Abstract

   The phytohormone brassinosteroid (BR) regulate various developmental
   and physiological processes in plants. However, the function of BR
   during early seedling development stage in rapeseed is largely unknown.
   To understand the effects of exogenous BR during early seedling
   development, the ZS11 and BR-INSENSITIVE (bin2) mutants were treated
   with BR before seed sowing and seed germination stage under 16/8 hours
   light/dark cycle. The phenotype results indicated that BR promotes only
   seedling establishment but not seed germination stage in ZS11, while no
   function in bin2 mutants. Since BRs play a crucial role in regulation
   of developmental transition between growth in the dark
   (skotomorphogenesis) and growth in the light (photomorphogenesis), the
   ZS11 and bin2 mutants were treated with BR under continuous light and
   dark. The BR treatment also showed the same functions as 16/8 hours
   light/dark cycle. To understand the function of BR on expression
   levels, the differentially expressed genes (DEGs) and differentially
   expressed metabolites (DEMs) between mock- and BR-treated seedlings
   were explored. A total of 234 significantly DEGs were identified
   between the mock- and BR-treated groups by transcriptomic analyses.
   These DEGs were markedly enriched in BR biosynthesis, pentose and
   glucuronate interconversions and plant hormone signal transduction
   pathways. Meanwhile, a total of 145 DEMs were identified through
   metabolomics analyses, with a significant enrichment in lipid
   substances. Interestingly, some genes and metabolites associated with
   auxin pathway were identified, which exhibited up-regulation in both
   DEGs and DEMs after BR treatment. Subsequently, functional enrichment
   analyses revealed that the majority of DEGs and DEMs were primarily
   enriched in ascorbate and aldehyde metabolism, arginine and proline
   metabolism, tryptophan metabolism (the main route for auxin synthesis)
   and cyanogenic amino acid metabolism. Furthermore, it was found that
   glutamate was up-regulated in nitrogen metabolism, glyoxylate and
   dicarboxylate metabolism, and arginine and proline metabolism pathways.
   These indicated that the glutamate signaling pathway was a key
   regulatory pathway for exogenous BR to induce seedling establishment.
   These evidence implied that exogenous BR treatment lead to
   up-regulation of auxin-related genes expression, then promoted seedling
   establishment in rapeseed.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12870-025-06223-4.

   Keywords: Brassinosteroid, Early seedling development, Auxin, Rapeseed

Introduction

   Rapeseed (Brassica napus L.) is formed through natural hybridization
   between B. rapa and B. oleracea approximately 7,500 years ago [[36]1].
   It is the second largest oilseed crop in China, which produces over 40%
   of domestic vegetable oil [[37]2]. Phytohormones play a paramount roles
   in regulating the growth, development and responses to environmental
   stresses in plants [[38]3, [39]4]. For instance, abscisic acid (ABA) is
   capable of facilitating and maintaining seed dormancy, whereas
   gibberellic acid (GA) can induce seed germination [[40]5]. Salt stress
   induces the expression of ethylene (ET) biosynthesis genes, thereby
   inhibiting root growth and flowering in plants [[41]6, [42]7]. The
   synergistic effect of exogenous auxin and jasmonic acid (JA) stimulates
   ABA signaling, resulting in a reduction in seed germination rate
   [[43]8]. However, fewer publications have been reported on the
   mechanism and regulatory networks of BR during seed germination and
   seedling establishment in rapeseed.

   BRs are a group of polyhydroxylated plant steroids. It was identified
   as the sixth major plant hormone in the 1970s. BR play a crucial role
   in plant growth and development, environmental adaptability, regulating
   gene expression and cellular activities through various mechanisms,
   thereby affecting plant morphogenesis and physiological functions
   [[44]9, [45]10]. The BR signal is recognized by the cell surface
   receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), thereby triggering the
   expression of BR-related genes [[46]11]. Research has also indicated
   that BRASSINAZOLE RESISTANT1 (BZR1) and BRI1-EMS-SUPPRESSOR1 (BES1)
   play roles in the BR signaling pathway [[47]12]. BZR1 and BES1 act as
   transcription factors to regulate the expression of BR response genes
   [[48]13]. The recent study has demonstrated the structure and function
   of ATP-BINDING CASSETTE subfamily B19 (ABCB19) is capable of exporting
   BR molecules to the outside of cells and positively regulates the BR
   signaling in arabidopsis [[49]14]. After the activation of the BR
   signaling pathway, it trigger a series of changes and play a certain
   biological function, thereby affecting the growth and development in
   plants. Early studies have indicated that BES1 accumulates in the
   nucleus in response to brassinosteroids, regulating gene expression and
   promoting stem elongation [[50]15]. Recently, BR also can activate the
   expression of cell wall-related genes and promote elongation.
   Meanwhile, WRKY46/54/70, as an important signaling component, is
   positively involved in BR regulated growth and negatively regulated
   drought response in arabidopsis [[51]16, [52]17]. In summary, BRs play
   an irreplaceable role in plant growth and development.

