1. Introduction
Plants develop dormancy in seeds and bulbs to resist adverse climatic conditions [1,2]. The lily (Lilium spp.) is a typical perennial herbaceous bulb ornamental crop worldwide that needs to undergo physiological dormancy and dormancy release to flower normally [3]. The complete dormancy release of lily bulbs directly affects the bulb sprouting speed and affects the commercial characteristics of cut flowers [4]. It is important to explore the molecular mechanisms of lily bulb dormancy release for the development of industrial lily bulb production and for breaking the market monopoly in China by the Netherlands and other countries [5,6,7,8]. During dormancy of bulbs, the meristematic or embryonic organ is in a stage of eco-developmental arrest, and the development of an apical bud reflects the activity of dormancy release in lily bulbs. When the apical bud is approximately 1.0 cm away from the plateau of the bulb, or when the growing point of the bud is located at two-thirds of the vertical height of the bulb, this is the morphological index at which the lily bulb breaks dormancy at low temperature [9,10,11]. To date, many factors, such as growth conditions, storage temperature, photoperiod of bulbs, and internal genetic factors, have been reported to influence lily bulb dormancy [12]. L. longiflorum has a short growth period, flowering within one year of sowing and tolerates high temperatures, but it is more sensitive to light, whereas the L. Oriental bulb is sensitive to temperature, intolerant of heat, and enters dormancy during summer’s high temperatures to maintain normal growth and flowering. Therefore, the L. Oriental is used as the material for studying the impact of dormancy characteristics on the quality of lily cut flowers. A series of physiological changes promote bulb dormancy release, such as reduced soluble sugar content [11], increased hydrogen peroxide levels and APX activity, activation of the pentose phosphate pathway, and decreased SOD and POD activity [13,14]. In addition, the interaction and signal transduction of the ABA and GA metabolism are important regulatory factors of plant dormancy release and germination [15,16]. However, studies on the gene expression and regulatory mechanisms involved in the dormancy release of lily bulbs are scarce.
Riboflavin is a novel physiological regulatory factor for plant growth and organ morphogenesis and is the synthetic precursor of the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), and it often participates in many REDOX reactions in the form of FAD and FMN in organisms, participating in cell growth and metabolism, nutrient regulation, organ development, etc. However, until now, no evidence has shown that riboflavin regulates dormancy release of bulb. Previous studies have shown that riboflavin promotes early flowering [17,18] and inhibits browning in fresh-cut apples by inhibiting the phenolic metabolism and enhancing the antioxidant system [19]. Exogenous riboflavin application activated oxidative and ionic stress tolerance to ameliorate salinity stress [20]. In addition, FAD acts as a prosthetic group of the FAD-dependent amine oxidase LSD to catalyze the demethylation of H3K4, and H3K4me3 directly affects the expression of cell cycle-related genes and regulates cell division and apoptosis [21]. Riboflavin also affects the mitochondrial TCA cycle and glycolysis and influences the cell cycle by regulating H3K4me3 modification in maize [22]. FMN can be used as an artificial demethylase to participate in the demethylation of m6A under visible light irradiation [23]. In Dimocarpus longan, miR408 targets the riboflavin pathway gene (DlNUDT23), which mediates m6A modification and subsequently promotes cell division and differentiation [24]. A lack of riboflavin reduces the activity of acetyl-CoA dehydrogenase, affects the synthesis of acetyl-CoA [25,26], and further decreases histone acetylation and chromatin remodeling [27]. In the lily, LoNFYA7 enhances H3K27me3 at the LoCALS3 locus and is involved in histone acetylation during the bud growth following dormancy release [28]. Moreover, LoCALS3 was identified as a marker for dormancy maintenance in lilies [29]. These studies indicated that riboflavin regulates the antioxidant system and photosynthetic pathway and affects epigenetic modifications during plant growth and development. Therefore, we speculated that riboflavin may regulate microRNAs (miRNAs) and their target genes and may be involved in acetyl-CoA biosynthesis in the dormancy release of lily bulbs.
miRNAs are a class of endogenous regulatory noncoding small RNAs with a length of 21–24 nt that regulate the transcription of target genes at the transcriptional and posttranscriptional levels to achieve the molecular regulation of hormone signaling, growth and development, dormancy and germination, organ morphogenesis, and various biotic and abiotic stresses [30]. Increasing evidence has demonstrated that miRNAs play a crucial role in plant dormancy release. In Chimonanthus praecox, miR157-x, miR167-x, miR167-y, miR390-y, and miR395-y are highly conserved and downregulated after dormancy release and may mediate ABA biosynthesis and auxin signal transduction to regulate the dormancy release of floral buds [31]. The PsmiR172b-PsTOE3-PsEBB1 module has dual functions in dormancy release and flowering in tree peonies [32]. In Arabidopsis thaliana, DOG1–miR156–miR172 interactions mediate the release of seed dormancy and the transformation of flowering development processes [33]. Currently, during cold storage in Lilium pumilum bulbs, miR168 targets AGO1 to regulate the homeostasis and feedback regulation of active miRNAs; miR395, miR396, and miR166 regulate lateral organs and meristem formation; and miR159, miR164, and miR160 maintain the energy supply [34]. miRNAs and their target genes related to DNA replication and photosynthetic metabolism may be activated to regulate potato tuber dormancy release [35]. miR156s-LfSPL2, miR172a-LfAP2, miR164a-LfNAC, and miR159a-LfSPL2 integrate into multiple flowering pathways and regulate vegetative phase change and flowering induction in Lilium × formolongi [36]. Therefore, exploring the mechanism of action of miRNAs and their targets in response to riboflavin is conducive to further revealing the molecular switches regulating the expression of key genes involved in the dormancy release of lily bulbs.
In this study, a hypothesis is provided that riboflavin might promote the dormancy release of lily bulbs by influencing the differential expression of miRNAs and target genes. The histomorphology, cell morphology, and endogenous physiological content of exogenous riboflavin-treated lily bulbs were analyzed, and mRNA-seq and sRNA-seq transcriptome analysis and RT-qPCR were performed. Gene cloning, bioinformatics analysis, and protein subcellular localization were verified. Our findings provide new insights into the potential regulatory mechanisms of miRNAs and how their targets respond to riboflavin and promote bulb plant dormancy release, and provide a feasible basis for shortening the dormancy-release time of lilies and promoting their flowering through the artificial application of riboflavin in the field.
2. Materials and Methods
2.1. Plant Materials
Lilium ‘Siberia’ is a cultivated hybrid species of Oriental lily. Lilium ‘Siberia’ bulbs (14–16 cm in size) were obtained from the Nanping Lily Germplasm Resource Nursery. After harvesting and storage at 4 °C for two months, different concentrations of riboflavin (0, 0.1, and 0.5 mM) were used to treat the Lilium ‘Siberia’ bulbs; they were soaked for 24 h and then stored at 4 °C. Riboflavin is absorbed into the bulb cells through the basal plate. According to the morphological index of lily bulb dormancy release at low temperatures, the apical bud was approximately 1.0 cm away from the top of the bulb [10]. Then, the apical buds of lily bulbs were obtained, and the bulbs were treated with different concentrations of riboflavin (0, 0.1, and 0.5 mM) and stored at 4 °C for 30 d. At this time, the bulbs treated with 0 mM riboflavin did not appear to undergo dormancy release, but the bulbs treated with 0.1 and 0.5 mM riboflavin began to rapidly sprout and appear with flower bud differentiation. Similarly, the apical buds of lily bulbs were obtained, and the bulbs were treated with different concentrations of riboflavin (0, 0.1, and 0.5 mM) and stored at 4 °C for 60 d. At this time, the sprouting percentage of the bulbs treated with different concentrations of riboflavin was higher than that of those stored at 4 °C for 30 d, and the bulbs treated with 0.1 and 0.5 mM riboflavin sprouted more than those with 0 mM riboflavin treatment. Next, the apical buds were used as the experimental samples, which were immediately frozen in liquid nitrogen and stored at −80 °C for transcriptome sequencing analysis, and the appropriate samples were used for histological analysis. Furthermore, the remaining bulbs of treatment continues to field planting, as shown in Figure 1. In our study, the sizes of lily bulbs were 14–16 cm, each lily bulb had 2–3 flower buds, and this criterion applied to all buds of a bulb. After 110 d, Lilium ‘Siberia’ was flowering, and the numbers of buds and blooms were counted.
