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The effect of Zn on Cd accumulation in rice varies under flooding and drainage conditions, and the underlying mechanism during uptake and transport from the soil to grains remains unclear. Isotope fractionation and gene expression were investigated using pot experiments under distinct water regimes and with Zn addition to gain a deeper understanding of the molecular effects of Zn on Cd uptake and transport in rice. The higher OsHMA2 expression but constitutively lower expression of zinc-regulated, iron-regulated transporter-like protein ( ZIP ) family genes in roots under the drainage regime than the flooding regime caused the enrichment of nonheavy Zn isotopes in the shoots relative to roots but minimally affected Cd isotopic fractionation. Drainage regime seem to exert a striking effect on the root-to-shoot translocation of Zn rather than Cd, and increased Zn transport via OsHMA2. The changes in expression patterns in response to Zn addition were similar to those observed upon switching from the flooding to drainage regime, except for OsNRAMP1 and OsNRAMP5 . However, soil solution-to-rice plants and root-to-shoot fractionation toward light Zn isotopes with Zn addition (Δ 66 Zn rice plant-soil solution = -0.49 to -0.40‰, Δ 66 Zn shoot-root = -0.36 to -0.27‰) indicated that Zn transport occurred via nonspecific uptake pathways and OsHMA2, respectively. Accordingly, the less pronounced and minimally varied Cd isotope fractionation suggested that OsNRAMP5 and OsHMA2 are crucial for Cd uptake and root-to-shoot transport, respectively, facilitating Cd accumulation in grains. This study demonstrated that a high Zn supply promotes Cd uptake and root-to-shoot transport in rice by sharing distinct pathways, and by utilizing a non-Zn-sensitive pathway with a high affinity for Cd.

Mercury (Hg) contamination in paddy soils poses a health risk to rice consumers and the environmental behavior of Hg determines its toxicity. Thus, the variations of Hg speciation are worthy of exploring. In this study, microcosm and pot experiments were conducted to elucidate Hg transformation, methylation, bioaccumulation, and risk coupled with biogeochemical cycling of key elements in a Hg-polluted alkaline paddy soil. In microcosm and pot experiments, organic- and sulfide-bound and residual Hg accounted for more than 98% of total Hg, and total contents of dissolved, exchangeable, specifically adsorbed, and fulvic acid-bound Hg were less than 2% of total Hg, indicating a low mobility and environmental risk of Hg. The decrease of pH aroused from Fe(III), SO 4 2- , and NO 3 - reduction promoted Hg mobility, whereas the increase of pH caused by Fe(II), S 2- , and NH 4 + oxidation reduced available Hg contents. Moreover, Fe-bearing minerals reduction and organic matter consumption promoted Hg mobility, whereas the produced HgS and Fe(II) oxidation increased Hg stability. During flooding, a fraction of inorganic Hg (IHg) could be transported into methylmercury (MeHg), and during drainage, MeHg would be converted back into IHg. After planting rice in an alkaline paddy soil, available Hg was below 0.3 mg kg -1 . During rice growth, a portion of available Hg transport from paddy soil to rice, promoting Hg accumulation in rice grains. After rice ripening, IHg levels in rice tissues followed the trend: root > leaf > stem > grain, and IHg content in rice grain exceed 0.02 mg kg -1 , but MeHg content in rice grain meets daily intake limit (37.45 μg kg -1 ). These results provide a basis for assessing the environmental risks and developing remediation strategies for Hg-contaminated redox-changing paddy fields as well as guaranteeing the safe production of rice grains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Iron (Fe) isotopes were utilized to provide insights into the temporal changes underlying Fe uptake and translocation during rice growth (tillering, jointing, flowering, and maturity stages) in soil-rice systems under typical flooding-drainage alternation. Fe isotopic composition (δ 56 Fe values) of the soil solution generally decreased at vegetative stages in flooding regimes but increased during grain-filling. Fe plaques were the prevalent source of Fe uptake, as indicated by the concurrent increase in the δ 56 Fe values of Fe plaques and rice plants during rice growth. The increasing fractionation magnitude from stem/nodes I to flag leaves can be attributed to the preferred phloem transport of light isotopes toward grains, particularly during grain-filling. This study demonstrates that rice plants take up heavy Fe isotopes from Fe plaque and soil solution via strategy II during flooding and the subsequent drainage period, respectively, thereby providing valuable insights into improving the nutritional quality during rice production.

