internode length of slr1 mutants were significantly reduced by 33.38, 40.82, 37.11, and 50%, respectively (Fig. S1e-h, see online supplementary material).However, no significant difference was observed in the leaf length-width ratio (Fig. S1i, see online supplementary material).Since cellulose, hemicellulose, and lignin are critical components in lodging resistance [3,4], the contents of cell components in the stems of WT and slr1 were measured.Compared to WT, the cellulose, hemicellulose, lignin, and crude fiber contents of slr1 mutants significantly decreased by 49.75, 36.81,24.36, and 21.89%, respectively (Fig. 1c-f).
In addition, SEM was performed to compare the difference in stem structure in WT and slr1 mutant.The results revealed that the hollow vessels within the xylem of the slr1 mutant was obvious presence, whereas the WT exhibited more xylem fiber cells (XFCs) (Fig. 1g).Conversely, in the WT, XFCs with thickened SCWs were observed at the periphery of secondary xylem, while parenchymalike cells (PCs) were prevalent in the corresponding tissues of slr1 (Fig. 1h).These findings suggest that the abnormal development of xylem led to stem lodging in the slr1 mutant.

Identification of the target gene in slr1 mutant
To map the target genes associated with stem lodging, we created an F 2 segregating population with slr1 as the female parent and WT as the male parent for crossbreeding.We obtained a total of 483 F 2 individuals, comprising 402 wild-type phenotypes and 81 mutant phenotypes.The Chi-square test revealed that the F 2 population significantly deviated from the Mendelian 3:1 segregation ratio (Table S2, see online supplementary material).To preliminarily map genes related to stem lodging, we conducted Mutmap analysis using the parents and two mixed DNA pools from the F 2 population (Table S3, see online supplementary material).Based on the identified homozygous SNP loci, the frequency of allele gene SNPs was assessed by analysing DNA sequencing variations, and ΔSNP index values were obtained.A distinct peak on chromosome 8 was observed (126314622-165 820 095; approximately 39.50 Mb), indicating significant differences in the ΔSNP index (Fig. 2a).Further, a series of KASP (Competitive Allele-Specific PCR) markers were developed using SNPs information from the candidate region for genotyping (Table S1, see online supplementary material).Next, based on phenotypic data of individual plants from the F 2 population, we performed genotyping to identify candidate region.The result indicated that the candidate region was located in SNP153164345 and SNP154539577 markers, with a physical distance of about 1.37 Mb. (Fig. 2b; Table S4, see online supplementary material).

CaSLR1 mediates stem lodging by affecting SCW thickness and stem strength in peppers and tomatoes
MYB61 has been identified as a regulator inf luencing cellulose and SCW formation [47].Consequently, CaZ8g15240 was recognized as a crucial candidate gene and designated CaSLR1.Utilizing VIGS technology to silence CaSLR1 in WT plants resulted in noticeable twisting and lodging characteristics in their stems (Fig. 3a).RT-qPCR analysis confirmed a significant downregulation of CaSLR1 expression in the stems, validating the successful silencing of CaSLR1 (Fig. 3b).These findings elucidated the role of CaSLR1 in mediating stem lodging development in pepper.
Given that SCW component biosynthesis affects plant mechanical support [3,4], SEM analysis of the stems from CaSLR1-silenced and control (pTRV2) plants (Fig. 3c) revealed a denser mechanical structure in the xylem of control plants, whereas the mechanical tissue of the xylem in CaSLR1-silenced plants exhibited cell wall collapse (Fig. 3d).Particularly, the cell walls of xylem fiber cells in CaSLR1-silenced plants appeared thinner compared to controls (Fig. 3e), indicating that silencing CaSLR1 leads to differences in xylem development.Moreover, the contents of the SCW components were significantly reduced (Fig. 3f).Stem strength analysis demonstrated that control plants exhibited a puncture strength of 1222.97 mg, whereas CaSLR1-silenced plants could withstand only 501.47 mg (Fig. 3g), indicating a reduction in stem strength due to the thinning of SCW.To uncover the functional conservation of CaSLR1, we used the CRISPR/Cas9 system to knock out SlMYB61 (Solyc01g102340), a homolog of CaSLR1, in tomatoes.Sequencing of the PCR product from the SlMYB61 knockout (Cri-SlMYB61) lines revealed a seven-base edit at the target site (Fig. 3h).The Cri-SlMYB61 lines were self-crossed to obtain a stable genetic T 1 generation, referred to as Cri-SlMYB61-T 1 .Compared to the wild-type (NC-T 1 ), Cri-SlMYB61-T 1 plants exhibited stem lodging (Fig. 3i).Additionally, the Cri-SlMYB61-T 1 plants had fewer root branches (Fig. 3j) and shorter inf lorescence branches (Fig. 3k) compared to NC-T 1 .
SEM analysis of cross-sectioned stems from NC-T 1 and Cri-SlMYB61-T 1 plants revealed significant differences.The pith (Pi) area in Cri-SlMYB61-T 1 was enlarged compared to NC-T 1 (Fig. 3l), with stems from the latter containing abundant and well-developed granular plastids, while those from Cri-SlMYB61-T 1 exhibited abnormal development (Fig. 3m).Moreover, the secondary cell walls (SCWs) of xylem fiber cells (XFCs)   in NC-T 1 were thicker, whereas those in Cri-SlMYB61-T 1 were thinner (Fig. 3n), indicating that the edited SlMYB61 affected stem structure, resulting in thinner SCWs in XFCs in tomatoes.These results suggest that SlMYB61 knockout in tomatoes leads to alterations in plant structure, notably in stem strength and stability, indicating a conserved function of SLR1 in both peppers and tomatoes.

