Light-induced anthocyanin biosynthesis in Lilium brownii is mediated by the LbrHY5–LbrMYB6 module

Light-induced anthocyanin biosynthesis in Lilium brownii is mediated by the LbrHY5–LbrMYB6 module | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Light-induced anthocyanin biosynthesis in Lilium brownii is mediated by the LbrHY5–LbrMYB6 module Yuwei Cao, Yuqian Zhang, Huiwen Yang, Yuwen Ye, Hui Lai, Shanghui Yin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9289041/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Lilium brownii is a valuable monocotyledonous plant with ornamental and medicinal importance, whose purplish-red pigmentation is attributed to anthocyanin accumulation under light exposure. However, the molecular mechanisms underlying light-regulated anthocyanin biosynthesis in lily remain unclear. Here, we demonstrate that light significantly enhances anthocyanin content in lily and upregulates key structural and LbrMYB6 regulatory genes. We identified LbrHY5 as a light-induced bZIP transcription factor localized to the nucleus. Transcriptomic analysis positioned LbrHY5 and LbrMYB6 at the central hubs of a light-responsive co-expression network. Moreover, molecular assays confirmed that LbrHY5 binds directly to the G-box motifs in promoters of LbrCHS and LbrMYB6, thereby activating their transcription. Overexpression of LbrHY5 and LbrMYB6 significantly increased anthocyanin accumulation and structural genes expression, with co-overexpression exhibiting synergistic effects. VIGS-mediated silencing of LbrMYB6 reduced anthocyanin contents and structural genes expression, while simultaneous silencing of both genes produced similar downregulation. Notably, individual silencing of LbrHY5 unexpectedly increased anthocyanin accumulation, suggesting complex regulatory mechanisms involving light-induced COP1 inactivation and potential feedback regulation. Collectively, our findings elucidated the role of a novel LbrHY5–LbrMYB6 module that mediates light-regulated anthocyanin biosynthesis in L. brownii, providing significant insights into the regulatory networks controlling pigmentation in monocots and offering potential strategies for improving color traits in ornamental lilies. Lilium brownii Light signaling LbrHY5 Anthocyanin biosynthesis Transcriptional regulation LbrMYB6 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Key message The LbrHY5–LbrMYB6 transcriptional module mediates light-induced anthocyanin biosynthesis in Lilium brownie by directly activating LbrCHS and downstream structural genes. 1 Introduction Lilium brownii is a monocotyledonous plant with considerable ornamental, edible, and medicinal value. The purplish-red pigments in its floral organs and bulbs are primarily anthocyanins, which are flavonoids that contribute to pollination, stress resistance, and human health (Grotewold, 2006 ). Anthocyanin biosynthesis is controlled by both developmental programs and environmental cues, with light being one of the most influential factors (Winkel-Shirley, 2001 ). Light represents a primary environmental determinant of anthocyanin accumulation across the plant kingdom. In dicotyledonous crops including tomato ( Solanum lycopersicum ) and grape ( Vitis vinifera ), light exposure coordinately induces expression of the core flavonoid biosynthetic genes —PAL , CHS , F3H , DFR , ANS , and UFGT— thereby promoting pigment synthesis in reproductive and vegetative tissues (Matus et al., 2009 ; Kim et al., 2021 ). Similarly, light exposure enhances the expression of anthocyanin-related genes in Chrysanthemum morifolium (Hong et al., 2016 ) and Lilium regale (Yamagishi, 2016 ), underscoring the conserved nature of light responses across species (Wei et al., 2011 ; Zhang et al., 2018 ; Zhou et al., 2022 ). Among the central regulators mediating light signal transduction, ELONGATED HYPOCOTYL5 (HY5)—a bZIP transcription factor—stands out for its light-dependent accumulation and subsequent activation of genes governing photomorphogenesis and flavonoid metabolism (Feng et al., 2013 ; Wang et al., 2021 ). Under light, HY5 accumulates and binds to G-box or ACE-box elements in the promoters of target genes in the anthocyanin pathway, thereby activating transcription (Bursch et al., 2021 ; Liu et al., 2019 ; Maier et al., 2013 ). Loss-of-function mutations in HY5 have been linked to reduced pigmentation and transcriptional suppression of core anthocyanin biosynthetic genes in model species such as Arabidopsi and tomato (Li et al., 2018 ; Qiu et al., 2019 ; Wang et al., 2021 ). HY5 often functions alongside other factors, such as B-box (BBX) proteins and MYB transcription factors, forming complexes that fine-tune anthocyanin biosynthesis in response to light quality and intensity (Bai et al., 2019a ; Fang et al., 2019 ; An et al., 2020 ). For example, PpHY5 and PpBBX16 form a complex that can binds directly to the PpCHS or PpMYB10 promoter (Bai et al., 2019a ). Additionally, HY5 stability is post-transcriptionally regulated by CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), which targets HY5 for degradation in darkness. Upon illumination, photoreceptors inhibit COP1, allowing HY5 to accumulate and bind to promoter elements of anthocyanin-related genes (Maier et al., 2013 ; Bursch et al., 2021 ). MYB6, a key R2R3-MYB transcription factor, participate in light signal-mediated secondary metabolism across diverse plant species, particularly playing a critical role in the flavonoid and anthocyanin biosynthetic pathways. In C. morifolium , CmMYB6 functions as a central activator of anthocyanin biosynthesis and floral pigmentation (Xia et al., 2026 ). In Populus tomentosa , MYB6 positively regulates the accumulation of both anthocyanins and proanthocyanidins (PAs) (Wang et al., 2019 ). Similarly, in Sanhua plum’s flesh ( Prunus salicina ), MYB6 is associated with C4H expression and anthocyanin accumulation (Xiang et al., 2023 ). Moreover, MYB6 transcription factors consistently show positive responsiveness to light signals. In Fagopyrum tataricum , the FtMYB6 promoter displays strong light-inducible activity, and its overexpression significantly enhances flavonol accumulation in transgenic tobacco and hairy roots of tartary buckwheat (Yao et al., 2020 ). However, LhMYB6 has been implicated in lily tepal pigmentation, and the mechanistic basis of its involvement in light-regulated anthocyanin biosynthesis remains elusive. Although lily is a shade-loving plant, the mechanism underlying its flower coloration, especially its rich and bright colors in high-altitude areas with strong ultraviolet radiation, depends on the light signaling pathway. The role of HY5 has been extensively characterized in dicots, but its function in monocots, especially ornamental species (e.g., lily), remains poorly understood. L. brownii exhibits light-dependent pigmentation, but the regulatory network controlling this process has not been comprehensively elucidated. In this study, we functionally characterized LbrHY5 in terms of its role in light-induced anthocyanin biosynthesis. We determined that LbrHY5 is a nuclear protein that binds to the promoters of key anthocyanin-related genes. The target genes of LbrHY5, LbrMYB6 and LbrCHS , were identified by combining transcriptome analysis with TF-centered Y1H screening. Moreover, LbrHY5 and LbrMYB6 overexpression can enhance pigment accumulation. Our results provide the first evidence of the regulatory effects of the LbrHY5–LbrMYB6 module on anthocyanin biosynthesis in lily, offering new insights into light-mediated coloration in monocots. 2 Materials and methods 2.1 Light treatment of lily scales L. brownii bulbs were purchased from Wanzai County (Yichun, Jiangxi, China). Light treatment was performed under cool white fluorescent light at an intensity of 100 µmol·m⁻²·s⁻¹, with a constant temperature of 22 ± 2°C. Scales in the darkness group (D) were kept in complete darkness under the same temperature. For the light-treated groups, scales were exposed to continuous light for 1 day (L1) or 2 days (L2), respectively. Each treatment and subsequent phenotypic analysis were completed using 48 scales. 2.2 Anthocyanin quantification and gene expression analysis Anthocyanins were extracted from scales after treatment with different light. The absorbances of the extracts at 530 nm, 620 nm and 650 nm were measured using a UV-2600 spectrophotometer (Shimadzu, Japan). The anthocyanin content was calculated as 0.1 OD (unit×10 3 g·FW − 1 ), and the calculation method was OD = (A₅₃₀ - A₆₂₀) − 0.1 × (A₆₅₀ - A₆₂₀). Following different light treatments, L. brownii scales were harvested for total RNA extraction, after which complementary DNA (cDNA) was synthesized via reverse transcription. Gene-specific quantitative real-time PCR (qRT-PCR) primers were designed according to non-conserved gene sequences, with Lilyactin used as the internal reference. A qRT-PCR analysis was performed using SYBR Green master mix and a CFX96 system (Bio-Rad, USA), with relative expression levels determined according to the 2 −ΔΔCt method. 2.3 Transcriptome sequencing and bioinformatic analysis An Illumina platform was used for a transcriptome sequencing analysis of L. brownii scales following different light treatments (D, L1, and L2), with three biological replicates per treatment. After quality control and alignment steps, DEGs were identified. GO and KEGG pathway enrichment analyses were conducted to determine the biological functions of DEGs. 2.4 Function identification of LbrHY5 in L. brownii The full-length LbrHY5 cDNA sequence was cloned via rapid amplification according to the transcriptome data. Conserved domains were predicted using the NCBI Conserved Domain Database. In addition, a phylogenetic tree was constructed using the neighbor-joining method and MEGA 11 software. 2.5 Subcellular localization analysis To generate the LbrHY5-GFP fusion construct, the coding sequence of LbrHY5 (excluding the termination codon) was ligated in-frame upstream of the GFP coding region within the pCAMBIA2300 backbone. The resulting plasmid was introduced into Agrobacterium tumefaciens GV3101, and bacterial suspensions were infiltrated into Nicotiana benthamiana leaves for transient expression assays. After 3 days, GFP fluorescence was observed using a laser scanning confocal microscope, with nuclei counterstained by mCherry. 2.6 TF-centered Y1H assay to identify target genes The key light-induced transcription factor gene LbrHY5 was inserted into the pGADT7 vector to construct the bait vector. A library of yeast cells containing candidate cis -motifs was screened. Positive clones were selected on SD/−Trp/−Leu/−His medium supplemented with 100 mM 3-AT and then sequenced. 2.7 Promoter cloning The promoter sequences of LbrCHS and LbrMYB6 were isolated using the Genome Walking Kit (Clotech, China). Putative cis -acting elements were identified using the PlantCARE database. 2.8 Dual-luciferase reporter assay The promoters of LbrCHS and LbrMYB6 were inserted into the pGreenII 0800-LUC vector, whereas the LbrHY5 CDS was cloned into the pGreenII 62-SK expression vector. Agrobacterium suspensions harboring the promoter reporter and transcription factor effector constructs were combined at a 10:1 volumetric ratio prior to co-infiltration into N. benthamiana leaves. Luciferase activities were measured after 3 days and then the firefly luciferase: Renilla luciferase ratio was calculated to determine transcriptional activation. The strain containing the empty expression vector was mixed with the reporter strain as a negative control. The firefly luciferase: Renilla luciferase ratio was set to 1, and 10 biological replicates were prepared for each experiment. 2.9 EMSA detection The LbrHY5 CDS was inserted into the pET-22b(+) expression vector. Recombinant LbrHY5-HIS was produced in Escherichia coli cells and purified. A western blot was used to detect the purified recombinant protein. Biotin-labeled double-stranded oligonucleotides containing the wild-type or mutated G-box motif were used as EMSA probes. Binding reactions were analyzed using a native polyacrylamide gel, with shifts detected using a chemiluminescent EMSA kit (Beyotime, China). 2.10 Overexpression and phenotypic analysis The LbrHY5 CDS was incorporated into the pCAMBIA1305 plant transformation vector. The resulting recombinant plasmid was inserted into A. tumefaciens strain EHA105 cells for the transformation of ‘Robina’ lily tepals and L. brownii scales via vacuum injection. MMA resuspension buffer [4.43 g/L MS (without vitamins), 20 g/L sucrose, 1.95 g/L MES, 10 mM MgCl 2 , 100 µM acetosyringone, and 10 µM paclobutrazol, pH 5.6] was adjusted for an OD 600 of 1.0. Tissues transformed with EV were used as the control. Anthocyanin accumulation and the expression of related genes were compared overexpression and control lines following light treatments. 