Mercury (Hg) is one of the most dangerous contaminants and has sparked global concern since it poses a health risk to humans when consumed through rice. Sulfur (S) is a crucial component for plant growth, and S may reduce Hg accumulation in rice grains. However, the detailed effects of S and the mechanisms underlying S-mediated responses in Hg-stressed rice plants remain unclear. Currently, to investigate the effects of S addition on rice growth, Hg accumulation, physiological indexes, and gene expression profiles, rice seedlings were hydroponically treated with Hg (20 µmol/L HgCl 2 ) and Hg plus elemental sulfur (100 mg/L). S application significantly reduced Hg accumulation in Hg-stressed rice roots and alleviated the inhibitory effects of Hg on rice growth. S addition significantly reduced Hg-induced reactive oxygen species generation, membrane lipid peroxidation levels, and activities of antioxidant enzymes while increasing glutathione content in leaves. Transcriptomic analysis of roots identified 3,411, 2,730, and 581 differentially expressed genes in the control (CK) vs. Hg, CK vs. Hg + S, and Hg vs. Hg + S datasets, respectively. The pathway of S-mediated biological metabolism fell into six groups: biosynthesis and metabolism, expression regulation, transport, stimulus response, oxidation reduction, and cell wall biogenesis. The majority of biological process-related genes were upregulated under Hg stress compared with CK treatment, but downregulated in the Hg + S treatment. The results provide transcriptomic and physiological evidence that S may be critical for plant Hg stress resistance and will help to develop strategies for reduction or phytoremediation of Hg contamination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Anions accompanying inorganic fertilizers, such as chloride and sulfate ions, potentially affect the solubility, uptake, and transport of Cd to rice grains. However, the role of anions in controlling Cd transport in the soil-soil solution-Fe plaque-rice plant continuum remains poorly understood. Cd isotope ratios were applied to Cd-contaminated soil pots, hydroponic rice, and adsorption experiments with or without KCl and K 2 SO 4 treatments to decipher transport processes in the complex soil-rice system. The chloride and sulfate ions increased the Cd concentrations in the soil solution, Fe plaque, and rice plants. Accordingly, the magnitude of positive fractionation from soil to the soil solution was less pronounced, but that between soil and Fe plaque or rice plant is barely varied. The similar isotope composition of Fe plaque and soil, and the similar fractionation magnitude between Fe plaque and the solution and between goethite and the solution, suggested that desorption-sorption between iron oxides and the solution could be important at the soil-soil solution-Fe plaque continuum. This study reveals the roles of chloride and sulfate ions: (i) induce the mobility of light Cd isotopes from soil to the soil solution, (ii) chloro-Cd and sulfato-Cd complexes contribute to Cd immobilization in the Fe plaque and uptake into roots, and (iii) facilitate second leaves/node II-to-grain Cd transport within shoots. These results provide insights into the anion-induced Cd isotope effect in the soil-rice system and the roles of anions in facilitating Cd migration and transformation.

Cd speciation in soil and its transport to rice roots are influenced by the soil pH, oxidation-reduction potential, and mineral transformation; however, the immobilization and migration of Cd in soil-rice systems with different pH values under distinct water regimes remain unclear. This study used Cd isotope fractionation, soil physical analysis, and root gene quantification to elucidate the immobilization and transport of Cd in different soil-rice systems. In drainage soils, the high soil pH enhanced the transformation and magnitude of negative fractionation of Cd from MgCl 2 extract to FeMn oxide-bound pool; however, it favored Cd uptake and root-to-grain transport. Compared with drainage regimes, the flooding regimes shifted fractionation toward heavy isotopes from MgCl 2 -extracted Cd to FeMn oxide-bound Cd in acidic soils (∆ 114/110 Cd MgCl2 extract - FeMn oxide-bound Cd  = -0.09 ± 0.03 ‰) and to light isotopes from MgCl 2 -extracted Cd to carbonate-bound Cd in neutral and alkaline soils (∆ 114/110 Cd MgCl2 extract - carbonate-bound Cd  = 0.29-0.40 ‰). The submerged soils facilitated the forming of carbonate and poorly crystalline minerals (such as ferrihydrite), which were transformed into highly crystalline forms (such as goethite). These results demonstrated that the dissolution-precipitation process of iron oxides was essential for controlling soil Cd availability under flooding regimes, and the relative contribution of carbonate minerals to Cd immobilization was promoted by a high soil pH. Flooding regimes induced lower expressions of OsNRAMP1 and OsNRAMP5 to limit the uptake of light Cd isotopes from MgCl 2 -extract pool, whereas a teeter-totter effect on gene expression patterns in roots (including those of OsHMA3 and OsHMA2) limited the transport of heavy Cd isotopes from root to grain. These findings demonstrate that flooding regimes could exert multiple effects on soil Cd immobilization and Cd transport to grain. Moreover, alkaline