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Combined transcriptional and metabolomic analysis of flavonoids in the regulation of female flower bud differentiation in Juglans sigillata Dode

BMC Plant Biology volume 25, Article number: 168 (2025) Cite this article

Abstract

Juglans sigillata Dode is rich in flavonoids, but the low ratio of female to male flower buds limits the development of the J. sigillata industry. While the abundance of flavonoids in J. sigillata is known, whether flavonoids influence female flower bud differentiation has not been reported. In this study, we explored the regulatory mechanisms of gene expression and metabolite accumulation during female flower bud differentiation through integrated transcriptomic and metabolomic analyses. Our findings revealed that flavonoid biosynthesis is a key pathway influencing female flower bud differentiation, with metabolites primarily shifting towards the isoflavonoid, flavone, and flavonol branches. Structural genes such as chalcone synthase, dihydroflavonol 4-reductase, flavonol synthase, and flavonoid 3',5'-hydroxylase were identified as playing crucial regulatory roles. The expression of these genes promoted the accumulation of flavonoids, which in turn influenced female flower bud differentiation by modulating key regulatory genes including Suppressor of Overexpression of Constans1, Constans, Flowering Locus T, and APETALA1. Furthermore, transcription factors (TFs) highly expressed during the physiological differentiation of female flower buds, particularly M-type MADS, WRKY, and MYB, were positively correlated with flavonoid biosynthesis genes, indicating their significant role in the regulation of flavonoid production. These results offer valuable insights into the mechanisms of female flower bud differentiation in J. sigillata and highlight the regulatory role of flavonoids in plant bud differentiation.

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Introduction

Flower bud differentiation is a critical phase in the development of flowering plants, as the quantity and quality of flower bud differentiation directly determines the the plant’s yield at the later stage. The physiological differentiation period plays a key role in regulating this process [1, 2]. In nature, plants such as Zea mays, Citrullus lanatus, and Lilium brownie are able to produce male and female flowers separately on the same plant, and they account for about 5% of flowering plants. The sex of single flowers in these plants is either female or male [3], and many of these plants have unequal male and female flowers and most of them have a low proportion of female/male [4]. To enhance yield in these species, promoting female flower production and suppressing male flower development is often necessary. Since flower bud differentiation is essential for yield formation, understanding its molecular mechanisms holds significant value for improving crop yields and advancing molecular-assisted plant breeding.

Flavonoids are a large group of secondary metabolites commonly found in various plant tissues, including roots, stems, leaves, flowers, and fruits. These compounds encompass chalcones, flavonoids, flavonols, isoflavones, dihydroflavonols, flavanols, and anthocyanins [5]. Flavonoids are important in floral organs and tissues, and can influence the formation and opening of plant flowers, pollen tube growth, as well as fruit formation and seed maturation processes [6,7,8]. During Freesia refracta flower development, FhFLS1, and FhFLS2 function to promote flavonol biosynthesis at both early and late stages [9]. In Camellia sinensis, flavonol compounds increase as flowers open, with petals and stamens serving as the primary sites for flavonoid and flavonol accumulation. The CsFLSb gene regulates flavonol biosynthesis, affecting fertility [10]. The exogenous application of the flavonoid compound hyperoside not only extends the flowering period of okra but also enhances its reproductive development and fruit setting [11, 12]. These findings suggest that flavonoid substances play a pivotal role in flower development in plants. However, literature on the relationship between plant flavonoid compounds and flower bud differentiation remains limited. Investigating the regulatory role of flavonoids in the process of plant bud differentiation will facilitate further investigation of the role of flavonoids in plant reproductive growth and facilitate the subsequent development and utilization of flavonoids.

Walnut is a tree species integrating economic benefits, ecological benefits and artistic value, playing a crucial role in the development of agriculture and forestry economy, increasing the income of working people and improving the ecological environment. Walnut female flower buds are mixed buds containing leaves and female flowers, and female flower buds can take more than one year from the beginning of differentiation to the full establishment of the floral organ [13], which is affected by genetic material, endogenous plant hormones [14, 15], nutrients [16], external conditions and other aspects. Juglans sigillata Dode, a species within the Juglandaceae family, is a typical monoecious plant with separate male and female flowers on the same individual. The differentiation of male flower buds occurs earlier than that of female flower buds. Recent studies have indicated that this flowering pattern is regulated by a 20 kb genomic region [17]. The ratio of female to male buds in J. sigillata ranges from 1:30 to 1:100 [16], giving male buds a competitive advantage in nutrient allocation. This disadvantage for female flower buds in nutrient competition negatively impacts their differentiation and development, resulting in a significantly lower yield per unit area for J. sigillata compared to common walnut trees of the same age [16]. J. sigillata are rich in flavonoids, and studies have shown that flavonoids accumulate in the kernel, kernel membrane, green skin, leaves, and branches [18, 19]. Studies in pecan have shown that flavonoids are organ-specific in pecan, with flavonols accumulating in the bark, branches, and leaves [20]. However, there is currently no research examining the role of flavonoids in the differentiation of walnut buds or the development of floral organs. Whether there is a link between the enrichment of flavonoids in walnut and the differentiation of female flower buds remains an open question.

