Transcriptomic and targeted metabolomic analyses provide insights into the flavonoids biosynthesis in the flowers of Lonicera macranthoides.

BACKGROUND: Flavonoids are one of the bioactive ingredients of Lonicera macranthoides (L. macranthoides), however, their biosynthesis in the flower is still unclear. In this study, combined transcriptomic and targeted metabolomic analyses were performed to clarify the flavonoids biosynthesis during flowering of L. macranthoides. RESULTS: In the three sample groups, GB_vs_WB, GB_vs_WF and GB_vs_GF, there were 25, 22 and 18 differentially expressed genes (DEGs) in flavonoids biosynthetic pathway respectively. A total of 339 flavonoids were detected and quantified at four developmental stages of flower in L. macranthoides. In the three sample groups, 113, 155 and 163 differentially accumulated flavonoids (DAFs) were detected respectively. Among the DAFs, most apigenin derivatives in flavones and most kaempferol derivatives in flavonols were up-regulated. Correlation analysis between DEGs and DAFs showed that the down-regulated expressions of the CHS, DFR, C4H, F3'H, CCoAOMT_32 and the up-regulated expressions of the two HCTs resulted in down-regulated levels of dihydroquercetin, epigallocatechin and up-regulated level of kaempferol-3-O-(6''-O-acetyl)-glucoside, cosmosiin and apigenin-4'-O-glucoside. The down-regulated expressions of F3H and FLS decreased the contents of 7 metabolites, including naringenin chalcone, proanthocyanidin B2, B3, B4, C1, limocitrin-3,7-di-O-glucoside and limocitrin-3-O-sophoroside. CONCLUSION: The findings are helpful for genetic improvement of varieties in L.macranthoides.

Exogenous melatonin alleviates sodium chloride stress and increases vegetative growth in Lonicera japonica seedlings via gene regulation.

Melatonin (Mt) functions as a growth regulator and multifunctional signaling molecule in plants, thereby playing a crucial role in promoting growth and orchestrating protective responses to various abiotic stresses. However, the mechanism whereby exogenous Mt protects Lonicera japonica Thunb. (L. japonica) against salt stress has not been fully elucidated. Therefore, this study aimed to elucidate how exogenous Mt alleviates sodium chloride (NaCl) stress in L. japonica seedlings. Salt-sensitive L. japonica seedlings were treated with an aqueous solution containing 150 mM of NaCl and aqueous solutions containing various concentrations of Mt. The results revealed that treatment of NaCl-stressed L. japonica seedlings with a 60 µM aqueous solution of Mt significantly enhanced vegetative plant growth by scavenging reactive oxygen species and thus reducing oxidative stress. The latter was evidenced by decreases in electrical conductivity and malondialdehyde (MDA) concentrations. Moreover, Mt treatment led to increases in the NaCl-stressed L. japonica seedlings' total chlorophyll content, soluble sugar content, and flavonoid content, demonstrating that Mt treatment improved the seedlings' tolerance of NaCl stress. This was also indicated by the NaCl-stressed L. japonica seedlings exhibiting marked increases in the activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) and in photosynthetic functions. Furthermore, Mt treatment of NaCl-stressed L. japonica seedlings increased their expression of phenylalanine ammonia-lyase 1 (PAL1), phenylalanine ammonia-lyase 2 (PAL2), calcium-dependent protein kinase (CPK), cinnamyl alcohol dehydrogenase (CAD), flavanol synthase (FLS), and chalcone synthase (CHS). In conclusion, our results demonstrate that treatment of L. japonica seedlings with a 60 µM aqueous solution of Mt significantly ameliorated the detrimental effects of NaCl stress in the seedlings. Therefore, such treatment has substantial potential for use in safeguarding medicinal plant crops against severe salinity.

High-resolution genome mapping and functional dissection of chlorogenic acid production in Lonicera maackii.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of L. maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in CGA biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

[Transcriptional regulation mechanism of differential accumulation of flavonoids in different varieties of Lonicera macranthoides based on metabonomics and transcriptomics].