soil was conducive to forming carbonate minerals to sequester Cd., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Paddy fields located around estuaries suffer from seawater intrusion, and how and to what extent salinity levels influence Cd accumulation in rice grains is still unclear. Pot experiments were carried out by cultivating rice under alternating flooding and drainage conditions with different salinity levels (0.2‰, 0.6‰ and 1.8‰). The Cd availability was greatly enhanced at 1.8‰ salinity due to the competition for binding sites by cations and the formation of Cd complexation with anions, which also contributed to Cd uptake by rice roots. The soil Cd fractions were investigated and found that the Cd availability significantly decreased during flooding stage, while it rapidly increased after soil drainage. During drainage stage, Cd availability was greatly enhanced at 1.8‰ salinity mainly attributed to the formation of CdCl n 2-n . The kinetic model was established to quantitatively evaluate Cd transformation, and it found that the release of Cd from organic matter and Fe-Mn oxides was greatly enhanced at 1.8‰ salinity. The results of pot experiments showed that there was a significant increase in Cd content in rice roots and grains in the treatment of 1.8‰ salinity, because the increasing salinity induced an increase in Cd availability and upregulation of key genes regulating Cd uptake in rice roots. Our findings elucidated the key mechanisms by which high salinity enhanced Cd accumulation in rice grains, and more attention should be given to the food safety of rice cultivated around estuaries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Arsenic (As) from mine wastewater is a significant source for acidic paddy soil pollution, and its mobility can be influenced by alternating redox conditions. However, mechanistic and quantitative insights into the biogeochemical cycles of exogenous As in paddy soil are still lacking. Herein, the variations of As species in paddy soil spiking with As(III) or As(V) were investigated in the process of 40 d of flooding followed 20 d of drainage. During flooding process, available As was immobilized in paddy soil spiking As(III) and the immobilized As was activated in paddy soil spiking As(V) owing to deprotonation. The contributions of Fe oxyhydroxides and humic substances (HS) to As immobilization in paddy soil spiking As(III) were 80.16% and 18.64%, respectively. Whereas the contributions of Fe oxyhydroxides and HS to As activation in paddy soil spiking As(V) were 47.9% and 52.1%, respectively. After entering drainage, available As was mainly immobilized by Fe oxyhydroxides and HS and adsorbed As(III) was oxidized. The contribution of Fe oxyhydroxides to As fixation in paddy soil spiking As(III) and As(V) was 88.82% and 90.26%, respectively, and of HS to As fixation in paddy soil spiking As(III) and As(V) was 11.12% and 8.95%, respectively. Based on the model fitting results, the activation of Fe oxyhydroxides and HS bound As followed with available As(V) reduction were key processes during flooding. This may be because the dispersion of soil particles and release of soil colloids activated the adsorbed As. Immobilization of available As(III) by amorphous Fe oxyhydroxides followed with adsorbed As(III) oxidation were key processes during drainage. This may be ascribe to the occurrence of coprecipitation and As(III) oxidation mediated by reactive oxygen species from Fe(II) oxidation. The results are beneficial for a deeper understanding of As species transformation at the interface of paddy soil-water as well as an estimation pathway for the impacts of key biogeochemical cycles on exogenous As species under a redox-alternating condition., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Grain filling is the key period that causes excess cadmium (Cd) accumulation in rice grains. Nevertheless, uncertainties remain in distinguishing the multiple sources of Cd enrichment in grains. To better understand the transport and redistribution of Cd to grains upon drainage and flooding during grain filling, Cd isotope ratios and Cd-related gene expression were investigated in pot experiments. The results showed that the Cd isotopes in rice plants were much lighter than those in soil solutions (∆ 114/110 Cd rice-soil solution  = -0.36 to -0.63 ‰) but moderately heavier than those in Fe plaques (∆ 114/110 Cd rice-Fe plaque  = 0.13 to 0.24 ‰). Calculations revealed that Fe plaque might serve as the source of Cd in rice (69.2 % to 82.6 %), particularly upon flooding at the grain filling stage (82.6 %). Drainage at the grain filling stage yielded a larger extent of negative fractionation from node I to the flag leaves (∆ 114/110 Cd flag leaves-node I  = -0.82 ± 0.03 ‰), rachises (∆ 114/110 Cd rachises-node I  = -0.41 ± 0.04 ‰) and husks (∆ 114/110 Cd rachises-node I  = -0.30 ± 0.02 ‰), and significantly upregulated the OsLCT1 (phloem loading) and CAL1 (Cd-binding and xylem loading) genes in node I relative to that upon flooding. These results suggest that phloem loading of Cd into grains and transport of Cd-CAL1 complexes to flag leaves, rachises and husks were simultaneously facilitated. Upon flooding of grain filling, the positive fractionation from the leaves, rachises and husks to the grains (∆ 114/110 Cd flag leaves/rachises/husks-node I  = 0.21 to 0.29 ‰) is less pronounced than those upon drainage (∆ 114/110 Cd flag leaves/rachises/husks-node I  = 0.27 to 0.80 ‰). The CAL1 gene in flag leaves is down-regulated relative to that upon drainage. Thus, the supply of Cd from the leaves, rachises and husks to the grains is facilitated during flooding. These findings demonstrate that the excess Cd was purposefully transported to grain via xylem-to-phloem within nodes I upon the drainage during grain filling, and the expression of genes responsible for encoding ligands and transporters together with isotope fractionation could be used to tracking the source of Cd transported to rice grain., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

