杨燕君、徐茂军、沈晨佳 (生命与环境科学学院) Industrial Crops and Products——Integrated mRNA and miRNA omics reveal t....pdf

Industrial Crops & Products 197 (2023) 116657 Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Integrated mRNA and miRNA omics reveal the regulatory role of UV-B radiation in active ingredient biosynthesis of Chrysanthemum morifolium Ramat Yanjun Yang a, b, 1, Jie Liu a, b, 1, Taiyao Yi a, b, Yao Li a, b, Mengyuan Li a, b, Haidi Liu c, Lijun Zheng a, b, Zhehao Chen c, Juan Hao a, b, Maojun Xu a, b, *, Chenjia Shen a, b, * a Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, Hangzhou, China Key Laboratory of Hangzhou City for Quality and Safety of Agricultural Products, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China c College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China b A R T I C L E I N F O A B S T R A C T Keywords: Bioactive ingredient Chlorogenic acid Differential expressed genes Flavonoid MicroRNA Ultraviolet-B radiation Ultraviolet-B (UV-B) is an effective elicitor that enhances bioactive ingredient biosynthesis in different plants. Chrysanthemum morifolium Ramat (C. morifolium) is a well-known herbal medicine and beverage material, which is cultivated in many Asian countries and regions. The C. morifolium inflorescences are the main accumulation sites for active ingredients, which include flavonoids and chlorogenic acid. However, the molecular mechanisms underlying UV-B-mediated bioactive ingredient biosynthesis in C. morifolium inflorescences remain largely un­ known. High-throughput sequencing technologies have been applied to identify the messenger RNAs (mRNAs) and microRNAs (miRNAs) responsive to UV-B radiation in C. morifolium inflorescences. Many UV-B responsive genes are enriched in flavonoid, unsaturated fatty acid, phenylpropanoid, and carotenoid biosynthesis pathways. In this study, we identified 169 miRNAs belonging to 38 typical families were detected in our constructed li­ braries. In total, 1954 unigenes were predicted to be targets of 118 miRNAs. Six miRNAs, PC-5p-7136_1319, PC3p-78379_156, ath-MIR414-p5_2ss2CT21AC, PC-3p-154888_69, nta-miR171a_2ss8GT18GA and htu-miR403a were involved in the regulation of active ingredient biosynthesis by targeting 4CL-like 5/9, CYP81E8, UGT91D1 and UGT72B1 genes. Five miRNAs, PC-5p-79_56613, ath-miR858a_L-1, ath-MIR414-p5_2ss15AC18AT, ath-MIR414p5_2ss2CT21AC, and aly-miR393a-5p were involved in regulation of flavonoid biosynthesis by targeting MYBbHLH complex-related transcription factor genes. Our data provides a platform for identifying mRNAs and miRNAs involved in the UV-B-induced accumulation of bioactive ingredients in C. morifolium inflorescences. 1. Introduction Chrysanthemum belongs to the Asteraceae family and is widely domesticated and cultivated in Southeast Asia (Su et al., 2019; Teixeira da Silva, 2003). As a major commercial cut flower, many Chrysanthemum species are used for gardening and landscaping (Anderson, 2006). In addition to their ornamental value, Chrysanthemum morifolium in­ florescences are harvested for beverage and medicine utilizations (Yuan et al., 2020). C. morifolium inflorescences contain different phyto­ chemicals, such as flavonoids, anthocyanins, and chlorogenic acid, which exert many medical effects, including antioxidant effects and preventing anti-inflammatory visceral injury (Lu et al., 2016). Conse­ quently, commercial demand for C. morifolium inflorescences has increased year on year (Kazeroonian et al., 2018). Plant secondary metabolism generates abundant active ingredients for human health, and is not only regulated by genetic factors but also environmental factors (Li et al., 2020; Zhan et al., 2022). Different environmental factors, including UV-B, latitude, extreme temperature and nitric oxide, function as effective elicitors to enhance active ingre­ dient biosynthesis (Manivannan et al., 2016; Takshak and Agrawal, * Corresponding authors at: Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, Hangzhou, China E-mail addresses: xumaojunhz@163.com (M. Xu), shencj@hznu.edu.cn (C. Shen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.indcrop.2023.116657 Received 10 November 2022; Received in revised form 1 March 2023; Accepted 29 March 2023 Available online 1 April 2023 0926-6690/© 2023 Elsevier B.V. All rights reserved. Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 2016; Zheng et al., 2022). High UV-B radiation doses are negative fac­ tors involved in plant growth and productivity (Yao et al., 2013). However, phytochemical studies have revealed that low UV-B radiation doses exert beneficial effects during plant secondary metabolism (Chen et al., 2020). Recently, UV-B radiation was applied as a simple and environmentally-friendly method to enhance medicinal plant active ingredient biosynthesis (Hao et al., 2022; Jaiswal et al., 2022; Pandey et al., 2021). Low UV-B doses enhanced saponin accumulation, which is a major phytochemical compound in Chlorophytum borivillianum (Jaiswal et al., 2022). In the medicinal plant Adhatoda vasica, UV-B helped to facilitate oridonin oxide and α-bisabolol oxide-B biosyn­ thesis (Pandey et al., 2021). Also, UV-B regulated active compound accumulation in Dendrobium officinale was confirmed by high perfor­ mance liquid chromatography analysis (Chen et al., 2020). However, the regulatory function of UV-B radiation in active ingredient biosynthesis from C. morifolium inflorescences remain unclear. Many transcriptomic analyses have identified secondary metabolism-related C. morifolium genes. CmTPS family genes were identified, with CmTPS5 and CmTPS8 encoding multi-product enzymes in monoterpenoid and sesquiterpenoid biosynthesis (Zhang et al., 2022). Whole-transcriptome analyses of mutant and normal C. morifolium capitula identified many transcription factors (TFs) related to anthocy­ anin biosynthesis (Liu et al., 2021a). Transcriptomic profiling revealed that down-regulation of F-box genes significantly increased diverse flavonoid levels (Jo et al., 2020). Comparative transcriptomic analyses revealed several TF genes, including CmMYB305, CmMYB29, CmRAD3, CmbZIP61, CmAGL24, and CmNAC1, that play important roles in carotenoid accumulation (Lu et al., 2019). Transcriptomic analysis of healthy and Spodoptera litura-infested C. morifolium plants identified 11 TPS candidates that were involved in herbivore-induced volatile terpene emission (Xu et al., 2021). A transcriptomic analysis of five cDNA li­ braries identified three CmMYB genes and one CmbHLH gene which were implicated in light-induced anthocyanin biosynthesis (Hong et al., 2015). MicroRNAs are the most abundant class of endogenous noncoding small RNA molecules and play roles in the regulation of many plant biological processes (Dong et al., 2022). In C. morifolium, some TF family-related miRNAs have been identified and functionally analyzed. For example, a transcriptome-wide survey identified HD-ZIP, DOF, SBF-like, and Trihelix family-related miRNAs (Song et al., 2016a,b,c,d). MiRNA expression analyses identified several aphid-infestation-responsive C. morifolium miRNAs (Xia et al., 2015). Embryo-abortion-associated Chrysanthemum miRNAs and their targets were identified during a cross breeding process (Zhang et al., 2015). However, the secondary metabolism-related miRNAs remain largely unknown. To reveal UV-B mediated regulatory complexity in C. morifolium secondary metabolism, integrated transcriptomic and miRNAomic analysis was performed. Using C. morifolium leaves, we previously identified many genes and miRNAs which were responsive to UV-B exposure (Yang et al., 2020; Yang et al., 2018). Although easy to har­ vest, leaves are not the main edible portion of C. morifolium. In the present study, inflorescences were harvested to reveal expression changes in active ingredient biosynthesis-related genes and miRNAs. Our data will give an opportunity to reveal the function of UV-B radi­ ation in bioactive ingredient biosynthesis in Chrysanthemum plants. (U3), 6 h (U6), and 12 h (U12), respectively. Inflorescences were collected and frozen rapidly in liquid N2 until used. 2. Materials and methods The raw small RNA sequences were filtered to produce clean reads. All clean reads were searched against the GenBank (http://www.ncbi. nlm.nih.gov/genbank/) and Rfam 12.1 (https://xfam.wordpress.com) to annotate sRNAs and remove known non-coding RNAs (Yang et al., 2020, 2018). Protein-coding sequences were removed based on the C. morifolium reference transcriptome. The remaining sequences were used to predict miRNA secondary structure, and aligned to miRBase 21.0 to obtain the known miRNAs (Haunsberger et al., 2017). Novel miRNAs were predicted using miRDeep2 software (ver.2.0.5). 2.2. cDNA library construction Total RNAs were extracted and purified using TRIzol reagent (Invi­ trogen, Shanghai, China) according to manufacturer’s instructions. RNA quality was analyzed using an Agilent NanoDrop spectrophotometer (Santa Clara, CA, USA). A total of 10 mg RNAs were subjected to mag­ netic beads with oligo-dT (Thermo Fisher Scientific, Shanghai, China). RNA was cut into small fragments using divalent cations at elevated temperatures. RNA fragments were used for first strand cDNA synthesis using a cDNA preparation kit (Illumina, San Diego, CA, USA). Frag­ mentary RNAs were reverse-transcribed to create a cDNA library using an Illumina cDNA library construction kit (San Diego, USA). After pu­ rification, DNA fragments with sequencing adapters were generated using the Illumina Hiseq2500 platform according to its protocols (LCBio, Hangzhou, China) (Feng et al., 2023). Three repeats for each sample of mRNA sequencing were performed. 2.3. De novo assembly and gene annotation Raw read quality was evaluated using in-house Perl scripts (LC-Bio, Hangzhou, China). Low quality reads were omitted and clean reads were assembled into contigs as previously described (Yang et al., 2018). Contig properties were calculated using Trinity software, and the longest sequences were selected as unigenes. The resulting sequences were searched against various databases for functional annotation. Using the Arabidopsis proteins as the query sequences, the homologous sequences of the C. morifolium inflorescences were searched by local BLASP pro­ gram, with an E-value threshold of 10− 10. 