Arsenite Antiporter PvACR3 Driven by Its Native Promoter Increases Leaf Arsenic Accumulation in Tobacco.

Pteris vittata is the first-reported arsenic (As) hyperaccumulator, which has been applied to phytoremediation of As-contaminated soil. PvACR3, a key arsenite (AsIII) antiporter, plays an important role in As hyperaccumulation in P. vittata. However, its functions in plants are not fully understood. In this study, the PvACR3 gene was heterologously expressed in tobacco, driven by its native promoter (ProPvACR3). After growing at 5 µM AsIII or 10 µM AsV in hydroponics for 1-5 days, PvACR3-expression enhanced the As levels in leaves by 66.4-113 and 51.8-101%, without impacting the As contents in the roots or stems. When cultivated in As-contaminated soil, PvACR3-expressed transgenic plants accumulated 47.9-85.5% greater As in the leaves than wild-type plants. In addition, PvACR3-expression increased the As resistance in transgenic tobacco, showing that enhanced leaf As levels are not detrimental to its overall As tolerance. PvACR3 was mainly expressed in tobacco leaf veins and was likely to unload AsIII from the vein xylem vessels to the mesophyll cells, thus elevating the leaf As levels. This work demonstrates that heterologously expressing PvACR3 under its native promoter specifically enhances leaf As accumulation in tobacco, which helps to reveal the As-hyperaccumulation mechanism in P. vittata and to enhance the As accumulation in plant leaves for phytoremediation.

Using Fe biofortification strategies to reduce both Ni concentration and oral bioavailability for rice with high Ni.

Due to naturally high Ni or soil Ni contamination, high Ni concentrations are reported in rice, raising a need to reduce rice Ni exposure risk. Here, reduction in rice Ni concentration and Ni oral bioavailability with rice Fe biofortification and dietary Fe supplementation was assessed using rice cultivation and mouse bioassays. Results showed that for rice grown in a high geogenic Ni soil, increases in rice Fe concentration from ∼10.0 to ∼30.0 µg g-1 with foliar EDTA-FeNa application led to decreases in Ni concentration from ∼4.0 to ∼1.0 µg g-1 due to inhibited Ni transport from shoot to grains via down-regulated Fe transporters. When fed to mice, Fe-biofortified rice was significantly (p < 0.01) lower in Ni oral bioavailability (59.9 ± 11.9% vs. 77.8 ± 15.1%; 42.4 ± 9.81% vs. 70.4 ± 6.81%). Dietary amendment of exogenous Fe supplements to two Ni-contaminated rice samples at 10-40 µg Fe g-1 also significantly (p < 0.05) reduced Ni RBA from 91.7% to 61.0-69.5% and from 77.4% to 29.2-55.2% due to down-regulation of duodenal Fe transporter expression. Results suggest that the Fe-based strategies not only reduced rice Ni concentration but also lowered rice Ni oral bioavailability, playing dual roles in reducing rice-Ni exposure.

Novel PvACR3;2 and PvACR3;3 genes from arsenic-hyperaccumulator Pteris vittata and their roles in manipulating plant arsenic accumulation.

Arsenite (AsIII) antiporter ACR3 is crucial for arsenic (As) translocation and sequestration in As-hyperaccumulator Pteris vittata, which has potential for phytoremediation of As-contaminated soils. In this study, two new ACR3 genes PvACR3;2 and PvACR3;3 were cloned from P. vittata and studied in model organism yeast (Saccharomyces cerevisiae) and model plant tobacco (Nicotiana tabacum). Both ACR3s mediated AsIII efflux in yeast, decreasing its As accumulation and enhancing its As tolerance. In addition, PvACR3;2 and PvACR3;3 were expressed in tobacco plant. Localized on the plasma membrane, PvACR3;2 mediated both AsIII translocation to the shoots and AsIII efflux from the roots in tobacco, resulting in 203 - 258% increase in shoot As after exposing to 5 µM AsIII under hydroponics. In comparison, localized to the vacuolar membrane, PvACR3;3 sequestrated AsIII in tobacco root vacuoles, leading to 18 - 20% higher As in the roots and 15 - 36% lower As in the shoots. Further, based on qRT-PCR, both genes were mainly expressed in P. vittata fronds, indicating PvACR3;2 and PvACR3;3 may play roles in AsIII translocation and sequestration in the fronds. This study provides not only new insights into the functions of new ACR3 genes in P. vittata, but also important gene resources for manipulating As accumulation in plants for phytoremediation and food safety.