In order to investigate the role of flavonoid metabolic biosynthesis pathway in female flower bud differentiation, this study used the undifferentiated, physiologically differentiated and morphologically differentiated female flower buds of J. sigillata, as well as leaf buds from the same developmental stages, as research materials. By employing RNA-seq and metabolomic analyses, we identified key pathways involved in the differentiation of female flower buds. We examined the expression patterns of metabolites and structural genes associated with the flavonoid biosynthesis pathway. Through correlation analysis, we explored the relationships between flavonoid synthesis metabolites and structural genes, and further investigated the connection between the flavonoid biosynthesis pathway, flower formation genes, and TFs influencing flavonoid biosynthesis. Based on the analysis, we constructed a regulatory network of female flower bud differentiation in J. sigillata, and the results are expected to provide a theoretical foundation for understanding the mechanism of female flower bud differentiation in J. sigillata, enhance the understanding of the role of flavonoids in plant reproductive growth, provide new insights into the regulatory mechanism of flower differentiation in monoecious and dioecious plants, and provide a basis for the further utilization and conservation of this species.

Materials and methods

Plant materials and processing

The material for this study was harvested in 2023 from Hezhang County, Guizhou Province (County27.10°N, 106.73°E, altitude of 1100 m), and the J. sigillata variety was 'Qianhe7', selecting trees in full fruition, robust, and uniformly long consistent, highly grafted 7 years as material. The top 1–2 full buds of the current year's short fruiting branches in the four directions of the periphery of the tree were female flower buds, and 1–2 leaf buds at the bottom of the branches. Samples were taken on April 20,2023 (female flower bud undifferentiated stage, F-UD), April 27, May 4 (physiological differentiation stage, F-PD), and June 5 (morphological differentiation stage, F-MD). Female flower buds were sampled four times. Sampling began on April 20 (L1), and leaf buds were sampled three times on May 4 (L2) and June 5 (L3). 10 trees as a replication, three replications, 1.5 g sample per replicate. The removed material was snap frozen in liquid nitrogen and then placed in dry ice and brought back to the laboratory at -80℃ for backup.

Anatomical analysis

The samples were fixed in FAA solution (formalin (37%): glacial acetic acid: anhydrous ethanol (50%), with a volume ratio of 5:5:90), dehydrated through gradient ethanol (70–100%), embedded in paraffin [21], and the samples were cut into a thickness of 7 μm using a HistoCare BIOCUTR manual rotary microtome. They were stained with a combination of safranin and fast green and mounted with neutral gum. All sections were observed and photographed using a biological microscope (BX3-CBH, Olympus, Japan). The samples were fixed in 2.5% glutaraldehyde solution, dehydrated through gradient ethanol (30%-100%) [22], freeze-dried and sprayed with gold, and the morphology of the samples was analyzed using a scanning electron microscope (SEM, JEOL 7800 F, Japan), with a scanning voltage of 10–20 kV.

Transcriptome and data analysis

RNA sequence data that support the findings of this study have been deposited in the https://www.ncbi.nlm.nih.gov/bioproject/?, with the primary accession code PRJNA1162697. A total of 21 samples of undifferentiated period, physiological differentiation period and morphological differentiation period, as well as leaf buds from the same period, were selected for transcriptome sequencing, the sample information is shown in Table S1. RNA-seq and analysis were performed by Shenzhen Microclass Kemin Biologicals Co Ltd (Shenzhen, China: https://www.bioincloud.tech/). All subsequent analyses were based on high-quality analysis of clean data. The genome database of J. sigillata was downloaded from the Genome Database for Juglandaceae (http://gigadb.org/search/new?keyword=+Juglans+sigillata) [23], NCBI: txid224355. HISAT2v2.0.5 was used to build the index of the reference genome and to align the paired-end clean reads with the reference genome. Differential expression analysis between two comparison groups was performed using DESeq2 software (1.16.1), the selection criteria for differentially expressed genes (DEGs) being |log2(FoldChange)|> 1 and padj < 0.05. GO and KEGG enrichment analysis of DEGs was performed by clusterProfiler (3.4.4) software, and padj < 0.05 was used as the criterion for significant enrichment. Network correlation analysis was completed using the Wekemo Bioincloud (https://www.bioincloud.tech), the correlation coefficient is calculated as pearson [24].

Metabolomics and data analysis

The metabolomic samples are consistent with the RNA-seq. The extraction and analysis of metabolites were performed by Shenzhen Microcourse Kemei Biotechnology Co., Ltd. (Shenzhen, China). Tissue samples (100 mg) ground in liquid nitrogen were placed in EP tubes and 500 μL of 80% methanol–water solution was added. After vortexing and incubating in an ice bath for 5 min, the samples were centrifuged at 15,000 g and 4 °C for 20 min. A certain amount of supernatant was diluted with mass spectrometry-grade water to achieve a methanol content of 53%. After another centrifugation at 15,000 g and 4 °C for 20 min, the supernatant was collected and subjected to LC–MS analysis [25]. Using a custom-built database, the experimental samples were analyzed using multiple reaction monitoring (MRM) mode. Compounds were quantified based on Q3 (daughter ions), and qualitative analysis was performed using Q1 (parent ions), Q3 (daughter ions), retention time (RT), declustering potential (DP), and collision energy (CE). The SCIEX OSV1.4 software was used to open the raw mass spectrometry files for peak integration and calibration. Peaks were filtered based on minimum peak height (500), signal-to-noise ratio (5), and smoothing points (1). The integrated peak area of each daughter ion within the chromatographic retention time represented the relative content of the corresponding substance, resulting in qualitative and quantitative information for the metabolites. PCA was performed using the Wekemo Bioincloud (https://www.bioincloud.tech) [24]. Orthogonal partial least squares discriminate analysis (OPLS-DA) was adopted for statistical analysis that determined global metabolic changes between comparable groups. The OPLS-DA model was used for the calculation of the variable importance in the projection (VIP). p-value were projected with paired Student's t-test on single dimensional statistical analysis. To identify differentially abundant metabolites (DAMs), a combination of fold change, p-value, and VIP values was used in the OPLS-DA model. The following criteria were set for screening: fold change > 2, p-value < 0.05 and VIP > 1. DAMs were annotated using KEGG databases (https://www.genome.jp/kegg/pathway.html), Lipidmaps database (http://www.lipidmaps.org/). For bioinformatics analysis of the metabolites was completed using the Wekemo Bioincloud (https://www.bioincloud.tech).

