Transcriptome Analysis Reveals Molecular Signatures of Luteoloside Accumulation in Senescing Leaves of Lonicera macranthoides.

Lonicera macranthoides is an important medicinal plant widely used in traditional Chinese medicine. Luteoloside is a critical bioactive compound in L. macranthoides. To date, the molecular mechanisms underlying luteoloside biosynthesis are still largely unknown. In this work, high performance liquid chromatography (HPLC) was employed to determine the luteoloside contents in leaves, stems, and flowers at different developmental stages. Results showed that senescing leaves can accumulate large amounts of luteoloside, extremely higher than that in young and semi-lignified leaves and other tissues. RNA-Seq analysis identified that twenty-four differentially expressed unigenes (DEGs) associated with luteoloside biosynthesis were significantly up-regulated in senescing leaves, which are positively correlated with luteoloside accumulation. These DEGs include phenylalanine ammonia lyase 2, cinnamate 4-hydroxylase 2, thirteen 4-coumarate-CoA ligases, chalcone synthase 2, six flavonoid 3'-monooxygenase (F3'H) and two flavone 7-O-ß-glucosyltransferase (UFGT) genes. Further analysis demonstrated that two F3'Hs (CL11828.Contig1 and CL11828.Contig2) and two UFGTs (Unigene2918 and Unigene97915) might play vital roles in luteoloside generation. Furthermore, several transcription factors (TFs) related to flavonoid biosynthesis including MYB, bHLH and WD40, were differentially expressed during leaf senescence. Among these TFs, MYB12, MYB75, bHLH113 and TTG1 were considered to be key factors involved in the regulation of luteoloside biosynthesis. These findings provide insights for elucidating the molecular signatures of luteoloside accumulation in L. macranthoides.

[Cloning and expression analysis of F3'H and quantification of downstream products in Chrysanthemum morifolium under flooding stress].

To investigate the effects of the expression of flavonoid 3' hydroxylase gene (F3'H) and active ingredients in Chrysanthemum morifolium under flooding stress, we cloned F3'H from Hangju (temporarily named CmF3'H) and conducted bioinformatics analysis. During the flower bud differentiation stage, we flooded the Ch. morifolium and then used the Real-time PCR to detect the relative expression of CmF3'H; Finally, active ingredients of the inflorescence were measured by HPLC.The sequencing results showed that 1 562 bp sequence was acquired with the largest open reading frame of 1 527 bp, which encoded 508 amino acids. The phylogenetic tree found that CmF3'H was highly homologous to other species of Compositae. Real-time PCR results showed that CmF3'H had a significant response to flooding stress and had the highest expression level after flooding for 24 h, which was about 9 times as that of the control group. The results of HPLC showed that luteolin and luteoloside, the downstream products catalyzed by the F3'H, were significantly higher than those in the control group. It was also found that the contents of chlorogenic acid and 3,5-O-di-caffeoylquinic acid were also significantly higher than those of the control group. Therefore, Ch. morifolium regulates the synthesis of downstream products by regulating the expression of CmF3'H in the flavonoid synthesis pathway under flooding stress, thereby responding to flooding stress. The flooding stress during flower bud differentiation can significantly enhance the accumulation of active ingredients.

[Research of regulating synthesis of luteolin and luteoloside of Lonicera japonica by LjFNS â ¡ 1.1 and LjFNS â ¡ 2.1 treated with 5-azaC].

This study is aimed to explore the mechanism of catalyzing the synthesis of luteolin and luteoloside by LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1.The leaves of Lonicera japonica were treated with different concentrations of 5-azaC(20,40,60,80,100 µmol•L-1) for three periods（1,2,3 d）. Firstly, we cloned LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1. Secondly, we analyzed the expression levels of LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1 by Real-Time PCR and the contents of luteolin and luteoloside determined by UPLC-MS/MS. The results explained the expression levels of LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1 consistent with the content variation of luteolin in general, but there was no significant correlation with the contents of luteoloside. And we found the expression levels of LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1 were slightly different. The research indicated that the contents of luteolin and luteoloside got higher by improving the expression levels of LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1. This will provide technical support and lay a theoretical foundation for regulating the synthesis of luteolin and luteoloside by LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1.