2.2. Histological Assays
To investigate the histological traits of the three stages of Lilium ‘Siberia’ bulb dormancy release, different concentrations of riboflavin (Mock (0), 0.1, and 0.5 mM) were used to treat Lilium ‘Siberia’ bulbs for 24 h (Figure 1). The bulbs’ apical buds were cut into small pieces approximately 0.5 cm in length and fixed in a modified Carnoy’s solution (formalin/acetic acid/50% ethanol solution (5:5:90, v/v/v)) (Servicebio, Wuhan, China). Tissue paraffin embedding, paraffin sectioning with thickness of 5 μm, hematoxylin-eosin (HE) staining, and iodide–potassium iodide staining were performed at Servicebio Co. (Wuhan, China). Each treatment included three replicates.
2.3. Physiological Determination
The biochemical kits were used to investigate the soluble sugar contents and H2O2 (Comin Biotech Co., Ltd., Suzhou, China). The total soluble sugar contents were determined using anthrone colorimetry method. The details of this method are referenced to Ou et al. [37]. Briefly, about 0.1 g of samples were homogenized in 1 mL of distilled water. The suspension was then centrifuged at 10,000 rpm for 10 min. Afterward, the sulfuric acid and anthrone colorimetry were subsequently added and incubated at 95 °C for 10 min. Then, the optical density of solutions was immediately measured at 620 nm to assay soluble sugar contents.
The hydrogen peroxide content assay kit was used to determine the level of H2O2 (Comin Biotech Co., Ltd., Suzhou, China). The assay protocol is referenced to Mei et al. and Li et al. [38,39]; titanium sulfate reacts with H2O2 to produce a compound with color (λmax = 415 nm).
The acetyl-coenzyme A (acetyl-CoA) content determination was performed according to BCA assay kit for protein content and acetyl-CoA assay kit (Comin Biotech Co., Ltd., Suzhou, China) with malate dehydrogenase-coupled assay system [40]. The content of acetyl-CoA was proportional to the rate of NADH production, and the increase rate of absorption value at 340 nm reflected the content of acetyl-CoA [41]. Calculation of acetyl-CoA content (nmol/g prot) = [(3280 × ΔA + 0.024) × V1]/(V1 × Cpr) = (3280 × ΔA + 0.024)/Cpr. (V1: Sample volume added to the reaction system, 0.025 mL; Cpr: Sample protein concentration, mg/mL; W: Sample quality, g).
The riboflavin content was measured via HPLC according to previous studies [42]. HPLC conditions: RIGOL L3000 high-performance liquid chromatograph, Compass-C18 (250 mm × 4.6 mm, 5 μm); mobile phase preparation: 0.05 mol/L sodium acetate solution and 1000 mL methanol were filtered with 0.45 μm filter membrane to remove impurities in the solvent and prevent blockage of the column. Methanol: 0.05 mol/L sodium acetate solution = 35:65. The sample size was 10 μL, the flow rate was 1 mL/min, the column temperature was 30 °C, the retention time was 30 min, and the detection wavelength was 270 nm.
2.4. Small RNA and mRNA Libraries Construction and Bioinformatics Analysis
The full-length transcriptome of Lilium ‘Siberia’ (NCBI: PRJNA1179346) was used as a reference dataset to construct the small RNA (sRNA) and mRNA transcriptome databases of apical buds treated with different concentrations of riboflavin [43,44,45]. Firstly, total RNA was extracted from Lilium ‘Siberia’ bulb apical buds subjected to different riboflavin treatments (0, 0.1 mM, and 0.5 mM; including three biological replicates) using TransZol Up reagent (TransGen Biotech) following the manufacturer’s instructions. MiRNAs and mRNAs were extracted from the same total RNA of the same bud so that there was a pair of libraries for each biological sample. Then, an NEBNext Small RNA Library Preparation Kit (#E7300) (New England Biolabs, Ipswich, MA, USA) was used to construct the libraries. Nine sRNA libraries (0 (Mock), 0.1, and 0.5 mM, including three biological replicates) were constructed. The size range of the RNA molecules was 18–30 nt, and the RNA was enriched by polyacrylamide gel electrophoresis (PAGE). Then, 3′ adapters and 5′ adapters were added. The ligation products were reverse transcribed by PCR amplification, and the 140–160 bp PCR products were enriched to generate a cDNA library and sequenced on the Illumina HiSeq Xten platform by Gene Denovo Biotechnology Co. (Guangzhou, China). The raw reads included adapters or low-quality sequences and were further filtered to obtain clean tags. The GenBank database (Release 209.0) and Rfam database (Release 11.0) were used for alignment with small RNAs from the clean tags, and rRNA, scRNA, snoRNA, snRNA, and tRNA were removed. Next, the remaining miRNA tags were aligned with miRbase (Release 22) by bowtie (version1.1.2) software, and the species studied miRNAs were identified. To study the miRNAs of the species not included in miRBase, it is necessary to use the conserved forms of miRNA in different species and find known miRNAs of the species by comparison. The naming conventions of known miRNAs are x, y, which indicate processing from the 5′ arms and 3′ arms of miRNA precursors, respectively. All of the unannotated tags were aligned with reference dataset. According to their positions and hairpin structures predicted by software miRdeep2 [46] (Mackowiak, 2011), the novel miRNA candidates were identified.
Next, nine mRNA transcriptome libraries (0 (Mock), 0.1, and 0.5 mM, including three biological replicates) were constructed. Then, agarose gel electrophoresis, Nanodrop microspectrophotometry Meter detection, and Agilent 2100 detection were performed to detect RNA quality. Next, transcriptome library construction including polyA+RNA captured, RNA fragmented and primed, first strand cDNA synthesized, second strand cDNA synthesized, 3′ ends adenylated and 5′ end repaired, DNA sequencing adapters ligated, and ligated fragments PCR amplified. Finally, DNA 1000 assay kit (Agilent Technologies 5067-1504) (Agilent Technologies, Santa Clara, CA, USA) and High-Sensitivity DNA assay kit (Agilent Technologies 5067-4626) were used to detect sample fragment sizes ranging from 25 to 1000 bp and from 50 to 7000 bp.
