Comparative metabolomics analysis revealed biomarkers and distinct flavonoid biosynthesis regulation in Chrysanthemum mongolicum and C. rhombifolium.

INTRODUCTION: Chrysanthemums are traditional flowers that originated in China and have high ornamental, economic and medicinal value. They are widely used as herbal remedies and consumed as food or beverages in folk medicine. However, little is known about their metabolic composition. OBJECTIVES: The aims of this work were to determine the metabolic composition of and natural variation among different species of Chrysanthemum and to explore new potential resources for drug discovery and sustainable utilisation of wild Chrysanthemum. METHODS: The metabolomes of Chrysanthemum mongolicum (Ling) Tzvel. and Chrysanthemum rhombifolium H. Ohashi & Yonek. were compared using a widely targeted metabolomics approach based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: In total, 477 metabolites were identified, of which 72 showed significant differences in expression between C. mongolicum and C. rhombifolium, mainly in flavonoids, organic acids and nucleotides. The flavone and flavonol biosynthesis pathway showed significant enrichment among the differentially expressed metabolites. The contents of genkwanin, trigonelline, diosmin, narcissoside, 3,4-dihydroxyphenylacetic acid, linarin, N',N'-p-coumarin, C-hexosyl-tricetin O-pentoside, chrysoeriol, acacetin and kaempferol-3-O-gentiobioside were significantly different between the two species and represent potential biomarkers. CONCLUSION: The types of flavonoid-related metabolites in the flavonoid biosynthesis pathway differed between C. mongolicum and C. rhombifolium. The mechanisms underlying the unique adaptations of these two species to their environments may involve variations in the composition and abundance of flavonoids, organic acids, and nucleotides. These methods are promising to identify functional compounds in Chrysanthemum species and can provide potential resources for drug discovery and the sustainable utilisation of Chrysanthemum plants.

Isolation, Structural Elucidation, Optical Resolution, and Antineuroinflammatory Activity of Phenanthrene and 9,10-Dihydrophenanthrene Derivatives from Bletilla striata.

A phytochemical investigation of the aqueous EtOH extract of Bletilla striata tubers afforded 34 phenanthrene and 9,10-dihydrophenanthrene derivatives, including four new compounds, 1-4. These compounds were identified using physicochemical analyses and various spectroscopic methods. Twelve of these compounds were resolved into their enantiomers, and the absolute configurations were determined by comparison of experimental and calculated ECD spectra. The antineuroinflammatory activities were evaluated by measuring the inhibition of nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells. Compounds 7, 32, and 33 displayed inhibitory activities, with IC50 values of 1.9, 5.0, and 1.0 µM, respectively, suggesting that they should be subjected to development as potential inhibitors of neuroinflammation.

Floral morphology and morphogenesis in Camptotheca (Nyssaceae), and its systematic significance.

Background and Aims: Camptotheca is endemic to China and there are limited data about the breeding system and morphogenesis of the flowers. Camptotheca is thought to be related to Nyssa and Davidia in Nyssaceae, which has sometimes been included in Cornaceae. However, molecular phylogenetic studies confirmed the inclusion of Camptotheca in Nyssaceae and its exclusion from Cornaceae. The aim of this study was to reveal developmental features of the inflorescence and flowers in Camptotheca to compare with related taxa in Cornales. Methods: Inflorescences and flowers of Camptotheca acuminata at all developmental stages were collected and studied with a scanning electron microscope and stereo microscope. Key Results: Camptotheca has botryoids which are composed of several capitate floral units (FUs) that are initiated acropetally. On each FU, flowers are grouped in dyads that are initiated acropetally. All floral organs are initiated centripetally. Calyx lobes are restricted to five teeth. The hypanthium, with five toothed calyx lobes, is adnate to the ovary. The five petals are free and valvate. Ten stamens are inserted in two whorls around the central depression, in which the style is immersed. Three carpels are initiated independently but the ovary is syncarpous and unilocular. The ovule is unitegmic and heterotropous. Inflorescences are functionally andromonoecious varying with the position of the FUs on the inflorescence system. Flowers on the upper FU often have robust styles and fully developed ovules. Flowers on the lower FU have undeveloped styles and aborted ovules, and the flowers on the middle FU are transitional. Conclusions: Camptotheca possesses several traits that unify it with Nyssa, Mastixia and Diplopanax. Inflorescence and floral characters support a close relationship with Nyssaceae and Mastixiaceae but a distant relationship with Cornus. Our results corroborate molecular inferences and support a separate family Nyssaceae.