2.11 VIGS-mediated infection of lily tepals Using the previously established petal VIGS system in lily (Bi et al., 2023 ), we cloned the LbrHY5 and LbrMYB6 genes and inserted them into the pTRV2 vector. The recombinant vectors were transformed into Agrobacterium tumefaciens strain EHA105 competent cells and resuspended in MMA buffer to an OD 600 of 1.0. The Agrobacterium suspensions containing the recombinant pTRV2 and pTRV1 were mixed at a 1:1 ratio and infiltrated into tepals using a 0.05 MPa vacuum infiltration method for 5 min at room temperature. A mixture of Agrobacterium suspensions containing empty pTRV2 and pTRV1 was used as the control for anthocyanin content and structural gene expression analyses. 2.12 Statistical analysis All experiments were performed with at least three independent biological replicates. Data are presented as means ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, USA). For comparisons between two groups, Student’s t-test was applied. For comparisons among multiple groups, ordinary one-way ANOVA was performed, followed by Dunnett’s multiple comparisons test when comparing all groups to a single control group. Statistical significance was set at p < 0.05, p < 0.01, and p < 0.001, as indicated in the figures. Different letters or asterisks denote statistically significant differences as described in the figure legends. 3 Results 3.1 Light promotes anthocyanin accumulation and related genes expression in L. brownii To clarify the effects of light on L. brownii bulb coloration, we exposed scales to light for different durations (no light, 1 day of light, and 2 days of light) (Fig. 1 A). In L. brownii bulbs, the anthocyanin content was almost undetectable in darkness. However, anthocyanin accumulated significantly as the duration of the light treatment increased, demonstrating a clear positive correlation between light exposure duration and anthocyanin production (Fig. 1 B). To elucidate the molecular basis of this light-induced pigmentation, we analyzed the expression levels of key structural and regulatory genes involved in anthocyanin biosynthesis. Notably, LbrCHS1 , LbrCHIb , LbrF3H , LbrFLS , LbrDFR , LbrANS , LbrANR and LbrMYB6 expression levels were consistently upregulated in response to light (Fig. 1 C). Collectively, these findings indicate that an exposure to light not only promotes anthocyanin accumulation in L. brownii but also enhances the expression of anthocyanin-related genes. To comprehensively identify key transcription factors involved in light-induced anthocyanin biosynthesis in L. brownii , we performed transcriptomic analysis on scales subjected to different light treatments (D, L1, L2). 3.2 Transcriptomic analysis reveals anthocyanin-related gene expression changes in response to light To elucidate the global changes in gene transcription levels in light-treated lily plants, we performed a transcriptome sequencing analysis of scales exposed to different light conditions. As shown in Fig. 2 and Supplementary Fig. 1, a Gene Ontology (GO) enrichment analysis revealed that differentially expressed genes (DEGs) after 1- and 2-day light treatments were significantly enriched in biological processes related to anthocyanin biosynthesis and light responses (Fig. 2A), thereby confirming that light is a key environmental signal activating anthocyanin biosynthesis. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis further demonstrated that these DEGs were specifically associated with the anthocyanin biosynthesis pathway (Fig. 2B), reflecting the coordinated activation of the complete biosynthetic network by light. A Venn diagram-based analysis identified both unique and shared genes among comparison groups (Fig. 2C), indicating that light-induced transcriptional reprogramming is a dynamic and complex process. Furthermore, there were significantly more upregulated genes than downregulated genes after light treatments (Fig. 2D), implying that light mainly activated anthocyanin biosynthesis-related gene expression. Furthermore, to investigate the key DEGs associated with anthocyanin biosynthesis and their regulatory networks, we selected 31 DEGs in KEGG pathways related to anthocyanin and flavonoid biosynthesis for further analyses. A subcluster matrix revealed the consistent expression pattern among these genes, with an initial increase followed by stabilization (Fig. 3 A). To identify potential core regulators, we constructed a co-expression network of the 31 anthocyanin-related DEGs. Notably, LbrHY5 and LbrMYB6 were positioned at the central hubs of this network (Fig. 3 B), suggesting that they function as key transcription factors governing the light-responsive anthocyanin regulatory network. Based on these transcriptomic results, we selected LbrHY5 and LbrMYB6 as candidate genes for further functional characterization. Additionally, a cluster heatmap confirmed the significant upregulation of anthocyanin biosynthesis structural genes, such as CHS , DFR , and ANS , after an exposure to light (Fig. 3 C). Figure 2 Transcriptome analysis of L. brownii scales exposed to darkness (D), 1 day of light (L1), and 2 days of light (L2). (A) Enriched GO terms related to anthocyanin biosynthesis and light among differentially expressed genes (DEGs) in the D, L1, and L2 groups. (B) Enriched KEGG pathways related to anthocyanin biosynthesis among DEGs in the D, L1, and L2 groups. (C) Venn diagram of the overlapping DEGs among the three groups. (D) Results of the statistical analysis of upregulated and downregulated genes in the three groups. 3.3 LbrHY5 is a light-induced nuclear-localized transcription factor Given that LbrHY5 was identified as a core transcription factor from the transcriptomic co-expression network (Fig. 3 B), we next performed bioinformatics analysis to characterize its sequence features and expression patterns. To clarify the biological function of LbrHY5 in L. brownii , we isolated and cloned the full-length LbrHY5 sequence. The encoded protein was revealed to contain 154 amino acids, with a calculated molecular weight of 17.4 kDa and a theoretical isoelectric point of 9.91. Additionally, the 810 bp full-length LbrHY5 sequence included a 464 bp coding sequence (CDS), 80 bp 5’- untranslated region (UTR), and 306 bp 3’-UTR. LbrHY5 expression levels were significantly upregulated following an exposure to continuous light for 2 days (relative to the expression in darkness), suggesting that LbrHY5 is a light-responsive gene (Fig. 4A). To analyze the subcellular localization of LbrHY5, the LbrHY5 CDS with the termination codon removed was cloned and inserted into the pCAMBIA2300-eGFP vector to generate the 35S :: LbrHY5 - GFP fusion construct, with 35S :: GFP serving as the control construct. The LbrHY5-GFP fusion protein was exclusively localized in the nucleus (Fig. 4B), which is consistent with the prediction that LbrHY5 is a transcription factor. To explore evolutionary relationships, MEGA 7.0 software was used to construct a phylogenetic tree for LbrHY5 and published HY5s according to the neighbor-joining method (with 1,000 bootstrap replicates). According to the results of the phylogenetic analysis, LbrHY5 was grouped in the HY5 subfamily (Fig. 4C). Furthermore, an amino acid sequence alignment using DNAMAN confirmed that LbrHY5 contains a conserved bZIP HY5-like domain included in the NCBI Conserved Domain Database, making it a member of the bZIP transcription factor family (Fig. 4D). Figure 4 Bioinformatics analysis of LbrHY5 in lily. (A) LbrHY5 expression levels in darkness and under light. Scales were treated with darkness (D) or continuous light for 2 days (L2). Data are presented as the mean ± SEM (n = 3). Statistically significant differences were determined using Student’s t- test (** p < 0.01). (B) Subcellular localization of the LbrHY5-GFP fusion protein, with GFP alone used as a negative control. Bar, 100 µm. (C) Neighbor-joining phylogenetic tree derived from deduced amino acid sequences of LbrHY5 and other reported HY5s. Numbers next to nodes are bootstrap values from 1,000 replications. Amino acid sequences were retrieved from GenBank databases. (D) Amino acid sequence alignment of LbrHY5 and other reported HY5s. The bZIP HY5-like domain is indicated by a black line. 3.4 LbrHY5 binds to multiple types of cis -elements To identify the cis -elements targeted by the LbrHY5 transcription factor, we performed a transcription factor-centered yeast one-hybrid (TF-centered Y1H) assay. We first constructed the LbrHY5 gene into the pGADT7 vector to generate the bait vector and then conducted a background screening test. As shown in Supplementary Fig. 2, yeast growth was undetectable at a concentration of 100 mM 3-AT, reflecting minimal auto-activation and validating the suitability of the system for library screening. The pGADT7-LbrHY5 bait vector was then transformed into a yeast library containing candidate cis -elements, with positive clones initially screened on SD/–Trp/–Leu/–His medium supplemented with 100 mM 3-AT. Selected clones were sequenced and analyzed using BLAST, which was following by a one-to-one verification of the confirmed clones. According to the results, LbrHY5 can bind to various cis -elements, including AT-rich motifs, CACT motifs, and RAV1-binding sites (Fig. 5 A and Supplementary Table 1). A combined analysis of transcriptome data and TF-centered Y1H assay results identified several potential targets of LbrHY5, including the key anthocyanin biosynthesis-related genes LbrCHS and LbrMYB6 . We subsequently isolated their promoter sequences by genome walking, obtaining 764 bp and 680 bp fragments, respectively (Fig. 5 B). Notably, using PlantCARE to examine LbrCHS and LbrMYB6 promoter sequences, we detected the presence of various cis -elements, including AT-rich motifs, CACT motifs, RAV1-binding sites, and G-box elements (Fig. 5 C). 3.5 LbrHY5 directly activates LbrCHS and LbrMYB6 transcription To confirm the direct binding of LbrHY5 to target gene promoters, we conducted a series of molecular assays. Considering the G-box element, which is a conserved promoter motif targeted by HY5, was detected in the LbrCHS and LbrMYB6 promoters, we examined LbrHY5 in terms of its ability to bind to the G-box sequence. On the basis of an electrophoretic mobility shift assay (EMSA), purified LbrHY5 can bind specifically to LbrCHS and LbrMYB6 promoter fragments containing the G-box motif (Fig. 6 A and B). The specificity of this binding was confirmed by competition assays, in which protein–DNA interactions were effectively disrupted by an excess of unlabeled wild-type probe, but remained unaffected by a mutated probe. Hence, the G-box promoter element was identified as a binding site for LbrHY5. Furthermore, to determine whether this binding leads to transcriptional activation, we performed a dual-luciferase (LUC) reporter assay using N. benthamiana leaves. Specifically, we constructed effector vectors expressing LbrHY5 as well as reporter vectors for the expression of the firefly luciferase gene under the control of the LbrCHS or LbrMYB6 promoter (Fig. 6 C). Compared with the control, the co-expression of LbrHY5 resulted in a significant increase in luciferase activity (Fig. 6 D and E), indicating that LbrHY5 can directly activate LbrCHS and LbrMYB6 transcription by binding to their promoters. 3.6 Overexpression of LbrHY5-LbrMYB6 enhances anthocyanin biosynthesis To explore the specific biological functions of LbrHY5 and LbrMYB6 in lily, we constructed the pCAMBIA1305- LbrHY5 and pCAMBIA1305- LbrMYB6 recombinant overexpression vector and inserted it into ‘Robina’ tepals. All transgenic plants were incubated in darkness for 2 days and then exposed to light for 1 day. Compared with EV ‘Robina’ tepals, the tepals of transgenic plants were more extensively colored (Fig. 7 A) and had higher total anthocyanin contents (Fig. 7 B). Additionally, key structural and regulatory genes in the anthocyanin biosynthesis pathway ( LbrCHS , LbrCHI , LbrDFR , LbrANS , and LbrMYB6 ) were expressed at significantly higher levels in OE- LbrHY5 transgenic tepals than in EV control tepals. Further analysis revealed that OE- LbrMYB6 treatment also promoted the expression of these structural genes, and their expression levels were positively correlated with anthocyanin content. Notably, when LbrHY5 and LbrMYB6 were overexpressed simultaneously, the transcriptional levels of the structural genes were more significantly upregulated compared with either single overexpression treatment, indicating a synergistic effect of these two factors in activating the transcription of structural genes (Fig. 7 C). Considered together, these observations suggest that LbrHY5 and LbrMYB6 positively regulates anthocyanin biosynthesis in lily, enhancing pigment accumulation through the transcriptional activation of key structural and regulatory genes in the anthocyanin biosynthesis pathway. Notably, while co-overexpression of LbrHY5 and LbrMYB6 resulted in higher expression levels of structural genes compared with single overexpression (Fig. 7 C), the total anthocyanin content in the co-overexpression group was slightly lower than that in the OE- LbrMYB6 group (Fig. 7 B). This discrepancy may be attributed to post-transcriptional regulatory mechanisms or rate-limiting steps in the anthocyanin biosynthetic pathway that are not fully reflected at the transcript level. Alternatively, excessive activation of the pathway by simultaneous overexpression of two strong activators might trigger feedback inhibition or metabolic flux redistribution, leading to attenuated anthocyanin accumulation. 