Transcriptome and metabolome analyses reveal novel insights into the seed germination of Michelia chapensis, an endangered species in China.

Michelia chapensis Dandy, a well-known medicinal woody plant endemic to China, is endangered and seriously constricted by seed dormancy-induced low-regeneration in natural conditions. Cold stratification can effectively reduce seed dormancy and promote the seed germination of M. chapensis. However, the molecular events and systematic changes that occurred during seed germination in M. chapensis remain largely unknown. In this study, we carried out transcriptomic and metabolomic analyses to elucidate the potential molecular mechanisms underlying seed germination in M. chapensis under cold stratification. The results showed that the embryo cells became bigger and looser with increasing stratification time. Moreover, the endosperm appeared reduced due to the consumption of nutrients. Seventeen phytohormones were examined by the metabolome targeted for hormones. Compared with the ES (no stratification), the levels of indole-3-acetic acid (IAA) and gibberellin A3 (GA3) were increased in the MS (stratification for 45 days), while the abscisic acid (ABA) was downregulated in both MS and LS (stratification for 90 days). The transcriptome profiling identified 24975 differentially expressed genes (DEGs) in the seeds during germination. The seed germination of M. chapensis was mainly regulated by the biological pathways of plant hormone signal transduction, energy supply, secondary metabolite biosynthesis, photosynthesis-related metabolism, and transcriptional regulation. This study reveals the biological evidence of seed germination at the transcriptional level and provides a foundation for unraveling molecular mechanisms regulating the seed germination of M. chapensis.

Gene identification and tissue expression analysis inform the floral organization and color in the basal angiosperm Magnolia polytepala (Magnoliaceae).

MAIN CONCLUSION: In Magnolia polytepala, the formation of floral organization and color was attributed to tissue-dependent differential expression levels of MADS-box genes and anthocyanin biosynthetic genes. In angiosperms, the diversity of floral morphology and organization suggests its value in exploring plant evolution. Magnolia polytepala, an endemic basal angiosperm species in China, possesses three green sepal-like tepals in the outermost whorl and pink petal-like tepals in the inner three whorls, forming unique floral morphology and organization. However, we know little about its underlying molecular regulatory mechanism. Here, we first reported the full-length transcriptome of M. polytepala using PacBio sequencing. A total of 16 MADS-box transcripts were obtained from the transcriptome data, including floral homeotic genes (e.g., MpAPETALA3) and other non-floral homeotic genes (MpAGL6, etc.). Phylogenetic analysis and spatial expression pattern reflected their putative biological function as their homologues in Arabidopsis. In addition, nine structural genes involved in anthocyanin biosynthesis pathway had been screened out, and tepal color difference was significantly associated with their tissue-dependent differential expression levels. This study provides a relatively comprehensive investigation of the MADS-box family and anthocyanin biosynthetic genes in M. polytepala, and will facilitate our understanding of the regulatory mechanism underlying floral organization and color in basal angiosperms.

Chitosan packaging functionalized with Cinnamodendron dinisii essential oil loaded zein: A proposal for meat conservation.

The nanoencapsulation of essential oils for biodegradable films functionalization is a viable alternative for the production of active food packaging. In this study, the Cinnamodendron dinisii Schwanke essential oil was nanoencapsulated using zein as wall material, and applied in chitosan matrix to produce an active nanocomposite film packaging for food conservation. The chemical composition of the Cinnamodendron dinisii Schwanke essential oil showed a variety of unexplored bioactive compounds, and 1,8-cineole was the major compound. The oil nanoencapsulation produced stable and homogeneous nanoparticles with zeta potential close to 30 mV and polydispersity index lower than 0.2. The nanoparticles size showed a size variation between 70 and 110 nm. The chitosan films obtained functionalized with nanoparticles demonstrated antioxidant activity and antimicrobial activity. The active packaging containing zein nanoparticles was efficient in the conservation of ground beef, stabilizing the deterioration reactions and preserving the color.

Preparation and quality evaluation of a volatile oil microemulsion from Flos magnoliae and Centipeda minima.