2.5. Differentially Expressed miRNAs and Identification of Their Target Genes
To reveal the biological function of miRNAs and their targets during dormancy release in Lilium ‘Siberia’ bulbs, we identified differentially expressed (DE) miRNAs across different riboflavin treatments. First, tags per million (TPM) values were used to calculate and normalize the miRNA expression levels. The formula is TPM = T × 106/N (T represents miRNA tags, N represents total miRNA tags), and the expression profiles of all miRNAs in all samples were obtained. Next, we filtered out miRNAs whose TPM is less than 1; R (
2.6. GO and KEGG Functional Enrichment Analyses of the Target Genes of the DEmiRNAs
We further assessed the functional enrichment of Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of miRNA target genes in the network. In the GO database (
2.7. RNA Isolation and RT-qPCR
Firstly, total RNA was extracted from Lilium ‘Siberia’ bulb apical buds subjected to different riboflavin treatments and different Lilium ‘Siberia’ organs (leaves, stamen stalks, petals, styles, anthers, and ovaries) using TransZol Up reagent (TransGen Biotech) following the manufacturer’s instructions. These total RNAs were used to perform mRNA RT-qPCR. Reverse transcription was performed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech, Nanjing, China) for gene RT-qPCR. Gene qPCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) in a 96-well plate on a QuantStudioTM 1 Plus (Thermo Fisher Scientific Applied Biosystems, Shanghai, China). For each gene, the qPCR system consisted of 5 μL of SYBR, 2 μL of cDNA template, 0.4 μL of primer at 10 nM, and 2.2 μL of ddH2O. The gene qPCR procedure was as follows: initial denaturation for 60 s at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s, and extension at 72 °C for 10 s. Each reaction was performed in triplicate. Lilium ‘Siberia’ Actin was used as the single internal control for calculating the relative expression of genes [49]. Secondly, total RNA was extracted from Lilium ′Siberia’ bulb apical buds subjected to different riboflavin treatments (0, 0.1, and 0.5 mM). These total RNAs were used to perform miRNA RT-qPCR. TransScript® miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Fuzhou, China) was used for miRNA RT-qPCR; miRNA qPCR was performed using Perfect Start Green qPCR Supermix (TransGen Biotech, Fuzhou, China). Each miRNA qPCR system consisted of 5 μL of 2×Perfect Start® Green qPCR SuperMix, 1 μL of cDNA template, 0.4 μL of primer at 10 nM, and 3.2 μL of ddH2O. The miRNA qPCR procedure was as follows: initial denaturation for 30 s at 95 °C, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 10 s. miR156-y was used as the single internal control to calculate the relative expression of miRNAs. The 2−ΔΔCT method was used to calculate the relative expression. All primers used in this research are listed in Table S1.
2.8. Cloning, Bioinformatics, and Subcellular Localization Analysis of LoPAP17
According to the full-length transcriptome of Lilium ‘Siberia’, the sequence of LoPAP17 was cloned from the transcriptome, including an open reading frame (ORF) of 990 bp. The LoPAP17-F/R primers used for gene cloning are listed in Table S1. DNAMAN 6.6 was used to compare the amino acid sequence of LoPAP17 with that of its homologs, and the neighbor-joining method was used to construct a phylogenetic tree in MEGA-5.0 [50]. String version 12.0 was used to predict the protein and protein interaction network of LoPAP17, and the Arabidopsis thaliana protein database was used as a reference database.
The online software YLoc (
2.9. Statistical Analysis
Statistical analyses were performed using Graphpad prism8.0 statistical software. One-way ANOVA was performed, and Dunnett’s test was used for multiple comparisons. The error bars show the means ± SDs of three replicates (n = 3). Asterisks indicate a significant difference (* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.001).
3. Results
3.1. Riboflavin Treatment Promotes the Dormancy Release of Lilium ‘Siberia’ Bulbs
To understand the role of riboflavin in the dormancy release of lilies, the changes in the morphology and endogenous physiological content of bulb apical bud cells were further analyzed after cold storage. The results show that 0.1 and 0.5 mM riboflavin promoted the release of dormancy in the bulb apical bud during cold storage for 30 d compared with the control (Mock) (Figure 2A–C). At 0 d, the morphology of the bulbs treated with different concentrations of riboflavin is shown in Figure S1A. At this time, the shoot apical meristem (SAM) overlaps with the basal plate of bulb; this indicates that the bulb has not broken dormancy, as shown in Figure S1B. During cold storage for 30 d, the sprouting percentages of the 0.1 mM riboflavin treatment were highest, at 58.3%. The 0.5 mM riboflavin treatment was 28.8%, while the sprouting percentage of the control was only 20.9% (Table 1). This indicates that an appropriate concentration (0.1 mM) of riboflavin promotes the release of dormancy in Lilium ‘Siberia’ bulbs, while a higher concentration (0.5 mM) of riboflavin reduces the effectiveness of promoting dormancy release during cold storage for 30 d. Similarly, during cold storage for 60 d, the sprouting percentages were 73.3% and 66.6% under 0.1 and 0.5 mM riboflavin treatment, respectively, while the sprouting percentage of the control was 40.0% (Table 2). These findings suggest that the appropriate concentration (0.1 mM) of riboflavin had the best effect on promoting the dormancy release of Lilium ‘Siberia’ bulbs during cold storage for 30 d, followed by the 0.5 mM riboflavin treatment. Furthermore, the results of the paraffin section analysis show that 0.1 and 0.5 mM riboflavin promoted floral primordium differentiation of Lilium ‘Siberia’ apical buds earlier than control during cold storage for 30 d (Figure 2D). After planting, it is interesting that lily bulbs treated with 0.1 mM riboflavin had the highest number of buds and blooms (Table 3, Figure 1), while those treated with 0.5 mM riboflavin had the lowest number of buds and blooms. This indicates that 0.1 mM riboflavin might promote the release of dormancy in Lilium ‘Siberia’ bulbs and promote floral bud differentiation.
Previous studies have shown that the precipitous endogenous physiological change is an important sign of lily bulb dormancy release during the cold storage process [11,53], and riboflavin often participates in many REDOX reactions. We wanted to know how riboflavin is involved in promoting the dormancy-release process of the lily at the physiological level. Hence, we further performed the assay of endogenous soluble sugar content and H2O2 content determination. The results show that the soluble sugar content of the apical bud of the bulb dramatically decreased and the H2O2 content significantly increased after 0.1 mM riboflavin treatment during cold storage for 30 d. Under 0.5 mM riboflavin treatment, the soluble sugar content had no significant difference, and the H2O2 content significantly increased (Figure 2E). This further indicated that 0.1 mM riboflavin treatment might have a better effect on the dormancy release of lily bulbs than 0.5 mM riboflavin treatment compared with control. During cold storage for 60 d, the soluble sugar content and H2O2 were decreased significantly under 0.1 and 0.5 mM riboflavin treatments compared with control (Figure 2F). From the perspective of absolute content, the soluble sugar content and H2O2 content of all treatment groups at the 60th day were higher than those at the 30th day. At this time, the apical buds all grew rapidly, and the lengths of the first internodes of the apical buds treated with different riboflavin concentrations were still higher than those of the controls (Table 2). These results indicate that 0.1 and 0.5 mM riboflavin could promote bulb dormancy release after 30 d of cold storage, but 0.5 mM riboflavin was less effective than 0.1 mM riboflavin. Moreover, soluble sugar content and hydrogen peroxide content increased with the extension of refrigeration time but showed fluctuations under riboflavin treatment. These data suggested that riboflavin promoted dormancy release in the Lilium ‘Siberia’ bulb after 30 d of cold storage and might influence the soluble sugar and H2O2 content.