3.7 Analysis of VIGS-mediated silencing of LbrHY5 and LbrMYB6 To further validate the roles of LbrHY5 and LbrMYB6 in anthocyanin biosynthesis in lily, VIGS technology was employed to perform gene silencing in ‘Robina’ lily tepals. The results showed that, compared with the empty vector (pTRV1 + pTRV2) control, tepals with individual silencing of LbrHY5 (pTRV1 + pTRV2- LbrHY5 ) exhibited significantly increased anthocyanin content (Fig. 8 A and B), along with significantly upregulated expression levels of structural genes ( LbrCHS , LbrCHI , LbrDFR , LbrANS ) and LbrMYB6 in the anthocyanin biosynthesis pathway (Fig. 8 C). In contrast to LbrHY5 silencing, tepals with individual silencing of LbrMYB6 (pTRV1 + pTRV2- LbrMYB6 ) and simultaneous silencing of both LbrHY5 and LbrMYB6 (pTRV1 + pTRV2- LbrHY5 + pTRV2- LbrMYB6 ) showed significantly reduced anthocyanin content (Fig. 8 B), and the expression levels of the structural genes were all significantly downregulated (Fig. 8 C). 3.8 A Working model for the LbrHY5-mediated regulatory effects of light on anthocyanin biosynthesis Based on our findings, we propose a comprehensive working model that elucidates the central role of LbrHY5 in light-induced anthocyanin biosynthesis in L. brownii (Fig. 9 ). Under dark conditions, LbrHY5 transcription is maintained at low levels, resulting in minimal expression of its target genes LbrCHS and LbrMYB6 , and consequently, low anthocyanin accumulation. Following an exposure to light, LbrHY5 transcription is strongly activated. The LbrHY5 protein then directly binds to the G-box motifs in the promoters of LbrCHS (a key structural gene) and LbrMYB6 (a regulatory transcription factor), activating their transcription. The upregulation of LbrMYB6 further promotes the expression of downstream anthocyanin structural genes ( LbrCHS , LbrCHI , LbrDFR , LbrANS ), establishing a transcriptional cascade that amplifies the light signal. This coordinated activation of the anthocyanin biosynthetic pathway ultimately leads to substantial anthocyanin accumulation and the characteristic pigmentation in lily scales. (Yamagish et al., 2010). In summary, the synergistic effects of two processes (i.e., direct activation by LbrHY5 and amplified regulation via LbrMYB6) lead to a robust and coordinated upregulation of the anthocyanin biosynthesis pathway. The associated transcriptional cascade ultimately leads to substantial anthocyanin accumulation in lily, resulting in a characteristic red coloration. 4 Discussion 4.1 Light as a key environmental signal promoting anthocyanin biosynthesis in Lilium brownii Light is an indispensable factor affecting plant coloration. Lily is a shade-loving plant that is highly sensitive to light. Insufficient light affects plant growth, while also influencing flower bud differentiation, resulting in flower and fruit drop. During the dicots of tomato ( S. lycopersicum ) and grape ( Vitis vinifera ), bags are often used to cover developing fruits, but they are removed to stimulate the rapid anthocyanin biosynthesis and accumulation at the mature stage. Moreover, the expression levels of anthocyanin biosynthesis-related structural genes ( PAL , CHS , CHI , F3H , DFR , ANS and UFGT ) are significantly upregulated after an exposure to light (Matus et al., 2009 ; Kim et al., 2021 ). In C. morifolium , anthocyanin biosynthesis genes ( CmCHS , CmF3H , CmANS , CmDFR and Cm3GT ) are reportedly expressed at lower levels in shaded plants than in plants exposed to normal light conditions (Hong et al., 2016 ). Another study showed that shading L. regale plants results in downregulated LrCHSa , LrCHSb , LrF3H , LrF3'H , LrDFR and LrANS expression levels, but the expression of anthocyanin biosynthesis genes is upregulated after light treatments, leading to increased tepal color formation (Yamagishi, 2016 ), which was consistent with the results of the current study. Our study demonstrates that an exposure to light significantly promotes anthocyanin accumulation in L. brownii , accompanied by the upregulation expression of structural and regulatory genes in anthocyanin pathway, including LbrCHS , LbrCHIb , LbrF3H , LbrFLS , LbrDFR , LbrANS , LbrANR and LbrMYB6 . 4.2 Transcriptomic insights into the LbrHY5–LbrMYB6 co-regulatory network Transcriptome profiling further revealed broad transcriptional reprogramming under light, emphasizing the role of LbrHY5 as a master regulator of the anthocyanin pathway. The induction of LbrMYB6 expression by LbrHY5 reflects the potential crosstalk between light and stress signaling pathways because LbrMYB6 is a transcription factor that modulates flavonoid biosynthesis in response to environmental stimuli. In conclusion, we propose a model in which light stabilizes LbrHY5, which in turn activates the expression of LbrMYB6 and structural genes, such as LbrCHS , leading to anthocyanin accumulation in lily. To the best of our knowledge, this study is the first to functionally characterize LbrHY5 in lily, which is a monocotyledonous ornamental species. The study data may form the theoretical basis for improving color traits in L. brownii via light treatment or genetic engineering, with implications for the optimizing L. brownii . 4.3 LbrHY5 functions as a central light-responsive transcription factor in lily We identified LbrHY5 as a bZIP transcription factor localized in the nucleus, wherein it positively regulates anthocyanin biosynthesis. HY5, which is a well-known integrator of light signals in plants, is relatively stable under light and activates the expression of downstream target genes involved in photomorphogenesis and pigment synthesis (Chen et al., 2013 ; Wang et al., 2021 ). Earlier research showed that HY5 activates the transcription of anthocyanin biosynthesis structural genes in response to light signals by binding to the G-box element in target gene promoter regions (Chen et al., 2022 ; Liu et al., 2019 ). Under light conditions, HY5 promotes anthocyanin biosynthesis and accumulation in plants. Accordingly, deleting the hy5 gene in plants leads to decreased anthocyanin contents. The positive regulatory role of HY5 in anthocyanin biosynthesis appears evolutionarily conserved across dicot species. In Arabidopsis , HY5 directly transactivates structural anthocyanin genes while also partnering with BBX proteins to amplify transcriptional output (Bursch et al., 2021 ). Similarly, in tomato, SlHY5 regulates the expression of CHS , F3H , DFR , ANS , and 3-GT compared with wild-type controls, hy5 mutants have lower anthocyanin contents (Qiu et al., 2019 ; Wang et al., 2021 ). In the present study, both LbrHY5 and LbrMYB6 overexpression increased anthocyanin levels and upregulated LbrCHS , LbrCHI, LbrDFR and LbrANS expression, suggesting that LbrHY5 encodes a conserved positive regulator of anthocyanin accumulation. Moreover, VIGS silencing experiments confirmed these findings while revealing complex regulation of LbrHY5. Unexpectedly, individual silencing of LbrHY5 significantly increased anthocyanin content and structural gene expression. This unexpected phenomenon may be attributed to complex regulatory feedback mechanisms. In many plant species, HY5 stability is post-translationally regulated by COP1 in darkness, and light exposure inactivates COP1, allowing HY5 accumulation (Zhao et al., 2022 ; Bi et al., 2025 ). Although similar mechanisms may operate in lily, further experiments are needed to elucidate the precise post-transcriptional regulation of LbrHY5 and the feedback responses triggered by partial silencing. Additionally, this partial silencing may have activated a feedback mechanism that enhances its own transcription, or directly increased anthocyanin biosynthesis via a compensatory upregulation of its target, LbrMYB6 . However, co-silencing LbrHY5 and LbrMYB6 similarly downregulated anthocyanin-related genes, consistent with their synergistic effect observed in overexpression assays. Taken together, these findings suggest that LbrHY5 exhibits more complex regulation involving light signaling and potential feedback mechanisms. 4.4 Distinct regulatory mechanisms of LbrHY5 in lily compared to dicot models Previous studies showed that HY5 regulates plant anthocyanin accumulation in response to light, and requires BBX factor to mediate and promote plant photomorphogenesis, but the regulatory mechanisms are different (Fang et al., 2019 ; Liu et al., 2022 ). In addition, studies have shown that HY5 can also regulate the transcriptional expression of downstream genes by interacting with MYB transcription factors and affecting the stability of MBW complex. The interaction between HY5 and MIR858a in A. thaliana promotes anthocyanin accumulation in seedlings, inhibits the expression of MYBL2 , a negative regulator of anthocyanin biosynthesis, and activates the expression of anthocyanin biosynthesis structural genes in response to light signals (Wang et al., 2016 ). In pear, it was found that PpBBX16 cannot directly bind to the PpCHS or PpMYB10 promoters, but the PpBBX16–PpHY5 complex can enhance the activity of MdMYB10 promoter (Bai et al., 2019a ). Moreover, PpHY5 alone cannot activate the transcription of target genes, and inhibits PpMYB10 expression with the help of PpBBX18 and PpBBX21, thereby regulating the biosynthesis of anthocyanin (Bai et al., 2019b ). In apple calli, MdBBX22 binds to MdHY5 to form a complex that activates the anthocyanin biosynthesis pathway, ultimately promoting anthocyanin accumulation and coloration under UV-B light (Bai et al., 2014 ; An et al., 2019 ). Conversely, MdBBX37 suppresses anthocyanin accumulation by binding to the MdHY5 promoter without interacting with the protein (An et al., 2020 ). Notably, in the current study, we determined that LbrHY5 can bind to the promoters of LbrCHS and LbrMYB6 in yeast one-hybrid assays, although it did not interact with LbrMYB6 in yeast two-hybrid assays. This suggests that LbrHY5 may directly activate the expression of these genes without forming stable complexes with the encoded proteins. The lack of LbrHY5–LbrMYB6 interaction in lily may reflect species-specific regulatory mechanisms, with LbrHY5 functioning independently of LbrMYB6. This differs from the regulatory mechanism in pear and apple, wherein HY5 forms complexes with MYB or BBX. Hence, compared with other plants, lily may have a more direct transcriptional activation mechanism. 5 Conclusions Our study provides a systematically regulatory mechanism of light-induced anthocyanin biosynthesis in Lilium brownii . We demonstrated that light exposure significantly promotes anthocyanin accumulation and upregulates the expression of key structural genes ( LbrCHS , LbrCHI , LbrF3H , LbrFLS , LbrDFR , LbrANS , LbrANR ) and the regulatory gene LbrMYB6 . LbrHY5 was identified as a light-responsive bZIP transcription factor localized to the nucleus, with its expression significantly induced by light. Transcriptome analysis revealed that LbrHY5 and LbrMYB6 serve as central hubs in a light-responsive co-expression network regulating anthocyanin biosynthesis. Electrophoretic mobility shift and dual-luciferase reporter assays collectively demonstrated that LbrHY5 engages G-box cis -elements within the LbrCHS and LbrMYB6 promoters, thereby driving their transcriptional activation. Functional analyses through overexpression and VIGS silencing demonstrated that both LbrHY5 and LbrMYB6 positively regulate anthocyanin biosynthesis, with synergistic effects observed upon co-overexpression. The unexpected increase in anthocyanin accumulation following individual LbrHY5 silencing may be attributed to light-induced COP1 inactivation and potential feedback regulatory mechanisms. In conclusion, our findings establish that the LbrHY5–LbrMYB6 transcriptional module plays a critical role in mediating light-induced anthocyanin biosynthesis in L. brownii , providing new insights into the regulatory networks controlling pigmentation in monocotyledonous plants and offering potential targets for genetic improvement of color traits in ornamental lilies. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding Declaration This work was supported by the National Natural Science Foundation of China (32402611); Jiangxi Provincial Department of the National Natural Science Foundation (20232BAB215044) and Key Science and Technology Research Project in Jiangxi Province Department of Education (GJJ2201232). Acknowledgments This study was conducted at the Ganzhou Key Laboratory of Nanling Plant Resources Protection and Utilization and Jiangxi Provincial Key Laboratory of Pest and Disease Control of Featured Horticultural Plants, China. Data availability Data will be made available on request. 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Supplementary Files Fig.S1.png Fig.S2.png TableS1.xlsx AppendixA.Supplementarydata.