   Transcriptome analysis through high-throughput sequencing technologies
   (such as RNA-seq) and bioinformatics analysis, is an important tool for
   studying gene expression and regulation. It plays a vital role in
   studying cells, physiology, biochemistry, and biological systems
   [[53]18, [54]19]. Metabolomics provides functional readouts of cellular
   biochemistry and is commonly used for phenotype prediction and disease
   prevention [[55]20]. Integration analysis of the metabolome and
   transcriptome can better explore the regulatory patterns between genes
   and metabolites, revealing the regulatory mechanisms of metabolic
   pathways within organisms [[56]21]. Recent studies have revealed the
   inhibitory mechanism of exogenous ABA treatment on seed germination in
   rapeseed through transcriptomics and metabolomics analysis [[57]22]. It
   has discovered that AgNPs-triggered metabolomic and transcriptomic
   analysis enhances salt tolerance in rice [[58]23]. Integrated
   transcriptomic and metabolomic analysis of leaves from wild and
   cultivated soybean seedlings under phosphorus deficiency reveals
   mechanisms for their tolerance to low-phosphorus stress [[59]24].

   In this study, transcriptomic and metabolomic analyses were used to
   infer the mechanism by which BR affects seedling establishment. This
   study investigated the impact of BR treatment on the germination of
   rapeseed, aiming to elucidate the relationship between gene expression
   and metabolites associated with early seedling development. In
   addition, through the combined analysis of metabolomics and
   transcriptomics, we investigated the correlation between DEMs and DEGs
   during seed germination promoted by exogenous BR. Further explores the
   potential role of the glutamate signaling pathway as a key regulatory
   mechanism induced by exogenous BR in seedling establishment, as well as
   the significant involvement of auxin in the response of rapeseed to BR.
   It would contribute to a comprehensive investigation and a better
   understanding of the impact of BR treatment on the accumulation of
   factors promoting rapeseed germination in rapeseed. Simultaneously,
   this study provides reliable information for subsequent rapeseed
   germplasm improvement and the study of gene regulatory networks.
   Therefore, this study provides a novel insight into the BR regulatory
   networks during early seedling development and lays an important
   foundation for rapeseed breeding.

Materials and methods

Plant cultivation and BR treatment

   In this study, the experiment was conducted using ZS11 and
   BR-INSENSITIVE 2 (BnaC01.bin2, with ZS11 background) with the same size
   as the experimental materials, which described in previous studies
   [[60]21, [61]25]. Using 30 mm culture dishes (with two layers of
   absorbent paper under the bottom), 50 seeds were added along with 2.0
   mL of distilled water and placed in an artificial climate chamber for
   germination (conditions: 16 h of light/8 hours of darkness, light
   intensity of 80%, humidity of 70%, temperature of 25℃). The mock and BR
   treatment was conducted 24 h after seeds sowing, the time point of most
   seeds have germinated. The experimental group was immersed with
   approximately 2.0 mL of 0.5 µM, 1.0 µM, 1.5 µM (Bomei; lot
   no.[62]BY042219) solution, while the control group was treated with 2.0
   mL of distilled water. For skotomorphogenesis and photomorphogenesis
   phenotype observation, ZS11 and bin2 mutant were incubated under 24
   continues light and dark. The other conditions and 1.0 µM BR treatment
   were the same as 16/8 hours light/dark cycle. For BR treatment before
   seed sowing assay, ZS11 and bin2 mutant were treated with 1.0 µM BR,
   then incubated at 16 h light/8 hour dark condition. The mock- and
   BR-treated samples under 16 h of light/8 hours of dark were harvested
   for transcriptome and metabolome sequencing three hours after
   treatment.