In order to improve the water solubility of the volatile oils extracted from Flos magnoliae (FM) and Centipeda minima (CM), they were prepared as a microemulsion (ME), which were then used in the development of an FM and CM volatile oil ME for the treatment of allergic rhinitis (AR). ME was prepared by phase inversion emulsification, and the prescription factors such as emulsifier, co­emulsifier, oil phase, Km, which represents the ratio of the mass of emulsifier to that of the co­emulsifier, and preparation factors such as temperature affecting the formation of the ME were selected according to the formation area of ME in a pseudo­ternary phase diagram. The quality of the ME was evaluated based on its appearance, particle size, Zeta potential and stability. The content of eucalyptol in ME was determined by gas chromatography­mass spectrometry (GC­MS). The cumulative permeability of the ME within 24 h was measured with a transdermal diffusion tester. The results revealed that the best formula for preparation of the ME was as follows: Castor oil polyoxyethylene ether (EL­40) was the emulsifier; the co­emulsifier was anhydrous ethanol; the Km was 2:1; the mixed phase of volatile oil and isopropyl myristate with mass ratio of 1:1 was used as oil phase; and the preparation temperature was 25˚C. The content of eucalyptol in the ME was 2.57 mg/g, and the cumulative permeability of the ME in 24 h was significantly increased compared with that of the reference oil solution. The appearance of the ME was uniform, and the solution was transparent. In conclusion, compared with traditional preparations, FM and CM volatile oil ME is a novel, improved and more effective preparation for the treatment of AR.

Metabolic and transcriptomic analysis related to flavonoid biosynthesis during the color formation of Michelia crassipes tepal.

Michelia crassipes is the only plant with purple flowers amongst Michelia species, and its tepals exhibit an obvious color change from green to purple. In this study, a combination of metabolic and transcriptomic analyses was conducted at three stages of tepals in Michelia crassipes: green tepal, purple spot-containing tepal, and totally purple tepal. Several classes of flavonoid compounds were detected and cyanidin 3-rutinoside and delphinidin 3-glucoside were the major anthocyanins underlying the purple color formation, along with co-pigmentation of flavone compounds represented by luteolin derivatives and flavonol compounds represented by kaempferol and quercetin derivatives. Transcriptome analysis revealed up-regulation of genes encoding enzymes involved in the conversion of phenylpropanoid for flavonoid biosynthesis in Stage 1 vs. Stage 2, whereas up-regulation of most flavonoid biosynthesis genes was observed in Stage 1 vs. Stage 3. MYB, bHLH, and WD40 isoforms, as well as other classes of transcriptional factors, also exhibited differential expression. In addition, differentially expressed genes putatively related to the transport of flavonoids were also identified. The results of the current study provide insight into the regulatory mechanism underlying the color transition from green to purple in Michelia crassipes tepals and describe a complicated network involving PAL, transporter genes, and transcription factors, specifically responsible for the emergence of purple color in Stage 1 vs. Stage 2.

Four New Lignans and IL-2 Inhibitors from Magnoliae Flos.

Four new lignans, a furofuran lignan medioresinol B (10) and three tetrahydrofuran lignans kobusinol C (16), 7'-methoxy magnostellin A (21), and mangnostellin D (23), along with 19 known lignans, were isolated from the flower buds of Magnolia biondii PAMP. The structures of the isolates were elucidated using spectroscopic analysis, mainly one- and two-dimensional NMR, high resolution-MS, and circular dichroism techniques as well as Mosher's esterification method. The anti-allergic effects of the isolated compounds were evaluated by analyzing the inhibition of interleukin-2 (IL-2) expression in Jurkat T-cells. Compounds 11-14 reduced IL-2 expression in a dose-dependent manner.

Phytoremediation of abandoned crude oil contaminated drill sites of Assam with the aid of a hydrocarbon-degrading bacterial formulation.

