Identification of anthocyanin biosynthesis related microRNAs and total microRNAs in Lonicera edulis by high-throughput sequencing.

miRNAs are important regulators of plant gene expression. There are few studies on the regulation of miRNAs in Lonicera edulis. We used high-throughput sequencing technology to analyse miRNAs in L. edulis, aiming to identify miRNAs and elucidate their function in L. edulis. In the present study, we employed the high-throughput sequencing technology to profile miRNAs in L. edulis. A total of 51,819,072 small RNA tags with sizes ranging from 18 to 30 nt were obtained, indicating that L. edulis have a large and diverse small RNA population. Bioinformatic analysis identified 507 mature miRNAs, and 16 predicted novel miRNAs that are likely to be unique to L. edulis. Three miRNAs related to anthocyanin biosynthesis were locked by gene ontology (GO) analysis and target gene analysis. The selected three miRNAs are relatively high in the expression of L. edulis. Some of the previous studies have studied these types of miRNAs involved in the anthocyanin metabolism pathway in fruits. Among them, expression profiles of three conserved miRNAs were validated by stem loop qRT-PCR. Further, the potential target genes of conserved and novel miRNAs were predicted and subjected to GO annotation. Enrichment analysis of the GO-represented biological processes and molecular functions revealed that these target genes were potentiallyinvolved in a wide range of metabolic pathways and developmental processes. In particular, different families of miRNAs can directly or indirectly regulate anthocyanin biosynthesis. In recent years, the research on miRNAs has become more and more clear, but the research on miRNAs involved in the regulation of anthocyanin synthesis of L. edulis is still lagging. This study provides a useful resource for further elucidation of the functional roles of miRNAs during fruit development and ripening.

Flavone synthases from Lonicera japonica and L. macranthoides reveal differential flavone accumulation.

Flavones are important secondary metabolites found in many plants. In Lonicera species, flavones contribute both physiological and pharmaceutical properties. However, flavone synthase (FNS), the key enzyme responsible for flavone biosynthesis, has not yet been characterized in Lonicera species. In this study, FNSII genes were identified from Lonicera japonica Thunb. and L. macranthoides Hand.-Mazz. In the presence of NADPH, the recombinant cytochrome P450 proteins encoded by LjFNSII-1.1, LjFNSII-2.1, and LmFNSII-1.1 converted eriodictyol, naringenin, and liquiritigenin to the corresponding flavones directly. The different catalytic properties between LjFNSII-2.1 and LjFNSII-1.1 were caused by a single amino acid substitution at position 242 (glutamic acid to lysine). A methionine at position 206 and a leucine at position 381 contributed considerably to the high catalytic activity of LjFNSII-1.1. In addition, LjFNSII-1.1&2.1 and LmFNSII-1.1 also biosynthesize flavones that were further modified by O-glycosylation in transgenic tobacco. The expression levels of the FNSII genes were consistent with flavone accumulation patterns in flower buds. Our findings suggested that the weak catalytic activity of LmFNSII-1.1 and the relatively low expression of LmFNSII-1.1 in flowers might be responsible for the low levels of flavone accumulation in flower buds of L. macranthoides.

[Bioinformatics analysis and expressed level of histone methyltransferase genes in Lonicera japonica].

Twenty-three histone methyltransferase genes were obtained from transcriptome dataset of Lonicera japonica. The nucleotide and proteins characteristics, subcellular localization, senior structural domains and conservative forecasting were analyzed. The result of phylogenetic tree showed that 23 histone methyltransferases were mainly divided into two groups: lysine methyltransferase and arginine methyltransferases. The result of gene expression showed that 23 histone methyltransferases showed preference in terms of interspecies and organs. They were more expressed in buds of L. japonica than in L. japonica var. chinensis and lower in leaves of L. japonica than in L. japonica var. chinensis. Eight genes were specific expressed in flower. These results provided basis for further understanding the function of histone methyltransferase and epigenetic regulation of active ingredients of L. japonica.

Transcriptome Analysis Reveals the Mechanism Underlying the Production of a High Quantity of Chlorogenic Acid in Young Leaves of Lonicera macranthoides Hand.-Mazz.

Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in southern China. The biosynthetic and metabolic pathways for a core secondary metabolite in L. macranthoides, chlorogenic acid (CGA), have been elucidated in many species. However, the mechanisms of CGA biosynthesis and the related gene regulatory network in L. macranthoides are still not well understood. In this study, CGA content was quantified by high performance liquid chromatography (HPLC), and CGA levels differed significantly among three tissues; specifically, the CGA content in young leaves (YL) was greater than that in young stems (YS), which was greater than that in mature flowers (MF). Transcriptome analysis of L. macranthoides yielded a total of 53,533,014 clean reads (average length 90 bp) and 76,453 unigenes (average length 703 bp). A total of 3,767 unigenes were involved in biosynthesis pathways of secondary metabolites. Of these unigenes, 80 were possibly related to CGA biosynthesis. Furthermore, differentially expressed genes (DEGs) were screened in different tissues including YL, MF and YS. In these tissues, 24 DEGs were found to be associated with CGA biosynthesis, including six phenylalanine ammonia lyase (PAL) genes, six 4-coumarate coenzyme A ligase (4CL) genes, four cinnamate 4-Hydroxylase (C4H) genes, seven hydroxycinnamoyl transferase/hydroxycinnamoyl-CoA quinate transferase HCT/HQT genes and one coumarate 3-hydroxylase (C3H) gene.These results further the understanding of CGA biosynthesis and the related regulatory network in L. macranthoides.

