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Carbon (C) and nitrogen (N) metabolism are essential for the growth and maintenance of submersed macrophytes. They are affected by nutrient and light availability and seasonal changes. In this study, we investigated the changes of C/N metabolism and growth of three natural submersed macrophyte populations (i.e., each in a different site), Potamogeton maackianus, in response to water depth gradients with 0.5 m interval in a freshwater lake for four seasons. Changes were identified by measuring the biomass of the sampled macrophytes and metabolite contents of soluble carbohydrate (SC), starch, and free amino acid (FAA) in their organs. Results showed that no significant and consistent changes were found in the SC and FAA contents in leaves and stems from 1.0 m to 5.5 m water depth, as compared to previous results of controlled studies, suggesting a relative stable C/N metabolism in response to the depth gradients in studied natural populations of P. maackianus. The metabolite contents with the most variations appeared mainly because of seasonal changes. The metabolite contents of the macrophyte populations were more closely correlated with the chemical parameters than with the light parameters in water columns. No significant correlations were found between the metabolite contents with the light attenuation coefficient ($K$) and Secchi depth (SD) in water columns. The NO3-N and total phosphorus (TP) contents in water columns were positively correlated with the SC and starch contents in leaves, respectively. The NH4-N content in water columns was negatively related to the FAA content in leaves. Metabolite contents had weak correlations with depth gradients and light parameters, probably because of the physiological integration among individuals by developed stolons and the canopy-forming characteristic of this species. Our results highlighted the importance of water quality to the C/N metabolism in natural population of submersed macrophyte. (C) 2017 Elsevier B.V. All rights reserved.

Ammonium pulse attributed to runoff of urban surface and agriculture following heavy rain is common in inland aquatic systems and can cause profoundly effects on the growth of macrophytes, especially when combined with low light. In this study, three patterns of NH4-N pulse (differing in magnitude and frequency) were applied to examine their effects on the growth of three submersed macrophytes, namely, Myriophyllum spicatum, Potamogeton maackianus, and Vallisneria natans, in terms of biomass, height, branch/ramet number, root length, leaf number, and total branch length under high and low light. Results showed that NH4-N pulse caused negative effects on the biomass of the submerged macrphytes even on the 13th day after releasing NH4-N pulse. The negative effects on M. spicatum were significantly greater than that on V. natans and P. maackianus. The effects of NH4-N pulse on specific species depended on the ammonium loading patterns. The negative effects of NH4-N pulse on P. maackianus were the strongest at high loading with low frequency, and on V. natans at moderate loading with moderate frequency. For M. spicatum, no significant differences were found among the three NH4-N pulse patterns. Low light availability did not significantly aggregate the negative effects of NH4-N pulse on the growth of the submersed macrophytes. Our study contributes to revealing the roles of NH4-N pulse on the growth of aquatic plants and its species specific effects on the dynamics of submerged macrophytes in lakes.

For submersed macrophytes, carbon (C) and nitrogen (N) metabolism is a central factor affecting growth and survival under low light availability. Three common submersed macrophytes, Vallisneria natans, Potamogeton maackianus, and Potamogeton malaianus, were grown at five water depths (1.0, 2.5, 4.0, 5.5, and 7.0 m) to explore mechanism of low light adaptation in terms of C/N metabolism by examining relative growth rate (RGR) and contents of soluble carbohydrate (SC), starch, total carbon (TC), free amino acid (FAA) and total nitrogen (TN) in leaf, stem and root of the plants. With increasing water depth, P. malaianus, P. maackianus and V. natans initiated dying at 4.0 m, 5.5 m and 7.0 m water depths, respectively. natans showed higher RGR than the other two species. For all the plants, the FAA contents increased with increasing water depth, except for the roots of P. maackianus. The TN contents showed a unimodal curve along water depths with the highest in moderate water depth, except for P. malaianus and-the roots of V. natans. For V. natans and P. maackianus, the C metabolic indices (SC, starch, and TC) showed a unimodal curve along water depths with the lowest in moderate water depth in the leaves and stems, except for TC contents in the leaves of P. maackianus. Compared with P. maackianus and P. malaianus, lower SC and FAA contents and higher starch storage in V. natans contributed to its higher tolerance to deeper depths. The nonlinear changes in metabolite contents along water depths for V. natans and P. maackianus demonstrated complex mechanism for low light adaptation, and thus partly explained the wider ranges of colonizing water depths than P. malaianus. (C) 2015 Elsevier B.V. All rights reserved.