This study aims to explore the molecular regulatory mechanism of the differential accumulation of flavonoids between 'Xianglei' and the wild type of Lonicera macranthoides. The flowers, stems, and leaves of the two varieties of L. macranthoides were collected. Ultra-performance liquid chromatography-mass spectrometry(UPLC-MS) and high-throughput sequencing(RNA-seq) were employed to screen out the differential flavonoids, key differentially expressed genes(DEGs) and transcription factors(TFs). Fourteen DEGs were randomly selected for verification by qRT-PCR. The results showed that a total of 17 differential flavonoids were obtained, including naringin chalcone, apigenin, and quercetin. The transcriptomic analysis predicted 19 DEGs associated with flavonoids, including 2 genes encoding chitin synthase(CHS) and 3 genes encoding chalcone isomerase(CHI). The regulatory network analysis and weighted gene co-expression network analysis(WGCNA) screen out the key enzyme genes CHS1, FLS1, and HCT regulating the accumulation of flavonoids. MYB12 and LBD4 may be involved in the biosynthesis of flavonoids by regulating the expression of key enzyme genes CHS1, FLS1, and HCT. The qRT-PCR and RNA-seq results were similar regarding the expression patterns of the 14 randomly selected DEGs. This study preliminarily analyzed the transcriptional regulatory mechanism for the differential accumulation of flavonoids in the two varieties of L. macranthoides and laid a foundation for further elucidating the regulatory effects of key enzyme genes and TFs on the accumulation of flavonoids.

Genome-wide analysis of the MADS-box gene family in Lonicera japonica and a proposed floral organ identity model.

BACKGROUND: Lonicera japonica Thunb. is widely used in traditional Chinese medicine. Medicinal L. japonica mainly consists of dried flower buds and partially opened flowers, thus flowers are an important quality indicator. MADS-box genes encode transcription factors that regulate flower development. However, little is known about these genes in L. japonica. RESULTS: In this study, 48 MADS-box genes were identified in L. japonica, including 20 Type-I genes (8 Mα, 2 Mß, and 10 Mγ) and 28 Type-II genes (26 MIKCc and 2 MIKC*). The Type-I and Type-II genes differed significantly in gene structure, conserved domains, protein structure, chromosomal distribution, phylogenesis, and expression pattern. Type-I genes had a simpler gene structure, lacked the K domain, had low protein structure conservation, were tandemly distributed on the chromosomes, had more frequent lineage-specific duplications, and were expressed at low levels. In contrast, Type-II genes had a more complex gene structure; contained conserved M, I, K, and C domains; had highly conserved protein structure; and were expressed at high levels throughout the flowering period. Eleven floral homeotic MADS-box genes that are orthologous to the proposed Arabidopsis ABCDE model of floral organ identity determination, were identified in L. japonica. By integrating expression pattern and protein interaction data for these genes, we developed a possible model for floral organ identity determination. CONCLUSION: This study genome-widely identified and characterized the MADS-box gene family in L. japonica. Eleven floral homeotic MADS-box genes were identified and a possible model for floral organ identity determination was also developed. This study contributes to our understanding of the MADS-box gene family and its possible involvement in floral organ development in L. japonica.

Comparative genomics of the medicinal plants Lonicera macranthoides and L. japonica provides insight into genus genome evolution and hederagenin-based saponin biosynthesis.

Lonicera macranthoides (LM) and L. japonica (LJ) are medicinal plants widely used in treating viral diseases, such as COVID-19. Although the two species are morphologically similar, their secondary metabolite profiles are significantly different. Here, metabolomics analysis showed that LM contained ~86.01 mg/g hederagenin-based saponins, 2000-fold higher than LJ. To gain molecular insights into its secondary metabolite production, a chromosome-level genome of LM was constructed, comprising 9 pseudo-chromosomes with 40 097 protein-encoding genes. Genome evolution analysis showed that LM and LJ were diverged 1.30-2.27 million years ago (MYA). The two plant species experienced a common whole-genome duplication event that occurred ∼53.9-55.2 MYA before speciation. Genes involved in hederagenin-based saponin biosynthesis were arranged in clusters on the chromosomes of LM and they were more highly expressed in LM than in LJ. Among them, oleanolic acid synthase (OAS) and UDP-glycosyltransferase 73 (UGT73) families were much more highly expressed in LM than in LJ. Specifically, LmOAS1 was identified to effectively catalyse the C-28 oxidation of ß-Amyrin to form oleanolic acid, the precursor of hederagenin-based saponin. LmUGT73P1 was identified to catalyse cauloside A to produce α-hederin. We further identified the key amino acid residues of LmOAS1 and LmUGT73P1 for their enzymatic activities. Additionally, comparing with collinear genes in LJ, LmOAS1 and LmUGT73P1 had an interesting phenomenon of 'neighbourhood replication' in LM genome. Collectively, the genomic resource and candidate genes reported here set the foundation to fully reveal the genome evolution of the Lonicera genus and hederagenin-based saponin biosynthetic pathway.