2.4. Analysis of differentially expressed genes (DEGs) The transcriptomes from all samples were used as a reference sequence for DEGs identification. Clean reads were mapped onto the reference sequence using Bowtie ver. 0.12.7 software. Genes with more than a two-fold change were selected as DEGs. Venn analysis was per­ formed to identify the DEGs in different samples. GO and KEGG terms with a P value lower than 0.05 were assigned as DEGs. 2.5. Small RNA libraries construction and sequencing Small RNA libraries preparation were carried out by LC-Bio (Hang­ zhou, China). About 1 μg of total RNAs from each sample were isolated to construct sRNA library. Briefly, the total RNAs were purified to pro­ duce sRNA fragments with lengths ranging from 18 to 30 nt. Then, the RNA fragments were ligated to the RNA sequencing adapter. cDNA li­ braries were generated by a reverse transcription reaction and PCR amplified. The PCR products were purified and processed by a HiSeq 2500 system (Illumina, San Diego, USA) according to the LC-Bio method. Three repeats for each sample of miRNA sequencing were performed. 2.6. Analysis of small RNA 2.1. Plant materials and treatments The C. morifolium cultivar ‘Xiaoyangju’ was grown in a plantation at the ‘Cangqian’ campus, Hangzhou Normal University. Mature flowers from C. morifolium seedings were harvested and separated into four classes (three replicates in each class). The seedings in flowering stage were treated with UV-B radiation (10 μmol m− 2 s− 1) for 0 h (CK), 3 h 2 Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 2.7. Analysis of differentially expressed miRNAs (DEMs) 2.10. Validation of DEMs The normalized expression value of different miRNA was calculated according to the Reads Per Kilobase Million method. DEGs and miRNAs (DEMs) between two sample groups were screened using the DESeq R package. The thresholds for a significant difference between sample groups were set as P < 0.01 and |log2(fold change) |> 1. The expression patterns of DEMs across four groups were performed using the K-mean method. Stem-loop quantitative reverse transcription PCR was used to confirm the expression levels of several miRNAs according to our pre­ vious method (Yang et al., 2020). For qPCR analysis of miRNAs, U6 gene was used as an internal standard. 2.11. Dual-luciferase report assay To create effector constructs, the mature sequences of PC-5p7136_1319 and aly-miR393a-5p were cloned into the pCAMBIA1301 vector. To create reporter constructs, the PC-5p-7136_1319 binding re­ gion of 4CL-like 9 and aly-miR393a-5p binding region of bHLH62 (DN38440) were cloned into the pGreenII 0800-Luc vector. The dualluciferase report experiment was used to confirm the relationship be­ tween miRNA and its target according to our previous method (Yang et al., 2020). 2.8. Enrichment analysis of target genes MiRNAs targets were predicted by psRobot and Target Finder using C. morifolium transcriptomic assembled sequence was used as a refer­ ence sequence (Sablok et al., 2019). The functional category analysis of miRNA target genes were performed using GO term and the KEGG pathway. The statistical significance of GO and KEGG enrichment analysis was measured by Fisher’s exact test under the condition of a P value at 0.05. 3. Results 3.1. Overview of C. morifolium transcriptomes 2.9. Quantitative real-time PCR (qRT-PCR) analysis To identify C. morifolium responsive genes during UV-B exposure, expression profiles were analyzed. A total of 118.06 Gb of raw data was obtained, of which 107.55 Gb was valid data. After filtering, approxi­ mately 98.14% of clean reads reached the threshold score of Q20, and approximately 93.9% reached Q30 (Table S1). Pearson correlation be­ tween different sample groups (three repeats for each group) were shown in Fig. S1. Reads from the C. morifolium inflorescences were assembled using Trinity software (Fig. 1a-c). In detail, the minimum A total of 3 μg RNAs were extracted and reverse transcribed to cDNA using ReverAid First Strand cDNA Synthesis Kit (Thermo Scientific, Shanghai, China). qRT-PCR was used to confirm the expression levels of several mRNAs according to our previous method (Yang et al., 2018). Three independent cDNA samples of each treatment group were used for the qRT-PCR validation. The C. morifolium TUBB gene was used as an internal standard. Fig. 1. Basic information for C. morifolium RNA sequencing. (a-b) Basic parameters of transcripts and unigenes of C. morifolium. (c) Length distribution of assembled transcripts and unigenes in C. morifolium. (d) Number of unigenes annotated by different databases, including Nr, eggNOG, Swissprot, Pfam, KEGG and GO databases. (e) Species distribution of all annotated unigenes. The numbers indicated the percentages of C. morifolium proteins similar to each public plant species. 