Characterization of bZIP Transcription Factors in Transcriptome of Chrysanthemum mongolicum and Roles of CmbZIP9 in Drought Stress Resistance.

bZIP transcription factors play important roles in regulating plant development and stress responses. Although bZIPs have been identified in many plant species, there is little information on the bZIPs in Chrysanthemum. In this study, bZIP TFs were identified from the leaf transcriptome of C. mongolicum, a plant naturally tolerant to drought. A total of 28 full-length bZIP family members were identified from the leaf transcriptome of C. mongolicum and were divided into five subfamilies based on their phylogenetic relationships with the bZIPs from Arabidopsis. Ten conserved motifs were detected among the bZIP proteins of C. mongolicum. Subcellular localization assays revealed that most of the CmbZIPs were predicted to be localized in the nucleus. A novel bZIP gene, designated as CmbZIP9, was cloned based on a sequence of the data of the C. mongolicum transcriptome and was overexpressed in tobacco. The results indicated that the overexpression of CmbZIP9 reduced the malondialdehyde (MDA) content and increased the peroxidase (POD) and superoxide dismutase (SOD) activities as well as the expression levels of stress-related genes under drought stress, thus enhancing the drought tolerance of transgenic tobacco lines. These results provide a theoretical basis for further exploring the functions of the bZIP family genes and lay a foundation for stress resistance improvement in chrysanthemums in the future.

Characterization of the Chloroplast Genome of Argyranthemum frutescens and a Comparison with Other Species in Anthemideae.

Argyranthemum frutescens, which belongs to the Anthemideae (Asteraceae), is widely cultivated as an ornamental plant. In this study, the complete chloroplast genome of A. frutescens was obtained based on the sequences generated by Illumina HiSeq. The chloroplast genome of A. frutescens was 149,626 base pairs (bp) in length, containing a pair of inverted repeats (IR, 24,510 bp) regions separated by a small single-copy (SSC, 18,352 bp) sequence and a large single-copy (LSC, 82,254 bp) sequence. The genome contained 132 genes, consisting of 85 coding DNA sequences, 37 tRNA genes, and 8 rRNA genes, with nineteen genes duplicated in the IR region. A comparison chloroplast genome analysis among ten species from the tribe of Anthemideae revealed that the chloroplast genome size varied, but the genome structure, gene content, and oligonucleotide repeats were highly conserved. Highly divergent regions, e.g., ycf1, trnK-psbK, petN-psbM intronic, were detected. Phylogenetic analysis supported Argyranthemum as a separate genus. The findings of this study will be helpful in the exploration of the phylogenetic relationships of the tribe of Anthemideae and contribute to the breeding improvement of A. frutescens.

Molecular cloning and functional analysis of a Chrysanthemum vestitum GME homolog that enhances drought tolerance in transgenic tobacco.

GDP-mannose 3, 5-epimerase (GME, EC 5.1.3.18), a key enzyme in the ascorbic acid synthesis pathway, catalyzes the conversion of GDP-D-mannose to GDP-l-galactose in higher plants. Here, a homolog of GME was isolated from Chrysanthemum vestitum. The cDNA sequence of CvGME was 1131 bp and contained a complete open reading frame encoding a protein comprising 376 amino acids. Quantitative real-time PCR analysis revealed that CvGME was most highly expressed in the stems and roots. Phylogenetic analysis showed that CvGME was closely related to LsGME from Lactuca sativa. Subcellular localization studies revealed that CvGME was localized in the nucleus. Heterologous expression of CvGME in transgenic tobacco plants increased the ascorbic acid content in the leaves. In addition, overexpression of CvGME reduced the malondialdehyde content and increased superoxide dismutase and peroxidase activity in tobacco leaves compared to those in the wild-type plants under drought stress conditions, explaining the increased drought tolerance of transgenic tobacco lines. These results suggest that CvGME can effectively enhance the tolerance of plants to drought by increasing the ascorbic acid content, which may help improve the drought tolerance of chrysanthemums through molecular breeding.

Chrysanthemumdabieshanense, a new name for Chrysanthemumvestitum var. latifolium (Asteraceae, Anthemideae).