Target sequence capture data shed light on the deeper evolutionary relationships of subgenus Chamaecerasus in Lonicera (Caprifoliaceae).

The genus Lonicera L. is widely distributed in the north temperate zone and is well-known for its high species richness and morphological diversity. Previous studies have suggested that many sections of Lonicera are not monophyletic and phylogenetic relationships within the genus are still poorly resolved. In this study, we sampled 37 accessions of Lonicera, covering four sections of subgenus Chamaecerasus plus six outgroup taxa, to recover the main clades of Lonicera based on sequences of nuclear loci generated by target enrichment and cpDNA from genome skimming. We found extensive cytonuclear discordance across the subgenus. Both nuclear and plastid phylogenetic analyses supported subgenus Chamaecerasus sister to subgenus Lonicera. Within subgenus Chamaecerasus, sections Isika and Nintooa were each polyphyletic. Based on the nuclear and chloroplast phylogenies, we propose to merge Lonicera korolkowii into section Coeloxylosteum and Lonicera caerulea into section Nintooa. In addition, Lonicera is estimated to have originated in the mid Oligocene (26.45 Ma). The stem age of section Nintooa was estimated to be 17.09 Ma (95% HPD: 13.30-24.45). The stem age of subgenus Lonicera was estimated to be 16.35 Ma (95% HPD: 14.12-23.66). Ancestral area reconstruction analyses indicate that subgenus Chamaecerasus originated in East Asia and Central Asia. In addition, sections Coeloxylosteum and Nintooa originated in East Asia, with subsequent dispersals into other areas. The aridification of the Asian interior likely promoted the rapid radiation of sections Coeloxylosteum and Nintooa within this region. Moreover, our biogeographic analysis fully supports the Bering and the North Atlantic Land Bridge hypotheses for the intercontinental migrations in the Northern Hemisphere. Overall, this study provides new insights into the taxonomically complex lineages of subgenus Chamaecerasus and the process of speciation.

A phylogenomic analysis of Lonicera and its bearing on the evolution of organ fusion.

PREMISE: The ~140 species of Lonicera are characterized by variously fused leaves, bracteoles, and ovaries, making it a model system for studying the evolution and development of organ fusion. However, previous phylogenetic analyses, based mainly on chloroplast DNA markers, have yielded uncertain and conflicting results. A well-supported phylogeny of Lonicera will allow us to trace the evolutionary history of organ fusion. METHODS: We inferred the phylogeny of Lonicera using restriction site-associated DNA sequencing (RADSeq), sampling all major clades and 18 of the 23 subsections. This provided the basis for inferring the evolution of five fusion-related traits. RESULTS: RADSeq data yielded a well-resolved and well-supported phylogeny. The two traditionally recognized subgenera (Periclymenum and Chamaecerasus), three of the four sections (Isoxylosteum, Coeloxylosteum, and Nintooa), and half of the subsections sampled were recovered as monophyletic. However, the large and heterogeneous section Isika was strongly supported as paraphyletic. Nintooa, a clade of ~22 mostly vine-forming species, including L. japonica, was recovered in a novel position, raising the possibility of cytonuclear discordance. We document the parallel evolution of fused leaves, bracteoles, and ovaries, with rare reversals. Most strikingly, complete cupules, in which four fused bracteoles completely enclose two unfused ovaries, arose at least three times. Surprisingly, these appear to have evolved directly from ancestors with free bracteoles instead of partial cupules. CONCLUSIONS: We provide the most comprehensive and well-supported phylogeny of Lonicera to date. Our inference of multiple evolutionary shifts in organ fusion provides a solid foundation for in depth developmental and functional analyses.