Sample collection and RNA-seq analyses

   In this study, samples were collected three hours after mock and BR
   treatment. Three independent biological replicates were performed for
   each treatment [[63]26]. The sequencing data were analyzed on the
   online platform of Majorbio ([64]www.majorbio. com). This study
   employed strand-specific library construction and Illumina sequencing.
   Initially, RNA was extracted from the seedlings of rapeseed samples of
   both the mock- and BR- treated groups, followed by quality assessment.
   The RNA was then reverse transcribed into cDNA using reverse
   transcriptase, and the RNA-DNA hybrids were synthesized into
   double-stranded DNA using DNA polymerase and purified using columns.
   Subsequently, sequencing adapters were ligated to the ends of cDNA.
   Then the cDNA library was amplified by polymerase chain reaction (PCR)
   [[65]27, [66]28]. Finally, the library was purified, measure the
   concentration, and sequence it. Clean reads mapping was conducted using
   the reference genome of Brassica napus ZS11
   ([67]http://cbi.hzau.edu.cn/cgi-bin/rape/download ext).

Metabolomics analysis

   The rapeseed sample (50 mg) was processed for metabolite extraction
   using a 6 mm grinding bead and 400 µL extraction solution (methanol:
   water = 4:1) with an internal standard. After grinding and ultrasonic
   extraction, the sample was centrifuged, and the supernatant was
   prepared for LC-MS/MS analysis. Six independent biological replicates
   were performed for each treatment. Raw data was processed with
   Progenesis QI for filtering, peak identification, and alignment,
   creating a data matrix with key parameters. MS and MS/MS spectra were
   matched against databases like Human Metabolome Database (HMDB,
   [68]http://www.hmdb.ca/), Metabolite Link (Metlin,
   [69]https://metlin.scripps.edu/), and a custom-built database for
   metabolite identification. The processed data was then uploaded to the
   Majorbio cloud platform (cloud.majorbio.com) for further analyses. This
   comprehensive procedure ensured efficient extraction and analysis of
   metabolites from the rapeseed sample, utilizing advanced techniques for
   accurate metabolite identification and data processing.

Correlation analysis of transcriptomic and metabolomic data

   IPath (Interactive Pathway Explorer) is a visual online tool for
   pathway analysis. By utilizing iPath 3.0 ([70]http://pathways.embl.de),
   we can obtain information about the up-regulated or down-regulated of
   metabolites in the entire system. IPath illustrates the different
   metabolites and metabolic pathways between the mock- and BR- treated
   group using nodes (metabolites) and edges (metabolic pathways) of
   different colors. Significantly differentially expressed genes (DEGs)
   are defined as those with a p-value < 0.05 and|log[2]FoldChange| ≥ 1.
   Metabolites with variable importance in projection (VIP) value ≥ 1 and
   p-value < 0.05 were identified as differential metabolites (DAMs) based
   on the OPLS-DA model. Metabolic pathway analysis is conducted based on
   Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Results

Phenotypical observation of rapeseed during seed germination and seedling
establishment stage under BR treatment

   In this study, the germinated ZS11 and BR mutant (bin2) seeds were
   treated with BR using 0.5 µM, 1.0 µM, and 1.5 µM BR treatments after
   24 h cultivation. Phenotypic observation revealed that the ZS11 seeds
   started germination after incubating for 24 h, while bin2 mutants show
   a several-hour delay in germination (Fig. [71]1A-B). One day after BR
   treatment, the BR-treated ZS11 seedlings started to open yellow
   cotyledons (Yellow cotyledons: when the seeds have just germinated, the
   chlorophyll in the body is not undergoing photosynthesis and is in a
   dormant state, also known as yellow-green substance. We refer to the
   cotyledons at this stage as yellow cotyledons), while the mock-treated
   ZS11 and mock/BR-treated bin2 remained at the seed germination stage
   (Fig. [72]1C). Two days after treatment, most cotyledons began to turn
   green in the BR-treated ZS11. The 1.0 µM BR treatment significantly
   promoted the growth of germinated ZS11 seedlings, while most cotyledons
   of seedlings remained yellow in the mock-treated ZS11 and
   mock/BR-treated bin2 groups (Fig. [73]1D). Then we chose 1.0 µM
   concentration of BR as the treatment for further studies.

Fig. 1.