To estimate the risks and developing remediation strategies for the mercury (Hg)-contaminated soils, it is crucial to understand the mechanisms of Hg transformation and migration in the redox-changing paddy fields. In present study, a Hg-spiked acidic paddy soil (pH 4.52) was incubated under anoxic conditions for 40 d and then under oxic conditions for 20 d. During anoxic incubation, the water-soluble, exchangeable, specifically adsorbed, and fulvic acid-complexed Hg decreased sharply, whereas the humic acid-complexed Hg, organic, and sulfide-bound Hg gradually increased, which were mainly ascribed to the enhanced adsorption on the surface of soil minerals with an increase in soil pH, complexation by organic matters, precipitation as HgS, and absorption by soil colloids triggered by reductive dissolution of Fe(III) oxides. By contrast, after oxygen was introduced into the system, a gradual increase in available Hg occurred with decreasing soil pH, decomposition of organic matters and formation of Fe(III) oxides. A kinetic model was established based on the key elementary reactions to quantitatively estimate transformation processes of Hg fractions. The model matched well with the modified Tessier sequential extraction data, and suggested that large molecular organic matter and humic acid dominated Hg complexation and immobilization in acidic paddy soils. The content of methylmercury increased and reached its peak on anoxic 20 d. Sulfate-reducing bacteria Desulfovibrio and Desulfomicrobium were the major Hg methylating bacteria in the anoxic stage whereas demethylating microorganisms Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_12 began to grow after oxygen was introduced. These new dynamic results provided new insights into the exogenous Hg transformation processes and the model could be used to predict Hg availability in periodically flooded acidic paddy fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Nitrate reduction coupled with arsenic (As) oxidation strongly influences the bioavailability and toxicity of As in anaerobic environments. In the present study, five representative paddy soils developed from different parent materials were used to investigate the universality and characteristics of nitrate reduction coupled with As oxidation in paddy soils. Experimental results indicated that 99.8 % of highly toxic aqueous As(III) was transformed to dissolved As(V) and Fe-bound As(V) in the presence of nitrate within 2-8 d, suggesting that As was apt to be reserved in its low-toxic and nonlabile form after nitrate treatment. Furthermore, nitrate additions also significantly induced the higher abundance of 16S rRNA and As(III) oxidase (aioA) genes in the five paddy soils, especially in the soils developed from purple sand-earth rock and quaternary red clay, which increased by 10 and 3-5 times, respectively, after nitrate was added. Moreover, a variety of putative novel nitrate-dependent As(III)-oxidizing bacteria were identified based on metagenomic analysis, mainly including Aromatoleum, Paenibacillus, Microvirga, Herbaspirillum, Bradyrhizobium, Azospirillum. Overall, all these findings indicate that nitrate reduction coupled with As(III) oxidation is an important nitrogen-As coupling process prevalent in paddy environments and emphasize the significance of developing and popularizing nitrate-based biotechnology to control As pollution in paddy soils and reduce the risk of As compromising food security., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The bioavailability of arsenic (As) is influenced by ammonium (NH 4 + -N) fertilization, but the underlying mechanisms controlling As transformation in soil-rice systems are still not fully understood. The effects of two NH 4 + -N fertilizers, urea and NH 4 HCO 3 , on the transformation of As in a paddy soil with low organic matter content and transfer in rice plants were investigated. Treatments with urea and NH 4 HCO 3 significantly increased arsenite (As(III)) concentration in porewater, bioavailable As in rhizosphere soil, and the relative abundance of the As(V) respiratory reductase gene ( arrA ) and As(III) methyltransferase gene ( arsM ). Furthermore, the relative expression of As transporter genes in rice roots, such as OsLsi1 , OsLsi2 , and OsLsi3 , was upregulated, and the translocation efficiency of As(III) from rice roots to brown rice was promoted. Subsequently, As(III) accumulation in brown rice significantly increased. Therefore, attention should be paid to As-contaminated paddy fields with NH 4 + -N fertilization.