Environmental deterioration due to crude oil contamination and abandoned drill sites is an ecological concern in Assam. To revive such contaminated sites, afield study was conducted to phytoremediate four crude oil abandoned drill sites of Assam (Gelakey, Amguri, Lakwa, and Borholla) with the aid of two hydrocarbon-degrading Pseudomonas strains designated N3 and N4. All the drill sites were contaminated with 15.1 to 32.8% crude oil, and the soil was alkaline in nature (pH8.0-8.7) with low moisture content, low soil conductivity and low activities of the soil enzymes phosphatase, dehydrogenase and urease. In addition, N, P, K, and C contents were below threshold limits, and the soil contained high levels of heavy metals. Bio-augmentation was achieved by applying Pseudomonas aeruginosa strains N3 and N4 followed by the introduction of screened plant species Tectona grandis, Gmelina arborea, Azadirachta indica, and Michelia champaca. The findings established the feasibility of the phytoremediation of abandoned crude oil-contaminated drill sites in Assam using microbes and native plants.

The constituents of Michelia compressa var. formosana and their bioactivities.

Phytochemical investigation of the heartwood of Michelia compressa afforded forty-four compounds, which were identified by comparison of experimental and literature analytical and spectroscopic data. Some compounds were evaluated for their anti-inflammatory and anticancer bioactivities. The result showed that soemerine (1) and cyathisterol (2) exhibited significant nitric oxide (NO) inhibition, with IC50 values of 8.5±0.3 and 9.6±0.5 µg/mL, respectively. In addition, liriodenine (3) and oliveroline (4) exhibited cytotoxicity to human nasopharyngeal carcinoma (NPC-TW01), non-small cell lung carcinoma (NCI-H226), T cell leukemia (Jurkat), renal carcinoma (A498), lung carcinoma (A549) and fibrosarcoma (HT1080) cell lines with IC50 values in the range of 15.7-3.68 µM.

Osteoblast-like cell response to macro- and micro-patterned carbon scaffolds obtained from the sea rush Juncus maritimus.

Carbon scaffolds with a directional patterned surface were obtained by pyrolysis of the sea rush Juncus maritimus. The structure of the scaffolds was investigated using scanning electron microscopy, mercury porosimetry and interferometric profilometry. X-ray diffraction and x-ray fluorescence were the techniques used for their chemical characterization. The alignment and differentiation of pre-osteoblasts (MC3T3-E1 cell line) incubated on the patterned scaffolds were evaluated by scanning electron microscopy, confocal laser scanning microscopy and by the quantification of the phosphatase alkaline activity and the osteocalcin synthesis. It was found that pyrolysis at 500 °C preserved and even enhanced the natural macro- and micro-patterning of the plant. The results obtained for porosity and chemical composition validated these structures as viable scaffolds for tissue engineering applications. Finally, the patterned surface was confirmed to promote the oriented growth of the pre-osteoblasts MC3T3-E1, not only after short periods of incubation (hours) but also after longer ones (several weeks). The quantification of the cell differentiation markers together with the evaluation of the cell layer morphology up to 28 days of incubation confirmed the differentiation of MC3T3-E1 cells to osteoblasts.

Costunolide causes mitotic arrest and enhances radiosensitivity in human hepatocellular carcinoma cells.

PURPOSE: This work aimed to investigate the effect of costunolide, a sesquiterpene lactone isolated from Michelia compressa, on cell cycle distribution and radiosensitivity of human hepatocellular carcinoma (HCC) cells. METHODS: The assessment used in this study included: cell viability assay, cell cycle analysis by DNA histogram, expression of phosphorylated histone H3 (Ser 10) by flow cytometer, mitotic index by Liu's stain and morphological observation, mitotic spindle alignment by immunofluorescence of alpha-tubulin, expression of cell cycle-related proteins by Western blotting, and radiation survival by clonogenic assay. RESULTS: Our results show that costunolide reduced the viability of HA22T/VGH cells. It caused a rapid G2/M arrest at 4 hours shown by DNA histogram. The increase in phosphorylated histone H3 (Ser 10)-positive cells and mitotic index indicates costunolide-treated cells are arrested at mitosis, not G2, phase. Immunofluorescence of alpha-tubulin for spindle formation further demonstrated these cells are halted at metaphase. Costunolide up-regulated the expression of phosphorylated Chk2 (Thr 68), phosphorylated Cdc25c (Ser 216), phosphorylated Cdk1 (Tyr 15) and cyclin B1 in HA22T/VGH cells. At optimal condition causing mitotic arrest, costunolide sensitized HA22T/VGH HCC cells to ionizing radiation with sensitizer enhancement ratio up to 1.9. CONCLUSIONS: Costunolide could reduce the viability and arrest cell cycling at mitosis in hepatoma cells. Logical exploration of this mitosis-arresting activity for cancer therapeutics shows costunolide enhanced the killing effect of radiotherapy against human HCC cells.