Pteris vittata coupled with phosphate rock effectively reduced As and Cd uptake by water spinach from contaminated soil.

Arsenic (As) and cadmium (Cd) are ubiquitous in the environment and they are both toxic to humans. When present in soils, they can enter food chain, thereby threatening human health. Water spinach (Ipomoea aquatica) is an important leafy vegetable, which is widely consumed in Asian countries. However, it is efficient in taking up As and Cd from soils and accumulating them in the edible parts. Therefore, it is of significance to reduce its As and Cd content, especially in contaminated soil. In this study, pot experiments were conducted to investigate the ability of As-hyperaccumulator Pteris vittata in reducing As and Cd uptake by water spinach under different phosphorus treatments. P. vittata was grown for 60 d in a contaminated-soil amended with P fertilizer (+P) or phosphate rock (+PR), followed by water spinach cultivation for another 30 d. Plant biomass, As and Cd contents in plants and soils, and soil pH were analyzed. We found that, P. vittata coupled with PR decreased the As concentration in water spinach shoots by 42%, probably due to As uptake by P. vittata. Moreover, P. vittata decreased the Cd accumulation in water spinach by 24-44%, probably due to pH increase of 0.47-0.61 after P. vittata cultivation. Taking together, the results showed that P. vittata coupled with PR decreased the As and Cd content in water spinach, which is of significance for improving food safety and protecting human health.

Expressing Arsenite Antiporter PvACR3;1 in Rice (Oryza sativa L.) Decreases Inorganic Arsenic Content in Rice Grains.

Rice (Oryza sativa) is a major food crop in the world, feeding half of the world's population. However, rice is efficient in taking up toxic metalloid arsenic (As), adversely impacting human health. Among different As species, inorganic As is more toxic than organic As. Thus, it is important to decrease inorganic As in rice to reduce human exposure from the food chain. The arsenite (AsIII) antiporter gene PvACR3;1 from As-hyperaccumulator Pteris vittata decreases shoot As accumulation when heterologously expressed in plants. In this study, three homozygous transgenic lines (L2, L4, and L7) of T3 generation were obtained after transforming PvACR3;1 into rice. At 5 µM of AsIII, PvACR3;1 transgenic rice accumulated 127%-205% higher As in the roots, with lower As translocation than wild type (WT) plants. In addition, at 20 µM of AsV, the transgenic rice showed similar results, indicating that expressing PvACR3;1 increased As retention in the roots from both AsIII and AsV. Furthermore, PvACR3;1 transgenic rice plants were grown in As-contaminated soils under flooded conditions. PvACR3;1 decreased As accumulations in transgenic rice shoots by 72%-83% without impacting nutrient minerals (Mn, Zn, and Cu). In addition, not only total As in unhusked rice grain of PvACR3;1 transgenic lines were decreased by 28%-39%, but also inorganic As was 26%-46% lower. Taken together, the results showed that expressing PvACR3;1 effectively decreased both total As and inorganic As in rice grain, which is of significance to breed low-As rice for food safety and human health.

Phosphate Transporter PvPht1;2 Enhances Phosphorus Accumulation and Plant Growth without Impacting Arsenic Uptake in Plants.

Phosphorus is an important macronutrient for plant growth and is acquired by plants mainly as phosphate (P). Phosphate transporters (Phts) are responsible for P and arsenate (AsV) uptake in plants including arsenic-hyperaccumulator Pteris vittata. P. vittata is efficient in AsV uptake and P utilization, but the molecular mechanism of its P uptake is largely unknown. In this study, a P. vittata Pht, PvPht1;2, was cloned and transformed into tobacco ( Nicotiana tabacum). In hydroponic experiments, all transgenic lines displayed markedly higher P content and better growth than wild type, suggesting that PvPht1;2 mediated P uptake in plants. In addition, expressing PvPht1;2 also increased the shoot/root 32P ratio by 69-92% and enhanced xylem sap P by 46-62%, indicating that PvPht1;2 also mediated P translocation in plants. Unlike many Phts permeable to AsV, PvPht1;2 showed little ability to transport AsV. In soil experiments, PvPht1;2 also significantly increased shoot biomass without elevating As accumulation in PvPht1;2 transgenic tobacco. Taken together, our results demonstrated that PvPht1;2 is a specific P transporter responsible for P acquisition and translocation in plants. We envisioned that PvPht1;2 can enhance crop P acquisition without impacting AsV uptake, thereby increasing crop production without compromising food safety.