Qualitative identification of lonicerae japonicae flos in traditional chinese medicine using metabarcoding combined with specific mini-barcodes.

BACKGROUND: Lonicerae japonicae flos, also known as Jinyinhua (JYH), is an important component of traditional Chinese patent medicine (TCPM) products. However, the potential for adulteration and substitution with low-quality materials highlights the need for a reliable and sensitive approach to identify the species composition of TCPM products for consumer safety. METHODS AND RESULTS: We used universal ITS2 primers to amplify TCPMs containing JYH. However, the results were inconclusive, as only one operational taxonomic unit (OTU) was identified as Lonicera sp., which could not be identified at the species level. To confirm the species identification of Lonicera sp. in TCPM, we developed a short mini-barcode primer based on the psbA-trnH region, which, in combination with DNA metabarcoding technology, allowed for qualitative and quantitative analysis of artificially mixed samples. We applied the mini-barcode to distinguish TCPMs containing JYH and demonstrated its relatively accurate quantitative ability in identifying two Lonicera species. CONCLUSIONS: Our study presents a method for qualitative and quantitative identification of JYH, providing a promising application of DNA metabarcoding technology in the quality control of TCPM products.

DNA Metabarcoding Reveals the Fungal Community on the Surface of Lonicerae Japonicae Flos, an Edible and Medicinal Herb.

Lonicerae Japonicae Flos (LJF) has been globally applied as an herbal medicine and tea. A number of reports recently revealed fungal and mycotoxin contamination in medicinal herbs. It is essential to analyze the fungal community in LJF to provide an early warning for supervision. In this study, the fungal community in LJF samples was identified through DNA metabarcoding. A total of 18 LJF samples were collected and divided based on the collection areas and processing methods. The results indicated that Ascomycota was the dominant phylum. At the genus level, Rhizopus was the most abundant, followed by Erysiphe and Fusarium. Ten pathogenic fungi were detected among the 41 identified species. Moreover, Rhizopus, Fusarium, and Aspergillus had lower relative abundances in LJF samples under oven drying than under other processing methods. This work is expected to provide comprehensive knowledge of the fungal community in LJF and a theoretical reference for enhanced processing methods in practical manufacturing.

[Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides].

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.

[Winter pruning boosts growth and yield of Lonicera japonica by regulating plant hormone content].

Lonicera japonica is a ubiquitous medicinal species in China.Winter pruning has long been used to improve its quality and yield, but the mechanism is rarely studied.Therefore, in this study, the growth phenotypes of L.japonica processed with different pruning methods were observed and the yield-and quality-boosting mechanism of pruning was analyzed.Specifically, the young shoots of the three-year old L.japonica were cut to different degrees(heavy pruning, mild pruning, and no pruning, respectively) in winter in 2020 and 2021, respectively, and the growth phenotypes, hormone content, and gene expression of the lateral buds at the sprouting stage and young shoots at the anthesis stage in the next year were analyzed.The result showed that the length, flower bud number, internode length, and node number of young shoots in the next year were in the order of heavy pruning>mild pruning>no pruning.The content of auxin and zeatin in apical buds of young shoots at the anthesis stage was the highest in the heavy pruning group, followed by the mild pruning group, and coming in the third was the no pruning group.The content of auxin and zeatin in lateral buds at the sprouting stage was in the order of no pruning>mild pruning>heavy pruning.Transcriptome analysis of the lateral buds at sprouting stage yielded the differentially expressed genes related to auxin and cytokinin, such as Lj1A1163T36, Lj3A719T115, Lj7C657T7, Lj9C505T15, and Lj9A505T70.In conclusion, the growth phenotypes of young shoots of L.japonica processed with different pruning methods in winter were related to the difference in hormone content in the apical buds.Therefore, winter pruning influenced the content of auxin and cytokinin in new shoots of L.japonica and further regulated the expression of hormone-related genes, thereby promoting shoot growth and increasing the yield of L.japonica.

Integrated metabolic profiling and transcriptome analysis of pigment accumulation in Lonicera japonica flower petals during colour-transition.