3.2. High-Throughput Sequencing and Identification of miRNAs and mRNAs in Lilium ‘Siberia’
To elucidate the regulatory mechanism of riboflavin-promoted Lilium ‘Siberia’ bulb dormancy release, small RNA and mRNA libraries of apical buds treated with different concentrations of riboflavin after cold storage for 30 d were constructed and sequenced (Mock, R1, and R2), and the three comparison groups were Mock-vs.-R1, Mock-vs.-R2, and R2-vs.-R1. The results of principal component analysis (PCA) showed good repeatability among the groups, which could be used for follow-up analysis (Figure S2A,B). A total of 233 known miRNAs from 189 miRNA gene families and 38 novel miRNAs were identified from the three groups. The most frequent length of valid reads among the libraries was 24 nt, followed by 21 nt for total reads and 22 nt for unique reads (Figure 3A). This result is consistent with that observed for most angiosperms [31,54,55,56]. In total, 21 differential expression (DE) known miRNAs and 6 DE novel miRNAs were obtained (Figure 3B, Table S3), and the DE known miRNAs were mainly 21 nt long. Among them, 4, 4, and 9 known miRNAs were upregulated in 3 treatment groups, respectively (Table S2), including miR390-x, miR391-x, miR396-y, miR399-y, miR5051-z, miR6470-z, miR1863-z, miR2275-y, miR171-x, miR160-y, and miR168-x (Table S3). There were 5 downregulated miRNAs in the three groups, respectively, including miR395-y, miR11525-z, miR5141-z, miR4414-z, miR9735-z, miR156-y, miR160-y, miR529-z, miR5225-z, miR6470-z, and miR164-y (Table S3). There were 1, 2, and 2 differentially upregulated novel miRNAs, respectively (Table S2), including novel-m0024-3p, novel-m0026-3p, novel-m0009-5p, and novel-m0010-5p (Table S3) and only 2 downregulated miRNAs (novel-m0002-3p and novel-m0008-5p) (Table S3). A total of 34,495 transcripts were annotated from the mRNA transcriptome of the three groups. After data filtering, the identified genes could be classified into two main categories: transcription factor genes and functional genes. Among them, the SAP, bHLH, GRAS, FAR1, and MYB gene families had the greatest number of transcription factors, followed by NF-YC, ERF, bZIP, and ARF (Figure S3). Compared with the Mock treatment group, the 0.1 mM riboflavin treatment group (R1) had 109 DE genes, and the 0.5 mM riboflavin treatment group (R2) had 131 DE genes; compared with the 0.5 mM riboflavin treatment group (R2), the 0.1 mM riboflavin treatment group (R1) had 134 DE genes. Among these genes, 78, 68, and 76 were upregulated, and 31, 63, and 58 were downregulated in Mock-vs.-R1, Mock-vs.-R2, and R2-vs.-R1 groups, respectively (Table S2). These results indicate that the number of upregulated genes is greater than the number of down-expressed genes after different concentrations of riboflavin treatment, and these genes might be regulated by miRNAs.
3.3. GO Analysis of DE mRNA and miRNA Target Genes
To further elucidate the biological functions of miRNAs and their target genes regulated by the riboflavin-mediated promotion of Lilium ‘Siberia’ bulb dormancy release, DE mRNAs and miRNAs were identified and annotated. Gene Ontology (GO) enrichment mainly includes molecular function, biological process, and cellular components. Among these, cellular components were mainly enriched in photosystem I/II and the Cdc73/Paf1 complex. In addition, compared with Mock, the 0.1 mM and 0.5 mM riboflavin (Mock-vs.-R1 and Mock-vs.-R2) significantly influenced sulfur adenylyltransferase activity, oxidoreductase activity, fatty acid synthase activity, acyltransferase activity, flavanone 4-reductase activity, cytokinin dehydrogenase activity, vitamin binding, and chlorophyll binding in terms of molecular function (Table S4). Biological processes such as sulfate reduction, the ethylene biosynthetic process, anthocyanin-containing compound metabolic process and flavonoid metabolic process, fatty acid biosynthetic process, photosynthesis, jasmonic acid response, and negative regulation of anion channel activity by blue light, were significantly enriched (Table S4). However, compared with the 0.5 mM riboflavin treatment, the 0.1 mM riboflavin treatment (R2-vs.-R1) mainly enriched oxidoreductase activity, NAD(P)H activity, adenylyl sulfate reductase activity, sulfate adenylyltransferase (ATP) activity, NAD binding, and galactinol-sucrose galactosyltransferase activity. Biological processes were enriched mainly in the flavonoid biosynthetic process, galactose metabolic process, sulfate reduction, mitotic cell cycle, DNA replication, phenylpropanoid metabolic process, and cold acclimation. These data indicate that exogenous riboflavin might regulate sulfur metabolism, oxidation–reduction reactions, the photosynthetic system, fatty acid metabolism, acyltransferase activity, flavonoid metabolic processes, cytokinin metabolism, galactose metabolism, ion channels under blue light, the cell cycle, and cold acclimation during promotion of dormancy release in lily bulbs.
3.4. KEGG Analysis of DE mRNA and miRNA Target Genes
To further reveal the regulatory network of miRNAs and their target genes regulated by the riboflavin-mediated promotion of Lilium ‘Siberia’ bulb dormancy release, KEGG pathway analysis was performed. The RNA-seq results show that the top 20 significantly enriched pathways of different expression genes in the three groups were photosynthetic antenna protein, flavonoid biosynthesis, sulfur metabolism, alkaloid synthesis, linoleic acid metabolism, flavonoid and flavonol biosynthesis, cysteine and methionine metabolism, fructose and mannose metabolism, riboflavin metabolism, fatty acid chain extension, pentose and glucuronic acid interconversion, and other metabolic pathways (Figure S4, Tables S5 and S6). The DE miRNA target genes of the three groups were mainly enriched in primary metabolic processes such as oxidative phosphorylation, starch and sucrose metabolic pathways, fatty acid degradation metabolism and unsaturated fatty acid synthesis, peroxisome and galactose metabolism, triglyceride metabolism, sulfur metabolism, and purine metabolism (0.1 mM riboflavin treatment) (Table S6, Figure S4). Among them, genes related to oxidative phosphorylation and starch and sucrose were significantly upregulated, and other pathway-related genes were significantly downregulated. miR-11525-z, miR-395-y, miR-4414-z, miR-5141-z, and miR-9735-z were significantly downregulated, and miR-399-y and miR-396-y were significantly upregulated (Table S3); these genes mainly targeted pgm, APS1, ATPA, and ACAA1 (Figure 4; Table S7). In the Mock-vs.-R2 comparison (0.5 mM riboflavin treatment), the top 20 significantly enriched KEGG pathways included fructose and mannose metabolism, the pentose–phosphate pathway, glycolysis, N-glycan biosynthesis, the proteasome, porphyrin metabolism, the circadian rhythm, and ubiquitin-mediated proteolysis (Table S6). Among them, genes related to porphyrin metabolism and circadian clock rhythm were upregulated; the FBA3 family mainly regulates carbohydrate and carbon metabolism, and some members were upregulated. miR-11525-z, miR156-y, miR5225-z, miR4414-z, and miR160-y were significantly downregulated, and miR1863-z, miR5051-z, miR529-z, and miR6470-z were significantly upregulated, which mainly targeted C3H, FBA3, CKB4, SPL14, SPL16, and HMG3 (Table S7). Similarly, in R2-vs.-R1, the KEGG enrichment pathway was similar to that in Mock-vs.-R2. The expression of miR156-y, miR399-y, miR396-y, and miR2275-y was significantly upregulated, and the expression of miR395-y, miR529-z, miR164-y, miR1863-z, and miR6470-z was significantly downregulated; these target genes mainly included CPL4, APS1, DIT1, CFDP1, SPL14, TRAF1A, and FB13 (Figure 4; Table S7). miR168-x was differentially upregulated only in the R2-vs.-R1 group and its targeted AGO1B gene family. RNA-seq and sRNA-seq association analyses revealed that miR395-y targeted APS1 to participate in sulfur metabolism during treatment with different concentrations of riboflavin. Riboflavin regulates primary metabolic processes, such as oxidative phosphorylation, sulfur metabolism, fatty acid metabolism, glucose metabolism, and amino acid metabolism during the promotion of dormancy release in the Lilium ‘Siberia’ bulb. Subsequently, the expression levels of miRNAs (miR395-y, miR396-y, miR399-y, miR529-z, miR11525-z, miR1863-z, miR164-y, miR5141-z, miR5051-z, miR5225-z, and novel-m0024-3p) and their target genes in Lilium ‘Siberia’ bulbs treated with different concentrations of riboflavin were investigated via RT-qPCR.