Subcellular localization and expression pattern of CaSLR1
A preliminary exploration of the function of CaSLR1, according to annotation information, revealed that CaSLR1 belongs to a typical MYB family with 1796 bp in length, consisting of three exons and two introns, and with a 1263 bp long CDS, encoding 421 amino acids (Fig. S2a, see online supplementary material).CaSLR1 (protein ID:XP_016538869) and SlMYB61 (protein ID: XP_004230371) were identified to be the most closely related based on the comparative analysis of a phylogenetic tree.Interestingly, the homologous proteins of CaSLR1 were identified from other species such as Mus musculus, Saccharomyces cerevisiae S288C, Caenorhabditis elegans, Chlamydomonas reinhardtii, Oryza sativa, Nicotiana tabacum, A. thaliana (Fig. S2b, see online supplementary material), indicating the evolutionary conservatism of SLR1.CaPIF3, CaFSCB, CaCE, and CaFXO are species-specific, lacking homologous genes in both animals and microorganisms (Fig. S3, see online supplementary material).Further, the sequence alignment analysis showed that two SANT/MYB domains in SLR1 were highly consistent between C. annuum (CaSLR1) and S. lycopersicum (SlMYB61), but they were slightly different among C. annuum (CaSLR1), O. sativa (OsMYB61), and A. thaliana (AtMYB61) (Fig. 4a), hinting at slightly different evolution.
To further elucidate the function of CaSLR1, subcellular localization was examined by transiently transforming Arabidopsis protoplasts with the 35S:CaSLR1-eGFP expression vector.Confocal microscopy revealed GFP f luorescence distributed in the cell membrane and nucleus.Co-localization analysis with the nuclear marker Ghd-RFP confirmed the presence of CaSLR1 in the nucleus (Fig. 4b), suggesting its role in gene regulation.
To investigate the organ-specific expression patterns of CaSLR1 in peppers, qRT-PCR analysis was conducted.The results revealed that the expression of CaSLR1 was highest in the stems, with levels 8.01, 138.80, 11.97, 4.06, and 98.27-fold higher compared to the roots, leaves, f lowers, fruits, and anthers, respectively, reaching significance (Fig. 4c).These results imply that CaSLR1 participates in regulating stem development.

RNA-seq analyses between WT and slr1
To delve into the molecular mechanisms underlying stem lodging in slr1 mutants, RNA-seq was conducted using stem samples from 25-and 45-day-old WT and slr1 mutants.After removing adaptor sequences, over 86% of clean reads were mapped to the reference genome 'Zunla_1', with a GC content of about 43% and a Q30 quality score exceeding 87% (Table S5, see online supplementary material).Differential expression analysis revealed 545 differentially expressed genes (DEGs) at 25 days and 1546 DEGs at 45 days in slr1 mutants compared to WT. Notably, 251 DEGs were common between the two time points (Fig. S4a, see online supplementary material).
The enrichment analysis of GO and KEGG uncovered that the DEGs were significantly enriched in pathways related to cell wall development, including plant-type SCW biogenesis and phenylpropanoid biosynthesis (Fig. S4b and c, Table S6, see online supplementary material).Protein-protein interaction (PPI) analysis showed that a regulatory network centered around CaSLR1 was formed during stem development (Fig. S4d, see online supplementary material).
To explore the co-expression patterns of CaSLR1, WGCNA was performed.The Principal Component Analysis model indicates a high consistency among the three repetitions (Fig. S5a, see online supplementary material).WGCNA revealed that 30 branches were clustered (Fig. S5b and c, see online supplementary material).Notably, the antiquewhite4 module, which encompassed the candidate gene CaSLR1 (Zunla_1 reference genomic gene ID: Capana08g001690), was delineated (Fig. 5a; Table S7, see online supplementary material).GO enrichment unveiled significant enrichment of genes within the antiquewhite4 module in various cellular components, particularly those related to SCW formation, plant-type SCW biogenesis, xylan O-acetyltransferase activity, and xylan biosynthetic process (Fig. 5b; Table S8, see online supplementary material).This highlights the potential role of CaSLR1 in orchestrating these crucial processes during stem development.

