Exploring ecological effects of arsenic and cadmium combined exposure on cropland soil: from multilevel organisms to soil functioning by multi-omics coupled with high-throughput quantitative PCR.

Arsenic (As) and cadmium (Cd) pose potential ecological threats to cropland soils; however, few studies have investigated their combined effects on multilevel organisms and soil functioning. Here, we used collembolans and soil microbiota as test organisms to examine their responses to soil As and Cd co-contamination at the gene, individual, and community levels, respectively, and further uncovered ecological relationships between pollutants, multilevel organisms, and soil functioning. At the gene level, collembolan transcriptome revealed that elevated As concentrations stimulated As-detoxifying genes AS3MT and GST, whereas the concurrent Cd restrained GST gene expression. At the individual level, collembolan reproduction was sensitive to pollutants while collembolan survival wasn't. At the community level, significant but inconsistent correlations were observed between the biodiversity of different soil keystone microbial clusters and soil As levels. Moreover, soil functioning related to nutrient (e.g., carbon, nitrogen, phosphorus, and sulfur) cycles was inhibited under As and Cd co-exposure only through the mediation of plant pathogens. Overall, these findings suggested multilevel bioindicators (i.e., AS3MT gene expression in collembolans, collembolan reproduction, and biodiversity of soil keystone microbial clusters) in cropland soils co-contaminated with As and Cd, thus improving the understanding of the ecotoxicological impact of heavy metal co-contamination on soil ecosystems.

Aluminum adsorption and antimonite oxidation dominantly regulate antimony solubility in soils.

Soil antimony (Sb) contamination occurs globally due to natural processes and human activities. Total Sb concentration in soils fails to assess its ecological risk, while determined by the concentration of available Sb, which is readily for biological uptake. Available Sb in different soils varied significantly according to soil properties. However, so far it is unknown how soil properties regulate Sb availability, and no model has been established to predict it through soil properties. In this study, 19 soils spiked with antimonite [Sb(III)] were used to identify the major factors controlling Sb availability and establish its predicting models. The results showed that available Sb in different soils varied largely depending on the contents of free aluminum (fAl), free iron (fFe) and electric conductivity (EC), which explained 33%, 27% and 24.9% of the total variation, respectively. During the first 42 days of soil aging, fAl and EC effectively predicted the concentrations of available Sb with R2 = 0.64, while during the later stages (70-150 d) of soil aging, fAl content was the unique parameter employed into the predicting model (R2 = 0.53). These results firstly demonstrate that the content of free aluminum (fAl) is the most important factor regulating Sb availability in soils, although the content of fAl is much lower than that of fFe. This finding can help to develop new remediation materials for Sb-contaminated soils. The prediction models can provide promising tools of assessing the ecological risk. In addition, Sb availability was also affected by the oxidation of Sb(III). After 150 days aging, 1-61% of Sb(III) was oxidized to pentavalent Sb [Sb(V)], which was significantly positively correlated with available Sb, suggesting that Sb(III) oxidization mobilizes Sb in soils. All these findings would help to understand Sb migration and transformation in soils, and to develop new strategies for remediating Sb-contaminated soils.

Coupling metabolisms of arsenic and iron with humic substances through microorganisms in paddy soil.

Humic acid (HA) and fulvic acid (FA) are dominating humic substances (HS) in soil. In this study, the effects of HA and FA addition (0.2%-1.5%) on arsenic (As) mobility and microbial community composition in paddy soil were investigated. FA significantly increased the concentrations of As (12-fold), iron (Fe; 20-fold), manganese (Mn; 3-fold) and acetic acid (3-fold) in soil porewater, and also caused significant enrichment of Desulfitobacterium (41-fold). Furthermore, the FA addition significantly increased the relative abundance of Bathyarchaeota (4-fold), a microorganism that is suggested to be important for FA degradation. In contrast, HA slightly increased As (1.2-fold) in porewater, had little effect on Fe, Mn and acetic acid, and 1.5% HA addition significantly decreased As in porewater at day 14 (45%). Both HA and FA addition promoted As methylation. HA increased dimethylarsenate concentration and FA increased monomethylarsenate concentration in porewater. These results highlight the contrasting effects of different (HA vs. FA) organic substances on As fate in paddy soil and advance our understanding of the associations among As, Fe and organic substances through microorganisms in paddy soil.

Microbe mediated arsenic release from iron minerals and arsenic methylation in rhizosphere controls arsenic fate in soil-rice system after straw incorporation.

Arsenic (As) contamination is a global problem. Straw incorporation is widely performed in As contaminated paddy fields. To understand how straw and straw biochar incorporation affect As transformation and translocation in the soil-microbe-rice system, a pot experiment was carried out with different dosages of rice straw and straw biochar application. Results showed that both straw biochar and straw application significantly increased As mobility. Straw biochar mobilized As mainly through increasing soil pH and DOM content. Straw incorporation mainly through enhancing As release from iron (Fe) minerals and arsenate (As(V)) reduction to arsenite (As(III)). Straw biochar didn't significantly affect As methylation, while straw incorporation significantly enhanced As methylation, elevated dimethylarsenate (DMA) concentration in soil porewater and increased As volatilization. Straw biochar didn't significantly change total As accumulation in rice grains, but decreased As(III) accumulation by silicon (Si) inhibition. Straw incorporation significantly increased DMA, but decreased As(III) concentration in rice grains. After biochar application, dissolved As was significantly positively correlated with the abundance of Bacillus, indicating that Bacillus might be involved in As release, and As(III) concentration in polished grains was negatively correlated with Si concentration. The significant positive correlation between dissolved As with Fe and the abundance of iron-reducing bacteria suggested the coupling of As and Fe reduction mediated by iron-reducing bacteria. The significant positive correlation between DMA in rice grains and the abundance of methanogenic bacteria indicated that methanogenic bacteria could be involved in As methylation after straw application. The results of this study would advance the understanding how rice straw incorporation affects As fate in soil-microbe-rice system, and provide some guidance to straw incorporation in As contaminated paddy soil. This study also revealed a wealth of microorganisms in the soil environment that dominate As mobility and transformation after straw incorporation.

