Spatial Metallomics Reveals Preferable Accumulation of Methylated Selenium in a Single Seed of the Hyperaccumulator Cardamine violifolia†.

Cardamine violifolia is a Se hyperaccumulator found in Enshi, China. In this study, spatial metallomics was applied to visualize the distribution and speciation of Se in a single seed of C. violifolia. It was found that Se reached 1729.89 ± 28.14 mg/kg and the main Se species were SeCys and SeMet in bulk seeds. Further in situ study on a single seed found that the methylated Se species located mostly in the episperm. This is the first visualized evidence of the in situ distribution of methylated Se species in the seeds of C. violifolia. In all, spatial metallomics finds a preferable accumulation of methylated Se species in the seed coat, which deepens the understanding of the tolerance of Se by C. violifolia. The protocol applied in this study may also be used for the understanding of the tolerance of heavy metals/metalloids in other hyperaccumulators.

Retardation factors in controlling the transport of inorganic, organic, and particulate phosphorus in fluvo-aquic soil.

Excessive application of fertilizers has caused a high load of phosphorus (P) in the North China Plain. The fate of P and its effects on aquatic ecosystems depend on its chemical speciation in soils. However, few studies systematically investigated the transport and retardation of different P species in the fluvo-aquic soil. In this study, the transport of inorganic P (orthophosphate, PO4), organic P (phytic acid, PA) and particulate P (hydroxyapatite nanoparticles, nHAP) in the fluvo-aquic soil were investigated by column experiments, and their retardation from major soil components such as kaolin, CaCO3, Al2O3, and goethite (GT) was also investigated by monitoring breakthrough curves and fitting transport models. The transport of P species in fluvo-aquic soil followed the order of PO4 > PA > nHAP. A high fraction of increased clay and mineral particle-associated P (P-E) was observed for PO4 and PA; while significant Ca-associated P (P-Ca) for nHAP. Under the experimental conditions, both CaCO3 and GT were the most influential factors for PO4, PA, and nHAP retention. Goethite strongly inhibited PO4 transport due to its high PO4 adsorption capacity, while CaCO3 strongly inhibited PA transport due to its strong association with PA under alkaline conditions. Both CaCO3 and GT can severely inhibit nHAP transport due to the favorable electrostatic conditions as well as the Ca2+ bridging effect. These results indicated that CaCO3 played a key role in regulating the retention of organic P and particulate P in the calcareous soil, and also suggested the important role of Fe (hydr)oxides in controlling the transport of inorganic P, which could out-compete that of CaCO3.

Expression and Functional Study of BcWRKY1 in Baphicacanthus cusia (Nees) Bremek.

Baphicacanthus cusia (Nees) Bremek (B. cusia) is an important medicinal plant. Its effective substances including indigo and indirubin are metabolites in indoleacetate metabolic pathway. Based on a previous transcriptome sequencing analysis, a WRKY transcription factor, BcWRKY1, in B. cusia was identified, showing significant correlation with effective substances from B. cusia. In this study, BcWRKY1 was cloned by reverse transcription-polymerase chain reaction (RT-PCR). Further analysis showed that the BcWRKY1 gene was 916 bp in length, containing three exons and two introns. The open reading frame (ORF) of BcWRKY1 was 534 bp in length and encoded a WRKY domain-containing protein with 177 amino acids residues. Subcellular localization showed that BcWRKY1 protein was mainly localized in the nucleus. It could bind to the W-box motif and its role in transcriptional activation was confirmed in yeast. The function of BcWRKY1 was investigated by overexpressing BcWRKY1 in Arabidopsis thaliana. Metabolic profiles in wild type and BcWRKY1-OX1 transgenic Arabidopsis thaliana were analyzed with LC-MS. Results showed that the metabolic profile was significantly changed in BcWRKY1-OX1 transgenic Arabidopsis thaliana compared with wild type. Furthermore, indole-related metabolites were significantly increased in BcWRKY1-OX1 transgenic Arabidopsis thaliana, and the metabolic pathway analysis showed that flavonoid biosynthesis was significantly enriched. Overexpression of BcWRKY1 significantly changed flavonoid and indole metabolism and indole-related metabolites were significantly upregulated. We postulated that the BcWRKY1 transcription factor might be involved in the regulation of effective substances metabolism in B. cusia.

Chemical behavior of fluorine and phosphorus in chemical looping gasification using phosphogypsum as an oxygen carrier.

In China, the amount of phosphogypsum (PG) has exceeded 250 million tons with more than 55 million tons of growth rates each year. As the micro constituent, fluorine and phosphorus restrict the resourceful disposal of PG. This paper focused on chemical looping gasification (CLG) which used PG as an oxygen carrier, systematically investigated the gasification performance and chemical behavior of fluorine and phosphorus contained in PG during CLG process. Main conclusions are as follows. The main pollutant of chemical looping gasification process was HF, which was transformed from NaF. Phosphorus transformed from water-soluble phosphorus (Ca(H2PO4)2, Ca(HPO4)) into insoluble Ca3(PO4)2.20 reducing-oxidizing cycles were investigated, and a less and less fluorine content in oxygen carrier was found because its phase transformation from solid NaF to gaseous HF, and the phosphorus content in oxygen carrier changed slightly under the current conditions. The Ca3(PO4)2 particle layers existed in both the middle of the reduced solid particles and the middle of the cycled oxygen carrier particles, confirmed to actually act as a glue between the particles. Furthermore, transformation routes of fluorine and phosphorus during the CLG process were discussed and the generation of syngas in CLG process needed to be purified.