   [74]Fig. 1
   [75]Open in a new tab

   Phenotypical observation of rapeseed responding to exogenous BR. (A, B)
   The 50 seeds of ZS11 and bin2 mutant with 2.0 mL distilled water were
   placed into a 30 mm petri dish and incubated in a growth chamber under
   the following conditions: 16 h light/8 hours dark cycle, light
   intensity of 80%, humidity of 70%, temperature of 25 °C for
   germination. (A) 0 day (B) 24 h. (C, D) The experimental group was
   treated with 2.0 mL of 0.5 µM, 1.0 µM, and 1.5 µM BR solutions (C)
   1 day and (D) 2 days, while the mock was treated with 2.0 mL distilled
   water for (C) 1 day (D) 2 days

   To detect the function of BR in seed germination stage, we prepared
   ZS11 and bin2 seeds incubated in 1.0 µM BR before sowing. The phenotype
   displayed the same as BR treatment after seed germination (Fig.
   [76]S1). These results suggested that BR mainly affects the seed
   development between seed germination and seedling establishment stage.
   Since BRs play a crucial role in regulation of developmental transition
   between growth in the dark (skotomorphogenesis) and growth in the light
   (photomorphogenesis), we also designed the similar experiments under
   dark and light conditions in combination with mock and BR treatment.
   The phenotypes under continuous light and dark condition displayed the
   same tendency as 16/8 hours light/dark cycle conditions, only the
   cotyledons cannot turn green under dark condition even with BR
   treatment in ZS11 (Fig. [77]S2; Fig. [78]S3). Therefore, our findings
   indicated that exogenous BR treatment can promote the early seedling
   development of rapeseed.

Identification of DEGs in response to BR treatment

   To explore the transcriptomic profiles of rapeseed under BR-treated
   group, we constructed cDNA libraries based on DNA from the BR-treated
   group and the mock group of rapeseed. A total of 95.67 Gb of clean data
   was obtained and clean data from each samples exceeding 10.02 Gb. To
   compare the gene expression profiles between the mock and BR-treated
   groups, a venn diagram reveals that 39,759 genes are expressed both in
   the mock and BR-treated groups (Fig. [79]2A). Our results indicated
   that a total of 234 DEGs, with 177 up-regulated and 57 down-regulated
   between mock and BR-treated groups, respectively (Fig. [80]2B; Table
   [81]S1).

Fig. 2.

   [82]Fig. 2
   [83]Open in a new tab

   Transcriptomic profiling of rapeseed in response to exogenous BR. (A)
   Venn diagram of differentially expressed genes in mock-and the
   BR-treated group. (B) The volcano plot of differentially expressed
   genes (DEGs) between the mock-and the BR-treated group. (C, D)
   Enrichment results plot of the top 20 DEGs between the mock- and
   BR-treated groups. (C) GO analyses based on up-regulated DEGs. (D) GO
   analyses based on down-regulated DEGs

   To detect the biological proceeds involving in BR transduction during
   seedling establishment in rapeseed, we conducted KEGG and Gene Ontology
   (GO) enrichment analyses. The results of the GO enrichment analysis
   indicated the DEGs were divided into 20 subcategories within 3 main
   categories, biological processes (BP) with 9 subcategories, cellular
   components (CC) with 4 subcategories, and molecular functions (MF) with
   3 subcategories (Fig. [84]S4; Table [85]S2). The biological process of
   GO enrichment analyses indicated significant enrichment in
   oxidoreductase activity, catabolic metabolic processes, organic
   substance catabolic processes and carbohydrate metabolic processes in
   the up-regulated DEGs (Fig. [86]2C). The biosynthesis of organic cyclic
   compounds, oxidoreductase activity in tetrapyrrole binding, as well as
   steroid hormones and phytosterols were significantly enriched in the
   down-regulated DEGs (Fig. [87]2D). The KEGG pathway enrichment results
   of significantly DEGs between the mock- and BR-treated groups indicate
   enrichment in plant hormone signal transduction, brassinosteroid
   biosynthesis, and pentose and glucuronate interconversions (Fig.
   [88]S5). Additionally, we found that several genes related to auxin
   expression were significantly up-regulated in DEGs (Table [89]S3), such
   as SMALL AUXIN UP-REGULATED RNA20 (SAUR20), ATP BINDING CASSETTE
   SUBFAMILY B MEMBER 4 (ABCB4), INDOLE-3-ACETIC ACID3/14/19/32
   (IAA3/14/19/32), and AUXIN TRANSPORT PROTEIN (BIG) (Fig. [90]3; Fig.
   [91]S6). Recently, a study found that the ABCB19, functions as a BR
   exporter and ABCB19 ATP hydrolysis activity is robustly activated by
   BR, and it has been demonstrated through transport assays that ABCB19
   facilitates the transport of BR [[92]14]. Therefore, we speculated that
   rapeseed could sense exogenous BR stimuli, then potentially leading to
   significant changes in the expression of auxin-related genes and signal
   transduction.