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 04 May, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 05 Apr, 2026 Editor assigned by journal 04 Apr, 2026 Submission checks completed at journal 04 Apr, 2026 First submitted to journal 01 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9289041","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619224929,"identity":"9a0661dd-18f2-4e00-862a-51d48f5d927f","order_by":0,"name":"Yuwei Cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYNCCCijNQ7yWMyRrYWwjRQt/++Fj0rzzavPMJRIYH7xtY5A3J6RF4kxamjTvtuPFljMSmA3ntjEY7mwgoMWAIcdMOnfbscQNNxLYpHnbGBIMDhDSwv8GqGUOWAv7b+K0SIBsaagB28JMlBaJG8+Srf8cO5C44czDZsk55yQMNxDSwt+ffPDmjJq6xA3Hkw9+eFNmI0/QFiBgkWBgOAykGRtAthJWDwTMHxgY6ohSOQpGwSgYBSMUAACPHUA0UxMtMQAAAABJRU5ErkJggg==","orcid":"","institution":"GanNan Normal University","correspondingAuthor":true,"prefix":"","firstName":"Yuwei","middleName":"","lastName":"Cao","suffix":""},{"id":619224930,"identity":"77c0eaaa-f9cf-444d-8f96-3c98a076d80b","order_by":1,"name":"Yuqian Zhang","email":"","orcid":"","institution":"GanNan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuqian","middleName":"","lastName":"Zhang","suffix":""},{"id":619224931,"identity":"9e09eca2-34fb-46c5-bddd-9b372e41465a","order_by":2,"name":"Huiwen Yang","email":"","orcid":"","institution":"GanNan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Huiwen","middleName":"","lastName":"Yang","suffix":""},{"id":619224932,"identity":"06e13e1f-ead5-4694-b806-4d5e9408945c","order_by":3,"name":"Yuwen Ye","email":"","orcid":"","institution":"GanNan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuwen","middleName":"","lastName":"Ye","suffix":""},{"id":619224933,"identity":"866fcf24-6b2c-4bcd-b0fa-bc848438086f","order_by":4,"name":"Hui Lai","email":"","orcid":"","institution":"GanNan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Lai","suffix":""},{"id":619224934,"identity":"60bb57ff-1dc5-4e51-9287-1c3b68ba4804","order_by":5,"name":"Shanghui Yin","email":"","orcid":"","institution":"GanNan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Shanghui","middleName":"","lastName":"Yin","suffix":""},{"id":619224935,"identity":"bbe47ead-f7f1-429e-b3a6-5c0777260c19","order_by":6,"name":"Wei Zhang","email":"","orcid":"","institution":"GanNan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":619224936,"identity":"1ac59f9b-1933-4c76-bbc6-45d09317f647","order_by":7,"name":"Huyue Li","email":"","orcid":"","institution":"GanNan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Huyue","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-04-01 08:38:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9289041/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9289041/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106562257,"identity":"2503dc65-74ad-46d5-b937-fd1632d0e188","added_by":"auto","created_at":"2026-04-10 00:00:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":344955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal anthocyanin content and related gene expression in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. brownii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e following different light treatments\u003c/strong\u003e. (A) Phenotypes of scales under different light conditions. Scale bar, 1 cm. (B) Total anthocyanin contents after light treatments. (C) Expression levels of genes related to anthocyanin biosynthesis after light treatments. Gene expression was normalized to the internal reference gene, and relative expression levels were calculated using the 2\u003csup\u003e-ΔCt\u003c/sup\u003e method. Data are presented as the means ± SEM (n = 3). Statistically significant differences were determined by ordinary one-way ANOVA followed by Dunnett’s \u003cem\u003et-\u003c/em\u003etest (* \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, ns: not significant).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/f7dfe7c36913a04671a5fd62.png"},{"id":106725414,"identity":"d925d8c7-a6f9-44ab-8758-03723cd511fa","added_by":"auto","created_at":"2026-04-12 18:32:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":591131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. brownii \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003escales exposed to darkness (D), 1 day of light (L1), and 2 days of light (L2). \u003c/strong\u003e(A) Enriched GO terms related to anthocyanin biosynthesis and light among differentially expressed genes (DEGs) in the D, L1, and L2 groups. (B) Enriched KEGG pathways related to anthocyanin biosynthesis among DEGs in the D, L1, and L2 groups. (C) Venn diagram of the overlapping DEGs among the three groups. (D) Results of the statistical analysis of upregulated and downregulated genes in the three groups.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/16911938cc03becb9cb857af.png"},{"id":106562265,"identity":"8bc32b2e-40b6-4423-9bbc-2f2f87b09891","added_by":"auto","created_at":"2026-04-10 00:00:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":534050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of differentially expressed genes (DEGs) among D, L1, and L2\u003c/strong\u003e. (A) Subcluster matrix presenting the expression trends of 31 anthocyanin-related DEGs. (B) Co-expression network of anthocyanin-related DEGs. Relationship threshold \u0026lt;0.6. (C) Cluster heatmap of 31 DEGs related to the anthocyanin biosynthesis and regulatory pathway.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/c30c8a36336de0e8e769fdd9.png"},{"id":106562268,"identity":"96622806-e67d-4425-80a6-5191b5c05673","added_by":"auto","created_at":"2026-04-10 00:00:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":856068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis of LbrHY5\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein lily. \u003c/strong\u003e(A) \u003cem\u003eLbrHY5\u003c/em\u003eexpression levels in darkness and under light. Scales were treated with darkness (D) or continuous light for 2 days (L2). Data are presented as the mean ± SEM (n = 3). Statistically significant differences were determined using Student’s \u003cem\u003et-\u003c/em\u003etest (** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01). (B) Subcellular localization of the LbrHY5-GFP fusion protein, with GFP alone used as a negative control. Bar, 100 μm. (C) Neighbor-joining phylogenetic tree derived from deduced amino acid sequences of LbrHY5 and other reported HY5s. Numbers next to nodes are bootstrap values from 1,000 replications. Amino acid sequences were retrieved from GenBank databases. (D) Amino acid sequence alignment of LbrHY5 and other reported HY5s. The bZIP HY5-like domain is indicated by a black line.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/2ac8a2256ac36cb936689082.png"},{"id":106727603,"identity":"1ed47fc9-07fb-4a95-8d43-fe8d14d49dc6","added_by":"auto","created_at":"2026-04-12 18:39:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":364723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLbrHY5 can bind to multiple types of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-elements in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrCHS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrMYB6 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromoters\u003c/strong\u003e. (A) Y1H assay results showing that LbrHY5 can bind to multiple types of \u003cem\u003ecis\u003c/em\u003e-elements. (B) Genome walking-based cloning of \u003cem\u003eLbrCHS\u003c/em\u003eand \u003cem\u003eLbrMYB6\u003c/em\u003e gene promoters. (C) Identified \u003cem\u003ecis\u003c/em\u003e-elements in \u003cem\u003eLbrCHS\u003c/em\u003eand \u003cem\u003eLbrMYB6 \u003c/em\u003epromoters\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/4b4b4b700b3f974972a62bf2.png"},{"id":106727606,"identity":"484befb6-577a-4202-acb1-5594713a9305","added_by":"auto","created_at":"2026-04-12 18:39:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":401740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLbrHY5 can directly bind to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrCHS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrMYB6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoters\u003c/strong\u003e. (A) EMSA showing the binding of LbrHY5 to \u003cem\u003eLbrCHS\u003c/em\u003e promoter fragments containing the G-box element. Unlabeled probes were used for the competition assay. (B) EMSA showing the binding of LbrHY5 to \u003cem\u003eLbrMYB6\u003c/em\u003e promoter fragments containing the G-box element. Unlabeled probes were used for the competition assay. The G-box motif and mutant site are indicated in red fonts. + and - represent presence and absence of the protein and probe, respectively. Arrows indicated the positions of protein–DNA complexes for the free probe. (C) Schematic representation of the luciferase reporter vectors containing the \u003cem\u003eLbrCHS\u003c/em\u003e or \u003cem\u003eLbrMYB6\u003c/em\u003e promoters as well as the effector vector containing \u003cem\u003eLbrHY5.\u003c/em\u003e (D) Dual-luciferase reporter assay showing LbrHY5 is a transcription factor that can activate the transcription of the \u003cem\u003eLbrCHS\u003c/em\u003e. (E) Dual-luciferase reporter assay showing LbrHY5 is a transcription factor that can activate the transcription of the \u003cem\u003eLbrMYB6\u003c/em\u003e. Each experiment was performed using 10 biological replicates. Data are presented as the mean ± SEM (n = 10). Significant differences were determined by Student’s \u003cem\u003et\u003c/em\u003e-test at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/ba77c7d683919cec52c065bd.png"},{"id":106725618,"identity":"54df7698-1304-4ce9-924b-823f1ff7a301","added_by":"auto","created_at":"2026-04-12 18:33:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":521171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotype and anthocyanin-related gene expression levels of ‘Robina’ tepals overexpressing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrHY5 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(OE-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrHY5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e), overexpressing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrMYB6 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(OE-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrMYB6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) and co-overexpressing both \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrHY5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrMYB6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (OE-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrHY5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e+OE-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrMYB6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e. (A) Phenotypic comparison of empty vector (EV) control tepals and overexpressing\u003cem\u003e \u003c/em\u003etransgenic tepals. (B) Total anthocyanin contents of EV and overexpressing\u003cem\u003e \u003c/em\u003etransgenic tepals. Three biological replicates were used for each sample. (C) Differences in the expression of structural and regulatory genes related to anthocyanin biosynthesis between EV and overexpressing plants as determined by qRT-PCR. Gene expression was normalized to the internal reference gene, and relative expression levels were calculated using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method, with the empty vector (EV) control group set to 1. Data are presented as the mean ± SEM of three replicates. Significant differences were determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisonstest (* \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/4b4bfda71f18560a74f97e3a.png"},{"id":106562270,"identity":"acb0f462-9d15-4498-8485-69809c855b73","added_by":"auto","created_at":"2026-04-10 00:00:30","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":515595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVIGS assay reveals the roles of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrHY5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLbrMYB6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in anthocyanin biosynthesis of ‘Robina’ lily\u003c/strong\u003e. (A) Visual phenotypes of tepals following VIGS treatment with empty vector (pTRV1+pTRV2), pTRV1+pTRV2-\u003cem\u003eLbrHY5\u003c/em\u003e, pTRV1+pTRV2-\u003cem\u003eLbrMYB6\u003c/em\u003e, or their combination (pTRV1+pTRV2-\u003cem\u003eLbrHY5\u003c/em\u003e+pTRV2-\u003cem\u003eLbrMYB6\u003c/em\u003e). Tissues were harvested after 1-day dark incubation and 2 days under light. (B) Quantification of total anthocyanin contents in the treated tepals (mean ± SEM, n = 3). (C) Transcript levels of key anthocyanin pathway genes measured by qRT-PCR. Gene expression was normalized to the internal reference gene, and relative expression levels were calculated using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method, with the empty vector (EV) control group set to 1. Data are shown as mean ± SEM (n=3). Statistically significant differences were determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) compared to EV control (ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/79402e75c5c249ca8900a57c.png"},{"id":106562266,"identity":"71b83391-dda2-4dbd-93a8-437cb37e19e9","added_by":"auto","created_at":"2026-04-10 00:00:30","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":244853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorking model for the effects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. brownii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e LbrHY5 on anthocyanin biosynthesis in darkness and under light.