Amborella trichopoda, plasmodesmata, and the evolution of phloem loading.

Phloem loading is the process by which photoassimilates synthesized in the mesophyll cells of leaves enter the sieve elements and companion cells of minor veins in preparation for long distance transport to sink organs. Three loading strategies have been described: active loading from the apoplast, passive loading via the symplast, and passive symplastic transfer followed by polymer trapping of raffinose and stachyose. We studied phloem loading in Amborella trichopoda, a premontane shrub that may be sister to all other flowering plants. The minor veins of A. trichopoda contain intermediary cells, indicative of the polymer trap mechanism, forming an arc on the abaxial side and subtending a cluster of ordinary companion cells in the interior of the veins. Intermediary cells are linked to bundle sheath cells by highly abundant plasmodesmata whereas ordinary companion cells have few plasmodesmata, characteristic of phloem that loads from the apoplast. Intermediary cells, ordinary companion cells, and sieve elements form symplastically connected complexes. Leaves provided with (14)CO(2) translocate radiolabeled sucrose, raffinose, and stachyose. Therefore, structural and physiological evidence suggests that both apoplastic and polymer trapping mechanisms of phloem loading operate in A. trichopoda. The evolution of phloem loading strategies is complex and may be difficult to resolve.

[Litter decomposition of dominant plant species in successional stages in mid-subtropical zone].

The litters of seven tree species representing the dominant plant species in three main successional stages in subtropical China, i.e., Pinus massoniana in early successional forests, Schima superba and Cinnamanun camphora in transitional forests, and Castanopsis eyeri, Cyclobalanopsis gracilis, Cyclobalanopsis glauca, and Michelia chapensis in late successional forests, were collected, and their decomposition rates were measured with litterbags. The results showed that M. chapensls and C. gracilis had the highest litter decomposition rate (k = 1.12 and 0.89, respectively), followed by C. camphora and S. superba (k = 0.61 and 0.55, respectively), and P. massoniana (k = 0.51), indicating that there was a trend of litter decomposition rate being increased with succession stage. Litter decomposition rate had significant correlations with the litter' s initial P, N, and lignin contents, lignin/N ratio (P < 0.01), and C/N ratio (P < 0.05), suggesting that the initial P, N, and lignin contents and lignin/N ratio of leaf litter could be the good indictors of litter decomposition rate.

Construction costs and physico-chemical properties of the assimilatory organs of Nepenthes species in Northern Borneo.

BACKGROUND AND AIMS: Species of the Nepenthaceae family are under-represented in studies of leaf traits and the consequent view of mineral nutrition and limitation in carnivorous plants. This study is aimed to complement existing data on leaf traits of carnivorous plants. METHODS: Physico-chemical properties, including construction costs (CC), of the assimilatory organs (leaf and pitcher) of a guild of lowland Nepenthes species inhabiting heath and/or peat swamp forests of Brunei, Northern Borneo were determined. KEY RESULTS: Stoichiometry analyses indicate that Nepenthes species are nitrogen limited. Most traits vary appreciably across species, but greater variations exist between the assimilatory organs. Organ mass per unit area, dry matter tissue concentration (density), nitrogen (N), phosphorus (P), carbon, heat of combustion (H(c)) and CC values were higher in the leaf relative to the pitcher, while organ thickness, potassium (K) and ash showed the opposite trend. Cross-species correlations indicate that joint rather than individual consideration of the leaf and the pitcher give better predictive relationships between variables, signalling tight coupling and functional interdependence of the two assimilatory organs. Across species, mass-based CC did not vary with N or P, but increases significantly with tissue density, carbon and H(c), and decreases with K and ash contents. Area-based CC gave the same trends (though weaker in strength) in addition to a significant positive correlation with tissue mass per unit area. CONCLUSIONS: The lower CC value for the pitcher is in agreement with the concept of low marginal cost for carnivory relative to conventional autotrophy. The poor explanatory power of N, P or N : P ratio with CC suggests that factors other than production of expensive photosynthetic machinery (which calls for a high N input), including concentrations of lignin, wax/lipids or osmoregulatory ions like K(+), may give a better explanation of the CC variation across Nepenthes species.