Recent phylogenetic analyses have revealed that Chrysanthemumvestitumvar.latifolium and C.vestitumvar.vestitum were placed in different clades based on their chloroplast genomes and nuclear LFAFY gene sequences. Accordingly, based on previous morphological analysis, molecular phylogenetic results, fieldwork, and herbarium studies, Chrysanthemumvestitumvar.latifolium should be raised to the species level. Considering the condition of the material found and Articles 6.9, 6.11, 41.2, 58.1 of the International Code of Nomenclature for Algae, Fungi, and Plants (Shenzhen Code) that is currently in force, Chrysanthemumdabieshanense Z.X.Fu, A.G.Zhen, & Y.P.Ma, nom. nov. is proposed as the new name for Chrysanthemumvestitumvar.latifolium J.Zhou & Jun Y.Chen. The detailed emended description, distribution map, insights into its habitat, and an updated comparative morphological study are presented in this study.

Two new polyacetylenic compounds from Atractylodes chinensis (DC.) Koidz.

Two new polyacetylenic compounds, (6E,12Z)-tetradecadiene-8,10-diyne-1,3-diol (1) and (6Z,12Z)-tetradecadiene-8,10-diyne-1,3-diol (2), were isolated from Atractylodes chinensis (DC.) Koidz. Their structures were established by analysis of spectroscopic data.

Characterizing the Leaf Transcriptome of Chrysanthemum rhombifolium (Ling et C. Shih), a Drought Resistant, Endemic Plant From China.

Chrysanthemum rhombifolium (Ling et C. Shih), an endemic plant that is extremely well-adapted to harsh environments. However, little is known about its molecular biology of the plant's resistant traits against stress, or even its molecular biology of overall plant. To investigate the molecular biology of C. rhombifolium and mechanism of stress adaptation, we performed transcriptome sequencing of its leaves using an Illumina platform. A total of 130,891 unigenes were obtained, and 97,496 (~74.5%) unigenes were annotated in the public protein database. The similarity search indicated that 40,878 and 74,084 unigenes showed significant similarities to known proteins from NCBI non-redundant and Swissprot protein databases, respectively. Of these, 56,213 and 42,005 unigenes were assigned to the Gene Ontology (GO) database and Cluster of Orthologous Groups (COG), respectively, and 38,918 unigenes were mapped into five main categories, including 18 KEGG pathways. Metabolism was the largest category (23,128, 59.4%) among the main KEGG categories, suggesting active metabolic processes in C. rhombifolium. About 2,459 unigenes were annotated to have a role in defense mechanism or stress tolerance. Transcriptome analysis of C. rhombifolium revealed the presence of 12,925 microsatellites in 10,524 unigenes and mono, trip, and dinucleotides having higher polymorphism rates. The phylogenetic analysis based on GME gene among related species confirmed the reliability of the transcriptomic data. This work is the first genetic study of C. rhombifolium as a new plant resource of stress-tolerant genes. This large number of transcriptome sequences enabled us to comprehensively understand the basic genetics of C. rhombifolium and discover novel genes that will be helpful in the molecular improvement of chrysanthemums.

AfLFY, a LEAFY homolog in Argyranthemum frutescens, controls flowering time and leaf development.

Flowering is important for plant propagation and survival, and it is also closely related to human life. Identifying the molecular mechanisms underlying flower development is essential for plant improvement and breeding. Flower development is a complex physiological process that is regulated by multiple genes. LFY genes play important roles in the floral meristem transition and act as crucial integrators in regulating the floral gene network. Argyranthemum frutescens is an ornamental species cultivated for floral displays, yet little is known about molecular mechanisms driving its flower development. In this study, the LEAFY gene homologue, AfLFY, was identified and cloned from A. frutescens, and its role and expression patterns were characterized. Two distinct copies of AfLFY were found in the A. frutescens genome and both sequences contained a 1248 bp open reading frame that encoded 415 amino acids. The putative protein sequences have a typical LFY family domain. In addition, AfLFY was expressed at the highest levels in young leaves of the vegetative stage and in the shoot apical bud meristem of the reproductive stage. Phylogenetic analysis showed that AfLFY was most closely related to DFL from Chrysanthemum lavandulifolium. Subcellular localization studies revealed that AfLFY localized to the nucleus. Heterologous expression of AfLFY in transgenic tobacco plants shortened its period of vegetative growth, converted the lateral meristems into terminal flowers and promoted precocious flowering. In addition, transgenic plants exhibited obvious morphological changes in leaf shape. qRT-PCR analysis indicated that the expression levels genes related to flowering, FT, SOC1, and AP1 were significantly upregulated in AfLFY transgenic plants. Our findings suggested that the AfLFY gene plays a vital role in promoting flowering and leaf development in A. frutescens. These results laid a foundation for us to understand the mechanism of AfLFY in regulation flowering, and the results will be helpful in improving A. frutescens through molecular breeding.