Fig. 3.

   [93]Fig. 3
   [94]Open in a new tab

   DEGs are associated with auxin pathway. (A-D) The relative expression
   of SAUR20 (A), ABCB4 (B), IAA32 (C) and BIG (D) between mock- and
   BR-treated seedlings. (A) SAUR20 is from SAUR-like auxin-responsive
   protein family. (B) ABCB4 encodes an auxin efflux transmembrane
   transporter that is a member of the multidrug resistance P-glycoprotein
   (MDR/PGP) subfamily of ABC transporters. (C) IAA32 belongs to auxin
   inducible gene family. (D) BIG is a calossin-like protein required for
   polar auxin transport. ***represents significant differences under
   p < 0.001

Metabolome profiles in rapeseed under BR treatment

   To understand the response of rapeseed to exogenous BR during early
   seedling development from a metabolic perspective, we conducted a
   broadly targeted metabolomics analyses using LC-MS. According to the
   expression levels of different metabolites among samples, we used a
   correlation heatmap analysis to evaluate the similarity within groups
   and the differences between mock- and BR-treated groups. Preliminary
   analysis revealed significant differences in metabolites between the
   mock- and BR-treated groups, as well as variation within each group.
   Partial least squares discrimination analysis (PLS-DA) resulted in the
   division of the total variation into two major components (Component 1
   and Component 2), which contributed 16.9% and 11.1% of the variation
   (Fig. [95]4A). By employing univariate statistical analysis (students’
   t test), as well as multivariate statistical analysis (OPLS-DA/PLS-DA)
   and fold change (FC) values, a total of 145 differential metabolites
   were identified between the mock- and BR- treated groups (Table
   [96]S4). The volcano plot of differential metabolite analysis revealed
   that 76 up-regulated and 69 down-regulated metabolites between mock-
   and BR-treated samples (Fig. [97]4B). The categorization of HMDB
   compounds is based on the biological functional hierarchy of
   metabolites. It indicated that DEMs were mainly belong to lipids and
   lipid-like molecules, organic heterocyclic compounds, organic oxygen
   compounds, phenylpropanoid and polyketones (Fig. [98]4C). These studies
   demonstrated that lipids and lipid-like molecules were significantly
   present in the BR-treated group. It also suggested that lipid
   metabolism plays a crucial role in the BR response (Table [99]S5).

Fig. 4.

   [100]Fig. 4
   [101]Open in a new tab

   Metabolic profiling of rapeseed in response to exogenous BR. (A)
   Principal Component Analysis (PCA) of the mock group and BR-treated
   group. (B) The volcano plot of DEMs between the mock- and the
   BR-treated group. (C) The categories of DEMs. (D) KEGG pathway
   enrichment analysis of DEMs

   To identify significant changes in metabolites under BR-treated group,
   we conducted analysis on DEMs to determine significantly enriched
   metabolic pathways. It indicated that the significantly enriched
   metabolic pathways include porphyrin and chlorophyll metabolism, ABC
   transporters, purine metabolism, phenylalanine metabolism, and
   flavonoid biosynthesis (Fig. [102]4D, Table [103]S6). Additionally, we
   found that the intermediate product of auxin synthesis, indole-3-acetic
   acid and indoleacetaldehyde in DEMs (Fig. [104]5; Fig. [105]S7).
   Indole-3-acetic acid is a common form of auxin in nature, while
   indoleacetaldehyde is a precursor of indole-3-acetic acid in the
   tryptophan pathway. We hypothesized that exogenous BR treatment of
   rapeseed can promote the formation of auxin internally, thereby
   enhancing seed germination and seedling development in rapeseed.

Fig. 5.