Mercury (Hg) is a toxic and nonessential element for organisms, and its contamination in the environment is a global concern. Previous research has shown that Hg stress may cause severe damage to rice roots; however, the transcriptomic changes in roots and physio-biochemical responses in leaves to different levels of Hg stress are not fully understood. In the present study, rice seedlings were exposed to 20, 80, and 160 μM HgCl 2 for three days in hydroponic experiments. The results showed that the majority of Hg was accumulated in rice roots after Hg exposure, and the 80- and 160-μM Hg stresses significantly increased the root-to-shoot translocation factors relative to 20-μM Hg stress, resulting in elevated Hg concentrations in rice shoots. Only the 160-μM Hg stress significantly inhibited root growth compared with the control, while photosynthesis capacity in leaves was significantly reduced under Hg stress. RNA transcriptome sequencing analyses of the roots showed that common responsive differentially expressed genes were strongly associated with glutathione metabolism, amino acid biosynthesis, and secondary metabolite metabolism, which may play significant roles in Hg accumulation by rice plants. Nine crucial genes identified by protein-protein interaction network analysis may be used as candidate target genes for further investigation of the detoxification mechanism, encoding proteins involved in jasmonic acid synthesis, sugar metabolism, allene oxide synthase, glutathione peroxidase, dismutase, and catalase. Furthermore, physio-biochemical analyses of the leaves indicated that higher production of reactive oxygen species was induced by Hg stress, while glutathione and antioxidant enzymes may play crucial roles in Hg detoxification. Our findings provide transcriptomic and physio-biochemical features of rice roots and shoots, which advance our understanding of the responsive and detoxification mechanisms in rice under different levels of Hg stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Seawater intrusion can cause environmental risks to paddy soils around estuaries, but the impacts on the availability of heavy metals are still unclear. River water and sea water were collected along the river of an estuary. A stirred-flow experiment was conducted to examine the Cd desorption behavior in Cd-contaminated paddy soil. While the pH increased with increasing salinity levels, more Cd was released with increasing salinity, suggesting that Cd competition by cations and complexation by anions, but not pH, dominated the release of Cd from soils. Moreover, paddy soil was incubated at different salinities under alternating redox conditions. The availability of Cd, as indicated by the diffusive gradients in thin film (DGT), became relatively high with increasing salinity levels during the initial anaerobic and later aerobic stages. The available Cd fractions substantially decreased under anaerobic condition, and then rapidly increased under aerobic condition. When oxygen was introduced into the system, Cd associated with organic matter and Fe-Mn oxides were released, and oxidative dissolution of Cd sulfides was observed, especially in the high salinity treatment. Seawater intrusion affects biogeochemical cycles and can promote rapid export of NH 4 + , Fe 2+ , and SO 4 2- in paddy soils, especially in soils with high salinity. Our findings demonstrated that the high salinity content in paddy soil significantly enhanced the availability of Cd, especially during the drainage stage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Low-lying paddy fields in estuaries can be affected by salt water intrusion; however, it remains unclear how salt water intrusion influences the availability of heavy metals in paddy soil. In this study, batch adsorption and incubation experiments of soil were conducted with different salt water sampled along the estuary to investigate the effects of salt water intrusion on cadmium (Cd) availability. The surface complexation model (SCM) was established to assess the effects of pH on Cd adsorption behavior, which presented typical pH-dependent characteristics. The results of SCM also showed that Cd-chloro complexes became the dominant species when the ionic strength increased. The results of Cd fractions in the incubation experiments revealed a significant increase in dissolved Cd with increasing ionic strength. This may be attributed to the increased point of zero charge (pH pzc ) in the presence of salt water with higher salinity, which likely formed more positive charges on soil surfaces, causing an inhibition of Cd adsorption via electrostatic repulsion. Moreover, higher concentrations of Cl - in salt water favored the formation of Cd-chloro complexes, facilitating Cd release from soil particles. This study provides mechanistic insights into the impact of salt water intrusion on Cd availability at the soil-water interface of paddy soil along the estuary., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rice can simultaneously absorb Fe 2+ via a strategy I-like system and Fe(III)-phytosiderophore via strategy II from soil. Still, it remains unclear which strategy and source of Fe dominate under distinct water conditions. An isotope signature combined with gene expression was employed to evaluate Fe uptake and transport in a soil-rice system under flooded and drained conditions. Rice of flooded treatment revealed a similar δ 56 Fe value to that of soils (Δ 56 Fe rice-soil = 0.05‰), while that of drained treatment was lighter than that of the soils (Δ 56 Fe rice-soil = -0.41‰). Calculations indicated that 70.4% of Fe in rice was from Fe plaque under flooded conditions, while Fe was predominantly from soil solution under drained conditions. Up-regulated expression of OsNAAT1 , OsTOM2 , and OsYSL15 was observed in the root of flooded treatment, while higher expression of OsIRT1 was observed in the drained treatment. These isotopic and genetic results suggested that the Fe(III)-DMA uptake from Fe plaque and Fe 2+ uptake from soil solution dominated under flooded and drained conditions, respectively.