qRT-PCR analysis

To validate the RNA-seq data, RNA was extracted using a plant RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing) and reverse transcribed using a Genstar cDNA kit (Kangrun Chengye Biotechnology Co., Ltd., Beijing). The qRT-PCR was performed on a real-time fluorescent quantitative PCR instrument (BIO-RAD, California, USA). The reaction system was 10 μL, including 0.4 μL of each forward primer and reverse primer, 1 μL of cDNA template, 5 μL of qPCR Master Mix premix, and 3.2 μL of ddH2O. Primers were designed using the Sangon primer design online platform (https:// www.sangon.com/newPrimerDesign), 18s gene as an internal reference gene. Primers are shown in the (Table S2).

Results

Anatomical analysis of bud differentiation

The tree conditions were as shown in (Fig. 1A), and the positions of male, female flower buds and leaf buds on fruiting branches were as shown in (Fig. 1B). By observing and recording the anatomy of female flower buds, the undifferentiated period, physiological differentiation period and morphological differentiation period of female flower buds of the J. sigillata were determined. On April 20th, the morphological differentiation of the female flower buds at the top had not yet started, with a diameter of 300 μm. The buds were tender green, the squama were tightly closed, soft in texture, and there was no significant difference in the external morphology between the flower buds and the leaf buds; from April 27th to May 4th, the top flower buds began to expand, the number of squama increased to 5, and villous squama were generated between the outer squama. The buds increased to 600 μm in length and 500 μm in width. On June 5, the size of the top flower buds had no obvious change, the number of squama slightly increased to about 11, and the buds were yellowish-green, which was significantly different from the leaf buds (Fig. 1C). Paraffin sections showed that on April 20th, two degenerated small leaves tightly wrapped the primordium of the female flower buds. The apical growth point was pointed and small, conical, the cells were closely arranged and regularly shaped, and the female flower buds were in the undifferentiated period; from April 20th to May 4th, the apical meristem gradually flattened, the cells were closely arranged, the primordial young leaves appeared and formed, the degree of scale closure decreased and could be stratified, being in the physiological differentiation period; on June 2nd, the flower buds differentiated the perianth primordium, and the perianth primordium was formed on the inside of the bract primordium, being in the morphological differentiation period (Fig. 1D).

Fig. 1
figure 1

Morphological characteristics of flower buds at different differentiation stages of J. sigillata. A J. sigillata trees top-grafted for 7 years. B Model diagram of the fruiting branch. C Photos of the external morphology of female flower buds and leaf buds. D Internal characteristic diagram of female flower buds and leaf buds, bar = 100 μm. Note: The order of the buds in figures C and D is from left to right: F-UD (20th April); F-PD1 ~ F-PD2 (27th April ~ 4th May); F-MD (4th June); L1 (20th April); L2 (4th May); L3 (5th June); lp (leaf primordium); pr (perianth); SAM (apical meristem); sq (squama); pi (primordial of inflorescence); pf (primordial of flower)

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Summary of metabolomic data analysis of female flower bud and leaf bud in J. sigillata

A total of 1338 metabolites were obtained by UPLC-MS. In PCA, PC1 was 33.6% and PC2 was 14.3%, indicating that female flower buds and leaf buds of J. sigillata at each stage were clearly distinguished, and within the same tissue, groups at different developmental stages were also clearly distinguished (Fig. 2A), which could be used for subsequent analysis. The 1338 metabolites were classified into 20 categories (Fig. 2B), among which the top five metabolites with the largest number were flavonoids, amino acid and its derivatives, organic acid and its derivatives, lipids, carbohydrates and its derivatives, with 242, 206, 117, 117 and 103 metabolites respectively. To understand the classification and functional characteristics of different metabolites, we annotated the identified metabolites. In the Lipidmaps database, the main annotations were flavonoids (98 metabolites), followed by fatty acids and conjugates (44 metabolites), and the least were monoradylglycerols, fatty aldehydes and secosteroids (Fig. 2C). To discover the metabolites that affect the differentiation process of female flower buds, we screened the DAMs in two aspects. Mainly, during the different differentiation periods of female flower buds, they were F-UD vs F-PD1, F-UD vs F-PD2, F-UD vs F-MD, F-PD1 vs F-PD2, F-PD1 vs F-MD, F-PD2 vs F-MD; Secondly, the DAMs between female flower buds and leaf buds in the same period were F-UP vs L1 in the undifferentiated period; F-PD2 vs L2 at the end of the physiological differentiation period; F-MD vs L3 in the morphological differentiation period. A total of 1125 DAMs were screened in the 9 comparison groups. Among them, 535, 587 and 592 DAMs were screened in the comparison groups of female flower buds at the undifferentiated period, physiological differentiated period and morphological differentiated period stage and leaf buds, respectively (Fig. 2D). This suggests that the process of differentiation between female flower buds and leaf buds is accompanied by an increase in the number of DAMs.