CaNAC6 binds to the promoter of CaSLR1 to affect the SCW biosynthetic pathway
NAC and MYB transcription factors (TFs) are recognized as the 'master switches' controlling SCW formation [8].Within the antiquewhite4 module, a CaNAC6 (Capana10g001138) TF was found to be co-expressed with CaSLR1 (Fig. S4d and Table S7, see online supplementary material).NAC29, a homologous protein of CaNAC6, has been implicated in regulating SCW deposition in rice [48].NAC TFs contribute to SCW biosynthesis by binding to SNBE elements that existed in MYB promoters [12].We identified three SNBEs elements within 2000 bp promoter in CaSLR1 (Fig. 6a).
To confirm the binding between CaNAC6 and the CaSLR1 promoter, a Y1H assay was conducted.Yeast cells were cotransformed with pAbAi-proSLR1 along with either pGADT7-NAC6 or pGADT7 vectors (NC) and cultured on SD/−Leu medium supplemented with 500 ng/mL of AbA.The results showed that the co-transformed pGADT7-NAC6 and pAbAi-proSLR1 were able to promote yeast cells' growth on the AbA selective medium, unlike the negative control cells (Fig. 6b).
Further, to confirm whether CaNAC6 could regulate CaSLR1 expression, we injected with 62SK-CaNAC6 and proSLR1-LUC into tobacco leaves via the LUC system.Compared to the negative control (62SK and proSLR1-LUC), the ratio of LUC to REN was 5.92-fold higher.However, substituting proSLR1-LUC with proSLR1mut-LUC resulted in a decreased ratio to 1.79, indicating a significant reduction in gene transcriptional activation (Fig. 6c-e).To demonstrate that CaNAC6 was directly bound to the three SNBE elements of CaSLR1, an EMSA assay was conducted using purified CaNAC6 recombinant protein fused with a GST tag (Fig. S6, see online supplementary material).The finding revealed that CaNAC6 was bound to the biotin-labeled SNBE1, 2, and 3 probes, with binding strength inversely proportional to the concentrations of competitor probes.However, mutation of the SNBEs core sequence abolished the binding, indicating the specificity of the interaction (Fig. 6f).Our results suggest that CaNAC6 positively regulated the expression of CaSLR1.

CaSLR1 regulates genes expression associated with SCW formation pathways in pepper stems
To further explore the underlying molecular mechanism of CaSLR1-mediated stem development in pepper, RNA-seq was used to analyse RNA samples extracted from stems of 45-day-old CaSLR1-silenced and control plants (pTRV2).After filtering out adaptor sequences, more than 87% clean reads were obtained, which were then mapped to the pepper 'Zunla_1' reference genome.The primary quality Q30 was above 92.9%(Fig. S10, see online supplementary material).In total, we identified 2105 DEGs, including 1132 up-regulated and 973 down-regulated genes (Fig. S7, see online supplementary material).The GO analysis revealed significant enrichment of DEGs in the cellular components associated with the plant-type cell wall and cellulose synthase complex.In terms of biological processes, enrichment was observed in cell wall organization, phenylpropanoid metabolism, and cellulose microfibril organization, indicating that CaSLR1-silencing affected the expression of genes related to cell wall components (Table S11, see online supplementary material).
To investigate the synergistic effects of DEGs in signal transduction and biochemical metabolism pathways, we performed KEGG enrichment analysis.The results revealed that DEGs were mainly enriched in 17 pathways, including hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis, etc. (Table S12, see online supplementary material), indicating that CaSLR1 affected lignin biosynthesis.Research indicates that the protein encoded by the auxin pathway gene ARF binds to the promoter regions of essential genes associated with lignin biosynthesis, including caffeoyl-CoA O-methyltransferase (CCoAOMT2) or 4-coumarate: coenzyme A ligase (4CL3, 4CL7, and 4CL9), and activates their expression thereby affecting mechanical bending in bamboo [29].
Additionally, we explored the gene expression of cellulose, cell wall structural proteins, and hemicellulose biosynthesis pathways in CaSLR1 silenced plants.Heatmap analysis revealed a significant down-regulation in numerous genes associated with cell wall structural protein biosynthesis, such as COBRA-like (COBL) and cellulose synthase A catalytic subunit (CESA) (Fig. 7a).Similarly, genes related to hemicellulose biosynthesis, including IRX9H, IRX9, and xyloglucan endotransglucosylase protein 30 (XTH30), were also down-regulated (Fig. 7b).XTH30 is recognized for its crucial role in building and restructuring xyloglucan cross-links [49].Notably, the expression of genes essential for lignin biosynthesis, including caffeoyl-CoA O-methyltransferase-like (CCoAOMTL), laccase-4-like, laccase 2-like, laccase 11-like, 4-coumarate-CoA ligaselike 1 (4CL1), and cinnamyl alcohol dehydrogenase 1 (CAD1) was also down-regulated (Fig. 7c), indicating that CaSLR1 may play a role in regulating genes expression associated with lignin biosynthesis.Furthermore, the down-regulated genes involved with cell wall formation were verified by qRT-PCR (Fig. 7d).This was consistent between the qRT-PCR results and transcriptomic sequencing data, validating the reliability of our findings.Therefore, we propose that CaSLR1 modulates the expression of secondary cell wall (SCW) biosynthesis genes, thereby inf luencing stem development in pepper.