BACKGROUND: Plants have remarkable diversity in petal colour through the biosynthesis and accumulation of various pigments. To better understand the mechanisms regulating petal pigmentation in Lonicera japonica, we used multiple approaches to investigate the changes in carotenoids, anthocyanins, endogenous hormones and gene expression dynamics during petal colour transitions, i.e., green bud petals (GB_Pe), white flower petals (WF_Pe) and yellow flower petals (YF_Pe). RESULTS: Metabolome analysis showed that YF_Pe contained a much higher content of carotenoids than GB_Pe and WF_Pe, with α-carotene, zeaxanthin, violaxanthin and γ-carotene identified as the major carotenoid compounds in YF_Pe. Comparative transcriptome analysis revealed that the key differentially expressed genes (DEGs) involved in carotenoid biosynthesis, such as phytoene synthase, phytoene desaturase and ζ-carotene desaturase, were significantly upregulated in YF_Pe. The results indicated that upregulated carotenoid concentrations and carotenoid biosynthesis-related genes predominantly promote colour transition. Meanwhile, two anthocyanins (pelargonidin and cyanidin) were significantly increased in YF_Pe, and the expression level of an anthocyanidin synthase gene was significantly upregulated, suggesting that anthocyanins may contribute to vivid yellow colour in YF_Pe. Furthermore, analyses of changes in indoleacetic acid, zeatin riboside, gibberellic acid, brassinosteroid (BR), methyl jasmonate and abscisic acid (ABA) levels indicated that colour transitions are regulated by endogenous hormones. The DEGs involved in the auxin, cytokinin, gibberellin, BR, jasmonic acid and ABA signalling pathways were enriched and associated with petal colour transitions. CONCLUSION: Our results provide global insight into the pigment accumulation and the regulatory mechanisms underlying petal colour transitions during the flower development process in L. japonica.

LjaFGD: Lonicera japonica functional genomics database.

Lonicera japonica Thunb., a traditional Chinese herb, has been used for treating human diseases for thousands of years. Recently, the genome of L. japonica has been decoded, providing valuable information for research into gene function. However, no comprehensive database for gene functional analysis and mining is available for L. japonica. We therefore constructed LjaFGD (www.gzybioinformatics.cn/LjaFGD and bioinformatics.cau.edu.cn/LjaFGD), a database for analyzing and comparing gene function in L. japonica. We constructed a gene co-expression network based on 77 RNA-seq samples, and then annotated genes of L. japonica by alignment against protein sequences from public databases. We also introduced several tools for gene functional analysis, including Blast, motif analysis, gene set enrichment analysis, heatmap analysis, and JBrowse. Our co-expression network revealed that MYB and WRKY transcription factor family genes were co-expressed with genes encoding key enzymes in the biosynthesis of chlorogenic acid and luteolin in L. japonica. We used flavonol synthase 1 (LjFLS1) as an example to show the reliability and applicability of our database. LjaFGD and its various associated tools will provide researchers with an accessible platform for retrieving functional information on L. japonica genes to further biological discovery.

[Different development phase of transcription proteomics and metabolomics of flower of Lonicera macranthoides].

In order to study the regulation mechanism of secondary metabolites biosynthesis in Lonicera macranthoides, the key genes involved in the regulation of biosynthesis and the mechanism of differential metabolites were explored. In this study, high-throughput sequencing technology was used for transcriptome sequencing of L. macranthoides at different development stages. By using Liquid chromatography-tandem mass spectrometry(LC-MS/MS) technology, the laws of qualitative, quantitative and synthetic accumulation of its metabolites were studied, and the key enzyme genes for the biosynthesis of phenolic acid and flavonoids were screened out according to the differentially expressed genes. A total of 111 differentially accumulate metabolites(DAM) and 6 653 differentially expressed genes(DGE) were obtained by metabonomics and transcriptomics analysis. The metabolites and key enzyme genes in the Erqing(KE) were significantly different from those in the Dabai(KD) and Yinhua(KY) stages. In the phenylalanine biosynthesis pathway, the ion abundance of chlorogenic acid, naringin, quercetin, rutin, coniferol and other metabolites decreased with the development of flowers, while the ion abundance of ferulic acid, coumarin and syringoside increased with the development of flowers. Key enzyme genes such as CHS, HCT, CCR, FLS and COMT positively regulate the downstream metabolites, while PAL, C4H and 4CL negatively regulate the downstream metabolites. This study provides candidate genes and theoretical basis for the further exploration of key enzymes in the biosynthesis of secondary metabolites and for the regulation of the accumulation of secondary metabolites in L. macranthoides by molecular biological methods.