3 Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 length of transcripts and unigenes is 201 bp. The median lengths of transcripts and unigenes are 469 bp and 508 bp, respectively. A large number of genes were annotated in various protein databases (Fig. 1d). The largest number of unigenes were annotated in NR database (42,451 unigenes) and smallest number of unigenes were annotated in Swissprot database (27,406 unigenes). Annotation information suggested that most C. morifolium genes showed high similarity to Artemisia annua, Helianthus annuus, and Cynara cardunculus genes (Fig. 1e). At 3 h, DEGs were significantly enriched in “temperature compen­ sation of the circadian clock”, “cysteine-type peptidase activity”, and “circadian rhythm” gene ontology (GO) terms; at 6 h, the DEGs are significantly enriched in “quercetin 7-O-glucosyltransferase activity”, and “flavonoid glucuronidation” GO terms; and at 12 h, the DEGs are significantly enriched in “flavonoid biosynthetic process”, and “circa­ dian rhythm” GO terms (Table S2-4). 3.3. Metabolic responses to UV-B radiation 3.2. Analysis of differentially expressed genes (DEGs) under UV-B treatment The effects of UV-B treatments on C. morifolium inflorescence me­ tabolisms were investigated. Significant P values for each primary and secondary metabolism-related Kyoto Encyclopedia of Genes and Ge­ nomes (KEGG) term was calculated. Metabolism-related KEGG terms were grouped into 11 classes and indicated by different color bars. Most significantly-changed KEGG terms belonged to flavonoid, lipid, phe­ nylpropanoid, and pigment metabolism processes (Fig. 3a). For flavo­ noid metabolism, genes belonging to the “flavone and flavonol biosynthesis” and “flavonoid biosynthesis” terms were significantly changed after UV-B treatment. For lipid metabolism, genes belonging to the “biosynthesis of unsaturated fatty acids” term were significantly changed during the UV-B treatment process. For phenylpropanoid metabolism, the genes belonging to the “phenylpropanoid biosynthesis” Many DEGs, including 1632 DEGs in the UV 3 h vs CK comparison, 2968 DEGs in the UV 6 h vs CK comparison, and 6438 DEGs in the UV 12 h vs CK comparison, were identified in different UV-B radiation treatment timepoints (Fig. 2a). Global gene expression profiles at different UV-B radiation timepoints are shown (Fig. 2b). Then, DEGs were grouped into different clusters. In detail, Cluster I included 1548 genes from the 6 h UV-B treatment group; Cluster III and VI included 3827 genes from 12 h UV-B treatment group; Cluster VII and VIII included 6791 genes significantly decreased during the UV-B treatment process; and Cluster III included 2547 genes significantly increased during UV-B treatment (Fig. 2c). Fig. 2. Analysis of DEGs under UV-B radiation treatment in C. morifolium. (a) Number of up- and down- regulated unigenes in different companions. (b) Heat map for cluster analysis of DEGs by K-means method. The expression levels of each gene were normalized among four sample groups, including CK, 3 h, 6 h and 12 h. The red color indicated high expression level, green color indicated low expression level, and while color indicated median expression level. (c) MeV cluster analysis of differentially expressed genes from the gene expression profiles. The heatmap scale ranges from − 1 to + 1 on a log2 scale of normalized expression levels. 4 Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 Fig. 3. Effects of UV-B treatment on primary and secondary metabolisms of C. morifolium. (a) KEGG enrichment analysis of DEGs related to primary and secondary metabolisms. The significant P value of each KEGG term in different comparisons are shown by a heatmap. All the KEGG terms were grouped into 11 metabolismrelated categories, which were indicated by different color bars. Red boxes indicated the most significant enriched KEGG terms. The number of up- and downregulated genes related to ‘Flavone and flavonol biosynthesis’ (b), ‘Flavonoid biosynthesis’ (c), ‘Biosynthesis of unsaturated fatty acids’ (d), ‘Phenylpropanoid biosynthesis’ (e), ‘Carotenoid biosynthesis’ (f), and ‘Tyrosine metabolism’ (g) terms. term were significantly changed during the UV-B treatment process. For pigment metabolism, genes belonging to the “carotenoid biosynthesis” term were significantly changed at 12 h after UV-B exposure. Notably, most DEGs related to flavonoid, unsaturated fatty acid, phenylpropanoid, and tyrosine metabolisms were induced by UV-B exposure. Only half of DEGs-related to carotenoid biosynthesis were induced by UV-B treatment at 3 h and 6 h, and most of the carotenoid biosynthesis-related genes were down-regulated at 12 h (Fig. 3b-g). 