   [106]Fig. 5
   [107]Open in a new tab

   The abundance of DEMs are associated with auxin pathway. (A) One of the
   intermediates in the tryptophan pathway for auxin formation. (B)
   Indole-3-acetic acid, also known as auxin. **represents significant
   differences under p < 0.01

Correlation and integrative analysis between metabolome and transcriptome

   By conducting correlation analyses of the metabolomic and
   transcriptomic profiles between the mock- and BR-treated groups, which
   is possible to identify key metabolic pathways and gene expression
   patterns related to the BR responses. We utilized bidirectional
   orthogonal partial least squares (O2PLS) to assess the inherent
   correlation between transcriptomic and metabolomic data, calculating
   scores for each sample to generate a combined score plot (Fig.
   [108]S8).We have constructed a joint score plot to visualize the
   relationship between the transcriptome and metabolome, selecting the
   top 15 DEGs and DEMs to build a histogram (Fig. [109]6A). A Venn
   diagram revealed 9 overlapping pathways between DEMs and DEGs,
   indicating a certain level of correlation between these two omics
   (Fig. [110]6B). Then we conducted KEGG pathway enrichment analysis,
   focusing on the enrichment of pathways both in the transcriptome and
   metabolome. By constructing a histogram, we visualized the enrichment
   levels of DEGs and DEMs pathways. DEMs and DEGs are mainly enriched in
   ascorbic acid and aldehyde metabolism (map00053), arginine and proline
   metabolism (map00330), tryptophan metabolism (map00380) and cyanogenic
   amino acid metabolism (map00460) (Fig. [111]6C; Table [112]S7).
   Notably, tryptophan metabolism, which was also the main biosynthetic
   pathway of auxin, was significantly enriched Transcriptome and
   metabolome analyses revealed up-regulation of auxin-related genes and
   metabolites in DEGs and DEMs, while indole-3-acetic acid and
   indoleacetaldehyde are important products in the auxin biosynthesis
   pathway of tryptophan.

Fig. 6.

   [113]Fig. 6
   [114]Open in a new tab

   Integration analysis of DEGs and DEMs. (A) Histogram of the top 15
   expressed genes and metabolites. Blue bars represent genes, while green
   bars represent metabolites. The horizontal axis represents the combined
   loading values pq1, while the vertical axis represents the interactions
   between metabolites and genes. (B) The venn diagram illustrates DEGs
   and DEMsinvolved in both the transcriptome and metabolome. (C)
   Transcriptome and metabolome identified the top 10 DEGs and DEMs
   metabolic pathway diagrams

   Utilizing iPath3.0 for visualizing metabolic pathways, comprehensive
   information on the entire metabolic pathway network of both DEGs and
   DEMs has been obtained (Fig. [115]7A). We visualized the regulatory
   interaction between DEGs and DEMs in a concentric form. Among the top
   20 DEGs, five were enriched among the total of 234 DEGs. These included
   24-epi-brassinolide, rupatadine, 2-(4-Methyl-5-thiazolyl)ethyl
   propionate, germacrone-13-al, and levan. Exception of levan, all of
   others showed up-regulated expression levels (Fig. [116]7B).
   Correlation analysis of the transcriptome and metabolome indicated that
   amino acid metabolism may play direct or indirect regulatory roles in
   key metabolic pathways.

Fig. 7.

   [117]Fig. 7
   [118]Open in a new tab

   Integration analysis of metabolomics data and transcriptomics data. (A)
   Analysis of differential expression DEGs and DEMs metabolic networks in
   KEGG biological systems, as well as related metabolic pathways. Red
   dots represent pathways annotated by DEMs, red and green lines
   represent pathways annotated by up-regulated and down-regulated genes
   respectively. Blue lines represent pathways annotated by genes common
   to both the mock- and BR-treated groups. (B) A graph showing the
   relationship between 234 significant DEGs and 5 of the top 20
   up-regulated DEGs. The outer circle represents significant DEGs, the
   inner circle represents DEMs.The depth of color represents the degree
   of significant difference

Glutamate metabolism under under BR treatment

   With the intention of the transcriptional regulatory network during
   seedling establishment under BR treatment, we further uncovered the
   significantly enriched amino acid metabolism of DEGs and DEMs. In the
   pathway of arginine and proline metabolism (map00910), up-regulation of
   glutamate expression was observed. Further analyses of the remaining
   enriched metabolic pathways revealed up-regulation of glutamate
   expression in nitrogen metabolism (map00330) and pyruvate metabolism
   (map00630) as well (Fig. [119]8; Table [120]S8-[121]9). Previous
   studies have shown glutamate plays a crucial role in plant seed
   germination, root growth, and flower development [[122]29]. Research
   indicates that glutamate can relieve the inhibitory effect of ABA on
   seed germination and seedling development [[123]30]. MAP KINASE 6
   (MPK6) and MAPK phosphatase 1 (MKP1) are involved in the composition of
   Arabidopsis root structure through the Glu signaling pathway [[124]31].
   Crosstalk network of related DEGs and DEMs in glutamate metabolism
   suggests an upregulation of DEGs controlling glutamate biosynthesis,
   leading to an increase in glutamate expression. Consequently, we
   hypothesized that exogenous BR treatment of rapeseed leads to
   up-regulation of endogenous glutamate expression, thereby promoting
   seed germination and growth.