Comparative transcriptomics analysis revealing flower trichome development during flower development in two Lonicera japonica Thunb. cultivars using RNA-seq.

BACKGROUND: Lonicera japonica Thunb. (L. japonica) has the functions of clearing away heat and detoxifying, broad-spectrum antibacterial and anti-virus, etc. More than 70% of anti-inflammatory and cold Chinese patent medicines contain L. japonica. Trichomes comprise specialized multicellular structures that have the capacity to synthesize and secrete secondary metabolites and protect plants from biotic and abiotic stresses. The extraction of trichome secretions has great commercial value. However, little is known about the trichome formation mechanism in L. japonica. Therefore, the study of trichome development between different varieties provides a basis for selecting suitable planting resources. RESULTS: Here, we present a genome-wide comparative transcriptome analysis between two L. japonica cultivars, toward the identification of biological processes and functional gene activities that occur during flowering stage trichome development. In this study, the density and average lengths of flower trichomes were at their highest during three-green periods (S2). Using the Illumina RNA-Seq method, we obtained 134,304 unigenes, 33,733 of which were differentially expressed. In an analysis of 40 differentially expressed unigenes (DEGs) involved in trichome development, 29 of these were transcription factors. The DEGs analysis of plant hormone signal transduction indicated that plant growth and development may be independent of gibberellin (GA) and cytokinine (CTK) signaling pathways, and plant stress may be independent of jasmonic acid (JA) and ethylene (ET) signaling pathways. We screened several genes involved in the floral biosynthesis of odors, tastes, colors, and plant hormones, and proposed biosynthetic pathways for sesquiterpenoid, triterpenoid, monoterpenoid, flavonoid, and plant hormones. Furthermore, 82 DEGs were assigned to cell cycles and 2616 were predicted as plant resistance genes (PRGs). CONCLUSIONS: This study provides a comprehensive characterization of the expression profiles of flower development during the seven developmental stages of L. japonica, thereby offering valuable insights into the molecular networks that underly flower development in L. japonica.

The honeysuckle genome provides insight into the molecular mechanism of carotenoid metabolism underlying dynamic flower coloration.

Lonicera japonica is a widespread member of the Caprifoliaceae (honeysuckle) family utilized in traditional medical practices. This twining vine honeysuckle also is a much-sought ornamental, in part due to its dynamic flower coloration, which changes from white to gold during development. The molecular mechanism underlying dynamic flower coloration in L. japonica was elucidated by integrating whole genome sequencing, transcriptomic analysis and biochemical assays. Here, we report a chromosome-level genome assembly of L. japonica, comprising nine pseudochromosomes with a total size of 843.2 Mb. We also provide evidence for a whole-genome duplication event in the lineage leading to L. japonica, which occurred after its divergence from Dipsacales and Asterales. Moreover, gene expression analysis not only revealed correlated expression of the relevant biosynthetic genes with carotenoid accumulation, but also suggested a role for carotenoid degradation in L. japonica's dynamic flower coloration. The variation of flower color is consistent with not only the observed carotenoid accumulation pattern, but also with the release of volatile apocarotenoids that presumably serve as pollinator attractants. Beyond novel insights into the evolution and dynamics of flower coloration, the high-quality L. japonica genome sequence also provides a foundation for molecular breeding to improve desired characteristics.

Regulation of chlorogenic acid, flavonoid, and iridoid biosynthesis by histone H3K4 and H3K9 methylation in Lonicera japonica.