3.5. Association Analysis of Riboflavin Metabolism, Oxidative Phosphorylation, and Primary Metabolism During Lilium ‘Siberia’ Dormancy Release
To further analyze the association of riboflavin metabolism with oxidative phosphorylation and primary metabolism during different concentration treatments of riboflavin in lily bulbs, the target genes of significantly enriched metabolic pathways and miRNAs were associated and analyzed (Figure 5A). First, the transcriptional abundance of LoPAP17 in the apical buds of Lilium ‘Siberia’ bulbs was significantly upregulated after treatment with different concentrations of riboflavin (Figure 5A), which might further promote an increase in the endogenous riboflavin content (Figure 5B,C) and affect the FMN levels (Figure 5A). Next, riboflavin is the precursor of FMN and FAD. FMN and FAD participate in the assembly of complex I and complex II in the oxidative phosphorylation process on the inner mitochondrial membrane, and NAD+ and H+ are produced. The upregulated expression of F-type ATPase in complex V activated the oxidative phosphorylation pathway to produce ATP and H+ (Figure 5A). The energy produced is involved in carbohydrate metabolism, glycolysis metabolism, amino acid metabolism, fatty acid metabolism, photosynthetic system reactions, and flavonoid metabolism in the cytoplasm. Second, riboflavin treatment significantly upregulated the expression of the light-harvesting complex I a/b binding protein (LHCA/B) genes related to photosynthetic systems I and II. The expression of genes related to flavonoid metabolism, such as CHS1, CHS5, ANS, and ANT18, was significantly upregulated; the expression of genes related to adenylyl sulfate reductase (APR) was significantly downregulated; and the expression of genes related to APS1, which is mainly targeted by miR395-y, was significantly downregulated. In addition, 0.1 mM riboflavin treatment significantly downregulated the expression of genes related to fatty acid β-oxidation and the ACAA1 gene family (Table S6). ACAA1 genes were mainly regulated by miR9735-z (Table S6), which is responsible for regulating the final step in fatty acid metabolism and the conversion of acetyl-CoA to fatty acids, and it might result in increased acetyl-CoA content (Figure 5C). We further determined the content of endogenous riboflavin and acetyl-CoA under different concentrations of riboflavin treatment in Lilium ‘Siberia’ apical buds after cold storage for 30 d. The contents of riboflavin and acetyl-CoA were all significantly increased with increased concentrations of exogenous riboflavin (Figure 5B,C). These results suggested that riboflavin treatment activated riboflavin metabolism and oxidative phosphorylation and influenced sulfur metabolism, fatty acid metabolism, amino acid metabolism, and primary metabolism. Moreover, riboflavin and acetyl-CoA accumulation increased while riboflavin promoted the release of dormancy in the Lilium ‘Siberia’ apical bud.
3.6. Gene Expression Network Analysis of miRNAs and Their Target Genes by RT-qPCR
To further verify the expression patterns of miRNAs and their target genes via miRNA-seq after treatment with different concentrations of riboflavin, 10 DE miRNAs and 8 target genes were randomly selected for RT-qPCR expression pattern verification. The results indicate that the expression patterns of 10 miRNAs in bulb apical bud dormancy-released plants treated with different concentrations of riboflavin were consistent with those in the transcriptome (Figure 6A), and they showed typical negative regulatory patterns with target genes at an individual stage. For example, compared with Mock, miR395-y was significantly upregulated after 0.5 mM riboflavin treatment, while the target gene LoAPS1 was significantly downregulated. Compared with that in the 0.1 mM riboflavin treatment group, the expression of miR529-z, which targets the gene LoSPL14, was significantly downregulated after 0.5 mM riboflavin treatment. Compared with Mock, miR396-y was significantly upregulated after 0.1 mM and 0.5 mM riboflavin treatments, and the target gene LoCFDP1 was significantly downregulated. Compared with Mock, miR1863-z negatively regulated LoFBA3, which was significantly upregulated after 0.1 mM and 0.5 mM riboflavin treatments, while the target gene LoFBA3 was significantly downregulated. Moreover, miR399-y might negatively regulate LoDIT1, miR11525-z might negatively regulate Lopgm, and miR5051-z might negatively regulate LoCKB4 (Figure 6B). In brief, our mRNA-seq and miRNA-seq data were effectively available. Moreover, several pivotal miRNAs and their target genes, such as miR395-y: LoAPS1, miR529-z: LoSPL14, miR396-y: LoCFDP1, miR1863-z: LoFBA3, miR399-y: LoDIT1, and miR11525-z: Lopgm, laid a foundation for further verification of the molecular mechanism of Lilium ‘Siberia’ bulb dormancy release from miRNAs and gene biological function.
3.7. The Dynamic Expression Patterns of Dormancy Release-Related Genes, Flowering-Related Genes, and Riboflavin Metabolism-Related Genes in Riboflavin Treatment and Different Organs of Lilium ‘Siberia’
To further explore the molecular mechanisms by which riboflavin affects the dormancy release of Lilium ‘Siberia’ bulbs, RT-qPCR was used to detect the expression patterns of genes related to the riboflavin metabolism pathway, seed dormancy release-related genes, flowering pathway-related genes, and H3K4me3 methylation-related genes under riboflavin treatment and in different organs of Lilium ‘Siberia’. The fold change (FC) value and TPM were used to normalize and select some significantly different expressions of miRNA and mRNA. The results show that the dormancy maintenance-related gene Locallose synthase 3 (LoCALS3) was significantly downregulated, the seed dormancy control gene LoTGA1 (transcript_HQ_X_transcript 16200/f5p0/1756) was significantly upregulated (Figure 7A), and the LoFlower locus CA (LoFCA) and LoFlowering Locus KH Domain (LoFLK) were significantly upregulated. These results indicate that riboflavin treatment promoted the dormancy release of Lilium ‘Siberia’ bulbs and promoted apical floral bud differentiation. In addition, the riboflavin metabolism pathway-related gene LoPAP17 was significantly upregulated, which activated the riboflavin pathway and promoted riboflavin accumulation. The expression of LoNUDT23 and LoLysine specific demethylase 1 (LoLSD1) increased with increasing exogenous riboflavin concentration (Figure 7A). In addition, compared with those in leaves, the expression patterns of miRNA target genes in different organs of Lilium ‘Siberia’ showed that only LoAPS1, the target gene of miR395-y, was significantly downregulated in different flower tissues (stalk of stamen, petal, style, anther, and ovary). The expressions of four other target genes (miR399-y: LoDIT1, miR1863-z: LoFBA3, miR529-z: LoSPL14, and miR396-y: LoCFDP1) were significantly upregulated in different flowering tissues. Among these genes, LoDIT1 and LoFBA3 had the highest expression in petals, LoCFDP1 had the highest expression in anthers, and LoSPL14 had the lowest expression in anthers (Figure 7B). However, LoCALS3 was significantly downregulated in different flowering tissues, and LoTGA1 was significantly downregulated in the stalks of stamens, petals, and ovaries but significantly upregulated in the styles and anthers. LoFCA was significantly upregulated in petals and significantly downregulated in anthers, and LoFLK was upregulated in ovaries and downregulated in the stalks of stamens and styles (Figure 7C). LoPAP17 was significantly upregulated in different flowering tissues, while LoNUDT23 was significantly upregulated only in petals, styles, and anthers; LoLSD1 was significantly upregulated in anthers and downregulated in the style and ovary. These results indicate that riboflavin treatment activated the expression of riboflavin metabolism pathway genes, H3K4me3 methylation-related genes, dormancy control-related genes, and flowering pathway-related genes and downregulated dormancy maintenance genes in the Lilium ‘Siberia’ bulb apical bud, promoting the dormancy release of the Lilium ‘Siberia’ bulb and promoting the differentiation of the Lilium ‘Siberia’ apical floral bud.