With extensive pharmacological actions, quercetin has anti‑oxidant, free radical scavenging, anti‑tumor, anti‑inflammatory, anti‑bacterial and anti‑viral activity. Quercetin also reduces blood glucose and reduces high blood pressure, and has immunoregulation and cardiovascular protection functions. Additionally, it has been reported that it can reduce depression. The current study evaluated whether quercetin protects against inflammation, matrix metalloproteinase‑2 (MMP‑2) activation and apoptosis induction in a rat model of cardiopulmonary resuscitation (CPR), and whether Bmi‑1 expression was involved in the effects. In CPR model rats, treatment with quercetin significantly recovered left ventricular ejection fraction, left ventricular fractional shortening, ejection fraction (%), and left ventricle weight/body weight. Treatment with quercetin significantly inhibited ROS generation, inflammation and MMP‑2 protein expression in the rat model CPR. Finally, quercetin significantly suppressed caspase‑3 activity and activated Bmi‑1 protein expression in the rat model of CPR. The results demonstrated that quercetin protects against inflammation, MMP‑2 activation and apoptosis induction in a rat model of CPR, and that this may be mediated by modulating Bmi‑1 expression.

Anthocyanins are responsible for the different colors of ornamental plants. Grape hyacinth (Muscari armeniacum), a monocot plant with bulbous flowers, is popular for its fascinating blue color. In the present study, we functionally characterized an R2R3-MYB transcription factor gene MaAN2 from M. armeniacum. Our results indicated that MaAN2 participates in controlling anthocyanin biosynthesis. Sequence alignment and phylogenetic analysis suggested that MaAN2 belonged to the R2R3-MYB family AN2 subgroup. The anthocyanin accumulation of grape hyacinth flowers was positively correlated with the expression of MaAN2. And the transcriptional expression of MaAN2 was also consistent with that of M. armeniacum dihydroflavonol 4-reductase (MaDFR) and M. armeniacum anthocyanidin synthase (MaANS) in flowers. A dual luciferase transient expression assay indicated that when MaAN2 was co-inflitrated with Arabidopsis thaliana TRANSPARENT TESTA8 (AtTT8), it strongly activated the promoters of MaDFR and MaANS, but not the promoters of M. armeniacum chalcone synthase (MaCHS), M. armeniacum chalcone isomerase (MaCHI), and M. armeniacum flavanone 3-hydroxylase (MaF3H). Bimolecular fluorescence complementation assay confirmed that MaAN2 interacted with AtTT8 in vivo. The ectopic expression of MaAN2 in Nicotiana tabacum resulted in obvious red coloration of the leaves and much redder flowers. Almost all anthocyanin biosynthetic genes were remarkably upregulated in the leaves and flowers of the transgenic tobacco, and NtAn1a and NtAn1b (two basic helix–loop–helix anthocyanin regulatory genes) were highly expressed in the transformed leaves, compared to the empty vector transformants. Collectively, our results suggest that MaAN2 plays a role in anthocyanin biosynthesis.

Grape hyacinth (Muscari spp.) is a popular ornamental bulbous perennial famous for its blue flowers. To understand the chemical basis of the rich blue colors in this plant, anthocyanin profiles of six blue flowering grape hyacinths as well as one pink and one white cultivar were determined using high-performance liquid chromatography and mass spectrometry. Along with two known compounds, eight putative anthocyanins were identified in the tepals of grape hyacinth for the first time. The accumulation and distribution of anthocyanins in the plant showed significant cultivar and flower development specificity. Violet-blue flowers mainly contained simple delphinidin-type anthocyanins bearing one or two methyl-groups but no acyl groups, whereas white and pink flowers synthesised more complex pelargonidin/cyanidin-derivatives with acyl-moieties but no methyl-groups. The results partially reveal why solid blue, orange or red flowers are rare in this plant in nature. In addition, pelargonidin-type anthocyanins were found for the first time in the genus, bringing more opportunities in terms of breeding of flower color in grape hyacinth.

