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.

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.

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.

Combinatorial transcriptomics and metabolomics analysis reveals the effects of the harvesting stages on the accumulation of phenylpropanoid metabolites in Lonicera japonica.

The flower buds of Lonicera japonica are widely used for its high medicinal value. It is reported that the accumulation of phenylpropanoids in the buds of L. japonica is affected by the stage at which it is harvested. However, the changes of active components and the underlying mechanisms in flower buds at different harvesting stages have not been reported. Integrative analyses of transcriptomics and metabolomics was used to explore the underlying mechanism of harvesting stages (green bud, GB; and white bud, WB) on the phenylpropanoids metabolites accumulation in L. japonica . The result showed that 3735 differentially expressed genes were identified, and the genes related to glycolysis/gluconeogenesis and phenylalanine biosynthesis pathway were significantly upregulated in GB stage. A total of 510 differential metabolites were identified in GB stage. Among them, 14 phenylpropanoids were changed during the GB and WB, seven of which increased in GB, including caffeic acid, sauchinone, coniferin, secoisolariciresinol diglucoside, scopolin, methyl cinnamate, chlorogenic acid, 7-hydroxycoumarin, while others such as sibiricose A6, coumarin, eleutheroside E decreased. Further correlation analysis showed that the unigenes for CSE, CAD, bg1, ADH, ALDH, DLAT and ENO significantly correlated with the 10 phenylpropanoid. The above results would provide basic data for the selection of harvesting stages in the production of L. japonica .

Integrated metabolic profiling and transcriptome analysis of Lonicera japonica flowers for chlorogenic acid, luteolin and endogenous hormone syntheses.

The active ingredients of many medicinal plants are the secondary metabolites associated with the growth period. Lonicera japonica Thunb. is an important traditional Chinese medicine, and the flower development stage is an important factor that influences the quality of medicinal ingredients. In this study, transcriptomics and metabolomics were performed to reveal the regulatory mechanism of secondary metabolites during flowering of L. japonica. The results showed that the content of chlorogenic acid (CGA) and luteolin gradually decreased from green bud stage (Sa) to white flower stage (Sc), especially from white flower bud stage (Sb) to Sc. Most of the genes encoding the crucial rate-limiting enzymes, including PAL, C4H, HCT, C3'H, F3'H and FNSII, were down-regulated in three comparisons. Correlation analysis identified some members of the MYB, AP2/ERF, bHLH and NAC transcription factor families that are closely related to CGA and luteolin biosynthesis. Furthermore, differentially expressed genes (DEGs) involved in hormone biosynthesis, signalling pathways and flowering process were analysed in three flower developmental stage.

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.

Transcriptomic and metabolomic analyses reveal molecular mechanisms associated with the natural abscission of blue honeysuckle (Lonicera caerulea L.) ripe fruits.

Blue honeysuckle (Lonicera caerulea L.) is rich in phenolic compounds and has an extremely high nutritional value. Fruit abscission in the ripe period significantly impacts production and economic benefits. However, the mechanism associated with the abscission of blue honeysuckle fruit remains largely unknown. The easy-abscission cultivar 'HSY' and the hard-abscission cultivar 'Berel' were selected as plant materials. Anatomical changes of the 'HSY' fruit abscission zone (FAZ) during the abscission mainly included cell expansion, detachment, and collapse. Active changes in cell wall-degrading enzyme activity between 39 days postanthesis (DPA) and 55 DPA in 'HSY' FAZ, but not in 'Berel', suggest a critical role for cell-wall-degrading enzymes in regulating abscission. Transcriptome and metabolome analyses revealed that the genes and metabolites responding to abscission mainly act on pathways such as plant hormone signal transduction, starch and sucrose metabolism, pentose and glucuronate interconversions, and phenylpropanoid biosynthesis. The regulatory pathways of fruit abscission are mainly summarized into two parts: phytohormone synthesis and signal transduction, FAZ cell wall metabolism. In this study, 46 key genes related to plant hormone response, 45 key genes involved in FAZ cell wall metabolism, and 73 transcription factors were screened. Quantitative real-time PCR (qRT-PCR) assessed the expression pattern of 12 selected candidate genes, demonstrating the accuracy of the transcriptome data and elucidating the expression patterns of key candidate genes during growth and development. This study will provide an essential resource for understanding the molecular regulatory mechanism of fruit abscission in the blue honeysuckle.

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.

A Comparative Analysis of the Chloroplast Genomes of Three Lonicera Medicinal Plants.