Multiple processes are involved in Cd transfer in rice plants, including root uptake, xylem loading, and immobilization. These processes can be mediated by membrane transporters and can alter Cd speciation by binding Cd to different organic ligands. However, it remains unclear which processes control Cd transport in rice in response to different watering conditions in soil. Herein, Cd isotope fractionation and Cd-related gene expression were employed to investigate the key regulatory mechanisms during uptake, root-to-shoot, and stem-to-leaf transport of Cd in rice grown in pot experiments with Cd-contaminated soil under flooded and non-flooded conditions, respectively. The results showed that soil flooding decreased the Cd concentration in soil porewater and, thereby, Cd uptake and transport in rice. Cd isotopes fractionated negatively from soil porewater to the whole rice (flooded: ∆ 114/110 Cd rice-porewater  = -0.15‰, non-flooded: ∆ 114/110 Cd rice-porewater  = -0.39‰), suggesting that Cd transporters preferentially absorbed light Cd isotopes. The non-flooded treatment revealed an upregulated expression of OsNRAMP1 and OsNRAMP5 genes compared to the flooded treatment, which may partially contribute to its more pronounced porewater-to-rice fractionation. Cd isotopes fractionated positively from roots to shoots under flooded conditions (∆ 114/110 Cd shoot-root  = 0.19‰). However, a reverse direction of fractionation was observed under non-flooded conditions (∆ 114/110 Cd shoot-root  = -0.67‰), which was associated with the substantial upregulation of CAL1 in roots, facilitating xylem loading of Cd-CAL1 complexes with lighter isotopes. After being transported to the shoots, the majority of Cd were detained in stems (44%-55%), which were strongly enriched in lighter isotopes than in the leaves (∆ 114/110 Cd leaf-stem  = 0.77 to 1.01‰). Besides the Cd-CAL1 transported from the roots, the expression of OsPCS1 and OsHMA3 in the stems could also favor the enrichment of Cd-PCs with lighter isotopes, leaving heavier isotopes to be transported to the leaves. The higher expression levels of OsMT1e in older leaves than in younger leaves implied that Cd immobilization via binding to metallothioneins like OsMT1e may favor the enrichment of lighter isotopes in older leaves. The non-flooded treatment showed lighter Cd isotopes in younger leaves than the flooded treatment, suggesting that more Cd-CAL1 in the stems and Cd-PCs in the older leaves might be transported to the younger leaves under non-flooded conditions. Our results demonstrate that isotopically light Cd can be preferentially transported from roots to shoots when more Cd is absorbed by rice under non-flooded conditions, and isotope fractionation signature together with gene expression quantification has the potential to provide a better understanding of the key processes regulating Cd transfer in rice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, a novel biodegradable PHBV/PLA/rice hulls (PPRH) composite was applied and tested as biofilm attachment carrier and carbon source in two bioreactors for biological denitrification process. The denitrification performance, effect of operational conditions and microbial community structure of PPRH biofilm were evaluated. The batch experiment results showed that PPRH-packed bioreactor could completely remove 50 mg L -1 of NO 3 - -N at natural pH (ca. 7.5) and room temperature. The continuous flow experiments indicated that high NO 3 - -N removal efficiency (77%-99%) was achieved with low nitrite (<0.48 mg L -1 ) and ammonia (<0.81 mg L -1 ) accumulation, when influent NO 3 - -N concentration was 30 mg L -1 and hydraulic retention time was 2-6 h. Furthermore, the microbial community analysis indicated that bacteria belonging to genus Diaphorobacter in phylum Proteobacteria were the most dominant and major denitrifiers in denitrification. In summary, PPRH composite was a promising carbon source for biological nitrate removal from water and wastewater., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The drainage of rice soils increases Cd solubility and results in high Cd concentrations in rice grains. However, plant Cd uptake is limited by sorption to iron plaques, and Cd redistribution in the plant is regulated by the nodes. To better understand the interplay of Cd uptake and redistribution in rice under drained and flooded conditions, we determined stable Cd isotope ratios and the expression of genes coding transporters that can transport Cd into the plant cells in a pot experiment. In soil, both water management practices showed similar patterns of isotope variation: the soil solution was enriched in heavy isotopes, and the root Fe plaque was enriched in light isotopes. In rice, the leaves were heavier (Δ 114/110 Cd leaf-shoot = 0.17 to 0.96‰) and the nodes were moderately lighter (Δ 114/110 Cd node-shoot = -0.26 to 0.00‰) relative to the shoots under flooded conditions, indicating preferential retention of light isotopes in nodes and export of heavy isotopes toward leaves. This is generally reversed under drained conditions (Δ 114/110 Cd leaf-shoot = -0.25 to -0.04‰, Δ 114/110 Cd node-shoot = 0.10 to 0.19‰). The drained treatment resulted in significantly higher expression of OsHMA2 and OsLCT1 (phloem loading) but lower expression of OsHMA3 (vacuolar sequestration) in nodes and flag leaves relative to the flooded treatment. It appeared that OsHMA2 and OsLCT1 might preferentially transport isotopically heavier Cd, and the excess Cd was purposefully retranslocated via the phloem under drained conditions when the vacuoles could not retain more Cd. Cd in seeds was isotopically heavier than that in stems under both water management practices, indicating that heavy isotopes were preferentially transferred toward seeds via the phloem, leaving light isotopes retained in stems. These findings demonstrate that the Fe plaque preferentially adsorbs and occludes light Cd isotopes on the root surface, and distinct water management practices alter the gene expression of key transporters in the nodes, which corresponds to a change in isotope fractionation between shoots and nodes/leaves.