Summary Through a combination of metabolite analysis with transcriptome sequencing, a new hypothesis was proposed to explain the lack of colour phenotype of the white variant of the blue grape hyacinth., Grape hyacinth (Muscari) is an important ornamental bulbous plant with an extraordinary blue colour. Muscari armeniacum, whose flowers can be naturally white, provides an opportunity to unravel the complex metabolic networks underlying certain biochemical traits, especially colour. A blue flower cDNA library of M. armeniacum and a white flower library of M. armeniacum f. album were used for transcriptome sequencing. A total of 89 926 uni-transcripts were isolated, 143 of which could be identified as putative homologues of colour-related genes in other species. Based on a comprehensive analysis relating colour compounds to gene expression profiles, the mechanism of colour biosynthesis was studied in M. armeniacum. Furthermore, a new hypothesis explaining the lack of colour phenotype of the grape hyacinth flower is proposed. Alteration of the substrate competition between flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) may lead to elimination of blue pigmentation while the multishunt from the limited flux in the cyanidin (Cy) synthesis pathway seems to be the most likely reason for the colour change in the white flowers of M. armeniacum. Moreover, mass sequence data obtained by the deep sequencing of M. armeniacum and its white variant provided a platform for future function and molecular biological research on M. armeniacum.

Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.

Purple turnip Brassica rapa ssp. rapa is highly appreciated by consumers but the metabolites and molecular mechanisms underlying the root skin pigmentation remain open to study. Herein, we analyzed the anthocyanin composition in purple turnip (PT) and green turnip (GT) at five developmental stages. A total of 21 anthocyanins were detected and classified into the six major anthocynanin aglycones. Distinctly, PT contains 20 times higher levels of anthocyanins than GT, which explain the difference in the root skin pigmentation. We further sequenced the transcriptomes and analyzed the differentially expressed genes between the two turnips. We found that PT essentially diverts dihydroflavonols to the biosynthesis of anthocyanins over flavonols biosynthesis by strongly down-regulating one flavonol synthase gene, while strikingly up-regulating dihydroflavonol 4-reductase (DFR), anthocyanidin synthase and UDP-glucose: flavonoid-3-O-glucosyltransferase genes as compared to GT. Moreover, a nonsense mutation identified in the coding sequence of the DFR gene may lead to a nonfunctional protein, adding another hurdle to the accumulation of anthocyanin in GT. We also uncovered several key members of MYB, bHLH and WRKY families as the putative main drivers of transcriptional changes between the two turnips. Overall, this study provides new tools for modifying anthocyanin content and improving turnip nutritional quality.

Flavonoid 3′, 5′-hydoxylase (F3′5′H) is a key enzyme for biosynthesis of the blue anthocyanin pigment delphinidin. A number of F3′5′H genes from dicots have been tested for their effects on flower pigmentation; here F3′5′H from a monocot was tested for its effect on delphinidin accumulation in petals. To this end, F3′5′H (PhF3′5′H) from the orchid Phalaenopsis was expressed under the control of the chalcone synthase promoter in petunia flowers. Quantitative RT-PCR showed that PhF3′5′H was expressed mainly in the petal limb; this expression produced an increase in dihydromyricetin and delphinidin and a change in petal color from pink to deeper pink. To increase the accumulation of delphinidin, Hyacinth HyDFR, which encodes dihydroflavonol 4-reductase, and petunia DifF, which encodes a cytochrome b 5 that is required for full activity of F3′5′H were overexpressed. The HyDFR petunia transformants had a deeper color petal limb, increased dihydromyricetin and delphinidin contents and adaxial petals with a number of blue cells. The flowers of the DifF petunia transformants also showed a slight color change. We also tested PhF3′5′H in Lilium oriental ‘Sorbonne’, where transient PhF3′5′H expression by particle bombardment resulted in purple cells in the petals. Production of blue flowers by Phalaenopsis F3′5′H and hyacinth DFR potentially enables manipulation of flower color in ornamental plants, including production of blue flowers.