3.5. Overview of miRNAomes Four C. morifolium small RNA libraries exposed to UV-B radiation at different time points were constructed and sequenced (Table S5). Pearson correlation between different sample groups (three repeats for each group) were shown in Fig. S2. The length distribution of most reads was 18–25 bp in length (Fig. 5a). Small RNAs were classified into different groups, including rRNA, tRNA, snoRNA, snRNA, and other Rfam RNA. The rRNA group accounted for the largest proportion in samples (Fig. 5b). Venn diagram pointed out that five miRNAs were specifically detected in the CK group, 11 miRNAs were mainly detected in the UV 6 h group, 10 in the UV 12 h group, and four in the UV 3 h (Fig. 5c). Interestingly, 169 miRNAs were detected in all groups. Based on their sequence features, miRNAs were classified into five groups: 1, 2a, 2b, 3, and 4. Group 4 miRNAs could not be mapped to selected pre-miRNAs in miRbase, and were thus considered novel miR­ NAs (Fig. 5d). C. morifolium miRNAs were classified into 38 typical families. The largest family was MIR156 (14 miRNAs), followed by MIR171_1 (11 miRNAs) and MIR414 families (nine miRNAs) (Fig. S3). Most detected miRNAs displayed high similarities to known miRNAs in other plants, such as 189 miRNAs were similar to miRNAs from Glycine max (Table S6). 3.4. The effects of UV-B treatment on flavonoid and chlorogenic acid metabolism Several flavonoid and chlorogenic acid biosynthesis-related DEGs were identified after UV-B radiation treatments. For precursor supply, eight phenylalanine ammonia-lyase (PAL) genes, two cinnamate 4monooxygenase (C4M) genes, and eight 4-coumarate–CoA ligase (4CL) genes were identified as DEGs under UV-B radiation treatments. For flavonoid biosynthesis pathway, three chalcone synthase (CHS) genes, three chalcone–flavonone isomerase (CHI) genes, four flavanone 3-hy­ droxylase (F3H) genes, five flavonoid 3′ -hydroxylase (F3′ H) genes, one flavonol synthase (FLS) genes, five UDP-glucosyltransferase (UGT75) genes, two anthocyanidin synthase (ANS) genes, and one dihydro­ flavonol 4-reductase gene were identified as DEGs under UV-B radiation treatments. For chlorogenic acid biosynthesis pathway, 13 hydrox­ ycinnamoyl CoA quinate transferase (HCT) genes, two hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) genes, two p-coumarate 3-hydroxylase (C3H) genes, and three cinna­ mate beta-D-glucosyltransferase (UGCT) genes were identified as DEGs after UV-B radiation treatment (Fig. 4b). 3.6. Analysis of differentially expressed miRNAs (DEMs) after UV-B treatment DEMs were identified after UV-B treatment (Fig. 6a). In total, 260 DEMs, including 22 DEMs in the UV 3 h/CK comparison, 15 DEMs in the UV 6 h/CK comparison, 21 DEMs in the UV 12 h/CK comparison, 25 DEMs in the UV 3 h/UV 6 h comparison, 22 DEMs in the UV 3 h/UV 12 h comparison, and 10 DEMs in the UV 6 h/UV 12 h comparison, were 5 Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 Fig. 4. Effects of UV-B treatment on flavonoid and chlorogenic acid metabolism. (a) Overview of flavonoid and chlorogenic acid metabolic pathway. Enzyme ab­ breviations: CHS: chalcone synthase; CHI: Chalcone–flavonone isomerase; F3H: flavanone 3-hydroxylase; F3′ H: flavonoid 3′ -monooxygenase; DFR: dihydroflavonol 4-reductase; FLS: flavonol synthase; UGT: UDP-glucosyltransferase; PAL: phenylalanine ammonia-lyase; ANS: anthocyanidin synthase; C4M: trans-cinnamate 4monooxygenase; 4CL: 4-coumarate–CoA ligase; HCT: hydroxycinnamoyl CoA quinate transferase; HQT: hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl­ transferase; and C3H: Coumaric acid-3-hydroxylase. (b) Expression changes of genes associated with flavonoid and chlorogenic acid metabolism under UV-B treatments in C. morifolium. The expression levels of each gene were normalized among four sample groups, including CK, 3 h, 6 h and 12 h. The red color indi­ cated high expression level, green color indicated low expression level, and while color indicated median expression level. The heatmap scale ranges from − 1 to + 1 on a log2 scale normalized expression levels. identified (Fig. 6b). UV-B responsive miRNAs were grouped into nine clusters, I to IX. Cluster IV, V, and VII contained most UV-B up-regulated miRNAs while Clusters III, VIII, and IX contained most UV-B downregulated miRNAs. Interestingly, UV-B rapid responsive miRNAs belonged to Cluster I, while UV-B later responsive miRNAs belonged to Cluster VII (Fig. 6c). UGT91D1, and UGT72B1. Specifically, 4CL-like 9 was a target of PC-5p7136_1319, 4CL-like 5 was a target of PC-3p-78379_156, CYP81E8 was a target of ath-MIR414-p5_2ss2CT21AC, and UGT91D1 was a target of PC3p-154888_69. Two target sites were identified in UGT72B1 for ntamiR171a_2ss8GT18GA and htu-miR403a (Fig. 7a). PC-5p-7136_1319 transcript levels were significantly up-regulated by UV-B radiation at U3 and U6 time points. The transcript of PC-3p-78379_156 was largely induced by UV-B radiation at all time points. The expression of athMIR414-p5_2ss2CT21AC was significantly reduced at the later stage of UV-B radiation. PC-3p-154888_69 and htu-miR403a transcript levels were induced at the early stage of UV-B radiation. 