Foliar application of Si can generally reduce As translocation from roots to shoots in rice; however, it does not always work, particularly under high As stress. Here, the effects of foliar application of nanoscale silica sol on As accumulation in rice were investigated under low (2 μmol/L) and high (8 μmol/L) arsenite stress. The results revealed that foliar Si application significantly decreased the As concentration in shoots under low arsenite stress, but showed different effects under high arsenite stress after 7 days of incubation. The reduction in root-to-shoot As translocation under the 2As+Si treatment was related to the down-regulation of OsLsi1 and OsLsi2 expression and up-regulation of OsABCC1 expression in roots. In the 8As+Si treatment, the expressions of OsLsi1, OsLsi2, and OsABCC1 were significantly promoted, which resulted in substantially higher As accumulation in both the roots and shoots. In the roots, As predominantly accumulated in the symplasts (90.6%-98.3%), in which the majority of As was sequestered in vacuoles (79.0%-94.0%) under both levels of arsenite stress. Compared with that of the 8As treatment, the 8As+Si treatment significantly increased the As concentration in cell walls, but showed no difference in the vacuolar As concentration, which remained constant at approximately 69.1-71.7 mg/kg during days 4-7. It appeared that the capacity of root cells to sequester As in the vacuoles had a threshold, and the excess As tended to accumulate in the cell walls and transfer to the shoots via apoplasts under high arsenite stress. This study provides a better understanding of the different effects of foliar Si application on As accumulation in rice from the view of arsenite-related gene expression and As subcellular distribution in roots., (Copyright © 2021. Published by Elsevier B.V.)

Nitrate (NO 3 - ) is known to be actively involved in the processes of mineralization and heavy metal transformation; however, it is unclear whether and how it affects the bioavailability of antimony (Sb) in paddy soils and subsequent Sb accumulation in rice. Here, the effects of NO 3 - on Sb transformation in soil-rice system were investigated with pot experiments over the entire growth period. Results demonstrated that NO 3 - reduced Sb accumulation in brown rice by 15.6% compared to that in the control. After amendment with NO 3 - , the Sb content in rice plants increased initially and then gradually decreased (in roots by 46.1%). During the first 15 days, the soil pH increased, the oxidation of Sb(III) and sulfides was promoted, but the reduction of iron oxide minerals was inhibited, resulting in the release of adsorbed and organic-bound Sb from soil. The microbial arsenite-oxidizing marker gene aoxB played an important role in Sb(III) oxidation. From days 15 to 45, after NO 3 - was partially consumed, the soil pH decreased, and the reductive dissolution of Fe(III)-bearing minerals was enhanced; consequently, iron oxide-bound Sb was transformed into adsorbed and dissolved Sb species. After day 45, NO 3 - was completely reduced, Sb(V) was evidently reduced to Sb(III), and green rust was generated gradually. Thus, the available Sb decreased due to its enhanced affinity for iron oxides. Moreover, NO 3 - inhibited the reductive dissolution of iron minerals, which ultimately caused low Sb availability. Therefore, NO 3 - can chemically and biologically reduce the Sb availability in paddy soils and alleviate Sb accumulation in rice. This study provides a potential strategy for decreasing Sb accumulation in rice in the Sb-contaminated sites., (Copyright © 2020. Published by Elsevier B.V.)

Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85 DUR1,2/DUR3 -c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85 DUR1,2/DUR3 -c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.