Both Lonicerae japonicae flos and Lonicerae similis flos are important components in traditional Chinese medicine (TCM) with precious medicinal value. However, the absence of studies on their chloroplast genomes and chromatography has considerably hindered the study of their evolutionary and phylogenetic relationships. In this study, the complete chloroplast (cp) genomes of Lonicera acuminata Wall. and Lonicera similis Hemsl. were sequenced using the Illumina sequencing platform and compared with that of Lonicera japonica Thunb., which has been previously reported. Furthermore, the chromatographic fingerprints of the three plants were constructed using HPLC and the content of quality marker (Q-Marker) was calculated. The annotation results showed that the two chloroplast genomes were typical quadripartite structures with lengths of 155,330 bp (L. acuminata) and 155,207 bp (L. similis). A total of 126 different genes were annotated, containing 82 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The expansion and contraction of the inverted repeat (IR) regions suggested that the boundary regions of IR/SC were comparatively conserved in the three species, and six regions (trnH-GUG-psbA, rps2-rpoC2, rbcL-psaI, trnN-GUU-ndhF, rps15-ycf1, and infA) with nucleotide diversity values (Pi) of variable sites higher than 1% were identified. Phylogenetic relation indicated that L. similis had a closer genetic relationship with L. japonica than L. acuminata. Additionally, the chromatographic fingerprints showed that the characteristic peaks of the three medicinal plants were similar, including Neochlorogenic acid, Chlorogenic acid, 4-Dicaffeoylquinic acid, Sweroside, Secoxyloganin, Luteoloside, Isochlorogenic acid A, Isochlorogenic acid B, and Isochlorogenic acid C. The content of chlorogenic acid and total phenolic acid in L. acuminata (7.4633 ± 0.4461%, 14.8953 ± 0.0728%) and L. similis (14.1055 ± 0.2566%, 21.9782 ± 0.1331%) was much higher than that of L. japonica (3.9729 ± 0.0928%, 6.0964 ± 0.1228%), respectively. This study provides appropriate information for species identification, phylogeny, quality assessment, and rational use of three medicinal plants of the genus Lonicera.

Light plays a critical role in the accumulation of chlorogenic acid in Lonicera macranthoides Hand.-Mazz.

Light has important effects on plant metabolism. However, the relationship between the chlorogenic acid (CGA) content and light in plants remains unclear. Here, we investigated the effects of shading treatment on gene expression and CGA content in Lonicera macranthoides Hand.-Mazz. (LM), a widely used medicinal plant. A total of 1891 differentially expressed genes (DEGs) were obtained in flower buds and 819 in leaves in response to light in shading treatment compared to the control sample by RNA-Seq. After shading treatment, the content of CGA in LM leaves decreased significantly by 1.78-fold, the carotenoid content increased, and the soluble sugar and starch contents significantly decreased. WGCNA and the expression of related genes verified by qRT‒PCR revealed that CGA synthesis pathway enzyme genes form a co-expression network with genes for carbohydrate synthesis, photosynthesis, light signalling elements, and transcription factor genes (TFs) that affect the accumulation of CGA. Through a virus-induced gene silencing (VIGS) system and CGA assay in Nicotiana benthamiana (NB), we determined that downregulation of NbHY5 expression decreased the CGA content in NB leaves. In this study, we found that light provides energy and material for the accumulation of CGA in LM, and light affects the expression of CGA accumulation-related genes. Our results show that different light intensities have multiple effects on leaves and flower buds in LM and are able to coregulate LmHY5 expression and CGA synthesis.

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.

Comparative transcriptome analysis to reveal key ethylene genes involved in a Lonicera macranthoides mutant.

BACKGROUND: Lonicera macranthoides Hand.-Mazz. is an important medicinal plant. Xianglei-type (XL) L. macranthoides was formed after many years of cultivation by researchers on the basis of the natural mutant. The corolla of L. macranthoides XL remains unexpanded and its flowering period is nearly three times longer than that of wild-type (WT) plants. However, the molecular mechanism behind this desirable trait remains a mystery. OBJECTIVE: To understand the floral phenotype differences between L. macranthoides and L. macranthoides XL at the molecular level. METHODS: Transcriptome analysis was performed on L. macranthoides XL and WT. One DEG was cloned by RT-PCR amplification and selected for qRT-PCR analysis. RESULTS: Transcriptome analysis showed that there were 5603 differentially expressed genes (DEGs) in XL vs. WT. Enrichment analysis of DEGs showed that pathways related to plant hormone signal transduction were significantly enriched. We identified 23 key genes in ethylene biosynthesis and signal transduction pathways. The most abundant were the ethylene biosynthesis DEGs. In addition, the open reading frames (ORFs) of WT and XL ETR2 were successfully cloned and named LM-ETR2 (GenBank: MW334978) and LM-XL-ETR2 (GenBank: MW334978), respectively. qRT-PCR at different flowering stages suggesting that ETR2 acts in the whole stage of flower development of WT and XL. CONCLUSIONS: This study provides new insight into the molecular mechanism that regulates the development of special traits in the flowers of L. macranthoides XL. The plant hormone ethylene plays an important role in flower development and flowering duration prolongation in L. macranthoides. The ethylene synthesis gene could be more responsible for the flower phenotype of XL. The genes identified here can be used for breeding and improvement of other flowering plants after functional verification.