Efficient Identification of Pulsatilla (Ranunculaceae) Using DNA Barcodes and Micro-Morphological Characters.

Pulsatilla (Ranunculaceae) comprises about 40 species, many of which have horticultural and/or medicinal importance. However, the recognition and identification of wild Pulsatilla species is difficult due to the presence of complex morphological characters. DNA barcoding is a powerful molecular tool capable of rapidly and accurately distinguishing between species. Here, we assessed the effectiveness of four commonly used DNA barcoding loci-rbcL (R), trnH-psbA ( T ), matK (M), and ITS (I)-to identify species of Pulsatilla from a comprehensive sampling group. Among the four barcoding single loci, the nuclear ITS marker showed the highest interspecific distances and the highest rate of correct identification. Among the eleven combinations, the chloroplast multi-locus R+T and R+M+T combinations were found to have the best species discrimination rate, followed by R+M. Overall, we propose that the R+M+T combination and the ITS marker on its own are, respectively, the best multi- and single-locus barcodes for discriminating among species of Pulsatilla. The phylogenetic analysis was able to distinguish species of Pulsatilla to the subgenus level, but the analysis also showed relatively low species resolution. This may be caused by incomplete lineage sorting and/or hybridization events in the evolutionary history of the genus, or by the resolution limit of the candidate barcodes. We also investigated the leaf epidermis of eight representative species using scanning electronic microscopy. The resulting micro-morphological characters were valuable for identification of related species. Using additional genome fragments, or even whole chloroplast genomes combined with micro-morphological data may permit even higher resolution of species in Pulsatilla.

The complete chloroplast genome of the marsh plant-Leucanthemella linearis (Asteraceae).

Leucanthemella linearis is an important marsh plant. The complete chloroplast genome sequence of L. linearis was obtained using next generation sequencing. It was 15,1401 bp in length, including a pair of inverted repeat (IR, 24,941 bp) regions separated by a small single copy (SSC, 18,392 bp) sequence and a large single copy (LSC, 83,127 bp) sequence. The cp genome contained 140 genes, consisting of 96 protein-coding genes, 8 rRNA genes and 36 tRNA genes. Twenty-two genes were present in the IR region. Thirty-four SSR sites were detected in the cp genome. Phylogenetic analysis with the reported chloroplast genomes revealed that L. linearis is most closely related to Tribe Heliantheae species. This new data will help to understand the phylogenetic position and biology of the Leucanthemella.

A Diversity-Enhanced Resource Allocation Strategy for Decomposition-Based Multiobjective Evolutionary Algorithm.

The multiobjective evolutionary algorithm (MOEA) based on decomposition transforms a multiobjective optimization problem into a set of aggregated subproblems and then optimizes them collaboratively. Since these subproblems usually have different degrees of difficulty, resource allocation (RA) strategies have been reported to enhance performance, attempting to dynamically assign proper amounts of computational resources for the solution of each of these subproblems. However, existing schemes for decomposition-based MOEAs fully rely on the relative improvement of the aggregated functions to do this. This paper proposes a diversity-enhanced RA strategy for this kind of MOEA, depending on both relative improvement on aggregated function value and solution density around each subproblem to assign computational resources. Thus, one subproblem surrounded with fewer solutions in its neighboring area and more relative improvement on the aggregated function value will be allocated a higher probability for evolution. Our experimental results show the advantages of our proposed strategy over two popular RA strategies available for decomposition-based MOEAs, on tackling a set of complicated benchmark problems.

Lico A Causes ER Stress and Apoptosis via Up-Regulating miR-144-3p in Human Lung Cancer Cell Line H292.