Fig. 2
figure 2

Metabolome data analysis. A Principal component analysis (PCA) of female flower bud and leaf bud samples of J.sigillata. B Classification of all metabolites in the samples. C Classification annotation of all metabolites in the Lipidmaps database. D Statistics of DAMs in each comparison group

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Flavonoids are the most abundant class of metabolites, and the results of the Lipidmaps database annotation also indicated that flavonoids were significantly higher than other metabolites (Fig. 2B, C), with flavonoid metabolites being significantly higher than other metabolite species in 1125 DAMs. We subjected all flavonoids in DAMs to KEGG enrichment analysis and found that these flavonoid DAMs were highly significantly enriched in flavonoid biosynthesis, flavonoid and flavonol biosynthesis, isoflavonoid biosynthesis, and anthocyanin biosynthesis pathways (Fig. 3A). We performed KEGG enrichment analysis of DAMs in all comparison groups separately, and the results showed that DAMs were highly significantly enriched in each comparison group (Fig. 3B). These results suggest that flavonoids may play an important role in female bud differentiation in J. sigillata and may be the key metabolites affecting female bud differentiation in J. sigillata.

Fig. 3
figure 3

KEGG enrichment analysis of DAMs. A KEGG enrichment analysis of all flavonoid differentially accumulating metabolites. B Enrichment of DAMs in the flavonoid biosynthetic pathway between each comparison group

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Summary of transcriptomic data analysis of female flower bud and leaf bud in J. sigillata

The 21 constructed libraries were subjected to whole transcriptome high-throughput RNA sequencing using the Illumina platform. The number of original read data of the 21 samples ranged from 5.95 Gb to 8.02 Gb. After filtering out the data with adapters, N bases and low sequencing quality, the clean data of each sample reached more than 5.68 Gb. In addition, the average percentages of Q20 and Q30 were 98.45% and 94.47% respectively. The Q20 of each sample was ≥ 98.31% and the Q30 was ≥ 94.24%. The GC range was 44.81%-45.28% (Table S3), indicating that the sequencing results met the test requirements and could proceed to the next step of analysis. Using the method of PCA analysis, the transcripts identified in 21 samples were analyzed. The PCA results showed that PC1 and PC2 explained 20.45% and 16.8% of the gene expression variation in all samples (Fig. 4A); the heatmap of sample correlation analysis showed that there was a high correlation among biological replicates of the samples (r2 > 0.92) (Fig. 4B). We screened the DEGs during the differentiation process of female flower buds, the screening targets were consistent with the metabolome. A total of 11,460 DEGs were screened in the 9 control groups and 5,343 DEGs were obtained after removing duplicates (Fig. 4C).

Fig. 4
figure 4

Transcriptome data analysis. A PCA of the identified genes in the samples. B Correlation heatmap in all samples. C Statistics of DEGs in each differentiation period of female flower buds and leaf buds of J. sigillata. D GO annotation of all DEGs. E KEGG annotation of all DEGs. In the GO and KEGG bubble charts, the size of the circle indicates the number of genes enriched to the pathway, and the color indicates -log10(pvalue), and the larger the -log10(pvalue), the smaller the pvalue, the more significant the pathway

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To elucidate the functions of DEGs, we determined the enrichment of DEGs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The GO database consists of three main categories: biological process, molecular function and cellular component. The results showed that the top 20 GO items enriched by DEGs belonged to biological process and molecular function. DEGs were mainly enriched in response to chitin, secondary metabolic process, secondary metabolite biosyntheic process, response to organonitrogen compound, and phenylpropanoid metabolic process (Fig. 4D). KEGG analysis showed that DEGs were highly significant enriched in the flavonoid biosynthesis pathway. The enrichment was in KEGG pathways such as phenylpropanoid biosynthesis, plant-pathogen interactions and plant hormone signal transduction (Fig. 4E). Notably, in addition to the KEGG pathways mentioned above, DEGs were also enriched in flavonoid biosynthesis and were extremely significantly enriched in this pathway, suggesting once again that flavonoids may be a key pathway affecting female bud differentiation in J. sigillata.