3.7. Enrichment analysis of DEM targets MiRNAs are implicated in many biological functions by regulating a series of downstream target genes. A total of 1954 unigenes were pre­ dicted to be targets of 118 miRNAs, including 36 novel miRNAs and 82 known miRNAs (Table S7). GO enrichment analyses grouped most target genes into three main categories. In the ‘biological process’ group, significantly enriched GO terms comprised “regulation of transcription” (P = 6.33E-14) and “tran­ scription” (P = 3.32E-10); in the ‘cellular component’ group, significantly enriched GO terms comprised the “nucleus” (P = 7.11E-13) and “SCF ubiquitin ligase complex” (P = 1.19E-07); and in the “molecular function” group, significantly enriched GO terms comprised “DNA-binding tran­ scription factor activity” (P = 2.22E-16) and ‘DNA binding’ (P = 1.62E12) (Table S8). The most significantly enriched KEGG terms were “plant hormone signal transduction” (P = 2.32E-08), and “ribosome biogenesis in eukaryotes” (P = 4.11E-04) terms (Fig. S4 and Table S9). 3.9. Flavonoid and chlorogenic acid metabolism-related TF-miRNA pairs The MYB-bHLH complex was considered to play roles in the regu­ lation of flavonoid and chlorogenic acid metabolism pathways (Xu et al., 2015). Among C. morifolium MYB genes, CmMYB4 (DN32646) and CmMYB12 (DN30067) were identified as targets of ath-miR858a_L_1, and CmMYB44 (DN28492) was identified as a target of PC-5p-79_56613 (Fig. 8a). The expression of PC-5p-79_56613 was significantly up-regulated by UV-B radiation treatment at U6 time point (Fig. 8b). Among C. morifolium bHLH genes, bHLH45 (DN46501) was the target of ath-MIR414-p5_2ss15AC18AT and ath-MIR414-p5_2ss2CT21AC. BHLH62 (DN38440) was predicted to be the target of aly-miR393a-5p (Fig. 8c). The expression of ath-MIR414-p5_2ss15AC18AT was down-regulated at U3 but returned to normal levels at U6 and U12. The expression of ath-MIR414-p5_2ss2CT21AC was down-regulated at the later stage of UV-B radiation treatment (U6 and U12). In contrast, the transcript of aly-mi393a-5p was induced at the later stage of UV-B treatment (U6 and U12) (Fig. 8d). 3.8. Flavonoid and chlorogenic acid metabolism-related gene-miRNA pairs DEM targets contained five flavonoid and chlorogenic acid metabolism-related genes, including 4CL-like 9, 4CL-like 5, CYP81E8, 6 Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 Fig. 5. Basic information for C. morifolium microRNAome. (a) Length distribution of all unique reads from four different groups. (b) Type classification of all sRNAs. The percentage of different RNA classes, including snRNA, snoRNA, tRNA, rRNA and other Rfam RNA, are shown in a histogram. (c) Venn diagrams of miRNAs expression overlap between UV-B treatment groups, including control, UV-B 3 h, UV-B 6 h, and UV-B 12 h. (d) Identification of known and novel miRNAs. All miRNAs were grouped into five groups, including gp1, gp2a, gp2b, gp3, and gp4, based on their sequence features. Gp1: Reads map to specific miRNAs/pre-miRNAs in miRbase and the pre-miRNAs further map to the reference sequence; gp2a: Reads map to selected miRNAs/pre-miRNAs in miRbase, and the mapped pre-miRNAs do not map to the reference sequence, but the reads map to the reference sequence; gp2b: Reads were mapped to miRNAs/pre-miRNAs of selected species in miRbase and the mapped pre-miRNAs were not further mapped to the reference sequence, but the reads were mapped to the reference sequence; gp3: Reads map to selected miRNAs/pre-miRNAs in miRbase, and the mapped pre-miRNAs do not map to the reference sequence, and the reads do not map to the reference sequence; and gp4: Reads do not map to selected pre-miRNAs in miRbase. 3.10. qRT-PCR confirmed mRNA and miRNA expression related to bioactive ingredient biosynthesis 4. Discussion In China, medicinal Chrysanthemum has a long history and is an important export medicine. Moreover, tea made using C. morifolium inflorescences exerts multiple pharmacological and clinical effects, including anti-inflammatory, detoxification, eyesight improvement, and liver-heat removal qualities (Ma et al., 2022). Due to their economic value, active ingredient biosynthesis and regulation processes have been comprehensively investigated in-depth in different Chrysanthemum species (Chen et al., 2019a; Zhou et al., 2021b). We focused on C. morifolium inflorescences to identify regulatory mechanisms of active ingredient biosynthesis. To date, several C. morifolium inflorescence transcriptomes have been published to reflect various development processes. For example, transcriptomic analyses provided new insights on petal senescence, ray floret morphogenesis, floral transition, and hooked petal morphogenesis (Ding et al., 2019; Pu et al., 2020; Ren et al., 2016; Yao et al., 2021). Transcriptomics analysis is used to screen environmental stimulus responsive genes. RNA-sequencing experiments identified 3858 hydro­ ponic responsive genes, 5279 Botrytis cinerea attack responsive genes, and 2135 light-induced genes in C. morifolium (Ai et al., 2021; Kumar et al., 2020). In our study, thousands