[Bioinformatics analysis of DNA demethylase genes in Lonicera japonica Thunb].

The DNA demethylase genes are widespread in plants. Four DNA demethylase genes (LJDME1, LJDME2, LJDME3 and LJDME4) were obtained from transcriptome dataset of Lonicera japonica Thunb by using bioinformatics methods and the proteins' physicochemical properties they encoded were predicted. The phylogenetic tree showed that the four DNA demethylase genes and Arabidopsis thaliana DME had a close relationship. The result of gene expression model showed that four DNA demethylase genes were different between species. The expression levels of LJDME1 and LJDME2 were even more higher in Lonicera japonica var. chinensis than those in L. japonica. LJDME] and LJDME2 maybe regulate the active compounds of L. japonica. This study aims to lay a foundation for further understanding of the function of DNA demethylase genes in L. japonica.

Predicting the function of 4-coumarate:CoA ligase (LJ4CL1) in Lonicera japonica.

4-Coumarate:CoA ligases (4CLs) are a group of essential enzymes involved in the pathway of phenylpropanoid-derived compound metabolisms; however it is still difficult to identify orthologs and paralogs of these important enzymes just based on sequence similarity of the conserved domains. Using sequence data of 20 plant species from the public databases and sequences from Lonicera japonica, we define 1252 adenosine monophosphate (AMP)-dependent synthetase/ligase sequences and classify them into three phylogenetic clades. 4CLs are in one of the four subgroups, according to their partitioning, with known proteins characterized in A. thaliana and Oryza sativa. We also defined 184 non-redundant sequences that encode proteins containing the GEICIRG motif and the taxonomic distribution of these GEICIRG-containing proteins suggests unique catalytic activities in plants. We further analyzed their transcription levels in L. japonica and L. japonica. var. chinensis flowers and chose the highest expressed genes representing the subgroups for structure and binding site predictions. Coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of the L. japonica flowers, the structural study on putative substrate binding amino acid residues, ferulate, and 4-coumaric acid of the conserved binding-site of LJ4CL1 leads to a conclusion that this highly expressed protein group in the flowers may process 4-coumarate that represents 90% of the known phenylpropanoid-derived compounds. The activity of purified crude LJ4CL1 protein was analyzed using 4-coumarate as template and high activity indicating that 4-coumarate is one of the substrates of LJ4CL1.

Transcriptome-wide analysis of SAMe superfamily to novelty phosphoethanolamine N-methyltransferase copy in Lonicera japonica.

The S-adenosyl-L-methionine-dependent methyltransferase superfamily plays important roles in plant development. The buds of Lonicera japonica are used as Chinese medical material and foods; chinese people began domesticating L. japonica thousands of years ago. Compared to the wild species, L. japonica var. chinensis, L. japonica gives a higher yield of buds, a fact closely related to positive selection over the long cultivation period of the species. Genome duplications, which are always detected in the domestic species, are the source of the multifaceted roles of the functional gene. In this paper, we investigated the evolution of the SAMe genes in L. japonica and L. japonica var. chinensis and further analyzed the roles of the duplicated genes among special groups. The SAMe protein sequences were subdivided into three clusters and several subgroups. The difference in transcriptional levels of the duplicated genes showed that seven SAMe genes could be related to the differences between the wild and the domesticated varieties. The sequence diversity of seven SAMe genes was also analyzed, and the results showed that different gene expression levels between the varieties could not be related to amino acid variation. The transcriptional level of duplicated PEAMT could be regulated through the SAM-SAH cycle.

[Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis].

OBJECTIVE: To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. METHOD: The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. RESULT: The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. CONCLUSION: The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

[Dynamic changes of enzyme activities and active component contents in Lonicera japonica during different blossoming stages].

OBJECTIVE: This paper aimed to study the dynamic changes of enzyme activities and active component contents in Lonicera japonica during different blossoming stages. METHOD: The enzyme activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD) and the contents of total phenol, total flavonoids, chlorogenic acid, anthocyanins in L. japonica during different blossoming stages were determined. RESULT: The contents of total phenolics, total flavonoids, anthocyanins decreased from the Sanqing stage to Jinhua stage while the content of chlorogenic acid increased slightly in white period, and then decreased gradually. The activities of three enzymes decreased gradually from Sanqing stage, and got to a minimum value in Yinhua stage, then increased slightly until the Jinhua stage. CONCLUSION: The enzyme activities of PPO and POD correlated the content of phenolic substances positively before the Jinhua stage in L. japonica. In the period of maturity, the POD activity was strengthened due to the induction of respiration and became the key enzyme to control active component content during the mature stage.