Research on the variations of organics and heavy metals in municipal sludge with additive acetic acid and modified phosphogypsum.

Concentration and fraction distribution of organics and heavy metals in municipal sludge treated by modified phosphogypsum and acetic acid (signed as MPG/HAC) were studied. The results showed that MPG/HAC conditioning significantly produce synergistic enhancement effect to dissolution of unstable heavy metals wrapped in the stable colloid network. Simultaneously, after conditioning, about 45.16% of organics such as proteins, polysaccharides and humic acid in supernatant was degraded, thus dissociating large amount of active group which accelerated immobilization of dissolved heavy metals and weaken its toxicity. In addition, MPG with a porous structure could adsorb unstable heavy metals and transform them into residual fraction, leading to a considerable decrease in their mobility risk level. Besides, linear regression models showed that a strong oxidizability of sludge, and destruction of colloidal network could greatly promote dissolution of unstable heavy metals. Simultaneously, sludge oxidizability and organics degradation rate, and disintegration of extracellular polymeric substances (EPS) layer highly accelerate immobilization of unstable metals. Excepting Cd, environmental risk of Cr, Cu, Pb, Zn, Ni and As can be effectively weakened after conditioning. Additionally, MPG/HAC conditioning might be appropriate for stabilization of Cd, Cr and Zn in water supply sludge, especially for Zn.

Targeted expression of Vitreoscilla hemoglobin improves the production of tropane alkaloids in Hyoscyamus niger hairy roots.

Under hypoxic conditions, the expression of Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68 µg/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.

Research on dewaterability and properties of sewage sludge under modified phosphogypsum and acetic acid pretreatments.

A combined modified phosphogypsum (MPG) and acetic acid (HAC) addition was performed to pretreat sewage sludge for better dewaterability, and dewatering performance, sludge flocs morphology, properties of treated sludge, composition and morphology distribution of extracellular polymeric substances (EPS), bound water release dynamic were investigated. Results suggested that combined addition could highly improve sludge dewatering compared to single addition. Furthermore, after pretreatment, stable sludge network was disintegrated and massive loosely bound EPS (LB-EPS) were destroyed into supernatant, thus increasing amount of organics with molecular weight between 1000 and 7000 Da in soluble EPS (SB-EPS). Simultaneously, about 9.5 kg kg-1 DS (Dry solids) of bound water was released from sludge network.

Investigation on extracellular polymeric substances, sludge flocs morphology, bound water release and dewatering performance of sewage sludge under pretreatment with modified phosphogypsum.

Modified phosphogypsum (MPG) was developed to improve dewaterability of sewage sludge, and dewatering performance, properties of treated sludge, composition and morphology distribution of EPS, dynamic analysis and multiple regression model on bound water release were investigated. The results showed that addition of MPG caused extracellular polymeric substances (EPS) disintegration through charge neutralization. Destruction of EPS promoted the formation of larger sludge flocs and the release of bound water into supernatant. Simultaneously, content of organics with molecular weight between 1000 and 7000 Da in soluble EPS (SB-EPS) increased with increasing of EPS dissolved into the liquid phase. Besides, about 8.8 kg•kg-1DS of bound water was released after pretreatment with 40%DS MPG dosage. Additionally, a multiple linear regression model for bound water release was established, showing that lower loosely bond EPS (LB-EPS) content and specific resistance of filtration (SRF) may improve dehydration performance, and larger sludge flocs may be beneficial for sludge dewatering.

Stable Internal Reference Genes for Normalizing Real-Time Quantitative PCR in Baphicacanthus cusia under Hormonal Stimuli and UV Irradiation, and in Different Plant Organs.

Baphicacanthus cusia (Nees) Bremek, the plant source for many kinds of drugs in traditional Chinese medicine, is widely distributed in South China, especially in Fujian. Recent studies about B. cusia mainly focus on its chemical composition and pharmacological effects, but further analysis of the plant's gene functions and expression is required to better understand the synthesis of its effective compounds. Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful method for gene expression analysis. It is necessary to select a suitable reference gene for expression normalization to ensure the accuracy of RT-qPCR results. Ten candidate reference genes were selected from the transcriptome datasets of B. cusia in this study, and the expression stability was assessed across 60 samples representing different tissues and organs under various conditions, including ultraviolet (UV) irradiation, hormonal stimuli (jasmonic acid methyl ester and abscisic acid), and in different plant organs. By employing different algorithms, such as geNorm, NormFinder, and BestKeeper, which are complementary approaches based on different statistical procedures, 18S rRNA was found to be the most stable gene under UV irradiation and hormonal stimuli, whereas ubiquitin-conjugating enzyme E2 was the best suitable gene for different plant organs. This novel study aimed to screen for suitable reference genes and corresponding primer pairs specifically designed for gene expression studies in B. cusia, in particular for RT-qPCR analyses.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 µM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.