of UV-B responsive genes were identified in C. morifolium inflorescences, suggesting our analyses had sufficient sequencing depth to screen for valuable genes. UV-B elicitor artificially induced flavonoid accumulation in plants The expression levels of eight key genes related to bioactive ingre­ dient biosynthesis were analyzed using qRT-PCR. The expression levels of MYB4, MYB44, CYP81E8 and UGT91D1 were significantly increased, and bHLH62, 4CL-like 9, and UGT72B1 were significantly decreased under UV-B radiation treatments (Fig. S5). The expression levels of six key miRNAs related to bioactive ingredient biosynthesis were analyzed using stem-loop PCR. The expression levels of PC-5p-7136_1319, PC-3p78379_156, PC-5p-79_56613, ath-miR858a_L-1, and aly-miR393a-5p were significantly increased under UV-B radiation treatments. The expression level of ath-MIR414-p5_2ss2CT21AC was significantly decreased under UV-B radiation treatments (Fig. S6). Expression levels of selected six miRNAs were basically consistent with the RNA sequencing data. 3.11. The target relationships between miRNA and mRNA Dual-luciferase reporter system was used to confirm the predicted interaction between two randomly selected miRNAs and their targets. Our data suggested that PC-3p-154888_69 and PC-5p-79_56613 sup­ pressed luciferase activity in WT-transfected cells, but not in mutanttransfected cells. UGT91D1 is a target of PC-3p-154888_69 and MYB44 is a target of PC-5p-79_56613 (Fig. S7). 7 Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 Fig. 6. Identification of UV-B responsive miRNAs in C. morifolium. (a) Heat map showing miRNAs expression pattern under different UV-B treatment conditions. (b) Number of up- and down-regulated miRNAs in different comparisons, including the UV 3 h vs CK, UV 6 h vs CK, UV12h vs CK, UV 3 h vs UV 6 h, UV 3 h vs UV 12 h, and UV 6 h vs UV 12 h. (c) MeV cluster analysis of differentially expressed miRNAs by the K-means method. All the identified miRNAs were grouped into nine Clusters. The heatmap scale ranges from − 1 to + 1 on a log2 scale. where redox active flavonoids have evolved to prevent damages that is caused by UV-B radiation (Shi and Liu, 2021). In Qi and Huai Chrysan­ themum inflorescences, UV-B radiation reportedly exerted significant effects active ingredient biosynthesis regulation (Yao et al., 2014). Chemical analysis pointed out that vitamin C, chlorogenic acid and flavones significantly accumulated in Qi Chrysanthemum inflorescences (Yao et al., 2015a). Enhanced UV-B treatment promoted various sec­ ondary metabolism pathways and facilitated active compound accu­ mulation in post-harvest Chrysanthemum inflorescences (Si et al., 2015; Yao et al., 2015b). Our transcriptomic data showed that most DEGs were enriched in flavonoid, unsaturated fatty acid, phenylpropanoid, and carotenoid biosynthesis pathways (Fig. 3a). Expression analysis further confirmed that most DEGs were induced under UV-B treatments, sug­ gesting a genetic basis for UV-B induced active ingredient biosynthesis. Gene function analysis can help to reveal UV-B role in active component accumulation in Chrysanthemum. UV-B exposure signifi­ cantly increased PLA and C4H enzyme activities in medicinal Chrysan­ themum inflorescences (Ma et al., 2016). C. morifolium flavonoid and chlorogenic acid biosynthesis pathways were described in previous works (Yang et al., 2020, 2018). In a flavonoid and chlorogenic acid biosynthesis pathway, a number of UV-B responsive genes were identi­ fied, providing potential explanation for UV-B induced flavonoid and chlorogenic acid accumulation in C. morifolium inflorescences. In Chrysanthemum plants, conserved miRNAs have various biological functions, including polyploidy breeding, embryo abortion, nitrogen starvation-responses, and salt and drought tolerance (Liu et al., 2021b; Song et al., 2015; Zhang et al., 2015; Zhang et al., 2017). Considerable evidence now suggests that plant miRNAs play roles in the responses of plant seedlings to various environmental signals by regulating the transcript levels of downstream target genes. In C. indicum, miR396a altered plant salt and drought tolerance by regulating two of its targets, GRF1 and GFR5 (Liu et al., 2021b). In C. morifolium, miR160 and its targets, auxin response factors, were reportedly involved in normal embryo development (Zhang et al., 2017). In C. nankingense, miR397 and its targets, LAC and CL11869 ABC1-like genes, were reported to be involved in nitrogen starvation responses (Song et al., 2015). In C. dichrum, miR398 appeared to enhance freezing tolerance by regu­ lating an Inducer of CBF Expression family gene (Chen et al., 2013). In our study, 4CL-like 5 and 9 were predicted as downstream targets of PC-5p-7136_1319 and PC-3p-78379_156, respectively (Fig. 7a). 