During our study on the bioactivities of natural flavonoids, we found that the total flavonoids (TFs) and the main constituent of it, licochalcone A (lico A), activated unfolded protein response (UPR) and induced autophagy and thereby apoptosis in H292 cells. MicroRNAs, such as the tumor repressor miR-144-3p, were reported to be differentially expressed in lung cancer cells and were linked to ER stress, autophagy, and apoptosis. However, the underlying miRNA-based mechanism for lico A modulating proliferation, autophagy and apoptosis in lung cancer cells is elusive. In this study, we found that miR-144-3p was down-regulated in H292 cells comparing to normal embryonic lung cells WI-38, and lico A (10 µM) could increase miR-144-3p level in H292 cells. Knockdown of miR-144-3p significantly abrogated the apoptosis and proliferation-inhibiting effects of lico A, and lico A could enhance the proliferation-inhibiting effect and apoptosis induced by miR-144-3p overexpression. Moreover, overexpression miR-144-3p could induce ER stress by down-regulating Nrf2, and lico A enhanced the Nrf2 down-regulation caused by miR-144-3p overexpression. Co-transfection experiments showed that lico A potentially increased the dicing of pre-miR-144 so as to increase the mature miR-144-3p level. Interestingly, high level of lico A (40 µM) up-regulated CHOP protein, but failed to increase the downstream genes levels of CHOP, including Bim and Bcl-2 in H292 cells. Docking studies indicated that CHOP-mediated pathway was potentially blocked by high dose of lico A. Our results suggested that lico A could cause UPR, autophagy and apoptosis, and the underlying mechanism involved up-regulation of miR-144-3p, and increased lico A level would also increase the potential for lico A inhibiting CHOP-dependent apoptosis in H292 cells.

Mitochondrial genome of the African lion Panthera leo leo.

In this study, the complete mitochondrial genome sequence of the African lion P. leo leo was reported. The total length of the mitogenome was 17,054 bp. It contained the typical mitochondrial structure, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region; 21 of the tRNA genes folded into typical cloverleaf secondary structure except for tRNASe. The overall composition of the mitogenome was A (32.0%), G (14.5%), C (26.5%) and T (27.0%). The new sequence will provide molecular genetic information for conservation genetics study of this important large carnivore.

Sesquiterpenoid derivatives from Ferula ferulaeoides (Steud.) Korov.

Eight sesquiterpenoids, named Ferulaeone A-H (1-8), and seven known sesquiterpenoid derivatives were isolated from the roots of Ferula ferulaeoides (Steud.) Korov. Their structures were established by comprehensive spectroscopic analysis, and biosynthetic pathways leading to these compounds were proposed. The cytotoxicity of all these isolates against HepG2, MCF-7, and C6 cancer cell lines was evaluated and compounds 6-11, 13 exihibited various degrees of cytotoxic effect. Among them, compounds 9-11 displayed the highest potency against C6 with IC(50) values 34, 36, and 31 µM, respectively.

Phenolic glycosides from Curculigo orchioides Gaertn.

Five new chlorophenolic glucosides, curculigine E (1), curculigine F (2), curculigine G (3), curculigine H (5), curculigine I (6) and one new phenolic glycoside, orcinoside H (4), together with eight known phenolic glycosides (7-14) were isolated from the Curculigo orchioides Gaertn. Their structures were established by spectroscopic techniques (IR, UV, MS, 1D and 2D NMR). The isolated phenolic glycosides were evaluated for antiosteoporotic activity against MC3T3-E1 cell line using MTT assays. Compounds 1, 2, 3, and 5 showed moderate antiosteoporotic activity with the proliferation rate of 10.1-14.1%.

DFL, a FLORICAULA/LEAFY homologue gene from Dendranthema lavandulifolium is expressed both in the vegetative and reproductive tissues.

FLO/LFY homologue genes were initially characterized as floral meristem identity genes and play a key role in flower development among diverse species. The inflorescence organization of chrysanthemum differs from typical dicotyledons such as Arabidopsis and Antirrhinum as clear sepals are absent, and instead, a pappus, a rudimentary sepal, is formed. To understand the mechanism of reproduction of chrysanthemum at the molecular level, DFL, a FLORICAULA/LEAFY homologous gene, was cloned from Dendranthema lavandulifolium, which is one of the original species of chrysanthemum. The DFL gene consists of a 1,236-bp open reading frame and encodes a putative protein of 412 amino acids, which is 63% identical to LFY and 70% to FLO. The expression patterns of DFL during the flower development were analyzed, and RT-PCR results showed that DFL was strongly expressed in the flower bud. In situ hybridization experiments showed that it is strongly expressed in the inflorescence bract, petal and stamen primordial tissues throughout the inflorescence development. Its expression signals were also detected in stems, leaf primordial tissues and developing inflorescence bracts.