Combined analysis of flavonoid biosynthesis-related DEGs and DAMs

Based on transcriptomic and metabolomic analyses, DEGs and DAMs were found to be highly significant enriched in flavonoid biosynthesis. To investigate the role of flavonoids in female flower bud differentiation of J. sigillata, we analyzed the expression pattern of DEGs and the accumulation pattern of DAMs in the flavonoid biosynthesis, and mapped the pathway diagram of this pathway (Fig. 5). PAL, 4CL, CHI, CHS and DFR had overall higher expression in female flower buds than in leaf buds and had relatively high expression during the physiological differentiation period of female flower buds. FLS genes had overall relatively high expression in F-UD, L1, and one FLS gene had high expression in F-UD and F-PD2, and there was one FLS gene was highly expressed in L2 and L3, these results indicated that different FLSs have different function in female bud differentiation in J. sigillata. F3H gene was highly expressed in the morphological differentiation period of female buds. F3′5'H was more highly expressed in L1. The two ANR genes were highly expressed in L1 and L3, respectively, and the accumulation of the downstream metabolites showed the same trend. Metabolites upstream of the flavonoid biosynthesis pathway accumulated relatively high during the physiological differentiation of female flower buds in J. sigillata, such as phenylalanine, cinnamic acid, coumaric acid. Dihydrokaempferol, kaemperol, rutin and apigenin several flavones and flavonols, which had a relatively high accumulation during the physiological differentiation of female flower buds. Isoflavonoids: higher accumulation in F-UD, which may be flower-forming induced accumulating metabolites in J. sigillata. Two flavanols, (-)-epigallocatechin, (-)-epicatechin, accumulated more in F-MD, L3 and F-MD, respectively. The relative accumulation of flavanones hesperetin and naringenin was higher in L3, while naringin accumulated more in L1 species. These results suggest that substance metabolic fluxes in the flavonoid pathway are more oriented toward the isoflavone, flavone and flavonols branches than the anthocyanin branch during female bud differentiation in J. sigillata.

Fig. 5
figure 5

The transcriptional profile of structural genes and metabolites in the flavonoid biosynthesis pathway of female flower buds and leaf buds of J. sigillata. Heat map of the expression level (TPM) of flavonoid biosynthesis structural genes during female flower bud differentiation, and the data were normalized from 0 to 1 in row normalization. The relative accumulation of flavonoid biosynthesis-related metabolites during the differentiation of female flower buds was sample-wise normalized. 4CL:4-Coumarate: Coenzyme A Ligase; CHS: chalcone synthase [EC:2.3.1.74]; F3`H: Flavonoid 3'-hydroxylase; CHI: chalcone isomerase [EC:5.5.1.6]; DFR: dihydroflavonol 4-Reductase; FLS: flavonol synthase; LAR: leucoanthocyanidin reductase; F3H: Flavonoid 3-hydroxylase; CYP75B1:flavonoid 3'-monooxygenase [EC:1.14.14.82]; PAL: phenylalanine ammonia-lyase; FNS: flavone synthase; LAR: leucoanthocyanidin reductase; ANS: anthocyanidin synthase; F3′5'H: flavonoid 3', 5'-hydroxylase

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Correlation analysis of flavonoid biosynthesis pathways with flower-forming genes

In order to investigate whether flavonoids interact with flower-forming genes to regulate the differentiation of female flower buds in J. sigillata, we conducted correlation analyses based on Pearson’s correlation coefficient between important flavonoids, important structural genes, and flower-forming genes. The results of flavonoid metabolites and flavonoid synthesis genes showed that CHS (OF28612, OF03554), FLS (OF04018, OF20658), DFR (OF17302), 4CL (OF05965), LAR (OF25127) and CHI (OF12477) had good correlations with most flavonoids. There was an extremely significant positive correlation between FLS (OF20658) and the high accumulation of apigenin and rutin in the female flower buds. FLS (OF20658) was highly expressed in F-UD, and the substances were highly accumulated during the physiological differentiation period, which could be attributed to the fact that translation requires a certain process, and the high expression of FLS gene in the undifferentiated stage promoted the accumulation of flavonol substances during the physiological differentiation period (Table 1).

Table 1 Pearson correlation analysis among important flavonoids with important structural genes in the flavonol synthesis pathway
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Flavonoids, and flavonoid synthesis genes correlated with flower-forming genes. Metabolites with high accumulation during the physiological differentiation period of female flower buds: phenylalanine, cinnamic acid, coumalic acid, apigenin, and rutin showed good correlation with SOC1 (OF12809), FLC (OF27904) and CO (OF25687). The metabolites chalcone and myricetin, which accumulated low during the physiological differentiation of female flower buds, were not positively correlated with the genes for flower-forming, and were only significantly negatively correlated with FLC (OF27904) and VRN1 (OF11156). In the flavonol biosynthesis pathway, CHS (OF28612, OF03554), DFR (OF17302), FLS (OF04018), 4CL (OF05965) and LAR (OF25127) showed highly significant correlations with several flower genes, and the correlations were positive (Table 2).

Table 2 Pearson correlation analysis among important flavonoids and structural genes in the flavonol synthesis pathway with flowering genes
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Differentially expressed transcription factors (TFs) and flavonoid biosynthesis