4CL-like proteins, which catalyze CoA ester formation, are key enzymes with important roles in flavonoid and lignin biosynthesis (Chen et al., 2019b). Interestingly, UGT72B1, a bifunctional O-glucosyltransferase encoding gene, was spliced by two miRNAs, nta-miR171a_2ss8GT18GA and miR403a (Brazier-Hicks et al., 2007). Our results suggested that several flavonoid and chlorogenic acid biosynthesis-related genes were regu­ lated by UV-B induced miRNAs. Several TFs, such as flavonoid metabolism-related MYB-bHLH-WDR 8 Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 Fig. 7. Identification of flavonoid and chlorogenic acid metabolism-related gene-miRNAs. (a) Target genes were identified for PC-5p-7136_1319, PC-3p-78379_156, ath-MIR414-p5_2ss2CT21AC, PC-3p-154888_69, nta-miR171a_2ss8GT18GA, and csi-miR403b-5p_2ss1AC21CG. (b) Expression changes of miRNAs associated with flavonoid and chlorogenic acid metabolism genes under UV-B treatment in Chrysanthemum. “* ” indicates a significant change (P < 0.05) in expression level between control and UV-B treatment. White circles indicated miss-matching pairs. Short vertical shoulders indicated matching pairs. complex components, have been functionally characterized in many plants (Xu et al., 2015). Previous studies identified a series of MYB family genes, such as CmMYB012, CmMYB21, and CmMYB7, in Chrysanthemum (Wang et al., 2022; Xiang et al., 2019; Zhou et al., 2021a). In C. morifolium inflorescences, miR858a_L-1 and its target gene, CmMYB4 (DN32646), and PC-5p-79_56613 and its target gene, CmMYB44 (DN28492), were identified. In model plants, orthologous CmMYB4/44 was reportedly involved in defense response (Yan et al., 2021). MiRNA-MYB path­ way-mediated flavonoid accumulation may have important roles in C. morifolium inflorescence responses to UV-B treatment. Moreover, ath-MIR414-p5_2ss15AC18AT/ath-MIR414-p5_2ss2CT21AC and their target gene, CmbHLH145 (DN46501), and aly-miR393a-5p and its target gene, CmbHLH62 (DN38440), were identified. A recent study investi­ gated how the activator complex CmMYB6-CmbHLH2 regulated petal anthocyanin homeostasis under different lights (Zhou et al., 2022). MiRNA-MYB pairs participate in a variety of biological processes, including growth, development, metabolism and other aspects. Our data suggested that miRNA-MYB pairs might play an essential role in regula­ tion of flavonoid and chlorogenic acid biosynthesis. In our study, UV-B responsive mRNAs and miRNAs in C. morifolium inflorescences were explored. Under UV-B radiation treatment, DEGs were enriched in flavonoid, unsaturated fatty acid, phenylpropanoid, and carotenoid biosynthesis pathways. Moreover, 169 miRNAs belonging to 38 typical families were detected. A total of 1954 unigenes were identified as target genes of 118 miRNAs. Six miRNAs were involved in active ingredient biosynthesis by regulating 4CL-like 5/9, CYP81E8, UGT91D1 and UGT72B1. Five miRNAs were involved in the transcriptional regulation of flavonoid biosynthesis by targeting MYB4/ 44 and bHLH45/62. Our results provide a comprehensive resource for screening mRNAs and miRNAs implicated in the UV-B-mediated biosynthesis of bioactive ingredients in C. morifolium inflorescences. Funding This study was funded by the National Natural Science Foundation of China (Grant No. 82274038, 32271905, and 81803655), the Zhejiang Provincial Natural Science Foundation of China (Grant No. LY18H280012, LY19C150005, LY19C020003, and LY23C160001), Major Increase Or Decrease Program In The Central Finance Level (Grant No. 2060302), Zhejiang Provincial Key Research & Development Project Grants (Grant No. 2017C02011, 2018C02030). CRediT authorship contribution statement Yanjun Yang: Conceptualization, Data curation, Resources, Funding acquisition, Validation, Writing-original draft. Jie Liu: Data curation, Investigation, Software. Taiyao Yi: Data curation, Investigation, Soft­ ware, Visualization. Yao Li: Formal analysis. Mengyuan Li: Software, Funding acquisition. Haidi Liu: Data curation, Resources, Investigation; Methodology. Lijun Zheng: Investigation; Methodology, Writingoriginal draft. Zhehao Chen: Data curation, Resources, Supervision, Visualization. Juan Hao: Data curation, Resources, Supervision, Visu­ alization. Maojun Xu: Conceptualization, Funding acquisition, Project administration, Writing - review & editing. Chenjia Shen: 9 Y. Yang et al. Industrial Crops & Products 197 (2023) 116657 Fig. 8. Identification of flavonoid and chlorogenic acid metabolism-related TFs-miRNAs. (a) MYB family members and their potential target miRNAs. (b) Expression changes of miRNAs associated with MYBs under UV-B treatment in Chrysanthemum. “* ” indicates significant a change (P < 0.05) in expression level between control and UV-B treatment. (c) bHLH family members and their potential target miRNAs. (d) Expression changes of miRNAs associated with bHLHs under UV-B treatment in Chrysanthemum. “*” indicates a significant change (P < 0.05) in expression level between control and UV-B treatment. White circles indicated miss-matching pairs. Short vertical shoulders indicated matching pairs. Conceptualization, Writing - review & editing. 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