Discussion
Stems serve as the primary load-bearing structure in plants, providing resistance against lodging.However, during stem development, cells within the vascular bundles, fibers, and vessels gradually lose their protoplasm, leaving only the cell walls to offer mechanical support [7].The mechanical strength and stability of the stem are largely inf luenced by cell wall components [50,51].Here, a typical pepper lodging mutant slr1, induced by EMS, exhibited constrictive xylem and defective secondary cell wall development, accompanied by a significant decrease in cellulose, hemicellulose, and lignin contents (Fig. 1c-h).Via fine mapping and chromosome structure variation analysis, a deletion region of approximately 105 Kb on chromosome No. 8 was identified as the candidate region, including CaZ8g15200, CaZ8g15210, CaZ8g15220, CaZ8g15230, and CaZ8g15240 five genes (Fig. 2b-c).Among five genes, CaZ8g15230 was not expressed in WT and slr1 mutants.Hence, we only focus on another four genes.CaZ8g15210 encodes carbohydrate esterase.Carbohydrate esterase family members have been suggested to aid in reducing cell wall rigidity by cleaving covalent linkages between lignin and glucuronoxylan [52].CaZ8g15240 encodes the transcription factor MYB61.Previous research has demonstrated that PbMYB308 binds with PbMYB61 to negatively regulate the synthesis of stone cell lignin in pear fruits [53].In A. thaliana, the AtMYB61 mutant exhibited reduced xylem formation and impaired xylem cell structure [54].
Our results reveal that CaSLR1 (CaZ8g15240) is homologous to AtMYB61 (Fig. S2b, see online supplementary material).Given that CaSLR1 knockdown lines exhibit a lodging phenotype similar to slr1, it is considered a candidate causal gene.In the study, notably, the F 2 population did not conform to the expected Mendelian 3:1 separation ratio, and only one peak was observed in the Mutmap analysis (Fig. 2a).Previous research indicated that germination and seedling establishment were compromised in Arabidopsis myb61 mutants [55].Our findings revealed that the CaSLR1 mutation significantly decreased the number of fruits and the seed germination percentage.Specifically, the seed germination rate was 99% in WT but only approximately 52% in slr1 mutants (Fig. S1a-c, see online supplementary material).We speculate that Caslr1 affects fruit and seed development, potentially explaining the abnormal F 2 segregation ratio.Additionally, the slr1 mutant is caused by a substantial mutation.Contrary to the predominant point mutations induced by EMS, as Xiong et al. [56] reported, the genomic mutagenesis library confirmed that EMS can also cause large fragment deletions [57].For instance, 870 large homozygous deletions were detected in tetraploid wheat and 7971 in hexaploid wheat [58], likely a consequence of using higher EMS dosage.
The softness of stems relies on the elastic behavior of fibers, which is determined by both the overall load and the composition of the SCW.Overexpressing plants with p1300-SND1P-XND1 exhibited recumbent stems, while stem length remained largely unaffected [59].Alterations in SCW formation and components within vascular cells have been shown to affect stem mechanical properties [34,35,60].Our study reveals that the cellulose content of CaSLR1-knockdown lines is significantly reduced by 36.39% compared to pTRV2 controls (Fig. 3f).While both CaSLR1 and AtMYB61 inf luenced cellulose content, notable differences in stem erectness were observed between peppers and Arabidopsis.CaSLR1-knockdown lines displayed complete lodging with a prostrate posture (Fig. 3a).However, the erectness of the inf lorescence stems of the Atmyb61 mutant was not affected [54].In rice lines where OsMYB61 was overexpressed, cellulose content increased, while total lignin and xylose (one of the main components of hemicellulose) contents remained unchanged at the basal internodes [47].In our study, CaMYB61-knockdown lines exhibited reductions in hemicellulose and lignin contents.Additionally, the stem strength of CaMYB61-silenced lines decreased by approximately 59%.The differing effects of MYB61 on the pathways of hemicellulose and lignin biosynthesis in various species could be a significant factor contributing to the alterations observed in stem development.