TFs are major regulators of gene expression. In this study, we screened 79 TFs from 5343 DEGs that were highly expressed during the physiological differentiation of female flower buds in J. sigillata, and the TFs were distributed in multiple families, among which the top three TF families with the highest number were NAC (NAM、ATAF1/2、CUC2) (11), ERF (ethylene responsive factor) (9), and C2H2 (Cys2His2) (9) (Fig. 6A). Notably, two TFs from the CO-like (CONSTANS-Like) family and five TFs from the MADS (MCM1, AGAMOUS, DEFICIENS, and SRF-like) family associated with flower development were also screened. To further explore the regulatory relationship between TFs and flavonoid biosynthesis-associated DEGs, Pearson correlation coefficients of TFs and flavonoid biosynthesis-associated DEGs were calculated, and the top 30 significantly related genes were selected to be linked and plotted as a network diagram (Fig. 6B). The results showed that most of these TFs showed positive correlation with the genes related to flavonoid biosynthesis pathway. M-type_MADS (OF04963), WRKY (OF10312), MYB (OF22990) and SBP(SQUAMOSA Promoter Binding Protein-like) (OF11497) had a high level of linkage and showed positive correlation, which was hypothesized to be a significant correlation of these TFs play an important role in female bud differentiation in J. sigillata. In the flavonoid biosynthesis pathway, PAL (OF12732), CHS (OF28612), DFR (OF17302), 4CL (OF05965), CHI (OF12477), F3′5'H (OF27121), LAR (OF03725) and LAR (OF03725) were highly and positively correlated with each other and TFs. While FLS (OF04018) showed negative correlation with most of the structural genes. It is speculated that these genes may be the key genes in each family that regulate flavonoid biosynthesis in female flower buds of J. sigillata. PAL (OF12732) showed high connectivity and positive correlation with the TFs TALE (OF15543), M-type_MADS (OF04963). CHS (OF28612) has strong positive correlation with DBB (OF21748), TALE (OF15543). DFR (OF17302) has strong positive correlation with DBB (OF21748). F3′5'H (OF27121) and 4CL (OF05965) both have strong positive correlation with M-type_MADS (OF04963). LAR (OF03725) is positively correlated with WRKY (OF10312). FLS (OF04018) has a strong negative correlation with TALE (OF15543). These results suggest that flavonoid biosynthesis is regulated by TFs. M-type_MADS (OF04963), MYB (OF22990), WRKY (OF10312), TALE (OF15543) and DBB (OF21748) have an important influence in the biosynthesis of flavonoids in the female buds of the J. sigillata. In order to verify the reliability of the transcriptome data, eight genes were selected for qRT-PCR experiments. qRT-PCR results showed that the expression patterns of these genes were consistent with the trend of the transcriptome data, indicating that the transcriptome data were highly reliable (Fig. 6C).

Fig. 6
figure 6

Correlation analysis of differentially expressed TFs with DEGs related to flavonoid biosynthesis. A Statistics of differentially highly expressed TFs during the physiological differentiation stage of female flower buds in J. sigillata. B network diagram of correlation between TFs and DEGs related to flavonoid synthesis. C qRT-PCR validation of the transcriptome. Note: The node color and size in the B graph indicate the DEG value; the larger the DEG value, the darker the color and the larger the point; the smaller the DEG value, the lighter the color and the smaller the point. The color of the connecting line indicates the correlation, red indicates positive correlation, green indicates negative correlation; the thickness of the connecting line indicates the correlation pvalue; the larger the correlation pvalue is, the thicker the connecting line is, and vice versa, the smaller the pvalue is RGLG2: E3 ubiquitin-protein ligase RGLG2

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Discussion

Structural changes in female flower bud differentiation in J. sigillata

The study of flower bud differentiation periods and the corresponding internal, and external morphological changes provides a fundamental basis for understanding flower bud development in plants. In recent years, differences in the reported differentiation periods of walnut female flower buds have been noted across various studies [16, 26]. Our research group previously analyzed the early stages of female flower bud differentiation in J. sigillata, detailing both the external morphology and internal structural characteristics [13]. The female flower buds of J. sigillata are mixed buds containing both female flowers and leaves, and their differentiation period is influenced by year-to-year climatic variations. To ensure precise sample collection and to explore the structural differences between female flower buds and leaf buds, this study conducted anatomical analyses of both structures. During the undifferentiated and physiological differentiation stages, there were no significant internal structural differences between female flower buds and leaf buds in J. sigillata. However, externally, female flower buds were larger in size compared to leaf buds. At the morphological differentiation stage, female flower buds differentiated into floral primordia, while leaf buds only formed young leaf structures (Fig. 1C, D).

Flavonoid biosynthesis is a key pathway during differentiation of female flower buds in J. sigillata

Flavonoids are among the most important secondary metabolites in plants, significantly impacting plant growth and development. In Jatropha, the flavonoid biosynthesis pathway is linked to male flower formation, while inhibition of flavonoid biosynthesis leads to male sterility in Petunia [27, 28]. Exogenous application of the flavonoid compound hyperoside not only extends the peak flowering period of okra but also promotes its reproductive development and fruiting [11, 12]. Upstream genes CHS2, CHS7, CHI, and DFR are highly expressed in female Broussonetia papyrifera and Ginkgo biloba leaves, with higher levels of catechins and epicatechins found in female Ginkgo saplings compared to males [29, 30]. In Chrysanthemum morifolium, high expression levels of flavonoid biosynthesis genes, including CHS, CHI, FNS, and F3H, during early flowering suggest that flavonoids may play a crucial role at the onset of flowering [31]. Moreover, flavonoid accumulation patterns in the leaves of conifers increase gradually as development progresses, with higher levels in female plants compared to males [30]. Similarly, flavonoid levels in the leaves and shoots of female Carica papaya are higher than in males, although the molecular mechanisms underlying this difference remain unclear [32]. Flavonols, a class of flavonoids, have a notable influence on floral organs. In Gentiana, apigenin and lignans, the primary flavonoids, accumulate during the early stages of flower development [33]. Additionally, two groups of flavonol derivatives have been identified: kaempferol derivatives, which increase during flower development, and quercetin derivatives, which decrease [9]. In this study, both DEGs and DAMs related to flavonoid biosynthesis were significantly enriched during the differentiation of female flower buds in J. sigillata (Figs. 3 and 4E). The upstream genes of flavonoid biosynthesis, such as CHI, CHS, and PAL, showed higher expression levels in female flower buds compared to leaf buds, especially during the undifferentiated and physiological differentiation stages of the female flower buds. Genes such as PAL (OF12732), FLS (OF20658), CHS (OF28612), DFR (OF17302), and F3′5’H (OF27121) exhibited high connectivity, which is similar to the results observed in Broussonetia papyrifera. The relative accumulation of phenylalanine, cinnamic acid, and coumaric acid is higher during F-PD. In the downstream metabolic branches, substances in the isoflavone, flavone, and flavonol branches accumulate more in the F-PD, indicating their more important roles. Among them, rutin and apigenin accumulate relatively higher in the differentiation of female flower buds. Rutin has been proven to accelerate cell division and growth, thereby promoting plant growth, which aligns with the cell division and growth accompanying the differentiation of female flower buds in J. sigillata. Although there are few reports directly linking female flower bud differentiation to flavonoids, the results of this study suggest that flavonoids not only vary between female flower and leaf buds but also fluctuate at different stages of flower development and differentiation. They may play a role in inducing female flower bud differentiation. Previous studies have reported interactions between flavonoid biosynthesis and phytohormones such as auxin [34, 35], gibberellins [36], jasmonic acid [37], and abscisic acid [38]. The effects of flavonoids on plant growth and development may be mediated by these interactions with phytohormone levels. A similar regulatory mechanism may exist during female flower bud differentiation in J. sigillata, though further investigation is needed to elucidate the specific mechanisms involved.