Lonicera japonica is used in Chinese herbal medicines with a wide spectrum of pharmacological properties associated with chlorogenic acid, flavonoid and iridoid. The biosynthesis of these compounds could be affected by genetic inheritance and epigenetic modification. However, the mechanisms that regulate the expression of genes involved in the biosynthesis of these compounds are rarely known. The results of qRT-PCR showed that the biosynthesis gene expression of these compounds was related to histone H3K4 and H3K9 methylation levels. These active compounds content of L. japonica were measured by UPLC-MS/MS. H3K4me3 showed a positive correlation with chlorogenic acid and loganic acid content, and H3K9me positively correlated with luteolin content. The correlation between histone methylation levels and the levels of luteolin and loganic acid in L. japonica from different producing areas validate the regulatory role of histone methylation in biosynthesis of bioactive compounds. Our study demonstrated a potential regulatory network of H3K9/H3K4 methylation to gene expression and content of secondary metabolites, and provided a basis for understanding the mechanism underlying the variation of major bioactive compounds in L. japonica.

Transcriptional regulation of Lonicera japonica Thunb. during flower development as revealed by comprehensive analysis of transcription factors.

BACKGROUND: Lonicera japonica Thunb. flower has been used for the treatment of various diseases for a long time and attracted many studies on its potential effects. Transcription factors (TFs) regulate extensive biological processes during plant development. As the restricted reports of L. japonica on TFs, our work was carried out to better understand the TFs' regulatory roles under different developmental stages in L. japonica. RESULTS: In this study, 1316 TFs belonging to 52 families were identified from the transcriptomic data, and corresponding expression profiles during the L. japonica flower development were comprehensively analyzed. 917 (69.68%) TFs were differentially expressed. TFs in bHLH, ERF, MYB, bZIP, and NAC families exhibited obviously altered expression during flower growth. Based on the analysis of differentially expressed TFs (DETFs), TFs in MYB, WRKY, NAC and LSD families that involved in phenylpropanoids biosynthesis, senescence processes and antioxidant activity were detected. The expression of MYB114 exhibited a positive correlation with the contents of luteoloside; Positive correlation was observed among the expression of MYC12, chalcone synthase (CHS) and flavonol synthase (FLS), while negative correlation was observed between the expression of MYB44 and the synthases; The expression of LSD1 was highly correlated with the expression of SOD and the total antioxidant capacity, while the expression of LOL1 and LOL2 exhibited a negative correlation with them; Many TFs in NAC and WRKY families may be potentially involved in the senescence process regulated by hormones and reactive oxygen species (ROS). The expression of NAC19, NAC29, and NAC53 exhibited a positive correlation with the contents of ABA and H2O2, while the expression of WRKY53, WRKY54, and WRKY70 exhibited a negative correlation with the contents of JA, SA and ABA. CONCLUSIONS: Our study provided a comprehensive characterization of the expression profiles of TFs during the developmental stages of L. japonica. In addition, we detected the key TFs that may play significant roles in controlling active components biosynthesis, antioxidant activity and flower senescence in L. japonica, thereby providing valuable insights into the molecular networks underlying L. japonica flower development.

Ectopic Expression of a R2R3-MYB Transcription Factor Gene LjaMYB12 from Lonicera japonica Increases Flavonoid Accumulation in Arabidopsis thaliana.

Lonicera japonica Thunb. is a widely used medicinal plant and is rich in a variety of active ingredients. Flavonoids are one of the important components in L. japonica and their content is an important indicator for evaluating the quality of this herb. To study the regulation of flavonoid biosynthesis in L. japonica, an R2R3-MYB transcription factor gene LjaMYB12 was isolated and characterized. Bioinformatics analysis indicated that LjaMYB12 belonged to the subgroup 7, with a typical R2R3 DNA-binding domain and conserved subgroup 7 motifs. The transcriptional level of LjaMYB12 was proportional to the total flavonoid content during the development of L. japonica flowers. Subcellular localization analysis revealed that LjaMYB12 localized to the nucleus. Transactivation activity assay indicated that LjaMYB12 was a transcriptional activator. Then, ectopic expression of LjaMYB12 in Arabidopsis could increase PAL activity and flavonoid content and promote transcription of a range of flavonoid biosynthetic genes. Interestingly, the fold changes of downstream genes in the flavonoid biosynthetic pathway were significantly higher than that of the upstream genes, which suggested that LjaMYB12 may have different regulatory patterns for the upstream and downstream pathways of flavonoid biosynthesis. The results provided here will effectively facilitate the study of subgroup 7 MYBs and transcriptional regulation of flavonoid biosynthesis in L. japonica.