Furthermore, MYB61, a crucial multifunctional protein, inf luences various metabolic processes in plant growth.OsMYB61 is directly regulated by Growth Regulator Factor 4 (GRF4), a known regulator of nitrogen using efficiency (NUE), which promotes biomass production in rice [61].In this study, biomass decreased in CaSLR1-silenced pepper lines and SlMYB61 knockout tomato lines (Fig. 3i-3k; Fig. S1f, see online supplementary material).As a regulator of resource allocation, AtMYB61 expression was observed in the metabolic sink, particularly in the xylem and roots.Mutants of AtMYB61 exhibited reductions in lateral roots and xylem vessels [54].In slr1 mutant and CaSLR1-knockdown lines, the decreased contents of cellulose, hemicellulose, and lignin revealed that the CaSLR1 affected the formation of structural carbohydrates in plants.These findings support the conclusion that MYB61 inf luences resource allocation.Furthermore, despite the homology between CaSLR1 and AtMYB61, there are notable differences in their phenotypic effects.These distinctions may arise from variations in stem types between pepper and Arabidopsis (b)   inf lorescences, as well as differences in amino acids sequences within conserved domains (Fig. 4a).Another contributing factor could be the variations in types of mutations, which lead to varying phenotypic outcomes.In this study, the deletion of the entire CaSLR1 gene exhibited a more pronounced effect (Fig. 1a), in contrast to the subtler phenotypes observed in AtMYB61knockout lines in Arabidopsis [54].An intriguing avenue for future research is to investigate whether mutations in different regions of CaSLR1 would impact the biosynthesis of cell wall components, thereby inf luencing the deposition of SCWs and potentially causing plant-specific variations.
A complex multilevel transcriptional network in plants acts on the biosynthetic genes of SCW components, thereby regulating the thickening process of SCWs [62].Studies on Arabidopsis mutants have established a clear consensus that NAC and MYB TFs act as switches in regulating SCW biosynthesis [62,63].MYB46 was one of the earliest discovered SCW master switches [64].The down-regulation of MYB46 and MYB83 drastically repressed SCW thickening in both vessels and fibers, thereby causing collapsed vessels and reduced plant growth [65].SND1 binds to the SNBE element in MYB46 promoter and activates its expression [11].In this study, WGCNA indicated that CaSLR1 and CaNAC6 clustered in the same module (Fig. 5a).CaNAC6 exhibited homology to NAC29, which regulated SCW deposition in rice [48].Overexpressing OsNAC29 lines exhibited thicker internodes and significantly increased cellulose content [47].Our study revealed that CaNAC6 specifically binds to SNBE elements in CaSLR1 promoter, thereby activating its expression (Fig. 6b-d).Therefore, CaNAC6 and CaSLR1 are important regulators of SCW formation in pepper.
Secondary wall-associated NACs TFs activate the secondary MYB master switch, leading to the expression of downstream SCW formation-related genes [63].In our study, CaSLR1 regulated the expression of downstream genes involved in cellulose, hemicellulose, and lignin biosynthesis, including CESA, COBL4, IRX9, XTH30, CCoAOMTL, laccase 2 L, 4CL1, CAD1, 4CCL7, IRX15LL (Fig. 7a-c).On the Golgi apparatus, CESAs assemble into cellulose synthase complexes, which are then transported to the plasma membrane for cellulose synthesis, ultimately depositing cellulose on one side of the cell wall [66].The spatial control of lignin chemistry depends on different combinations of laccases with nonredundant activities immobilized in cell wall layers [67].Given all this, CaSLR1 and CaNAC6 may be crucial master switches in the molecular network of regulating secondary cell wall formation in peppers.Based on our study, a hypothesis was proposed to elucidate the mechanism of stem lodging resistance in pepper.In the WT, CaNAC6 binds to the SNBEs in the promoter of CaSLR1, inducing the transcription of CaSLR1.Consequently, the expression of CaSLR1 promoted the accumulation of cellulose, hemicellulose, and lignin, resulting in thickened secondary cell walls (SCWs) and erect stems.Conversely, in the slr1 mutant, the complete deletion of CaSLR1 and its promoter prevents CaNAC6 from binding to the SNBE elements, reducing the accumulation of these structural components, leading to thinner SCW and stem lodging (Fig. 7e).These findings highlight the CaNAC6-CaSLR1 module that contributes to lodging resistance, underscoring the pivotal role of CaSLR1 within the regulatory network for lodging resistance.