Identification and characterization of reactive metabolites in myristicin-mediated mechanism-based inhibition of CYP1A2.

Myristicin belongs to the methylenedioxyphenyl or allyl-benzene family of compounds, which are found widely in plants of the Umbelliferae family, such as parsley and carrot. Myristicin is also the major active component in the essential oils of mace and nutmeg. However, this compound can cause adverse reactions, particularly when taken inappropriately or in overdoses. One important source of toxicity of natural products arises from their metabolic biotransformations into reactive metabolites. Myristicin contains a methylenedioxyphenyl substructure, and this specific structural feature may allow compounds to cause a mechanism-based inhibition of cytochrome P450 enzymes and produce reactive metabolites. Therefore, the aim of this work was to identify whether the role of myristicin in CYP enzyme inhibition is mechanism-based inhibition and to gain further information regarding the structure of the resulting reactive metabolites. CYP cocktail assays showed that myristicin most significantly inhibits CYP1A2 among five CYP enzymes (CYP1A2, CYP2D6, CYP2E1, CYP3A4 and CYP2C19) from human liver microsomes. The 3.21-fold IC50 shift value of CYP1A2 indicates that myristicin may be a mechanism-based inhibitor of CYP1A2. Next, reduced glutathione was shown to block the inhibition of CYP1A2, indicating that myristicin utilized a mechanism-based inhibition. Phase I metabolism assays identified two metabolites, 5-allyl-1-methoxy-2,3-dihydroxybenzene (M1) and 1'-hydroxymyristicin or 2',3'-epoxy-myristicin (M2). Reduced glutathione capturing assays captured the glutathione-M1 adduct, and the reactive metabolites were identified using UPLC-MS(2) as a quinone and its tautomer. Thus, it was concluded that myristicin is a mechanism-based inhibitor of CYP1A2, and the reactive metabolites are quinone tautomers. Additionally, the cleavage process of the glutathione-M1 adduct was analyzed in further detail. This study provides additional information on the metabolic mechanism of myristicin inhibition and improves risk evaluation for this compound.

Reactive metabolite activation by CYP2C19-mediated rhein hepatotoxicity.

1. Rhein, an active ingredient in the root of rhubarb, is used for its beneficial effects in a variety of clinical applications including the treatment of osteoarthritis and diabetic nephropathy. However, its hepatotoxicity has been reported in recent years. Rhein belongs to the conjugate structure which could be activated to reactive metabolites (RMs) inducing side-effects. This study is to explore the relationship between RMs and hepatotoxicity. 2. Based on the early detection of RMs, we have established a series of key technologies to research rhein hepatotoxicity mechanism: IC50 shift experiments and reduced glutathione (GSH) trapping experiment are adopted to identify RMs. The model of low activity of CYP450 enzymes (CYPs) in primary rat hepatocyte is constructed to analyze the relationship between the primary metabolic enzyme and hepatotoxicity of rhein better. 3. The IC50 shift value for CYP2C19 is 1.989, it suggests that CYP2C19 could activate rhein to RM. The structure of RM is epoxide intermediate. Besides, it is found that CYP2C19 is a primary metabolic enzyme for rhein. In the cytotoxicity assay, it is reported that rhein could cause mitochondrial dysfunction. Furthermore, mitochondrial membrane potential (Δψm) and AST levels could be restored by adding inhibitor of CYP2C19 together with rhein, which further shows that CYP2C19 could mediate the hepatotoxicity of rhein. 4. We put forward the possible mechanism that reactive metabolite activation by CYP2C19 mediated rhein hepatotoxicity, it provides important information on predicting in vivo drug-induced liver injury (DILI).