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/18ed69649ffd4b2e33731b25.png"},{"id":106959794,"identity":"a5902c02-c4c9-43b3-a207-ab592c8f38b4","added_by":"auto","created_at":"2026-04-15 09:15:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6127409,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/4cd5ae73-f996-427b-b043-faef8403383b.pdf"},{"id":106724981,"identity":"73d33433-bc3d-46f8-a803-1d5d0e0003c0","added_by":"auto","created_at":"2026-04-12 18:30:51","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":629429,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/1653bbd1d26885be35e01635.png"},{"id":106725412,"identity":"ab1592ff-742c-459e-8340-c10a05e60d8e","added_by":"auto","created_at":"2026-04-12 18:32:47","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":831941,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S2.png","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/6af433db9af251ca3e14eeff.png"},{"id":106562261,"identity":"305308ba-df7d-47da-a8a5-7b8c2ca63e2e","added_by":"auto","created_at":"2026-04-10 00:00:30","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9704,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/85c24480f05a81e6d1c2728d.xlsx"},{"id":106562262,"identity":"72d29f43-1274-462a-9b7b-97b5437c3e2a","added_by":"auto","created_at":"2026-04-10 00:00:30","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13887,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixA.Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-9289041/v1/af3779d57e867b53189083ba.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eLight-induced anthocyanin biosynthesis in Lilium brownii is mediated by the LbrHY5–LbrMYB6 module\u003c/p\u003e","fulltext":[{"header":"Key message","content":"\u003cp\u003e\u003cstrong\u003eThe LbrHY5\u0026ndash;LbrMYB6 transcriptional module mediates light-induced anthocyanin biosynthesis in \u003cem\u003eLilium brownie\u003c/em\u003e by directly activating \u003cem\u003eLbrCHS\u003c/em\u003e and downstream structural genes.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"1 Introduction","content":"\u003cp\u003e \u003cem\u003eLilium brownii\u003c/em\u003e is a monocotyledonous plant with considerable ornamental, edible, and medicinal value. The purplish-red pigments in its floral organs and bulbs are primarily anthocyanins, which are flavonoids that contribute to pollination, stress resistance, and human health (Grotewold, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Anthocyanin biosynthesis is controlled by both developmental programs and environmental cues, with light being one of the most influential factors (Winkel-Shirley, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLight represents a primary environmental determinant of anthocyanin accumulation across the plant kingdom. In dicotyledonous crops including tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) and grape (\u003cem\u003eVitis vinifera\u003c/em\u003e), light exposure coordinately induces expression of the core flavonoid biosynthetic genes\u003cem\u003e\u0026mdash;PAL\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e, and \u003cem\u003eUFGT\u0026mdash;\u003c/em\u003ethereby promoting pigment synthesis in reproductive and vegetative tissues (Matus et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, light exposure enhances the expression of anthocyanin-related genes in \u003cem\u003eChrysanthemum morifolium\u003c/em\u003e (Hong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and \u003cem\u003eLilium regale\u003c/em\u003e (Yamagishi, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), underscoring the conserved nature of light responses across species (Wei et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the central regulators mediating light signal transduction, ELONGATED HYPOCOTYL5 (HY5)\u0026mdash;a bZIP transcription factor\u0026mdash;stands out for its light-dependent accumulation and subsequent activation of genes governing photomorphogenesis and flavonoid metabolism (Feng et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Under light, HY5 accumulates and binds to G-box or ACE-box elements in the promoters of target genes in the anthocyanin pathway, thereby activating transcription (Bursch et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Maier et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLoss-of-function mutations in \u003cem\u003eHY5\u003c/em\u003e have been linked to reduced pigmentation and transcriptional suppression of core anthocyanin biosynthetic genes in model species such as \u003cem\u003eArabidopsi\u003c/em\u003e and tomato (Li et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Qiu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). HY5 often functions alongside other factors, such as B-box (BBX) proteins and MYB transcription factors, forming complexes that fine-tune anthocyanin biosynthesis in response to light quality and intensity (Bai et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Fang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; An et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, PpHY5 and PpBBX16 form a complex that can binds directly to the \u003cem\u003ePpCHS\u003c/em\u003e or \u003cem\u003ePpMYB10\u003c/em\u003e promoter (Bai et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Additionally, HY5 stability is post-transcriptionally regulated by CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), which targets HY5 for degradation in darkness. Upon illumination, photoreceptors inhibit COP1, allowing HY5 to accumulate and bind to promoter elements of anthocyanin-related genes (Maier et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bursch et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMYB6, a key R2R3-MYB transcription factor, participate in light signal-mediated secondary metabolism across diverse plant species, particularly playing a critical role in the flavonoid and anthocyanin biosynthetic pathways. In \u003cem\u003eC. morifolium\u003c/em\u003e, CmMYB6 functions as a central activator of anthocyanin biosynthesis and floral pigmentation (Xia et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). In \u003cem\u003ePopulus tomentosa\u003c/em\u003e, MYB6 positively regulates the accumulation of both anthocyanins and proanthocyanidins (PAs) (Wang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, in Sanhua plum\u0026rsquo;s flesh (\u003cem\u003ePrunus salicina\u003c/em\u003e), MYB6 is associated with \u003cem\u003eC4H\u003c/em\u003e expression and anthocyanin accumulation (Xiang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, MYB6 transcription factors consistently show positive responsiveness to light signals. In \u003cem\u003eFagopyrum tataricum\u003c/em\u003e, the FtMYB6 promoter displays strong light-inducible activity, and its overexpression significantly enhances flavonol accumulation in transgenic tobacco and hairy roots of tartary buckwheat (Yao et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, LhMYB6 has been implicated in lily tepal pigmentation, and the mechanistic basis of its involvement in light-regulated anthocyanin biosynthesis remains elusive.\u003c/p\u003e \u003cp\u003eAlthough lily is a shade-loving plant, the mechanism underlying its flower coloration, especially its rich and bright colors in high-altitude areas with strong ultraviolet radiation, depends on the light signaling pathway. The role of HY5 has been extensively characterized in dicots, but its function in monocots, especially ornamental species (e.g., lily), remains poorly understood. \u003cem\u003eL. brownii\u003c/em\u003e exhibits light-dependent pigmentation, but the regulatory network controlling this process has not been comprehensively elucidated. In this study, we functionally characterized LbrHY5 in terms of its role in light-induced anthocyanin biosynthesis. We determined that LbrHY5 is a nuclear protein that binds to the promoters of key anthocyanin-related genes. The target genes of LbrHY5, \u003cem\u003eLbrMYB6\u003c/em\u003e and \u003cem\u003eLbrCHS\u003c/em\u003e, were identified by combining transcriptome analysis with TF-centered Y1H screening. Moreover, \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e overexpression can enhance pigment accumulation. Our results provide the first evidence of the regulatory effects of the LbrHY5\u0026ndash;LbrMYB6 module on anthocyanin biosynthesis in lily, offering new insights into light-mediated coloration in monocots.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Light treatment of lily scales\u003c/h2\u003e \u003cp\u003e \u003cem\u003eL. brownii\u003c/em\u003e bulbs were purchased from Wanzai County (Yichun, Jiangxi, China). Light treatment was performed under cool white fluorescent light at an intensity of 100 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1;, with a constant temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Scales in the darkness group (D) were kept in complete darkness under the same temperature. For the light-treated groups, scales were exposed to continuous light for 1 day (L1) or 2 days (L2), respectively. Each treatment and subsequent phenotypic analysis were completed using 48 scales.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Anthocyanin quantification and gene expression analysis\u003c/h2\u003e \u003cp\u003eAnthocyanins were extracted from scales after treatment with different light. The absorbances of the extracts at 530 nm, 620 nm and 650 nm were measured using a UV-2600 spectrophotometer (Shimadzu, Japan). The anthocyanin content was calculated as 0.1 OD (unit\u0026times;10\u003csup\u003e3\u003c/sup\u003e g\u0026middot;FW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and the calculation method was OD = (A₅₃₀ - A₆₂₀)\u0026thinsp;\u0026minus;\u0026thinsp;0.1 \u0026times; (A₆₅₀ - A₆₂₀).\u003c/p\u003e \u003cp\u003eFollowing different light treatments, \u003cem\u003eL. brownii\u003c/em\u003e scales were harvested for total RNA extraction, after which complementary DNA (cDNA) was synthesized via reverse transcription. Gene-specific quantitative real-time PCR (qRT-PCR) primers were designed according to non-conserved gene sequences, with \u003cem\u003eLilyactin\u003c/em\u003e used as the internal reference. A qRT-PCR analysis was performed using SYBR Green master mix and a CFX96 system (Bio-Rad, USA), with relative expression levels determined according to the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Transcriptome sequencing and bioinformatic analysis\u003c/h2\u003e \u003cp\u003eAn Illumina platform was used for a transcriptome sequencing analysis of \u003cem\u003eL. brownii\u003c/em\u003e scales following different light treatments (D, L1, and L2), with three biological replicates per treatment. After quality control and alignment steps, DEGs were identified. GO and KEGG pathway enrichment analyses were conducted to determine the biological functions of DEGs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Function identification of \u003cem\u003eLbrHY5\u003c/em\u003e in \u003cem\u003eL. brownii\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe full-length \u003cem\u003eLbrHY5\u003c/em\u003e cDNA sequence was cloned via rapid amplification according to the transcriptome data. Conserved domains were predicted using the NCBI Conserved Domain Database. In addition, a phylogenetic tree was constructed using the neighbor-joining method and MEGA 11 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Subcellular localization analysis\u003c/h2\u003e \u003cp\u003eTo generate the LbrHY5-GFP fusion construct, the coding sequence of \u003cem\u003eLbrHY5\u003c/em\u003e (excluding the termination codon) was ligated in-frame upstream of the GFP coding region within the pCAMBIA2300 backbone. The resulting plasmid was introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101, and bacterial suspensions were infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves for transient expression assays. After 3 days, GFP fluorescence was observed using a laser scanning confocal microscope, with nuclei counterstained by mCherry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 TF-centered Y1H assay to identify target genes\u003c/h2\u003e \u003cp\u003eThe key light-induced transcription factor gene \u003cem\u003eLbrHY5\u003c/em\u003e was inserted into the pGADT7 vector to construct the bait vector. A library of yeast cells containing candidate \u003cem\u003ecis\u003c/em\u003e-motifs was screened. Positive clones were selected on SD/\u0026minus;Trp/\u0026minus;Leu/\u0026minus;His medium supplemented with 100 mM 3-AT and then sequenced.