3.8. Cloning, Bioinformatic Analysis, and Subcellular Localization of LoPAP17
As mentioned above, LoPAP17, whose expression was significantly activated in the apical buds of Lilium ‘Siberia’ bulbs after treatment with different concentrations of riboflavin, is a pivotal gene in the riboflavin metabolic pathway. To further explore the significance of riboflavin metabolism in the dormancy release process of Lilium ‘Siberia’, we cloned the whole open reading frame (ORF) sequence of LoPAP17 (transcript_HQ_X_transcript24827/f3p0/1280) by PCR. The total length of the sequence was 990 bp (Figure S5A), and the sequence encoded 329 aa. The LoPAP17 amino acid sequence was constructed as a molecular evolutionary tree. The results show that LoPAP17 is closely related to the orthologous genes of Acorus calamus, Asparagus officinalis, Zingiber officinale, and Musa acuminata on the same branch (Figure S5B). The prediction results of the protein interaction network show that the LoPAP17 protein might interact with other genes involved in riboflavin metabolism, such as FHY1, RIBF2, RIBF1, PAP8, PAP7, PAP4, and NUDT23 (Figure S5C). Multiple amino acid sequence alignments revealed that LoPAP17 has a conserved domain compared with other plants, such as Phoenix_dactylifera, Musa_acuminata, Cocos_nucifera, Asparagus officinalis, and Acorus_calamus, which share the same metallophosphatase domain and belong to the metallophosphatase superfamily (Figure S5D). Subcellular localization prediction revealed that the LoPAP17 protein was located in the nucleus. To clarify the specific localization of the LoPAP17 protein in cells, a 1302: GFP fusion expression vector was constructed after Agrobacterium transformation and injection into the inner epidermis of the onion. The results show that LoPAP17 is indeed located in the nucleus and is localized to the nucleus (Figure 8). SignalP-6.0 prediction showed that LoPAP17 protein was predicted as a signal peptide (Sec/SPI) and cleavage site between pos. 26 and 27, probability is 0.973517 (Figure S5E). TMHMM2.0 prediction showed that LoPAP17 has the possibility of being a transmembrane protein by 0.52556 (Figure S5F). These characteristics suggest that LoPAP17 may have abundant biological function, and suggest that LoPAP17 may have other molecular functions beyond promoting riboflavin biosynthesis, which we will explore further in subsequent projects. The above molecular information on the LoPAP17 gene, amino acid, and protein indicates that LoPAP17, a pivotal gene of the riboflavin metabolism pathway, might play an important role in the dormancy release process of Lilium ‘Siberia’ bulbs and provide a foundation for further functional verification.
4. Discussion
4.1. MiRNAs and Their Target Genes Responded to Riboflavin with Promotion of Dormancy Release and Flower Bud Differentiation of Lilium ‘Siberia’ Bulbs
Previously, the importance of riboflavin biosynthesis in plants received little attention. Recently, however, important progress has been made in the application of riboflavin in agricultural production and the elucidation of its epigenetic regulatory mechanisms [20,22,24]. The authors’ previous studies revealed that miRNAs targeted riboflavin metabolism, mediated m6A modification, regulated cell division and differentiation, and that 0.5 mM riboflavin promoted globular embryo (GE) and somatic embryogenesis in Longan [24]. However, no evidence indicating that riboflavin promotes bulb plant dormancy release is available. In our study, we first found that exogenous riboflavin promoted the dormancy release of Lilium ‘Siberia’ bulbs and promoted floral bud differentiation of Lilium ‘Siberia’ (Figure 1 and Figure 2, Table 1, Table 2 and Table 3). The miRNAs that promoted dormancy release and flower bud differentiation in response to riboflavin treatment were distributed mainly within 24 nt, followed by 21 nt and 22 nt (Figure 3A). This result is consistent with the findings that microRNAs respond to dormancy release during cold storage in Lilium pumilum bulbs and that miRNAs mediate the regulation of flowering induction [36]. Twenty-four-nt miRNAs were reported to participate in DNA methylation regulation [57]. In our study, the 21 DE miRNAs that responded to riboflavin were mainly 21 nt long, for example, the expression of miR395-y, miR9735-z, miR5141-z, miR11525-z, and miR4414-z decreased in response to 0.1 mM riboflavin treatment (Figure 3C), and these miRNAs mainly targeted LoAPS1, LoACAA1, LoATPA, Lopgm, and LoCOG3. This result is similar to that of a miRNA study on flower bud differentiation during pear dormancy release [58]. There were also 21 nt DE miRNAs in the pear buds. In contrast, the expression of miR390, miR391, miR396, miR399, miR529, miR1863, miR5051, miR168, miR156, and miR6470, which mainly target LoGAUT11, LoZC3H, LoCFDP1, LoDIT1, LoSPL14, LoFBA3, LoEXA1, LoAGO1B, and LoHMG3, was upregulated (Figure 4). In general, 15 miRNAs were considered putative key miRNAs involved in response to riboflavin-promoted dormancy release and flower bud differentiation in Lilium ‘Siberia’. A previous study showed that miR391 is positively upregulated during dormancy release and floral bud differentiation in sweet cherries [59]. miR168.2 upregulated and dominantly mediated AGO1 cleavage in Japanese pear flower buds and endo-dormancy release [58]. miR395 and miR396 participate in morphological changes in lateral organs and meristem formation and accumulate biomass during dormancy release in Lilium pumilum bulbs during cold storage [34]. In contrast to the findings of previous study [34], our study showed that miR395-y targeted LoAPS1 but not SPL and that miR396-y targeted LoCFDP1 but not GRFs. Therefore, we suggest that different regulators might induce different kinds of miRNAs targeting different genes to respond to Lilium ‘Siberia’ dormancy release and floral bud differentiation.