Plant materials and growth conditions
The elite inbred line '6421', which serves as a foundational breeding stock extensively utilized across China, was treated with 1% EMS (medial lethal dosage) to construct a mutant library [18].We had sown 10 000 mutants and 200 WT seeds, and then the individual plant was conducted self-cross.Next, we harvested the self-cross seeds from individual plants to construct the M1 generation, and selected M2 generation with stem lodging from M1 self-cross seeds.Using the same planting screening method as the M2 generation, we obtained a genetically stable stem lodging mutant slr1 M3 generation [19].WT and slr1 plants were planted in a greenhouse with 65% relative humidity.Various tissues were gathered from the growing plants for gene expression analysis, including roots, stems, leaves, blooms, fruits, and anthers.

Scanning electron microscopy (SEM) and determination of stem components
Fresh tissues were collected, taking care to minimize mechanical damage, such as bruising, pulling, and squeezing.Based on a previously described method [35], the tissue blocks were placed in electron microscopy fixative for 2 h and then preserved at 4 • C for transportation.The tissues were observed with an SEM, and images were taken.

Candidate gene mapping
We identified the chromosome region of candidate target genes using MutMap method [21].Leaf samples were taken from 30 of the WT and slr1 plants of the F 2 segregating population.We extracted the genomic DNA (DNA extraction kit, Product code: DP360, Tiangen Biotech Co. Ltd, Beijing, China) [22] of 30 upright and 30 lodging plants from the F 2 population, respectively.Next, we constructed two mixed DNA pools and performed the wholegenome resequencing on the HiSeq™ PE150 platform (Illumina, Inc., San Diego, CA, USA).Reads data were filtered by Fastp [23], and clean reads were mapped to the reference of 'Zhangshugang' (http://ted.bti.cornell.edu/ftp/pepper/genome/Zhangshugang/) genome [20] using bwa mem v0.7.12 [24].Duplicated reads derived from PCR were marked with samblaster.The generated BAM file was utilized to identify single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) using GATK [25].Structural variations were identified by manta [26].SNPs were filtered using bcftools [27] with the parameters QD < 2.0 || MQRankSum < −12.5 || ReadPosRankSum < −8.0.The SNP-index value was calculated to locate the traits, with the wild-type (WT) parent selected as the reference parent to compute the SNP-index of the two offspring.
To fine-map the candidate genes, we detected SNP cosegregation via the Kompetitive Allele-Specific PCR (KASP) genotyping technique.Based on a previously described method [28], we designed allele-specific primers based on the 200 bp sequence upstream and downstream of the SNPs, shown in Table S1 (see online supplementary material).A total of 483 F 2 individuals were used for KASP genotyping.The SNP genotyping detection system PARMS (penta-primer amplification refractory mutation system) (Gentides Biotech Co., Ltd) was used according to the manufacturer's protocol.The primers were synthesized by the Gentides Biotech Co. Ltd (Wuhan, China).A 10 μl reaction system was prepared, including 5uL 2× PARMS (containing f luorescent universal primers FAM, HEX), 0.7uL specific amplification primers, 1uL (50 ng) DNA template and 3.3 uL double distilled H 2 O. Signals

Virus-induced gene silencing (VIGS)
To decrease the expression of target genes in the WT, we conducted virus-induced gene silencing following a previously established method with slight modifications [35].We identified specific regions of CaSLR1 via online tool SGN VIGS (https://vigs.solgenomics.net/)[36,37].Unique primers were used to amplify the cDNA of pepper stems, yielding a product of approximately 300 base pairs.Next, we used the One Step Cloning Kit (Product code: C112-02) [32] to clone the fragment into the pTRV2 vector, which has been double-digested with EcoRI (Product code: FD0274) and BamHI (Product code: FD0054) (Thermo Fisher Scientific, Waltham, Massachusetts, USA).The vectors were then transformed into Agrobacterium strain GV3101.The whole cotyledons of ten-day-old seedlings were infected by this Agrobacterium strain GV3101.After six weeks, we collected the first and second internode stems above the cotyledons for scanning electron microscope analysis, and determined the contents of cellulose, lignin, and hemicellulose in same samples.The stem strength of the first internode was measured eight weeks post-infection.

CRISPR/Cas9-mediated CaSLR1 homolog encoding gene SlMY61 knockout in tomato
The homologous sequence of SlMYB61 (Solyc01g102340) was obtained by aligning CaSLR1 to the tomato genome (Solanum lycopersicum).Based on the CDS of SlMYB61, we designed CRISPR target sites with the assistance of the CRISPR-P 2.0 website [16].Two pairs of PCR primers for two CRISPR targets 'TTGAACTTCATGCAGTTCTT' and 'ATAACGAGATAAAGAATCTG' were designed (Table S1, see online supplementary material).Next, the PGTR plasmid served as a template for PCR, and the product was cloned into the CRISPR expression vector pKSE401 by Golden Gate cloning [68].The vector was used to transform Agrobacterium tumefaciens EHA105 cells, constructing pKSE401expressing strain Cri-SlMYB61 for inoculation.Tomato (Micro Tom) cotyledons were transformed using a previously established method [38].

Weighted gene co-expression network analysis (WGCNA)
Based on gene expression levels, we constructed a co-expression network using the WGCNA package in R. Firstly, we normalized expression data to FPKM +1 value and then transformed it to log2.The correlation between module genes was subsequently analysed using WGCNA [28].The WGCNA hierarchical network was utilized to construct a co-expressed gene network module.BLASTALL v2.2.26 (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/), with an e-value set to 1e-5, was used to annotate the module genes via GO and KEGG databases and Perl scripts.

Yeast one-hybrid assay (Y1H)
To explore the interactions of CaNAC6 with CaMYB61, Y1H assays were conducted.The ORFs of CaNAC6 were cloned into the pGADT7 backbone as prey [46].A tandem of three SNBEs of the CaMYB61 promoter was cloned into the pABAi backbone as bait.Both vectors were transformed into Y1H Gold yeast strain cells using Clontech's small-scale transformation method.Glass beads were used to coat SD/−Leu /AbA until no bacterial liquid f low was observed.Samples were then cultured on plates at 30 • C in an incubator for 5-7 days.