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Promoter cloning\u003c/h2\u003e \u003cp\u003eThe promoter sequences of \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e were isolated using the Genome Walking Kit (Clotech, China). Putative \u003cem\u003ecis\u003c/em\u003e-acting elements were identified using the PlantCARE database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Dual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe promoters of \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e were inserted into the pGreenII 0800-LUC vector, whereas the \u003cem\u003eLbrHY5\u003c/em\u003e CDS was cloned into the pGreenII 62-SK expression vector. Agrobacterium suspensions harboring the promoter reporter and transcription factor effector constructs were combined at a 10:1 volumetric ratio prior to co-infiltration into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Luciferase activities were measured after 3 days and then the firefly luciferase: \u003cem\u003eRenilla\u003c/em\u003e luciferase ratio was calculated to determine transcriptional activation. The strain containing the empty expression vector was mixed with the reporter strain as a negative control. The firefly luciferase: \u003cem\u003eRenilla\u003c/em\u003e luciferase ratio was set to 1, and 10 biological replicates were prepared for each experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 EMSA detection\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eLbrHY5\u003c/em\u003e CDS was inserted into the pET-22b(+) expression vector. Recombinant LbrHY5-HIS was produced in \u003cem\u003eEscherichia coli\u003c/em\u003e cells and purified. A western blot was used to detect the purified recombinant protein. Biotin-labeled double-stranded oligonucleotides containing the wild-type or mutated G-box motif were used as EMSA probes. Binding reactions were analyzed using a native polyacrylamide gel, with shifts detected using a chemiluminescent EMSA kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Overexpression and phenotypic analysis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eLbrHY5\u003c/em\u003e CDS was incorporated into the pCAMBIA1305 plant transformation vector. The resulting recombinant plasmid was inserted into \u003cem\u003eA. tumefaciens\u003c/em\u003e strain EHA105 cells for the transformation of \u0026lsquo;Robina\u0026rsquo; lily tepals and \u003cem\u003eL. brownii\u003c/em\u003e scales via vacuum injection. MMA resuspension buffer [4.43 g/L MS (without vitamins), 20 g/L sucrose, 1.95 g/L MES, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 100 \u0026micro;M acetosyringone, and 10 \u0026micro;M paclobutrazol, pH 5.6] was adjusted for an OD\u003csub\u003e600\u003c/sub\u003e of 1.0. Tissues transformed with EV were used as the control. Anthocyanin accumulation and the expression of related genes were compared overexpression and control lines following light treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 VIGS-mediated infection of lily tepals\u003c/h2\u003e \u003cp\u003eUsing the previously established petal VIGS system in lily (Bi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we cloned the \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e genes and inserted them into the pTRV2 vector. The recombinant vectors were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 competent cells and resuspended in MMA buffer to an OD\u003csub\u003e600\u003c/sub\u003e of 1.0. The \u003cem\u003eAgrobacterium\u003c/em\u003e suspensions containing the recombinant pTRV2 and pTRV1 were mixed at a 1:1 ratio and infiltrated into tepals using a 0.05 MPa vacuum infiltration method for 5 min at room temperature. A mixture of \u003cem\u003eAgrobacterium\u003c/em\u003e suspensions containing empty pTRV2 and pTRV1 was used as the control for anthocyanin content and structural gene expression analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed with at least three independent biological replicates. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, USA). For comparisons between two groups, Student\u0026rsquo;s t-test was applied. For comparisons among multiple groups, ordinary one-way ANOVA was performed, followed by Dunnett\u0026rsquo;s multiple comparisons test when comparing all groups to a single control group. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, as indicated in the figures. Different letters or asterisks denote statistically significant differences as described in the figure legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Light promotes anthocyanin accumulation and related genes expression \u003cem\u003ein L. brownii\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo clarify the effects of light on \u003cem\u003eL. brownii\u003c/em\u003e bulb coloration, we exposed scales to light for different durations (no light, 1 day of light, and 2 days of light) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In \u003cem\u003eL. brownii\u003c/em\u003e bulbs, the anthocyanin content was almost undetectable in darkness. However, anthocyanin accumulated significantly as the duration of the light treatment increased, demonstrating a clear positive correlation between light exposure duration and anthocyanin production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo elucidate the molecular basis of this light-induced pigmentation, we analyzed the expression levels of key structural and regulatory genes involved in anthocyanin biosynthesis. Notably, \u003cem\u003eLbrCHS1\u003c/em\u003e, \u003cem\u003eLbrCHIb\u003c/em\u003e, \u003cem\u003eLbrF3H\u003c/em\u003e, \u003cem\u003eLbrFLS\u003c/em\u003e, \u003cem\u003eLbrDFR\u003c/em\u003e, \u003cem\u003eLbrANS\u003c/em\u003e, \u003cem\u003eLbrANR\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e expression levels were consistently upregulated in response to light (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Collectively, these findings indicate that an exposure to light not only promotes anthocyanin accumulation in \u003cem\u003eL. brownii\u003c/em\u003e but also enhances the expression of anthocyanin-related genes. To comprehensively identify key transcription factors involved in light-induced anthocyanin biosynthesis in \u003cem\u003eL. brownii\u003c/em\u003e, we performed transcriptomic analysis on scales subjected to different light treatments (D, L1, L2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Transcriptomic analysis reveals anthocyanin-related gene expression changes in response to light\u003c/h2\u003e \u003cp\u003eTo elucidate the global changes in gene transcription levels in light-treated lily plants, we performed a transcriptome sequencing analysis of scales exposed to different light conditions. As shown in Fig.\u0026nbsp;2 and Supplementary Fig.\u0026nbsp;1, a Gene Ontology (GO) enrichment analysis revealed that differentially expressed genes (DEGs) after 1- and 2-day light treatments were significantly enriched in biological processes related to anthocyanin biosynthesis and light responses (Fig.\u0026nbsp;2A), thereby confirming that light is a key environmental signal activating anthocyanin biosynthesis. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis further demonstrated that these DEGs were specifically associated with the anthocyanin biosynthesis pathway (Fig.\u0026nbsp;2B), reflecting the coordinated activation of the complete biosynthetic network by light. A Venn diagram-based analysis identified both unique and shared genes among comparison groups (Fig.\u0026nbsp;2C), indicating that light-induced transcriptional reprogramming is a dynamic and complex process. Furthermore, there were significantly more upregulated genes than downregulated genes after light treatments (Fig.\u0026nbsp;2D), implying that light mainly activated anthocyanin biosynthesis-related gene expression.\u003c/p\u003e \u003cp\u003eFurthermore, to investigate the key DEGs associated with anthocyanin biosynthesis and their regulatory networks, we selected 31 DEGs in KEGG pathways related to anthocyanin and flavonoid biosynthesis for further analyses. A subcluster matrix revealed the consistent expression pattern among these genes, with an initial increase followed by stabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To identify potential core regulators, we constructed a co-expression network of the 31 anthocyanin-related DEGs. Notably, \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e were positioned at the central hubs of this network (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting that they function as key transcription factors governing the light-responsive anthocyanin regulatory network. Based on these transcriptomic results, we selected \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e as candidate genes for further functional characterization. Additionally, a cluster heatmap confirmed the significant upregulation of anthocyanin biosynthesis structural genes, such as \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, and \u003cem\u003eANS\u003c/em\u003e, after an exposure to light (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;2 Transcriptome analysis of\u003c/b\u003e \u003cb\u003eL. brownii\u003c/b\u003e \u003cb\u003escales exposed to darkness (D), 1 day of light (L1), and 2 days of light (L2).\u003c/b\u003e (A) Enriched GO terms related to anthocyanin biosynthesis and light among differentially expressed genes (DEGs) in the D, L1, and L2 groups. (B) Enriched KEGG pathways related to anthocyanin biosynthesis among DEGs in the D, L1, and L2 groups. (C) Venn diagram of the overlapping DEGs among the three groups. (D) Results of the statistical analysis of upregulated and downregulated genes in the three groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 LbrHY5 is a light-induced nuclear-localized transcription factor\u003c/h2\u003e \u003cp\u003eGiven that \u003cem\u003eLbrHY5\u003c/em\u003e was identified as a core transcription factor from the transcriptomic co-expression network (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), we next performed bioinformatics analysis to characterize its sequence features and expression patterns. To clarify the biological function of LbrHY5 in \u003cem\u003eL. brownii\u003c/em\u003e, we isolated and cloned the full-length \u003cem\u003eLbrHY5\u003c/em\u003e sequence. The encoded protein was revealed to contain 154 amino acids, with a calculated molecular weight of 17.4 kDa and a theoretical isoelectric point of 9.91. Additionally, the 810 bp full-length \u003cem\u003eLbrHY5\u003c/em\u003e sequence included a 464 bp coding sequence (CDS), 80 bp 5\u0026rsquo;- untranslated region (UTR), and 306 bp 3\u0026rsquo;-UTR.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLbrHY5\u003c/em\u003e expression levels were significantly upregulated following an exposure to continuous light for 2 days (relative to the expression in darkness), suggesting that \u003cem\u003eLbrHY5\u003c/em\u003e is a light-responsive gene (Fig.\u0026nbsp;4A). To analyze the subcellular localization of LbrHY5, the \u003cem\u003eLbrHY5\u003c/em\u003e CDS with the termination codon removed was cloned and inserted into the pCAMBIA2300-eGFP vector to generate the \u003cem\u003e35S\u003c/em\u003e::\u003cem\u003eLbrHY5\u003c/em\u003e-\u003cem\u003eGFP\u003c/em\u003e fusion construct, with \u003cem\u003e35S\u003c/em\u003e::\u003cem\u003eGFP\u003c/em\u003e serving as the control construct. The LbrHY5-GFP fusion protein was exclusively localized in the nucleus (Fig.\u0026nbsp;4B), which is consistent with the prediction that LbrHY5 is a transcription factor.\u003c/p\u003e \u003cp\u003eTo explore evolutionary relationships, MEGA 7.0 software was used to construct a phylogenetic tree for LbrHY5 and published HY5s according to the neighbor-joining method (with 1,000 bootstrap replicates). According to the results of the phylogenetic analysis, LbrHY5 was grouped in the HY5 subfamily (Fig.\u0026nbsp;4C). Furthermore, an amino acid sequence alignment using DNAMAN confirmed that LbrHY5 contains a conserved bZIP HY5-like domain included in the NCBI Conserved Domain Database, making it a member of the bZIP transcription factor family (Fig.\u0026nbsp;4D).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"1\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eFigure\u0026nbsp;4 Bioinformatics analysis of LbrHY5 in lily.\u003c/b\u003e (A) \u003cem\u003eLbrHY5\u003c/em\u003e expression levels in darkness and under light. Scales were treated with darkness (D) or continuous light for 2 days (L2). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;3). Statistically significant differences were determined using Student\u0026rsquo;s \u003cem\u003et-\u003c/em\u003etest (** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). (B) Subcellular localization of the LbrHY5-GFP fusion protein, with GFP alone used as a negative control. Bar, 100 \u0026micro;m. (C) Neighbor-joining phylogenetic tree derived from deduced amino acid sequences of LbrHY5 and other reported HY5s. Numbers next to nodes are bootstrap values from 1,000 replications. Amino acid sequences were retrieved from GenBank databases. (D) Amino acid sequence alignment of LbrHY5 and other reported HY5s. The bZIP HY5-like domain is indicated by a black line.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 LbrHY5 binds to multiple types of \u003cem\u003ecis\u003c/em\u003e-elements\u003c/h2\u003e \u003cp\u003eTo identify the \u003cem\u003ecis\u003c/em\u003e-elements targeted by the LbrHY5 transcription factor, we performed a transcription factor-centered yeast one-hybrid (TF-centered Y1H) assay. We first constructed the \u003cem\u003eLbrHY5\u003c/em\u003e gene into the pGADT7 vector to generate the bait vector and then conducted a background screening test. As shown in Supplementary Fig.