The inflorescence of the broad-leafed grape hyacinth, Muscari latifolium, shows an interesting, two-tone appearance with the upper flowers being pale blue and the lower ones purple. To elucidate the mechanism of the differential color development, anatomical research was carried out and a cytological study of the colored protoplasts in which the shapes of the cells accumulating anthocyanin were observed by scanning electron microscopy. Next, vacuolar pH was recorded using a pH meter with a micro combination pH electrode, and the sap's metal-ion content was measured by inductively coupled plasma mass spectrometry. The anthocyanin and co-pigment composition was determined by high-performance liquid chromatography (HPLC). Chemical analyses reveal that the difference in metal-ion content of the two parts was not great. The vacuolar pHs of the upper and lower flowers were 5.91 and 5.84, respectively, with the difference being nonsignificant. HPLC results indicate that the dihydroflavonol and flavonol contents are also very similar in the two sorts of flower. However, the upper flowers contained only delphinidin, whereas the lower flowers also contained cyanidin. The total anthocyanin content in the lower flowers was 4.36 mg g(-1), which is approximately seven times higher than in the upper flowers, while the delphinidin content is four times higher. Quantitative real-time PCR analysis established that the two-tone flower was a result of different expressions of the F3'5'H, F3'H and DFR genes, and these lead to different amounts of anthocyanin.

Previous studies have demonstrated that oxymatrine may inhibit ventricular remodeling and serves an important role in the treatment of cardiovascular disease. The present study investigated whether oxymatrine treatment protects against the effects of cardiopulmonary resuscitation (CPR) via regulation of the transforming growth factor‑β1 (TGF‑β1)/mothers against decapentaplegic (Smad) signaling pathway. A CPR model was established in Sprague‑Dawley (SD) rats by asphyxiation, and rats were subsequently anaesthetized by intraperitoneal injection of chloral hydrate. SD rats were then administered 25 or 50 mg/kg oxymatrine once a day for 4 weeks. Oxymatrine treatment significantly improved troponin I levels, the ejection fraction, hydroxyproline content and the myocardial performance index in model rats. However, treatment with oxymatrine significantly reduced arterial oxygen tension, arterial lactate levels and oxygen extraction. Treatment with oxymatrine following CPR significantly inhibited the protein expression levels of TGF‑β1, TGF‑β1 receptor type 1 and Smad homolog 3 (Smad3) in model rats. The results of this research indicated that oxymatrine treatment may protect against the effects of CPR via regulation of the TGF‑β1/Smad3 signaling pathway and may be a novel drug for CPR in a clinical setting.

The uses of laccase in the degradation and removal of antibiotics have recently been reported because of the high efficiency and environmental friendliness of laccase. However, these removal studies mostly refer to a limited number of antibiotics. In this study, soil adsorption was introduced into the laccase-oxidation system to assist the simultaneous removal of 14 kinds of sulfonamide, tetracycline, and quinolone antibiotics, which differed in structures and chemical properties. The complementary effects of laccase-mediated oxidation and soil adsorption enabled the simultaneous removal. Removal characteristics were determined by a comprehensive consideration of the separate optimum conditions for laccase oxidation and soil adsorption removal experiments. With concentrations of laccase, syringaldehyde (SA), and soil of 0.5mg/mL, 0.5mmol/L, and 50g/L, respectively, and at pH 6 and 25°C, the removal rates of each antibiotic exceeded 70% in 15min and were close to 100% in 180min. Sulfonamide antibiotics (SAs) were removed mainly by laccase oxidation and quinolone antibiotics (QUs) mainly by soil adsorption. Tetracycline antibiotics (TCs) were removed by both treatments in the coupled system, but laccase oxidation dominated. Electrostatic adsorption was speculated to be one of the adsorption mechanisms in soil adsorption with QUs and TCs.