Dual luciferase reporter analysis (LUC)
To validate the activation of CaMYB61 by CaNAC6, the coding sequence (CDS) of CaNAC6 was cloned into the pGreenII 62-SK backbone to construct an effector vector.The promoter sequence of CaMYB61 was then inserted into the pGreenII 0800-LUC (luciferase) backbone to drive the expression of LUC gene, creating a reporter vector.These vectors were transformed into GV3101 cells, which were subsequently injected into tobacco leaves and kept in the dark.After 2-3 days, we used a 5200 Imaging System (Tanon Science & Technology, Shanghai, China) to capture the f luorescence signal on leaves.Subsequently, we used a Dual-Luciferase ® Reporter Assay System (Product code: E1910, Promega, Madison, WI, USA) to measure the f luorescence values of LUC and REN (Renilla luciferase).Each sample underwent testing with a minimum of six biological replicates.

Electrophoretic mobility shift analysis (EMSA)
The CDS of CaNAC6 was inserted into the PGX-6P-1 GST backbone and then transformed into Escherichia coli BL21 (DE3).Expression induction resulted in the production of the NAC6-GST fusion protein.Following the manufacturer's instructions, the LightShift chemiluminescence EMSA kit (Product code: Bes5003, Biotechnology Co., Ltd, Guangzhou, China) was utilized to detect the interaction between CaNAC6 and the SNBE of the CaSLR1 promoter.The synthesized SNBE element was annealed with a biotin-labeled double-stranded DNA probe (Novagen Biotechnology Co., Ltd, Beijing, China).The mutant biotin-labeled and unlabeled probes were incubated with the NAC6-GST fusion protein to assess its binding capability.Following the binding reaction, the complex underwent electrophoresis on a 6% acrylamide gel and was subsequently transferred to a nylon membrane.After rinsing, the nylon was positioned in a membrane box, and the X-ray film was exposed for 5 minutes.Gel shift was facilitated using Anti-streptavidin-HRP (horseradish peroxidase) conjugated antibody (Product code: HRP-66001, Reda Henghui Technology Development Co. Ltd, Beijing, China).

Statistical analysis
The experimental data were analysed using Microsoft Excel 2013.A histogram was generated using GraphPad Prism v7.04.Statistical significance was evaluated using Student's t-test.All qRT-PCR (quantitative real-time-PCR) analyses were carried out with at least three replicates.

Figure 2 .
Figure 2. Mapping of the genes related to stem lodging using Mutmap.(a) Mutmap indicated a preliminary map region.The candidate chromosomal region of about 39.50 Mb was marked in a arrow.(b) Candidate regions were narrowed down by genotyping.NP, WT chromosomal .MP, slr1 chromosomal.The normal phenotype individual plants with chromosomal exchanges in the F 2 population., including WT-24 and WT-73.The chromosomal exchanges plants with a lodging phenotype in the F 2 population, were marked M-467, M-139, and M-242.The candidate region was identified as the chromosomal domain between the two SNP markers (SNP153164345 and SNP15453957).(c) Analysis of the chromosomal structural variation within the range of 153.16-154.53Mb.Vertical axis: Depth coverage rate between WT and slr1; Abscissa axis: chromosome position.(d) Approximately 105 Kb was deleted in the genome of slr1 mutant, including CaZ8g15200, CaZ8g15210, CaZ8g15220, CaZ8g15230, and CaZ8g15240.(e) The agarose gel electrophoresis was conducted using PCR products of five genes from both WT and slr1 plants.The genes and their respective PCR product sizes are as follows: CaZ8g15200 (CaPIF3, 2181 bp); CaZ8g15210 (CaFSCB, 2385 bp); CaZ8g15220 (CaCE, 810 bp); CaZ8g15230 (CaFXO, 1098 bp); and CaZ8g15240 (CaSLR1, 1263 bp).(f) The expression levels of the five genes in the stems of the WT and slr1 plants.The results are presented as mean ± SE (n = 3).* P < 0.05, * * P < 0.01, * * * P < 0.001, and n.s, not significant, as determined by the Student's t-test.