[Effects of Straw Incorporation on Cadmium Accumulation and Subcellular Distribution in Rice].

Cadmium (Cd) is classified as a Group-1 human carcinogen and rice consumption constitutes a major source of dietary intake of Cd for populations whose staple food is rice. Straw incorporation is widely performed in Cd-contaminated paddy fields, which may significantly affect the bioavailability of Cd in soil and the distribution of Cd in rice plants, consequently altering Cd accumulation in rice grains. In this study, both pot and field trials were conducted to investigate the effects of different amounts of straw incorporation (0.0%, 1.0%, 2.5%, and 5.0%) on Cd sub-cellular distribution in rice plants and Cd accumulation in rice grains. The results showed that Cd was mainly sequestered in cell wall, accounting for 86%-95% and 30%-51% of total cadmium in root and shoot cells, respectively. In shoot cells, about 35%-61% of Cd was distributed in cellular soluble fractions. When rice straw was incorporated at 1.0% and 2.5% levels, Cd sequestration in the cell wall significantly increased and Cd translocation from roots to shoots significantly decreased. However, when rice straw was incorporated at the 5% level, Cd sequestration in root cell walls significantly decreased and Cd translocation from roots to shoots significantly increased at the tillering stage. At the filling stage, 5% rice straw incorporation still significantly increased Cd sequestration in root cell walls and Cd translocation from roots to shoots did not significantly change. The rice straw and rape straw used for the field trail contained high concentrations of Cd (0.49 and 0.67 mg·kg-1, respectively). Rape straw incorporation alone or together with lime did not significantly affect Cd accumulation in brown rice or rice straw. Rice straw incorporation alone did not significantly affect Cd accumulation in brown rice or rice straw, while incorporation with lime significantly decreased Cd accumulation in both brown rice and rice straw. Biochar application can also significantly reduce Cd accumulation in rice and when biochar was added together with lime, the reduction in Cd accumulation in rice was more significant. Therefore, at Cd-contaminated paddy fields, rice straw or rape straw is not suggested to be returned directly; incorporation with lime would be better for reducing Cd accumulation in rice grains. The results of this study will provide theoretical and practical guidance for the safe production of rice and for straw recycling at Cd-polluted paddy fields.

Arsenic biotransformation and volatilization in transgenic rice.

• Biotransformation of arsenic includes oxidation, reduction, methylation, and conversion to more complex organic arsenicals. Members of the class of arsenite (As(III)) S-adenosylmethyltransferase enzymes catalyze As(III) methylation to a variety of mono-, di-, and trimethylated species, some of which are less toxic than As(III) itself. However, no methyltransferase gene has been identified in plants. • Here, an arsM gene from the soil bacterium Rhodopseudomonas palustris was expressed in Japonica rice (Oryza sativa) cv Nipponbare, and the transgenic rice produced methylated arsenic species, which were measured by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). • Both monomethylarsenate (MAs(V)) and dimethylarsenate (DMAs(V)) were detected in the roots and shoots of transgenic rice. After 12 d exposure to As(III), the transgenic rice gave off 10-fold greater volatile arsenicals. • The present study demonstrates that expression of an arsM gene in rice induces arsenic methylation and volatilization, theoretically providing a potential stratagem for phytoremediation.

A CDC25 homologue from rice functions as an arsenate reductase.

Enzymatic reduction of arsenate to arsenite is the first step in arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. The gene products were purified and demonstrated to reduce arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)(5)R motif led to nearly complete loss of both phosphatase and arsenate reductase activities. In planta expression of OsACR2.1 increased dramatically after exposure to arsenate. OsACR2.2 was observed only in roots following arsenate exposure, and its expression was less than OsACR2.1.

Characterization of arsenate reductase in the extract of roots and fronds of Chinese brake fern, an arsenic hyperaccumulator.

Root extracts from the arsenic (As) hyperaccumulating Chinese brake fern (Pteris vittata) were shown to be able to reduce arsenate to arsenite. An arsenate reductase (AR) in the fern showed a reaction mechanism similar to the previously reported Acr2p, an AR from yeast (Saccharomyces cerevisiae), using glutathione as the electron donor. Substrate specificity as well as sensitivity toward inhibitors for the fern AR (phosphate as a competitive inhibitor, arsenite as a noncompetitive inhibitor) was also similar to Acr2p. Kinetic analysis showed that the fern AR had a Michaelis constant value of 2.33 mM for arsenate, 15-fold lower than the purified Acr2p. The AR-specific activity of the fern roots treated with 2 mM arsenate for 9 d was at least 7 times higher than those of roots and shoots of plant species that are known not to tolerate arsenate. A T-DNA knockout mutant of Arabidopsis (Arabidopsis thaliana) with disruption in the putative Acr2 gene had no AR activity. We could not detect AR activity in shoots of the fern. These results indicate that (1) arsenite, the previously reported main storage form of As in the fern fronds, may come mainly from the reduction of arsenate in roots; and (2) AR plays an important role in the detoxification of As in the As hyperaccumulating fern.