4.2. Riboflavin Regulates Gene Expression in the Primary Metabolic Pathway and Promotes Dormancy Release and Flower Bud Differentiation in Lilium ‘Siberia’
Our data demonstrated that exogenous riboflavin treatment directly upregulated the expression of LoPAP17, which is located in the nucleus and phosphorylates riboflavin to form FMN (Figure 8). Simultaneously, in the oxidative phosphorylation process, riboflavin-activated FMN and FAD participate in the assembly of complex I and complex II and upregulate the F-type ATPase of complex V (Figure 5A). The energy produced is involved in carbohydrate metabolism, glycolysis metabolism, amino acid metabolism, sulfur metabolism, fatty acid metabolism, and photosynthetic system reactions. The content of soluble sugar decreased significantly during 0.1 mM riboflavin-promoted dormancy release (Figure 2E,F). Our results suggest that riboflavin metabolism plays an important role in the primary metabolic pathway. This result is theoretically consistent with those of previous studies. In animals, riboflavin deficiency causes disturbances in circulating glucose and insulin levels [60]. A lower content of flavin in leaves induces early flowering and photoperiod gene expression in Arabidopsis [17]. More importantly, in contrast to previous studies, we identified miRNAs and target genes in the primary metabolic pathway that respond to riboflavin-promoted dormancy release of Lilium ‘Siberia’. For example, miR395-y targeted LoAPS1, and miR5141-z targeted the LoATPA gene family and regulated sulfur metabolism (Figure 9). miR1863-z and miR6470-z targeted FBA3, and miR11525-z targeted pgm and regulated carbohydrate metabolism and glycolysis metabolism. miR9735-z targeted LoACAA1 and regulated fatty acid metabolism. miR2275-y targeted Locys12, which regulates cysteine biosynthetic processes (Figure 4). In addition, in our study, we found that exogenous riboflavin not only downregulated LoCAL3, upregulated LoTGA1, and promoted bulb dormancy release but also upregulated flowering pathway-related genes (LoFLK and LoFCA) and riboflavin pathway-related genes (LoPAP17 and LoNUDT23). Simultaneously, LoLSD1 was upregulated (Figure 7). Furthermore, all of these genes were significantly upregulated in petals, except for LoCASL3 and LoAPS1, which were significantly downregulated in different floral organs. In addition, after 0.1 mM riboflavin treatment and storage for 30 d, Lilium ‘Siberia’ bulb dormancy was promoted, and the riboflavin and H2O2 content significantly increased. This result was similar to that of a previous study in which riboflavin-binding protein (RfBP) modulated the leaf content of free riboflavin, after which H2O2 accumulated and pathogen defense was enhanced [61]. In Arabidopsis, increased H2O2 levels and enhanced chloroplastic lipoxygenase activity can promote flowering [17,62]. In our study, riboflavin regulated gene expression and affected the primary metabolic pathway such as oxidative phosphorylation, carbohydrate metabolism, glycolysis metabolism, amino acid metabolism, fatty acid metabolism, and photosynthetic system reactions. Previous research showed that these metabolic pathways provide the necessary substances and energy for REDOX reactions, RNA and DNA synthesis, protein synthesis, and the differentiation and growth of apical meristem cells involved in the process of bulb dormancy release [15]. The downregulated genes were mainly annotated as SULTR1; 3,5′-adenylylsulfate reductase 3, APS1, and SDI1 during dormancy release after 0.1 mM riboflavin treatment mainly participated in sulfur metabolism (Table S5). Arabidopsis seed germination experiments indicate a reduction in intracellular sulfate levels, leading to a decrease in cysteine precursor synthesis and a significant reduction in ABA content within the cells [63]. A previous report indicated that reduced ABA synthesis promotes dormancy release [16]. Contemporarily, riboflavin promoted H2O2 content, which improved the oxidation–reduction reaction during riboflavin-promoted dormancy release of Lilium ‘Siberia’ bulbs. Thus, the mechanism underlying the response of miRNAs and their targets to riboflavin-promoted Lilium ‘Siberia’ dormancy release has been investigated, which is conducive to further revealing the molecular switches regulating the expression of key genes.
4.3. Exogenous Riboflavin Promotes Endogenous Riboflavin and Acetyl-CoA Accumulation and Might Affect Protein Acetylation During Promoting Dormancy Release of Lilium ‘Siberia‘
Our studies showed that endogenous riboflavin and acetyl-CoA accumulation increased during exogenous riboflavin-promoted dormancy release of Lilium ‘Siberia’ bulbs (Figure 5). This is the first study to reveal a regulatory relationship between riboflavin and acetyl-CoA in plants. More and more studies have reported the relationship between endogenous riboflavin synthesis and cellular energy metabolism during exogenous riboflavin treatment. In Longan, an exogenous riboflavin feeding assay promoted the riboflavin content during early somatic embryogenesis and promoted global embryo development [24]. During maize seed development, O18 overexpression increased riboflavin production and influenced respiratory complexes I and II, subsequently regulating the mitochondrial tricarboxylic acid (TCA) cycle, glycolysis, lysine accumulation, and H3K4me3 levels. These results substantiated the key role of riboflavin in coordinating cellular energy and the cell cycle to regulate maize endosperm development [22]. In rice, exogenous riboflavin scavenges H2O2, improves oxidative stress tolerance and ionic stress tolerance, and ameliorates salinity stress [20]. In mice, clinical studies have shown that riboflavin and mitochondrial ROS eliminator co-therapy further restored mitochondrial enzyme complex I, II, III, and IV function [64]. In riboflavin-deficient rat liver mitochondria, severe losses of various acyl-CoA dehydrogenases and electron transfer flavoproteins occur [65]. However, the above studies have yet to elucidate the relationship between riboflavin and acetyl-CoA. Acetyl-CoA is not only the product of β-oxidation of fatty acids and oxidative decarboxylation of pyruvate from glycolysis, which is a pivotal substance in the metabolism of energy substances in plants. Acetyl-CoA also functions as a carbon-source rheostat that initiates the cellular growth program by directly influencing the activity of histone acetyltransferase (HAT) and histone deacetylase (HDAC) and then promoting the acetylation of histones [66]. Acetyl-CoA is mainly produced in the glycolytic pathway by the cleavage of citric acid by ATP citrate lyase (ACL) in the nucleus. In rice, ACLA2 interacts with HAT1, regulates the content of acetyl-CoA and the level of acetylation of histone H4K5, and promotes cell proliferation during the early stages of rice endosperm development [67]. In our study, we found that exogenous riboflavin promoted not only the synthesis of endogenous riboflavin but also the increase in acetyl-CoA content during cold storage for 30 d, suggesting that exogenous riboflavin is highly likely to promote histone acetylation during dormancy release in Lilium ‘Siberia’ (Figure 9).
Furthermore, we investigated the gene information, protein domains, protein interaction networks, and subcellular localization of the key gene in the riboflavin pathway, LoPAP17. LoPAP17 was found in the nucleus and upregulated in the anthers, petals, and ovary. In Arabidopsis thaliana, AtPAP17 encodes a purple acid phosphatase involved in phosphate metabolism [68]. In addition, we found that riboflavin also mediated the expression of several key miRNAs and target genes and regulated oxidative phosphorylation in mitochondria (Figure 9), consistent with the findings of Zhu’s study [69]. Therefore, we propose that riboflavin affects the metabolic level of REDOX reactions in mitochondria and dynamically regulates epigenetic modifications, regulating gene expression. Our results also make us consider whether there is a specific molecular mechanism of riboflavin metabolism regulating histone acetylation modification during bulb dormancy release and flower bud differentiation in Lilium ‘Siberia’.
5. Conclusions
In our study, several miRNAs and their targets were identified during riboflavin-promoted dormancy release of lily. In addition, riboflavin also upregulated genes related to the riboflavin pathway, H3K4me3 methylation, and the flowering pathway and downregulated genes related to dormancy maintenance, during the riboflavin-promoted dormancy release and flowering of Lilium ‘Siberia’. Interestingly, we first found that exogenous riboflavin promoted endogenous riboflavin and acetyl-CoA accumulation and influenced primary metabolic processes, including oxidative phosphorylation, sulfur metabolism, fatty acid metabolism, glucose metabolism, and amino acid metabolism (Figure 9). These metabolic pathways provide the necessary substances and energy and may influence ABA synthesis. Acetyl-CoA may directly influence the activity of histone acetyltransferase (HAT) and histone deacetylase (HDAC) and then promote the acetylation of histones [65]. Whether riboflavin promotes protein acetylation during riboflavin-promoted dormancy release in Lilium ‘Siberia’ is a topic worthy of further in-depth research. These findings provide new insights into miRNAs’ response to riboflavin-promoted Lilium ‘Siberia’ bulb dormancy release and lay a foundation for controlling the development of industrial Lilium ‘Siberia’ bulb production.
X.X.: Writing—original draft; designing and coordinating the work; project administration; investigation; funding acquisition. C.Y.: Project administration; prepared the materials. Y.Z.: Project administration; prepared the materials. W.G.: Prepared the materials. Z.L.: Conceptualization; experimental design; writing—review and editing. S.F.: Supervision; resources; conceptualization; methodology. All authors have read and agreed to the published version of the manuscript.