Flavonoids during female flower bud differentiation in J. sigillata can correlate well with genes for flower formation

Flower induction marks the plant's transition from vegetative to reproductive growth, regulated by both internal and external factors. Major pathways involved in flower induction include the photoperiodic pathway, vernalization pathway, gibberellin pathway, autonomous pathway, and the age pathway. The flowering regulatory mechanisms in Arabidopsis, primarily involving FT, SOC1, CO, LFY, and AP1, have provided significant reference value for the study of flowering in woody plants [39]. In recent studies, key gene interactions have been uncovered. For example, in stone garlic, MADS-box and MYB transcription factors interact with flower-inducible genes such as SPLs, CO14, and GI, impacting flower bud differentiation [40]. In pecan, AGL24 from the MADS-box TF family positively interacts with SOC1 and AP1, and all three play crucial roles in regulating flowering and promoting female flower formation [41, 42]. Moreover, overexpression of PvMADS56 in Arabidopsis resulted in early flowering, which complemented the delayed flowering phenotype of soc1 mutants [43]. In this study on J. sigillata, AG (OF00522) and SOC1 (OF12809) genes were found to be highly expressed during the physiological differentiation stage of female flower buds. Photoperiod is an important regulator of flowering, and the CONSTANS-like (COL) gene family plays a pivotal role in the photoperiodic flowering pathway [44]. The FT gene, a direct target of CO, integrates light signals and biological clock signals, which are then transmitted to downstream flowering genes, including SOC1 and FT, facilitating flowering [45]. These findings reinforce the crucial role of flower-inducing genes in flower bud differentiation. To explore potential relationships between flavonoid biosynthesis, structural genes involved in flavonoid production, and flowering genes, we conducted correlation analyses. The correlation analysis of DAMs and DEGs within the flavonoid biosynthesis pathway revealed that key genes such as CHS (OF28612, OF03554), FLS (OF04018, OF20658), DFR (OF17302), 4CL (OF05965), LAR (OF25127), and CHI (OF12477) showed strong correlations with a wide range of flavonoids (Table 1). Currently, no literature indicates an interaction between flavonoids and plant flowering genes. However, in this study, metabolites and structural genes in the flavonoid biosynthesis pathway showed significant positive correlations with flowering genes such as SOC1, FT, CO, and AP1. This suggests that flavonoids might play a role in the differentiation of female flower buds in Juglans J. sigillata by interacting with these flowering genes. This insight opens new avenues for understanding the intricate mechanisms of reproductive development in woody plants.

TFs associated with flavonoid synthesis in female flower bud of J. sigillata

The biosynthesis of flavonoids in plants is orchestrated by a synergistic interaction between structural genes and regulatory transcription factors. Key structural genes involved in the flavonoid pathway include PAL, C4H, CHS, CHI, F3H, FLS, and DFR, all of which are regulated by TFs such as MYB, WRKY, bHLH, and NAC [9, 46, 47]. In the process of female flower bud differentiation in J. sigillata, correlation analyses identified high connectivity for PAL (OF12732), FLS (OF04018), DFR (OF04017), CHI (OF12477), and CHS (OF28162) (Fig. 6B), indicating their crucial role in flavonoid biosynthesis in female flower buds. In Pyrus bretschneideri, PbMYB10b, PbMYB9, and PbMYB3 are activators of the flavonoid pathway, promoting the expression of key genes such as CHS, CHI, UFGT, and FLS [48, 49]. Similarly, in Freesia hybrida, FhMYBFs and FhMYB21 activate the expression of FhFLS1 and FhFLS2 during flower development, with distinct roles in different floral tissues [9]. In J. sigillata, MYB has been shown to enhance flavonoid accumulation by upregulating biosynthesis-related genes [50]. Likewise, in durian, DzMYB controls flavonoid biosynthesis by interacting with CHS, F3H, and CHI promoters [51]. In this study, MYB (OF22990) exhibited high connectivity and was positively correlated with the flavonoid biosynthesis genes PAL, CHI, and CHS (Fig. 5B), supporting its positive regulatory role in flavonoid biosynthesis in J. sigillata. Overexpression of MdWRKY11 in apple promotes flavonoid accumulation and upregulates the expression of genes like MdF3H, MdFLS, MdDFR, MdANS, and MdUFGT [52]. Similarly, VvWRKY70 in grapes and NtWRKY11b in tobacco have been shown to regulate flavonoid biosynthesis by inhibiting the promoter activity of flavonoid pathway genes [53, 54]. In this study, WRKY (OF10312) showed a strong positive correlation with 4CL (OF20609), LAR (OF03725), and F3′5'H (OF27121), suggesting its role in positively regulating the synthesis of flavonoids in J. sigillata (Fig. 5B). MADS-box transcription factors are well-known for their roles in floral organ development. In buckwheat, overexpression of FdMADS28 increases rutin content, a flavonoid, by modulating the metabolic pathway [55]. In this study, an M-type MADS transcription factor (OF04963) exhibited a high positive correlation with F3′5'H (OF27121, OF27123) and PAL (OF12732) (Fig. 5B), implying that it may positively regulate the flavonoid biosynthesis pathway in J. sigillata. These transcription factors regulate the expression of structural genes in the flavonoid biosynthesis pathway by binding to cis-elements in the gene promoters, effectively modulating flavonoid production. Future research can explore these regulatory relationships further, providing insights into the mechanisms underlying flavonoid synthesis and their role in flower bud differentiation.