\u0026nbsp;2, yeast growth was undetectable at a concentration of 100 mM 3-AT, reflecting minimal auto-activation and validating the suitability of the system for library screening. The pGADT7-LbrHY5 bait vector was then transformed into a yeast library containing candidate \u003cem\u003ecis\u003c/em\u003e-elements, with positive clones initially screened on SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His medium supplemented with 100 mM 3-AT. Selected clones were sequenced and analyzed using BLAST, which was following by a one-to-one verification of the confirmed clones. According to the results, LbrHY5 can bind to various \u003cem\u003ecis\u003c/em\u003e-elements, including AT-rich motifs, CACT motifs, and RAV1-binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eA combined analysis of transcriptome data and TF-centered Y1H assay results identified several potential targets of LbrHY5, including the key anthocyanin biosynthesis-related genes \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e. We subsequently isolated their promoter sequences by genome walking, obtaining 764 bp and 680 bp fragments, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Notably, using PlantCARE to examine \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e promoter sequences, we detected the presence of various \u003cem\u003ecis\u003c/em\u003e-elements, including AT-rich motifs, CACT motifs, RAV1-binding sites, and G-box elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 LbrHY5 directly activates \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e transcription\u003c/h2\u003e \u003cp\u003eTo confirm the direct binding of LbrHY5 to target gene promoters, we conducted a series of molecular assays. Considering the G-box element, which is a conserved promoter motif targeted by HY5, was detected in the \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e promoters, we examined LbrHY5 in terms of its ability to bind to the G-box sequence. On the basis of an electrophoretic mobility shift assay (EMSA), purified LbrHY5 can bind specifically to \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e promoter fragments containing the G-box motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). The specificity of this binding was confirmed by competition assays, in which protein\u0026ndash;DNA interactions were effectively disrupted by an excess of unlabeled wild-type probe, but remained unaffected by a mutated probe. Hence, the G-box promoter element was identified as a binding site for LbrHY5.\u003c/p\u003e \u003cp\u003eFurthermore, to determine whether this binding leads to transcriptional activation, we performed a dual-luciferase (LUC) reporter assay using \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Specifically, we constructed effector vectors expressing \u003cem\u003eLbrHY5\u003c/em\u003e as well as reporter vectors for the expression of the firefly luciferase gene under the control of the \u003cem\u003eLbrCHS\u003c/em\u003e or \u003cem\u003eLbrMYB6\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Compared with the control, the co-expression of \u003cem\u003eLbrHY5\u003c/em\u003e resulted in a significant increase in luciferase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and E), indicating that LbrHY5 can directly activate \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e transcription by binding to their promoters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Overexpression of \u003cem\u003eLbrHY5-LbrMYB6\u003c/em\u003e enhances anthocyanin biosynthesis\u003c/h2\u003e \u003cp\u003eTo explore the specific biological functions of \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e in lily, we constructed the pCAMBIA1305-\u003cem\u003eLbrHY5\u003c/em\u003e and pCAMBIA1305-\u003cem\u003eLbrMYB6\u003c/em\u003e recombinant overexpression vector and inserted it into \u0026lsquo;Robina\u0026rsquo; tepals. All transgenic plants were incubated in darkness for 2 days and then exposed to light for 1 day. Compared with EV \u0026lsquo;Robina\u0026rsquo; tepals, the tepals of transgenic plants were more extensively colored (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and had higher total anthocyanin contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Additionally, key structural and regulatory genes in the anthocyanin biosynthesis pathway (\u003cem\u003eLbrCHS\u003c/em\u003e, \u003cem\u003eLbrCHI\u003c/em\u003e, \u003cem\u003eLbrDFR\u003c/em\u003e, \u003cem\u003eLbrANS\u003c/em\u003e, and \u003cem\u003eLbrMYB6\u003c/em\u003e) were expressed at significantly higher levels in OE-\u003cem\u003eLbrHY5\u003c/em\u003e transgenic tepals than in EV control tepals. Further analysis revealed that OE-\u003cem\u003eLbrMYB6\u003c/em\u003e treatment also promoted the expression of these structural genes, and their expression levels were positively correlated with anthocyanin content. Notably, when \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e were overexpressed simultaneously, the transcriptional levels of the structural genes were more significantly upregulated compared with either single overexpression treatment, indicating a synergistic effect of these two factors in activating the transcription of structural genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Considered together, these observations suggest that LbrHY5 and LbrMYB6 positively regulates anthocyanin biosynthesis in lily, enhancing pigment accumulation through the transcriptional activation of key structural and regulatory genes in the anthocyanin biosynthesis pathway. Notably, while co-overexpression of \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e resulted in higher expression levels of structural genes compared with single overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), the total anthocyanin content in the co-overexpression group was slightly lower than that in the OE-\u003cem\u003eLbrMYB6\u003c/em\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). This discrepancy may be attributed to post-transcriptional regulatory mechanisms or rate-limiting steps in the anthocyanin biosynthetic pathway that are not fully reflected at the transcript level. Alternatively, excessive activation of the pathway by simultaneous overexpression of two strong activators might trigger feedback inhibition or metabolic flux redistribution, leading to attenuated anthocyanin accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Analysis of VIGS-mediated silencing of \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo further validate the roles of \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e in anthocyanin biosynthesis in lily, VIGS technology was employed to perform gene silencing in \u0026lsquo;Robina\u0026rsquo; lily tepals. The results showed that, compared with the empty vector (pTRV1\u0026thinsp;+\u0026thinsp;pTRV2) control, tepals with individual silencing of \u003cem\u003eLbrHY5\u003c/em\u003e (pTRV1\u0026thinsp;+\u0026thinsp;pTRV2-\u003cem\u003eLbrHY5\u003c/em\u003e) exhibited significantly increased anthocyanin content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and B), along with significantly upregulated expression levels of structural genes (\u003cem\u003eLbrCHS\u003c/em\u003e, \u003cem\u003eLbrCHI\u003c/em\u003e, \u003cem\u003eLbrDFR\u003c/em\u003e, \u003cem\u003eLbrANS\u003c/em\u003e) and \u003cem\u003eLbrMYB6\u003c/em\u003e in the anthocyanin biosynthesis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn contrast to \u003cem\u003eLbrHY5\u003c/em\u003e silencing, tepals with individual silencing of \u003cem\u003eLbrMYB6\u003c/em\u003e (pTRV1\u0026thinsp;+\u0026thinsp;pTRV2-\u003cem\u003eLbrMYB6\u003c/em\u003e) and simultaneous silencing of both \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e (pTRV1\u0026thinsp;+\u0026thinsp;pTRV2-\u003cem\u003eLbrHY5\u003c/em\u003e\u0026thinsp;+\u0026thinsp;pTRV2-\u003cem\u003eLbrMYB6\u003c/em\u003e) showed significantly reduced anthocyanin content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), and the expression levels of the structural genes were all significantly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.8 A Working model for the LbrHY5-mediated regulatory effects of light on anthocyanin biosynthesis\u003c/h2\u003e \u003cp\u003eBased on our findings, we propose a comprehensive working model that elucidates the central role of LbrHY5 in light-induced anthocyanin biosynthesis in \u003cem\u003eL. brownii\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Under dark conditions, \u003cem\u003eLbrHY5\u003c/em\u003e transcription is maintained at low levels, resulting in minimal expression of its target genes \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e, and consequently, low anthocyanin accumulation. Following an exposure to light, \u003cem\u003eLbrHY5\u003c/em\u003e transcription is strongly activated. The LbrHY5 protein then directly binds to the G-box motifs in the promoters of \u003cem\u003eLbrCHS\u003c/em\u003e (a key structural gene) and \u003cem\u003eLbrMYB6\u003c/em\u003e (a regulatory transcription factor), activating their transcription. The upregulation of \u003cem\u003eLbrMYB6\u003c/em\u003e further promotes the expression of downstream anthocyanin structural genes (\u003cem\u003eLbrCHS\u003c/em\u003e, \u003cem\u003eLbrCHI\u003c/em\u003e, \u003cem\u003eLbrDFR\u003c/em\u003e, \u003cem\u003eLbrANS\u003c/em\u003e), establishing a transcriptional cascade that amplifies the light signal. This coordinated activation of the anthocyanin biosynthetic pathway ultimately leads to substantial anthocyanin accumulation and the characteristic pigmentation in lily scales. (Yamagish et al., 2010).\u003c/p\u003e \u003cp\u003eIn summary, the synergistic effects of two processes (i.e., direct activation by LbrHY5 and amplified regulation via LbrMYB6) lead to a robust and coordinated upregulation of the anthocyanin biosynthesis pathway. The associated transcriptional cascade ultimately leads to substantial anthocyanin accumulation in lily, resulting in a characteristic red coloration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Light as a key environmental signal promoting anthocyanin biosynthesis in \u003cem\u003eLilium brownii\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eLight is an indispensable factor affecting plant coloration. Lily is a shade-loving plant that is highly sensitive to light. Insufficient light affects plant growth, while also influencing flower bud differentiation, resulting in flower and fruit drop. During the dicots of tomato (\u003cem\u003eS. lycopersicum\u003c/em\u003e) and grape (\u003cem\u003eVitis vinifera\u003c/em\u003e), bags are often used to cover developing fruits, but they are removed to stimulate the rapid anthocyanin biosynthesis and accumulation at the mature stage. Moreover, the expression levels of anthocyanin biosynthesis-related structural genes (\u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eCHI\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e and \u003cem\u003eUFGT\u003c/em\u003e) are significantly upregulated after an exposure to light (Matus et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In \u003cem\u003eC. morifolium\u003c/em\u003e, anthocyanin biosynthesis genes (\u003cem\u003eCmCHS\u003c/em\u003e, \u003cem\u003eCmF3H\u003c/em\u003e, \u003cem\u003eCmANS\u003c/em\u003e, \u003cem\u003eCmDFR\u003c/em\u003e and \u003cem\u003eCm3GT\u003c/em\u003e) are reportedly expressed at lower levels in shaded plants than in plants exposed to normal light conditions (Hong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Another study showed that shading \u003cem\u003eL. regale\u003c/em\u003e plants results in downregulated \u003cem\u003eLrCHSa\u003c/em\u003e, \u003cem\u003eLrCHSb\u003c/em\u003e, \u003cem\u003eLrF3H\u003c/em\u003e, \u003cem\u003eLrF3'H\u003c/em\u003e, \u003cem\u003eLrDFR\u003c/em\u003e and \u003cem\u003eLrANS\u003c/em\u003e expression levels, but the expression of anthocyanin biosynthesis genes is upregulated after light treatments, leading to increased tepal color formation (Yamagishi, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which was consistent with the results of the current study. Our study demonstrates that an exposure to light significantly promotes anthocyanin accumulation in \u003cem\u003eL. brownii\u003c/em\u003e, accompanied by the upregulation expression of structural and regulatory genes in anthocyanin pathway, including \u003cem\u003eLbrCHS\u003c/em\u003e, \u003cem\u003eLbrCHIb\u003c/em\u003e, \u003cem\u003eLbrF3H\u003c/em\u003e, \u003cem\u003eLbrFLS\u003c/em\u003e, \u003cem\u003eLbrDFR\u003c/em\u003e, \u003cem\u003eLbrANS\u003c/em\u003e, \u003cem\u003eLbrANR\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Transcriptomic insights into the LbrHY5\u0026ndash;LbrMYB6 co-regulatory network\u003c/h2\u003e \u003cp\u003eTranscriptome profiling further revealed broad transcriptional reprogramming under light, emphasizing the role of LbrHY5 as a master regulator of the anthocyanin pathway. The induction of \u003cem\u003eLbrMYB6\u003c/em\u003e expression by LbrHY5 reflects the potential crosstalk between light and stress signaling pathways because LbrMYB6 is a transcription factor that modulates flavonoid biosynthesis in response to environmental stimuli.