[Analysis of critical genes expression of chlorogenic acid and luteolin biosyntheses in Lonicera confusa].

This study analysed the tissue specific expression of critical genes involved in chlorogenic acid and luteolin biosynthesis, for exploiting the molecular mechanism of components biosynthesis in Lonicera confusa. Expression of PAL, 4CL, C4H, CHS, CHI, FNS and HQT gene families of chlorogenic acid and luteolin biosynthesis-related genes in buds and leaves of L. confusa were analyed by Real-time PCR. Expressions of PAL1, C4H1, 4CL1, CHS1, CHI3 and HQT2 in buds were lower than that in leaves, and expressions of PAL3, 4CL2, CHI2 and FNS2 in buds were higher than that in leaves. The results indicated that that PAL3 and 4CL2 may be associated with accumulation of chlorogenic acid, and the expression patterns of PAL1, CHS1, CHI3 and HQT2 in buds and leaves of L. confusa were different with L. japonica. This study provided some theoretical basis for the further research on genetic mechanism of active components differences in L. confusa and L. japonica.

Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes.

Chlorogenic acids (CGAs) and luteolin are active compounds in Lonicera japonica, a plant of high medicinal value in traditional Chinese medicine. This study provides a comprehensive overview of gene families involved in chlorogenic acid and luteolin biosynthesis in L. japonica, as well as its substitutes Lonicera hypoglauca and Lonicera macranthoides. The gene sequence feature and gene expression patterns in various tissues and buds of the species were characterized. Bioinformatics analysis revealed that 14 chlorogenic acid and luteolin biosynthesis-related genes were identified from the L. japonica transcriptome assembly. Phylogenetic analyses suggested that the function of individual gene could be differentiation and induce active compound diversity. Their orthologous genes were also recognized in L. hypoglauca and L. macranthoides genomic datasets, except for LHCHS1 and LMC4H2. The expression patterns of these genes are different in the tissues of L. japonica, L. hypoglauca and L. macranthoides. Results also showed that CGAs were controlled in the first step of biosynthesis, whereas both steps controlled luteolin in the bud of L. japonica. The expression of LJFNS2 exhibited positive correlation with luteolin levels in L. japonica. This study provides significant information for understanding the functional diversity of gene families involved in chlorogenic acid and the luteolin biosynthesis, active compound diversity of L. japonica and its substitutes, and the different usages of the three species.

Simultaneous determination of nine major active compounds in Dracocephalum rupestre by HPLC.

A new method was developed for the simultaneous determination of nine major constituents in Dracocephalum rupestre, including 5,7-dihydroxychromone (1), eriodictyol-7-O-beta-d-glucoside (2), luteolin-7-O-beta-d-glucoside (3), naringenin-7-O-beta-d-glucoside (4), apigenin-7-O-beta-d-glucoside (5), eriodictyol (6), luteolin (7), naringenin (8) and apigenin (9). The quantitative determination was conducted by reversed phase high-performance liquid chromatography with photodiode array detector (LC-PDA). Separation was performed on an Agilent Eclipse XDB-C(18) column (150 mm x 4.6 mm i.d., 5 microm) with gradient elution of acetonitrile and 0.5% aqueous acetic acid. The components were identified by retention time, ultraviolet (UV) spectra and quantified by LC-PDA at 260 nm. All calibration curves showed good linearity (r(2)>0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.0%. The recoveries were between 95.15 and 104.45%. The limits of detection (LOD) ranged from 0.002 to 0.422 microg/ml and limits of quantification (LOQ) ranged from 0.005 to 1.208 microg/ml, respectively. The identity of the peaks was further confirmed by high-performance liquid chromatography with triple-quadrupole mass spectrometry system coupled with electrospray ionization (ESI) interface. The developed method was applied to the determination of nine constituents in 14 samples of D. rupestre collected at various harvesting times. Most compounds accumulated at much higher amounts in about June-July. The satisfactory results indicated that the developed method was readily utilized as a quality control method for D. rupestre.