[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.

[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.

Chromosome-scale genome assembly of an important medicinal plant honeysuckle.

Lonicera japonica (honeysuckle) is one of the most important medicinal plants and widely utilized in traditional Chinese medicine. At present, there are many varieties of honeysuckle used in cultivation, among which Sijihua variety are widely cultivated due to its wide adaptability, stress resistance, early flowering and high yield. In this study, we assembled the genome of Sijihua, which was approximately 886.04 Mb in size with a scaffold N50 of 79.5 Mb. 93.28% of the total assembled sequences were anchored to 9 pseudo-chromosomes by using PacBio long reads and Hi-C sequencing data. We predicted 39,320 protein-coding genes and 92.87% of them could be annotated in NR, GO, KOG, KEGG and other databases. In addition, we identified 644 tRNAs, 2,156 rRNAs, 109 miRNAs and 5,502 pseudogenes from the genome. The chromosome-scale genome of Sijihua will be a significant resource for understanding the genetic basis of high stress-resistance, which will facilitate further study of the genetic diversity and accelerate the genetic improvement and breeding of L. japonica.

Genome-wide identification, molecular characterization, and gene expression analyses of honeysuckle NHX antiporters suggest their involvement in salt stress adaptation.

Background: Ion homeostasis is an essential process for the survival of plants under salt stress. Na+/H+ antiporters (NHXs) are secondary ion transporters that regulate Na+ compartmentalization or efflux reduce Na+ toxicity and play a critical role during plant development and stress responses. Methods and Results: To gain insight into the functional divergence of NHX genes in honeysuckle, a total of seven LjNHX genes were identified on the whole genome level and were renamed according to their chromosomal positions. All LjNHXs possessed the Na+/H+ exchanger domain and the amiloride-binding site was presented in all NHX proteins except LjNHX4. The phylogenetic analysis divided the seven NHX genes into Vac-clade (LjNHX1/2/3/4/5/7) and PM-clade (LjNHX6) based on their subcellular localization and validated by the distribution of conserved protein motifs and exon/intron organization analysis. The protein-protein interaction network showed that LjNHX4/5/6/7 shared the same putatively interactive proteins, including SOS2, SOS3, HKT1, and AVP1. Cis-acting elements and gene ontology (GO) analysis suggested that most LjNHXs involve in the response to salt stress through ion transmembrane transport. The expression profile analysis revealed that the expression levels of LjNHX3/7 were remarkably affected by salinity. These results suggested that LjNHXs play significant roles in honeysuckle development and response to salt stresses. Conclusions: The theoretical foundation was established in the present study for the further functional characterization of the NHX gene family in honeysuckle.

Comparative transcriptome analysis reveals variations of bioactive constituents in Lonicera japonica flowers under salt stress.

Lonicera japonica flowers (LJF) is a traditional Chinese medicine packed with phenols constituents and widely used in the treatments of various diseases throughout the world. However, there is still very little known on how LJF identifies and resists salt stress. Here in, we systematically investigated the effect of salt on the phenotypic, metabolite, and transcriptomic in LJF. During long term stress (35 days), 1055 differential expression genes (DEGs) involved in the biosynthesis of secondary metabolites were screened through transcriptome analysis, among which the candidate genes and pathways involved in phenols biosynthesis were highlighted; and performed by phylogenetic tree analysis and multiple nucleotide sequence alignment. Ninety compounds were identified and their relative levels were compared between the control and stressed groups based on the LC-MS analysis, Putative biosynthesis networks of phenolic acid and flavonoid were con-structed with structural DEGs. Strikingly, the expression patterns of structural DEGs were mostly consistent with the variations of phenols under salt stress. Notably, the upregulation of UDP-glycosyl transferases under salt stress indicated post-modification of glycosyl transferases may participate in downstream flavonoids synthesis. This study reveals the relationships of the gene regulation and the phenols biosynthesis in LJF under salt stress, paving the way for the use of gene-specific expression to improve the yield of biocomponent.