Fig. 8.

   [125]Fig. 8
   [126]Open in a new tab

   The expression profiles of DEGs and DEMs related to glutamate
   metabolism. (A) Revealing the regulatory network of glutamate
   metabolism through DEGs and DEMs. (B) DEGs related to glutamate
   metabolism. The red color indicates up-regulation of expression in
   rapeseed after BR treatment

Discussion

   Brassinosteroids (BR) are a class of steroid hormones that play a key
   role in the regulation of plant growth and development, exerting
   important effects on metabolic regulation in plants [[127]32]. Research
   has shown that BR regulates microtubule organization and cell wall
   composition, thereby controlling the growth of both longitudinal and
   radial root axes [[128]33]. Conversely, there is less studies that
   discuss the impact of BR on seedling establishment and its regulatory
   network through integrated analysis of transcriptome and metabolome.
   With the development of scientific technologies, the integration of
   transcriptomics and metabolomics analysis has become a standard tool,
   providing a reliable and effective method for identifying BR-responsive
   genes and exploring their related metabolites. In this study, a
   comprehensive analysis of transcriptome and metabolome was conducted on
   ZS11 treated with BR, identifying a total of 234 significantly DEGs and
   145 significantly DEMs (Figs. [129]2 and [130]4). Especially, we
   observed up-regulation of genes associated with auxin synthesis as well
   as metabolite expression in DEGs and DEMs (Figs. [131]3 and [132]5;
   Fig. [133]S6). These evidences suggested that exogenous BR treatment of
   rapeseed may promote germination and growth by enhancing auxin
   synthesis. The BR signaling pathway and related metabolic pathways in
   plants have been extensively studied, such as the important regulatory
   roles of BSU1 and BIN2 [[134]34], BZR1 and BES1 in the BR signaling
   pathway [[135]35, [136]36]. BIN2 functions in the cross-talk between
   auxin and BR signaling pathways. Studies have found that bin2 increases
   the expression of auxin-induced genes by directly inactivating the
   repressor Auxin Response Factor (ARF), thereby leading to a coordinated
   increase in transcription [[137]37]. Additionally, studies also have
   shown that BIN2-mediated phosphorylation can also enhance auxin
   signaling output during lateral root organogenesis in arabidopsis
   [[138]38]. In our study, we treated seeds of ZS11 and the bin2 mutant
   with different concentrations of BR before sowing and during the
   germination stage. We found that treatment with 0.5 µM, 1.0 µM, and 1.5
   µM BR promoted the establishment of ZS11 seeds, with the 1.0 µM BR
   treatment showing a significant effect. However, BR treatment did not
   promote the establishment of seeds from the bin2 mutant, and it also
   had no promoting effect on seed germination. On the other hand, we
   detected the effects of BR treatment on the seed establishment of ZS11
   and the bin2 mutant under different light and dark conditions. We
   treated seeds of ZS11 and the bin2 mutant with 1.0 µM BR after
   germination under three conditions (16 h light/8 h dark; dark;
   continuous light). The phenotypical observations showed that BR
   treatment promoted the establishment of ZS11 seeds under three
   conditions, but had no promoting effect on the bin2 mutant. Light
   mainly affected the synthesis of chlorophyll in cotyledons after seed
   establishment. Based on the growth conditions of rapeseed sowing, we
   ultimately chose the 16-hour light/8-hour dark cycle as the
   experimental condition for subsequent transcriptome and metabolome. The
   interplay of plant hormones has also been a focus of research in recent
   years. Our study contributes to laying the groundwork for understanding
   the regulatory network mechanism of exogenous BR-induced auxin
   synthesis, promoting germination and growth in Brassica napus.