\u003c/p\u003e \u003cp\u003eIn conclusion, we propose a model in which light stabilizes LbrHY5, which in turn activates the expression of \u003cem\u003eLbrMYB6\u003c/em\u003e and structural genes, such as \u003cem\u003eLbrCHS\u003c/em\u003e, leading to anthocyanin accumulation in lily. To the best of our knowledge, this study is the first to functionally characterize LbrHY5 in lily, which is a monocotyledonous ornamental species. The study data may form the theoretical basis for improving color traits in \u003cem\u003eL. brownii\u003c/em\u003e via light treatment or genetic engineering, with implications for the optimizing \u003cem\u003eL. brownii\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.3 LbrHY5 functions as a central light-responsive transcription factor in lily\u003c/h2\u003e \u003cp\u003eWe identified LbrHY5 as a bZIP transcription factor localized in the nucleus, wherein it positively regulates anthocyanin biosynthesis. HY5, which is a well-known integrator of light signals in plants, is relatively stable under light and activates the expression of downstream target genes involved in photomorphogenesis and pigment synthesis (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Earlier research showed that HY5 activates the transcription of anthocyanin biosynthesis structural genes in response to light signals by binding to the G-box element in target gene promoter regions (Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Under light conditions, HY5 promotes anthocyanin biosynthesis and accumulation in plants. Accordingly, deleting the \u003cem\u003ehy5\u003c/em\u003e gene in plants leads to decreased anthocyanin contents. The positive regulatory role of HY5 in anthocyanin biosynthesis appears evolutionarily conserved across dicot species. In \u003cem\u003eArabidopsis\u003c/em\u003e, HY5 directly transactivates structural anthocyanin genes while also partnering with BBX proteins to amplify transcriptional output (Bursch et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, in tomato, SlHY5 regulates the expression of \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e, and \u003cem\u003e3-GT\u003c/em\u003e compared with wild-type controls, \u003cem\u003ehy5\u003c/em\u003e mutants have lower anthocyanin contents (Qiu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, both \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e overexpression increased anthocyanin levels and upregulated \u003cem\u003eLbrCHS\u003c/em\u003e, \u003cem\u003eLbrCHI, LbrDFR\u003c/em\u003e and \u003cem\u003eLbrANS\u003c/em\u003e expression, suggesting that \u003cem\u003eLbrHY5\u003c/em\u003e encodes a conserved positive regulator of anthocyanin accumulation. Moreover, VIGS silencing experiments confirmed these findings while revealing complex regulation of LbrHY5. Unexpectedly, individual silencing of \u003cem\u003eLbrHY5\u003c/em\u003e significantly increased anthocyanin content and structural gene expression. This unexpected phenomenon may be attributed to complex regulatory feedback mechanisms. In many plant species, HY5 stability is post-translationally regulated by COP1 in darkness, and light exposure inactivates COP1, allowing HY5 accumulation (Zhao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Although similar mechanisms may operate in lily, further experiments are needed to elucidate the precise post-transcriptional regulation of LbrHY5 and the feedback responses triggered by partial silencing. Additionally, this partial silencing may have activated a feedback mechanism that enhances its own transcription, or directly increased anthocyanin biosynthesis via a compensatory upregulation of its target, \u003cem\u003eLbrMYB6\u003c/em\u003e. However, co-silencing \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e similarly downregulated anthocyanin-related genes, consistent with their synergistic effect observed in overexpression assays. Taken together, these findings suggest that LbrHY5 exhibits more complex regulation involving light signaling and potential feedback mechanisms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Distinct regulatory mechanisms of LbrHY5 in lily compared to dicot models\u003c/h2\u003e \u003cp\u003ePrevious studies showed that HY5 regulates plant anthocyanin accumulation in response to light, and requires BBX factor to mediate and promote plant photomorphogenesis, but the regulatory mechanisms are different (Fang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, studies have shown that HY5 can also regulate the transcriptional expression of downstream genes by interacting with MYB transcription factors and affecting the stability of MBW complex. The interaction between HY5 and MIR858a in \u003cem\u003eA. thaliana\u003c/em\u003e promotes anthocyanin accumulation in seedlings, inhibits the expression of \u003cem\u003eMYBL2\u003c/em\u003e, a negative regulator of anthocyanin biosynthesis, and activates the expression of anthocyanin biosynthesis structural genes in response to light signals (Wang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In pear, it was found that PpBBX16 cannot directly bind to the \u003cem\u003ePpCHS\u003c/em\u003e or \u003cem\u003ePpMYB10\u003c/em\u003e promoters, but the PpBBX16\u0026ndash;PpHY5 complex can enhance the activity of MdMYB10 promoter (Bai et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Moreover, PpHY5 alone cannot activate the transcription of target genes, and inhibits \u003cem\u003ePpMYB10\u003c/em\u003e expression with the help of PpBBX18 and PpBBX21, thereby regulating the biosynthesis of anthocyanin (Bai et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). In apple calli, MdBBX22 binds to MdHY5 to form a complex that activates the anthocyanin biosynthesis pathway, ultimately promoting anthocyanin accumulation and coloration under UV-B light (Bai et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; An et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Conversely, MdBBX37 suppresses anthocyanin accumulation by binding to the \u003cem\u003eMdHY5\u003c/em\u003e promoter without interacting with the protein (An et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, in the current study, we determined that LbrHY5 can bind to the promoters of \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e in yeast one-hybrid assays, although it did not interact with LbrMYB6 in yeast two-hybrid assays. This suggests that LbrHY5 may directly activate the expression of these genes without forming stable complexes with the encoded proteins. The lack of LbrHY5\u0026ndash;LbrMYB6 interaction in lily may reflect species-specific regulatory mechanisms, with LbrHY5 functioning independently of LbrMYB6. This differs from the regulatory mechanism in pear and apple, wherein HY5 forms complexes with MYB or BBX. Hence, compared with other plants, lily may have a more direct transcriptional activation mechanism.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eOur study provides a systematically regulatory mechanism of light-induced anthocyanin biosynthesis in \u003cem\u003eLilium brownii\u003c/em\u003e. We demonstrated that light exposure significantly promotes anthocyanin accumulation and upregulates the expression of key structural genes (\u003cem\u003eLbrCHS\u003c/em\u003e, \u003cem\u003eLbrCHI\u003c/em\u003e, \u003cem\u003eLbrF3H\u003c/em\u003e, \u003cem\u003eLbrFLS\u003c/em\u003e, \u003cem\u003eLbrDFR\u003c/em\u003e, \u003cem\u003eLbrANS\u003c/em\u003e, \u003cem\u003eLbrANR\u003c/em\u003e) and the regulatory gene \u003cem\u003eLbrMYB6\u003c/em\u003e. LbrHY5 was identified as a light-responsive bZIP transcription factor localized to the nucleus, with its expression significantly induced by light. Transcriptome analysis revealed that \u003cem\u003eLbrHY5\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e serve as central hubs in a light-responsive co-expression network regulating anthocyanin biosynthesis. Electrophoretic mobility shift and dual-luciferase reporter assays collectively demonstrated that LbrHY5 engages G-box \u003cem\u003ecis\u003c/em\u003e-elements within the \u003cem\u003eLbrCHS\u003c/em\u003e and \u003cem\u003eLbrMYB6\u003c/em\u003e promoters, thereby driving their transcriptional activation. Functional analyses through overexpression and VIGS silencing demonstrated that both LbrHY5 and LbrMYB6 positively regulate anthocyanin biosynthesis, with synergistic effects observed upon co-overexpression. The unexpected increase in anthocyanin accumulation following individual \u003cem\u003eLbrHY5\u003c/em\u003e silencing may be attributed to light-induced COP1 inactivation and potential feedback regulatory mechanisms. In conclusion, our findings establish that the LbrHY5\u0026ndash;LbrMYB6 transcriptional module plays a critical role in mediating light-induced anthocyanin biosynthesis in \u003cem\u003eL. brownii\u003c/em\u003e, providing new insights into the regulatory networks controlling pigmentation in monocotyledonous plants and offering potential targets for genetic improvement of color traits in ornamental lilies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32402611); Jiangxi Provincial Department of the National Natural Science Foundation (20232BAB215044) and Key Science and Technology Research Project in Jiangxi Province Department of Education (GJJ2201232).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted at the Ganzhou Key Laboratory of Nanling Plant Resources Protection and Utilization and Jiangxi Provincial Key Laboratory of Pest and Disease Control of Featured Horticultural Plants, China.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAn JP, Wang XF, Espley RV, Lin-Wang K, Bi SQ, You CX, Hao YJ (2020) An apple B-box protein MdBBX37 modulates anthocyanin biosynthesis and hypocotyl elongation synergistically with MdMYBs and MdHY5. 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Int J Mol Sci 23: 8357\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lilium brownii, Light signaling, LbrHY5, Anthocyanin biosynthesis, Transcriptional regulation, LbrMYB6","lastPublishedDoi":"10.21203/rs.3.rs-9289041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9289041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLilium brownii is a valuable monocotyledonous plant with ornamental and medicinal importance, whose purplish-red pigmentation is attributed to anthocyanin accumulation under light exposure. However, the molecular mechanisms underlying light-regulated anthocyanin biosynthesis in lily remain unclear. Here, we demonstrate that light significantly enhances anthocyanin content in lily and upregulates key structural and LbrMYB6 regulatory genes. We identified LbrHY5 as a light-induced bZIP transcription factor localized to the nucleus. Transcriptomic analysis positioned LbrHY5 and LbrMYB6 at the central hubs of a light-responsive co-expression network. Moreover, molecular assays confirmed that LbrHY5 binds directly to the G-box motifs in promoters of LbrCHS and LbrMYB6, thereby activating their transcription. Overexpression of LbrHY5 and LbrMYB6 significantly increased anthocyanin accumulation and structural genes expression, with co-overexpression exhibiting synergistic effects. VIGS-mediated silencing of LbrMYB6 reduced anthocyanin contents and structural genes expression, while simultaneous silencing of both genes produced similar downregulation. Notably, individual silencing of LbrHY5 unexpectedly increased anthocyanin accumulation, suggesting complex regulatory mechanisms involving light-induced COP1 inactivation and potential feedback regulation. Collectively, our findings elucidated the role of a novel LbrHY5–LbrMYB6 module that mediates light-regulated anthocyanin biosynthesis in L. brownii, providing significant insights into the regulatory networks controlling pigmentation in monocots and offering potential strategies for improving color traits in ornamental lilies.\u003c/p\u003e","manuscriptTitle":"Light-induced anthocyanin biosynthesis in Lilium brownii is mediated by the LbrHY5–LbrMYB6 module","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-10 00:00:23","doi":"10.21203/rs.3.rs-9289041/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-04T14:40:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29309266682070104829655173020119635621","date":"2026-04-07T16:59:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-05T19:40:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-04T06:42:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-04T05:47:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2026-04-01T08:34:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4de805b9-90ce-4cb5-b621-a50ae17427f9","owner":[],"postedDate":"April 10th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-04T14:40:02+00:00","index":20,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-10T00:00:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-10 00:00:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9289041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9289041","identity":"rs-9289041","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}