Figure 3 .
Figure 3. CaSLR1 mediates stem lodging by affecting SCW thickness and stem strength in peppers and tomatoes.(a) Virus-induced gene silencing of CaSLR1 in pepper, scale = 2.5 cm.(b) RT-qPCR analysis the relative expression levels of CaSLR1 in the CaSLR1-silenced line (pTRV2-CaSLR1) and control (pTRV2, NC) lines.(c) The stems of CaSLR1-knockdown and control lines; scale = 1 cm.(d) The xylem tissues from the stems of the CaSLR1-knockdown and control lines, respectively; scale = 100 μm.(e) The secondary xylem from the stems of CaSLR1-knockdown and control lines, respectively; scale = 30 μm. (f) The contents of cellulose, hemicellulose, and lignin from the stems of the CaSLR1-knockdown and control lines, respectively.(g) The stem strength of CaSLR1-knockdown and control lines.The results were expressed as the mean ± SE (n = 3).* * P < 0.01 and * * * P < 0.001, as determined by Student's t-test.(h) SlMBY61, encoding a homolog of CaSLR1, was edited at the third exon in tomato (Micro-Tom).PAM: protospacer-associated motif.Seven bases (TAAAGAA) were edited.Target bases: ATAACGAGATAAAGAATCTG. PAM: TGG.(i) The phenotype of T 1 generation from the knockout line (Cri-SlMYB61) and no knockout line (negative control, NC).Scale = 5 cm.(j) The root from T 1 generation.Scale = 5 cm.(k) The f lower branches and inf lorescences from T 1 generation.Scale = 2 cm.(l) Scanning electron microscopy analysis of T 1 generation stem.Scale = 500 μm.Xy: xylem; PC: parenchymal cell; Pi: pith.(m) The parenchymal cell closed to xylem of T 1 generation stem.Scale = 50 μm.PC: parenchymal cell.The arrow points to the granular body.(n) The xylem fiber cell closed to secondary xylem from T 1 generation stem.Scale = 50 μm.SCW: secondary cell wall; XFC: xylem fiber cell; Ve: vessel.The arrow points to SCW.

Figure 5 .
Figure 5. WGCNA analysis.(a) Genes cluster dendrogram analysis in which different gene modules are represented by branches and various colors.(b) GO enrichment analysis of the genes in the antiquewhite4 module.(c) The expression heatmap of SCW biosynthesis-related candidate genes in the antiquewhite4 module.(d) RT-qPCR analysis of four SCW biosynthesis-related genes in the antiquewhite4 module, CaNAC7: NAC transcription factor 7; CaGXM1: glucuronoxylan 4-O-methyltransferase 1; CaPE53: pectinesterase 53; and CaGRP1L: glycine-rich cell wall structural protein 1-like.The results were expressed as the mean ± SE (n = 3).* * * P < 0.001, as determined by the Student's t-test.

Figure 6 .
Figure 6.CaNAC6 binds to the promoter of CaSLR1 and induces its transcription.(a) The position of the three secondary wall NAC binding elements (SNBEs) in the 2000 bp long promoter of CaSLR1.(b) The potential binding of CaNAC6 with CaSLR1 promoter in yeast.SD/−Leu, medium lacking leucine.SD/−Leu/500 AbA, medium lacking leucine and added with 500 ng/mL aureobasidin A (AbA).(c) Schematic of the vector used for the Dual-luciferase reporter system.LUC, firef ly luciferase.REN, renilla luciferase.(d) LUC images of the tobacco leaves after transient infiltration.(e) The ratio of LUC to REN activity.The results were expressed as the mean ± SE (n = 6).* * * P < 0.001, as determined by the Student's t-test.(f) The binding of CaNAC6 with the SNBE elements of CaSLR1 was determined using an EMSA assay.

Figure 7 .
Figure 7. CaSLR1 regulates the expression of cellulose, hemicellulose, and lignin biosynthesis related genes in pepper stems.(a) The expression-related heatmap of genes involved in the cellulose biosynthesis pathway and cell wall structural proteins.(b) The expression-related heatmap of genes involved in the hemicellulose biosynthesis pathway.(c) The expression-related heatmap of genes involved in the lignin biosynthesis pathway.(d) qRT-PCR analysis verified the down-regulated genes related to SCW formation pathways using RNA-seq.The results were expressed as the mean ± SE (n = 3).* * P < 0.01; * * * P < 0.001, as determined by the Student's t-test.(e) A hypothesis suggests that CaSLR1 regulated the stem lodging resistance.In WT, CaNAC6 binds to SNBE elements in the promoter of CaSLR1, thereby inducing its transcription.As a result, the stem accumulated cellulose, hemicellulose, and lignin, which promotes the deposition of the SCW and increases stem strength, enabling the stem remain upright.In contrast, in the mutant slr1, a deletion of CaSLR1 and its promoter abolished the binding of CaNAC6 to the SNBEs.The transcription of CaSLR1 was inhibited, and the accumulation of cellulose, hemicellulose, and lignin in stems was reduced.Accordingly, it resulted in the thinning of the SCW, weakening of stem strength, and finally, stem lodging.'×' marked that the effect was cancelled.CESA: cellulose synthase; COBL4: COBRA-like 4; IRX9: irregular xylem 9; XTH30: xyloglucan endotransglucosylase protein 30; CCoAOMTL: caffeoyl-CoA O-methyltransferase 1ike; laccase 2 L: laccase 2-like; 4CL1: 4-coumarate-Co-A ligase 1; CAD1: cinnamyl alcohol dehydrogenase 1; 4CCL7: 4-coumarate-Co-A ligase like 7; AAE2: Acyl-activating enzyme 2; ACSL4: Long chain acyl-CoA synthetase 4-like; IRX15LL: IRX15 − LIKE − like.