Conclusion

In this study, we explored the regulatory patterns of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) during female flower bud differentiation in J. sigillata through integrated transcriptomic and metabolomic analyses. The results highlighted flavonoid biosynthesis as a key pathway involved in the differentiation process. Specifically, flavonoid metabolism was channeled toward the isoflavone, flavone, and flavonol branches during female flower bud differentiation. This process was primarily regulated by several critical structural genes, including PAL (OF12732), FLS (OF20658), CHS (OF28612), DFR (OF17302), and F3′5'H (OF27121). Moreover, these structural genes and associated flavonoid metabolites were found to have high correlation levels with flower-inducing genes such as SOC1 (OF12809), FT (OF15270), CO (OF25687), and AP1 (OF11596). The transcription factors M-type MADS (OF04963), MYB (OF22990), and WRKY (OF10312) also showed strong associations with structural flavonoid synthesis genes, suggesting a complex network regulating flavonoid biosynthesis in response to female flower bud differentiation in J. sigillata. Our findings indicate that flavonoid synthesis is tightly regulated by structural genes, while changes in flavonoid content can impact the expression of flower-forming genes. This interaction underscores the role of flavonoids in modulating the differentiation of female flower buds in J. sigillata. This study provides valuable candidate genes for future research on female flower bud differentiation in J. sigillata. The next step is to functionally validate key structural genes and transcription factors, and further investigate the relationship between flavonoid biosynthesis and flower gene regulation. This will deepen our understanding of how flavonoids contribute to plant reproductive development, particularly in J. sigillata (Fig 7).

Fig. 7
figure 7

Diagram of the pattern of flavonoid regulation of female flower bud differentiation in J. sigillata, the biosynthetic pathway of flavonoids was modified from [56]. Italics indicate structural genes, red and pink colors indicate positive regulation by genes or metabolites, green and blue colors indicate negative regulation by genes or metabolites, double arrows indicate the presence of interactions, and single arrows indicate positive regulation

Full size image

Data availability

RNA sequence data that support the findings of this study have been deposited in the https://www.ncbi.nlm.nih.gov/bioproject/, with the primary accession code PRJNA1162697.

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Funding

This research was funded by the National Natural Science Foundation of China [32460744] and Natural Science Foundation Key Project of Guizhou Province (ZK [2024] No. 012).

Author information

Authors and Affiliations

  1. College of Agriculture, Guizhou Engineering Research Center for Fruit Crops, Guizhou University, Guiyang, China

    Jinyan Chen, Wenwen Li, Chunxiang Li, Ruipu Wang & Xuejun Pan

  2. College of Agriculture, Guizhou University, Guiyang, 550025, China

    Jinyan Chen, Wenwen Li, Wen’ e Zhang, Chunxiang Li, Ruipu Wang & Xuejun Pan

  3. Forestry Bureau, Hezhang County, Guizhou, China

    Jian Peng

Authors
  1. Jinyan Chen
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  2. Wenwen Li
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  3. Wen’ e Zhang
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  4. Chunxiang Li
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  5. Ruipu Wang
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  6. Xuejun Pan
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  7. Jian Peng
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Contributions

Wen' e Zhang and Xuejun Pan: conceptualization; Jinyan Chen, Wenwen Li and Chunxiang Li: Conduct experiments; Jian Peng: investigation and resources; Jinyan Chen: data curation and writing—original draft; Wen' e Zhang and Xuejun Pan: awriting—review & editing; Ruipu Wang: Paper polishing; Xuejun Pan and Wen' e Zhang: funding acquisition.

Corresponding authors

Correspondence to Xuejun Pan or Jian Peng.

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Chen, J., Li, W., Zhang, W.e. et al. Combined transcriptional and metabolomic analysis of flavonoids in the regulation of female flower bud differentiation in Juglans sigillata Dode. BMC Plant Biol 25, 168 (2025). https://doi.org/10.1186/s12870-025-06121-9

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  • DOI: https://doi.org/10.1186/s12870-025-06121-9

Keywords

BMC Plant Biology

ISSN: 1471-2229

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