Cloning and characterization of Lonicera japonica p-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid.

Lonicera japonica is used in Chinese medicine as a source of antioxidants, primarily flavonoids, and a phenolic acid chlorogenic acid (CGA). Here we report the isolation and characterization of the full-length cDNA of LjC3H, a gene encoding p-coumaroyl ester 3-hydroxylase, an enzyme involved in CGA synthesis. Phylogenetic analysis indicated that is protein belongs to the CYP98A subfamily, and homology modeling revealed that its structure resembles that of other cytochrome P450 family proteins. Southern blot analysis indicated that more than one copy of sequences homologous to LjC3H is present in the L. japonica genome. Heterologous expression of LjC3H cDNA in Escherichia coli allowed an in vitro assay of LjC3H to be performed. This experiment revealed that the enzyme favors p-coumaroylshikimate over p-coumaroylquinate as substrate. LjC3H transcript abundance was increased both by treatment of the leaves with methyl jasmonate and by exposure to UV-B radiation. The CGA levels in the leaves of L. japonica were positively correlated with LjC3H transcript abundance.

Transcriptome analysis of buds and leaves using 454 pyrosequencing to discover genes associated with the biosynthesis of active ingredients in Lonicera japonica Thunb.

BACKGROUND: Lonicera japonica Thunb. is a plant used in traditional Chinese medicine known for its anti-inflammatory, anti-oxidative, anti-carcinogenic, and antiviral pharmacological properties. The major active secondary metabolites of this plant are chlorogenic acid (CGA) and luteoloside. While the biosynthetic pathways of these metabolites are relatively well known, the genetic information available for this species, especially the biosynthetic pathways of its active ingredients, is limited. METHODOLOGY/PRINCIPAL FINDINGS: We obtained one million reads (average length of 400 bp) in a whole sequence run using a Roche/454 GS FLX titanium platform. Altogether, 85.69% of the unigenes covering the entire life cycle of the plant were annotated and 325 unigenes were assigned to secondary metabolic pathways. Moreover, 2039 unigenes were predicted as transcription factors. Nearly all of the possible enzymes involved in the biosynthesis of CGA and luteoloside were discovered in L. japonica. Three hydroxycinnamoyl transferase genes, including two hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes and one hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) gene featuring high similarity to known genes from other species, were cloned. The HCT gene was discovered for the first time in L. japonica. In addition, 188 candidate cytochrome P450 unigenes and 245 glycosyltransferase unigenes were found in the expressed sequence tag (EST) dataset. CONCLUSION: This study provides a high quality EST database for L. japonica by 454 pyrosequencing. Based on the EST annotation, a set of putative genes involved in CGA and luteoloside biosynthetic pathways were discovered. The database serves as an important source of public information on genetic markers, gene expression, genomics, and functional genomics in L. japonica.

Purification and partial characterization of polyphenol oxidase from the flower buds of Lonicera japonica Thunb.

The purification and partial enzymology characteristics of polyphenol oxidase from Lonicera japonica (LjPPO) were studied in this paper. The crude enzyme solution was purified in turn by ammonium sulfate, dialysis, and DEAE-cellulose ion-exchange chromatography after preliminary treatments. Purification resulted in 31-fold enrichment and its molecular weight was estimated to be ~49 kDa exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH for optimal conditions of LjPPO was 7.5, and the temperature was 25 °C, in addition, the inhibitive effects of inhibitors were enhanced positively with increasing of the concentration. Moreover, crude enzyme solution showed diphenolase activity toward catechol, l-dopa and chlorogenic acid rather than monophenolase and triphenolase activity, and the best substrate was catechol because of the highest V(max)/K(m) value. However, the oxidation of diphenol related to browning significantly, so the data obtained in this research provided theoretical basis for the prevention of enzymatic browning of L. japonica during processing.

Molecular cloning and expression profiling of the first specific jasmonate biosynthetic pathway gene allene oxide synthase from Lonicera japonica.

In jasmonate biosynthetic pathway, allene oxide synthase (AOS, EC 4.2.1.92), which is a cytochrome P450 (CYP74A), catalyzes the first committed step. We herein cloned a novel cDNA from Lonicera japonica Thunb., named LjAOS (GenBank accession: DQ303120), which was homologous to other AOSs. Southern blot analysis revealed that it was a multi-copy gene. Real-time quantitative PCR analysis showed that LjAOS mRNA accumulated most abundantly in alabastrums, in which the content of chlorogenic acid (CA, the major important active ingredient indicator) was previously proven to be the highest.