   The response of rapeseed seeds to BR is a complex physiological process
   involving multiple genes and metabolites. Analysis of the enriched KEGG
   pathways related to the response mechanism of rapeseed to BR indicates
   that DEMs and DEGs are primarily involved in ascorbic acid and aldehyde
   metabolism, as well as arginine, proline, and tryptophan metabolism,
   among other amino acid metabolic pathways. Significantly, DEGs and DEMs
   are significantly enriched in the amino acid metabolism pathway
   (Fig. [139]6A-C). The tryptophan aminotransferase of maize (TAM)
   pathway is one of the important pathways for auxin synthesis [[140]39].
   Therefore, we hypothesized that exogenous BR treatment of Brassica
   napus may promote auxin synthesis through the TAM pathway. In addition,
   the NITRATE-PROTEIN SYMPORTER (NRT) gene associated with nitrogen
   metabolism and the PROLINE DEHYDROGENASE (PRODH) gene involved in
   arginine and proline metabolism, leading to the up-regulation of
   glutamate expression. NRT is a gene that encodes high-affinity nitrate
   transporter proteins [[141]40]. It has been revealed that under low
   nitrate conditions, the brassinosteroid transcription factor BES1
   regulates the expression of NRT2.1 and NRT2.2 in response to
   brassinosteroids [[142]41], and NRT2.1 is also capable of responding to
   NO by modulating auxin synthesis to influence root growth [[143]42].

   Glutamate plays multiple roles in plant growth and development: (1) as
   a nitrogen source, supporting plant growth and development, (2)
   promoting photosynthesis in plants, (3) enhancing plant tolerance to
   stress factors, (4) boosting plant disease resistance, among others.
   The glutamate-mediated Ca^2+ influx is involved in regulating seed
   germination under salt stress in arabidopsis [[144]43, [145]44].
   Glutamate can serve as a signaling molecule to enhance the activity of
   antioxidant enzymes in soybeans, thus increasing their tolerance to
   stress [[146]45]. Currently, some studies have also indicated the
   impact mechanism of glutamic acid and plant hormones on plant growth
   and development. For example, glutamate carboxypeptidase AMP1 regulates
   ABA oxidation, stress response, and participates in Arabidopsis
   thaliana amino acid metabolism [[147]46]. The binding of γ-polyglutamic
   acid with gibberellins (GA) can enhance seed germination rate
   [[148]47], while ethylene 3 (EIN3) can modulate glutamate dehydrogenase
   (GDH) activity [[149]48]. Foliar application of ABA effectively
   alleviated the adverse effects of freezing stress on K. obovata, such
   as proline, by activating antioxidant enzyme activity and increasing
   osmolyte accumulation [[150]49]. Therefore, it suggested that exogenous
   brassinosteroid treatment on rapeseed may enhance the glutamate
   signaling pathway by up-regulating glutamate levels, thereby promoting
   seedling establishment. These findings provide a novel insight into the
   interaction between plant hormones and amino acids. In conclusion, NRT
   and PRODH play crucial roles in the regulation of plant growth and
   development processes.Our findings suggest that exogenous application
   of BR up-regulates the expression of NRT and PRODH genes, leading to
   the up-regulation of glutamate expression, promoting the germination
   and growth of rapeseed seeds, and providing a novel gene network
   regulatory mechanism.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [151]Supplementary Material 1^ (279.2KB, jpg)
   [152]Supplementary Material 2^ (299.2KB, jpg)
   [153]Supplementary Material 3^ (307.7KB, jpg)
   [154]Supplementary Material 4^ (942.7KB, jpg)
   [155]Supplementary Material 5^ (98.7KB, jpg)
   [156]Supplementary Material 6^ (1.4MB, jpg)
   [157]Supplementary Material 7^ (952.6KB, jpg)
   [158]Supplementary Material 8^ (237.2KB, jpg)
   [159]Supplementary Material 9^ (46KB, xlsx)
   [160]Supplementary Material 10^ (35.2KB, xlsx)
   [161]Supplementary Material 11^ (12.1KB, xlsx)
   [162]Supplementary Material 12^ (46.2KB, xlsx)
   [163]Supplementary Material 13^ (9.9KB, xlsx)
   [164]Supplementary Material 14^ (10.8KB, xlsx)
   [165]Supplementary Material 15^ (13KB, xlsx)
   [166]Supplementary Material 16^ (9.9KB, xlsx)
   [167]Supplementary Material 17^ (9.7KB, xlsx)
   [168]Supplementary Material 18^ (5.4MB, docx)

Acknowledgements