Background: Genes associated with arsenite uptake and transport in rice plants (i.e., OsLsi1, OsLsi2, OsLsi3, OsLsi6 and OsABCC1) have been identified to date. However, their expression over time during the whole growth period of rice under arsenite stress conditions is still poorly understood. In this study, the dynamics of gene expression associated with arsenite transport and arsenic concentrations in different organs of rice were investigated to determine the critical period(s) of arsenite uptake and translocation regulated by gene expression during the whole growth period., Results: The relative expression of OsLsi2 and OsLsi1 in the roots was upregulated and reached its highest value (2 -∆∆Ct  = 4.04 and 1.19, respectively) at the jointing stage (9 weeks after transplantation), in which the arsenic concentration in roots also was the highest at 144 mg/kg. A range from 45.1 to 61.2% of total arsenic accumulated in the roots during seedling to heading stages (3-16 weeks), which was mainly associated with the relatively high expression of OsABCC1 (1.50-7.68), resulting in arsenic located in the vacuoles of roots. Subsequently, the As translocation factor from root to shoot increased over time from heading to milky ripe (16-20 weeks), and 74.3% of the arsenic accumulated in shoots at the milk stage. Such an increase in arsenic accumulation in shoots was likely related to the findings that (i) OsABCC1 expression in roots was suppressed to 0.14-0.75 in 18-20 weeks; (ii) OsLsi3 and OsABCC1 expression in nodes I, II, and III was upregulated to 4.01-25.8 and 1.59-2.36, respectively, in 16-20 weeks; and (iii) OsLsi6 and OsABCC1 expression in leaves and husks was significantly upregulated to 2.03-5.26 at 18 weeks., Conclusions: The jointing stage is the key period for the expression of arsenite-transporting genes in roots, and the heading to milky ripe stages are the key period for the expression of arsenite-transporting genes in shoots, both of which should be considered for regulation during safe rice production in arsenic-contaminated paddy soil.

Microbial arsenite (As(III)) oxidation associated with nitrate (NO 3 - ) reduction might be an important process in diminishing arsenic bioavailability and toxicity to rice when paddy soils are contaminated by arsenic. In a noncontaminated soil, however, the responses of bacterial communities and functional genes to As(III) under nitrate-reducing conditions are poorly understood. In this study, anaerobic paddy soil microcosms were established with As(III) and/or NO 3 - to investigate how the bacterial communities and their functional genes were stimulated during As(III) oxidation and nitrate reduction. Microbial oxidation of As(III) to As(V) was substantially accelerated by nitrate addition, while nitrate reduction was not affected by As(III) addition. Metagenomic analysis revealed that nitrate-reducing bacteria were principally affiliated with Pseudogulbenkiania , with narG , nirS , and norBC genes. Putative As(III)-oxidizing bacteria were dominated by an Azoarcus sp. with As(III) oxidase genes aioA and aioB detected in its draft genome, which also had complete sets of denitrification genes (mainly, napA , nirK , and nosZ ). Quantitive PCR analysis confirmed that the abundance of Azoarcus spp., aioA , and nosZ genes was enhanced by As(III) addition. These findings suggest the importance of Azoarcus - and Pseudogulbenkiania -related spp., both of which showed various physio-ecological characteristics for arsenic and nitrogen biogeochemistry, in coupling As(III) oxidation and nitrate reduction in flooded paddy soil.

Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ethyl carbamate is a well-known carcinogen and widely occurs in Chinese rice wine. To provide more clues to minimise ethyl carbamate accumulation, the levels of possible precursors of ethyl carbamate in Chinese rice wine were investigated by HPLC. Studies of the possible precursors of ethyl carbamate in Chinese raw rice wine with various additives and treatments indicated that significant amounts of urea can account for ethyl carbamate formation. It was also recognised that citrulline is another important precursor that significantly affects ethyl carbamate production during the boiling procedure used in the Chinese rice wine manufacturing process. Besides urea and citrulline, arginine was also found to be an indirect ethyl carbamate precursor due to its ability to form urea and citrulline by microorganism metabolism.

Ethyl carbamate (EC), which is present in Chinese rice wine, has a large potential for carcinogenicity and genotoxicity. EC is produced during the process of rice wine fermentation and storage. High concentrations of precursors, as well as high temperatures, will significantly accelerate the formation of EC. The present work aims to reduce EC formation by optimizing the production process, especially the boiling procedure. With various boiling sterilization temperatures, EC accumulated to different concentrations but the lower the temperature, the less EC was formed. To preserve the quality traits of Chinese rice wine, including biological and non-biological stability, as well as the sugar component, an 80°C boiling temperature is suggested. The present study provides direction for process optimization, which combined with improved production technology and metabolic engineered yeast strains, can reduce the content of EC in Chinese rice wine. Copyright © 2014 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]