Natural cis-solamin is a mixture of two tetra-epimeric diastereoisomers: biosynthetic implications for Annonaceous acetogenins.

The anti-tumour natural product cis-solamin has been shown to occur as a mixture two of tetra-epimeric diastereoisomers 1A and 1B, whereas solamin (6) was isolated as a single diastereoisomer; the biosyntheses of 1A/B and 6 are likely to involve enzyme-mediated cyclohydrations of the bis-epoxide acetogenins anti-diepomuricanin A2 and syn-diepomuricanin A1 respectively, where addition of water occurs regioselectively at either C15 or C20.

RNA editing of the ribosomal protein S13 transcripts in magnolia and sunflower mitochondria.

To obtain information on ribosomal protein gene content and RNA editing in plant mitochondria, we investigated the conservation of the gene for ribosomal protein S13 (rps13) in the mitochondrial genomes of Magnolia spp. and Helianthus annuus. A complete rps13 open reading frame was identified in the mitochondrial genome of Magnolia and Helianthus. Comparison of genomic and cDNA sequences revealed that transcripts from the mitochondrial rps13 locus in both Magnolia and Helianthus undergo RNA editing suggesting the functionality of the related gene. Six and four editing events in the rps13 transcript population of Magnolia and Helianthus mitochondria, respectively, cause critical changes of codons and, consequently, induce 5 and 3.4% amino acid modifications in the respective genomically-encoded S13 polypeptides. Both editing pattern and efficiency of RNA editing differ in the reverse transcription (RT)-PCR-derived cDNA populations from the rps13 locus from the analyzed plant species with only the Magnolia rps13 being fully edited.

Influence of elevated CO2 and mycorrhizae on nitrogen acquisition: contrasting responses in Pinus taeda and Liquidambar styraciflua.

An understanding of root system capacity to acquire nitrogen (N) is critical in assessing the long-term growth impact of rising atmospheric CO2 concentration ([CO2]) on trees and forest ecosystems. We examined the effects of mycorrhizal inoculation and elevated [CO2] on root ammonium (NH4+) and nitrate (NO3-) uptake capacity in sweetgum (Liquidambar styraciflua L.) and loblolly pine (Pinus taeda L.). Mycorrhizal treatments included inoculation of seedlings with the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith in sweetgum and the ectomycorrhizal (EM) fungus Laccaria bicolor (Maire) Orton in loblolly pine. These plants were then equally divided between ambient and elevated [CO2] treatments. After 6 months of treatment, root systems of both species exhibited a greater uptake capacity for NH4+ than for NO3-. In both species, mycorrhizal inoculation significantly increased uptake capacity for NO3-, but not for NH4+. In sweetgum, the mycorrhizal effect on NO3- and NH4+ uptake capacity depended on growth [C02]. Similarly, in loblolly pine, the mycorrhizal effect on NO3- uptake capacity depended on growth [CO2], but the effect on NH4+ uptake capacity did not. Mycorrhizal inoculation significantly enhanced root nitrate reductase activity (NRA) in both species, but elevated [CO2] increased root NRA only in sweetgum. Leaf NRA in sweetgum did not change significantly with mycorrhizal inoculation, but increased in response to [CO2]. Leaf NRA in loblolly pine was unaffected by either treatment. The results indicate that the mycorrhizal effect on specific root N uptake in these species depends on both the form of inorganic N and the mycorrhizal type. However, our data show that in addressing N status of plants under high [CO2], reliable prediction is possible only when information about other root system adjustments (e.g., biomass allocation to fine roots) is simultaneously considered.