The datasets of small RNA and RNA transcriptomes used in this study can be found in online repositories. The data view from
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.
Footnotes
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Enlarge this image.Figure 1. Riboflavin-treated 14–16 cm Lilium ‘Siberia’ bulbs, apical meristem observation, and Lilium ‘Siberia’ flowering. The arrow represents the relationships between processing steps.
Enlarge this image.Figure 2. Effects of riboflavin on the growth of the apical bud of the Lilium ‘Siberia’ bulb and changes in its endogenous physiological content. (A) The sprouting of Lilium ‘Siberia’ bulbs after 30 d of storage at 4 °C; (B) the apical bud of Lilium ‘Siberia’ bulbs stored at 4 °C for 30 d; (C) the apical bud of the bulbs that were stored for 60 d. The scale bar represents 1 cm. The red arrows and red boxes indicate the distance between the first growth point of the terminal bud and the tip of the bulb. (D) Paraffin section of the apical meristem at 20 × η. The red arrow indicates the floret primordia. (E,F) show the soluble sugar and H2O2 contents after 30 and 60 d of treatment with different concentrations of riboflavin (0, 0.1, 0.5 mM); the error bars show the means ± SDs of three replicates (n = 3). Asterisks indicate a significant difference (* p ≤ 0.05, ** p ≤ 0.01).
Enlarge this image.Figure 3. The DE miRNAs are involved in the promotion of dormancy release by riboflavin in Lilium ‘Siberia’ bulbs. (A) miRNA length distribution in the riboflavin-treated Lilium ‘Siberia’ bulbs. (B) Venn diagram of 21 DE miRNAs identified after treatment with different concentrations of riboflavin. (C) Analysis of TPM values of DE known miRNAs. (D) Analysis of novel DE miRNA TPM values in the three groups.
Enlarge this image.Figure 3. The DE miRNAs are involved in the promotion of dormancy release by riboflavin in Lilium ‘Siberia’ bulbs. (A) miRNA length distribution in the riboflavin-treated Lilium ‘Siberia’ bulbs. (B) Venn diagram of 21 DE miRNAs identified after treatment with different concentrations of riboflavin. (C) Analysis of TPM values of DE known miRNAs. (D) Analysis of novel DE miRNA TPM values in the three groups.
Enlarge this image.Figure 4. miRNA and target gene prediction after treatment of Lilium ‘Siberia’ bulbs with different concentrations of riboflavin. Pink indicates miRNA, and green indicates target genes. The patmatch software (version 1.2) and psRobot (version 1.2, https://omicslab.genetics.ac.cn/psRobot/, accessed on 26 December 2024) were used to predict miRNAs and their target genes.
Enlarge this image.Figure 5. Exogenous riboflavin treatment activated the endogenous riboflavin metabolic pathway, oxidative phosphorylation pathway, primary metabolic pathway, and acetyl-CoA content in the apical buds of Lilium ‘Siberia’ bulbs. (A) Association of riboflavin metabolism with oxidative phosphorylation and primary metabolism (purine metabolism, sulfur metabolism, amino acid metabolism, fatty acid metabolism, and glucose metabolism) during the dormancy release process of the Lilium ‘Siberia’ bulb apical bud. The red font represents upregulation or increased abundance in sRNA-seq and mRNA-seq data. (B,C) Acetyl-CoA content in the Lilium ‘Siberia’ bulb apical buds after treatment with different concentrations of riboflavin followed by storage at 4 °C for 30 d. The error bars show the means ± SDs of three replicates (n = 3). Asterisks indicate a significant difference (**** p ≤ 0.001).
Enlarge this image.Figure 5. Exogenous riboflavin treatment activated the endogenous riboflavin metabolic pathway, oxidative phosphorylation pathway, primary metabolic pathway, and acetyl-CoA content in the apical buds of Lilium ‘Siberia’ bulbs. (A) Association of riboflavin metabolism with oxidative phosphorylation and primary metabolism (purine metabolism, sulfur metabolism, amino acid metabolism, fatty acid metabolism, and glucose metabolism) during the dormancy release process of the Lilium ‘Siberia’ bulb apical bud. The red font represents upregulation or increased abundance in sRNA-seq and mRNA-seq data. (B,C) Acetyl-CoA content in the Lilium ‘Siberia’ bulb apical buds after treatment with different concentrations of riboflavin followed by storage at 4 °C for 30 d. The error bars show the means ± SDs of three replicates (n = 3). Asterisks indicate a significant difference (**** p ≤ 0.001).
Enlarge this image.Figure 6. RT-qPCR analysis of the expression of miRNAs and their target genes after riboflavin treatment: (A) represents the expression analysis of miRNAs; (B) represents the expression analysis of target genes. The error bars show the means ± SDs of three replicates (n = 3). Asterisks indicate a significant difference (* p ≤ 0.05, ** p ≤ 0.01).
Enlarge this image.Figure 7. The expression patterns of genes related to dormancy release, the flowering pathway, riboflavin metabolism, and H3K4me3 in different organs of Lilium ‘Siberia’ were analyzed under riboflavin treatment: (A) shows the expression of genes related to dormancy release, the flowering pathway, riboflavin metabolism, and H3K4me3 in Lilium ‘Siberia’ after treatment with different concentrations of riboflavin; (B) represents the expression of target genes in different organs of Lilium ‘Siberia’; (C,D) represent the expression of genes related to dormancy release, the flowering pathway, riboflavin metabolism, and H3K4me3 in different organs of the Lilium ‘Siberia’. The error bars show the means ± SDs of three replicates (n = 3). Asterisks indicate a significant difference (* p ≤ 0.05, ** p ≤ 0.01).
Enlarge this image.Figure 8. Subcellular localization of the LoPAP17 protein in the inner epidermis of the onion. The white scale bar in the figure represents 50 μm. The arrow represents fluorescent organelles.
Enlarge this image.Figure 9. Schematic diagram of the molecular mechanism by which riboflavin mediates miRNA-mediated regulation of target genes, promotes acetyl-CoA accumulation, is associated with primary metabolism, and promotes the release of dormancy in Lilium ‘Siberia’.
Morphological changes in the apical buds of the Lilium ‘Siberia’ bulbs 30 d after riboflavin treatment.
| Apical Bud Morphology | Mock | 0.1 mM | 0.5 mM |
|---|---|---|---|
| >1 cm | 9 | 28 | 15 |
| ≤1 cm | 37 | 20 | 37 |
| Total | 46 | 48 | 52 |
| Sprouting percentage (%) | 20.9 | 58.3 | 28.8 |
Morphological changes in the apical buds of the Lilium ‘Siberia’ bulbs 60 d after riboflavin treatment.
| Apical Bud Morphology | Mock | 0.1 mM | 0.5 mM |
|---|---|---|---|
| ≤1 cm | 9 | 4 | 5 |
| >1 cm | 6 | 11 | 10 |
| Total | 15 | 15 | 15 |
| Sprouting percentage (%) | 40 | 73.3 | 66.6 |
Flowering characteristics and date of Lilium ‘Siberia’ bulbs after riboflavin treatment.
| Riboflavin Treatment | Number of Plants | Number of Buds | 7 May 2023 |
|---|---|---|---|
| Mock | 15 | 36 | 11 |
| 0.1 mM | 15 | 40 | 20 |
| 0.5 mM | 15 | 34 | 8 |
Supplementary Materials
The following supporting information can be downloaded at:
References
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; Yang, Chenglong 1
; Zheng, Yiping 1 ; Guo, Wenjie 1 ; Lai, Zhongxiong 2
; Fang, Shaozhong 1 1 Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou 350001, China;
2 Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, China