Plant Defensin-Dissimilar Thionin OsThi9 Alleviates Cadmium Toxicity in Rice Plants and Reduces Cadmium Accumulation in Rice Grains.

Thionins are important antibacterial peptides in plants. However, the roles of plant thionins, especially the defensin-dissimilar thionins, in alleviating heavy-metal toxicity and accumulation remain unclear. Here, cadmium (Cd)-related functions and mechanisms of the defensin-dissimilar rice thionin OsThi9 were investigated. OsThi9 was significantly upregulated in response to Cd exposure. OsThi9 was localized to the cell wall and was shown to bind Cd; these characters help to increase Cd tolerance. In Cd-exposed rice plants, OsThi9 overexpression significantly increased cell wall Cd binding, decreasing upward Cd translocation and subsequent Cd accumulation in shoots and straw, while OsThi9 knockout had inverse effects. Importantly, in rice plants grown in Cd-contaminated soils, OsThi9 overexpression significantly reduced Cd accumulation in brown rice (decrease of ≥ 51.8%) without negatively impairing the crop yield and essential elements. Thus, OsThi9 plays an important role in the alleviation of Cd toxicity and accumulation and has significant potential for developing low-Cd rice.

Generation of low-cadmium rice germplasms via knockout of OsLCD using CRISPR/Cas9.

The OsLCD gene, which has been implicated in cadmium (Cd) accumulation in rice, might be a useful target for CRISPR/Cas9 editing. However, the effects of OsLCD gene editing on Cd accumulation, plant growth, and yield traits remain unknown. Here, we used CRISPR/Cas9 to generate oslcd single mutants from indica and japonica rice cultivars. We also generated osnramp5 single mutants and oslcd osnramp5 double mutants in the indica background. When grown in Cd-contaminated paddy soils, all oslcd single mutants accumulated less Cd than the wild types (WTs). Consistent with this, oslcd single mutants grown in Cd-contaminated hydroponic culture accumulated significantly less Cd in the shoots as compared to WTs. This decrease in accumulation probably resulted from the reduction of Cd translocation under Cd stress. Oxidative damage also decreased, and plant growth increased in all oslcd single mutant seedlings as compared to WTs in the presence of Cd. Plant growth and most yield traits, as well essential element concentrations in rice seedling shoots, brown rice, and rice straw, were similar between oslcd single mutants and WTs. In the presence of Cd, Cd concentrations in the brown rice and shoots of oslcd osnramp5 double mutants were significantly decreased compared with WTs as well as osnramp single mutants. Our results suggested that OsLCD knockout may reduce Cd accumulation alone or in combination with other knockout mutations in a variety of rice genotypes; unlike OsNramp5 mutations, OsLCD knockout did not reduce essential element contents. Therefore, OsLCD knockout might be used to generate low-Cd rice germplasms.

Transcriptomic and physio-biochemical features in rice (Oryza sativa L.) in response to mercury stress.

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 HgCl2 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.

Comparative transcriptomics provide new insights into the mechanisms by which foliar silicon alleviates the effects of cadmium exposure in rice.

Silicon (Si) has been shown to alleviate Cd stress in rice. Here, we investigated the beneficial effects of foliar Si in an indica rice Huanghuazhan (HHZ). Our results showed that foliar Si increases the dry weight and decreases Cd translocation in Cd-exposed rice at the grain-filling stage only, implying that the filling stage is critical for foliar Si to reduce Cd accumulation. We also investigated the transcriptomics in flag leaves (FLs), spikelets (SPs), and node Is (NIs) of Cd-exposed HHZ after foliar Si application at the filling stage. Importantly, the gene expression profiles associated with the Si-mediated alleviation of Cd stress were tissue specific, while shared pathways were mediated by Si in Cd-exposed rice tissues. Furthermore, after the Si treatment of Cd-exposed rice, the ATP-binding cassette (ABC)-transporters were mostly upregulated in FL and SP, while the bivalent cation transporters were mostly downregulated in FL and NI, possibly helping to reduce Cd accumulation. The genes associated with essential nutrient transporters, carbohydrate and secondary metabolite biosynthesis, and cytochrome oxidase activity were mostly upregulated in Cd-exposed FL and SP, which may help to alleviate oxidative stress and improve plant growth under Cd exposure. Interestingly, genes responsible for signal transduction were negatively regulated in FL, but positively regulated in SP, by foliar Si. Our results provide transcriptomic evidence that foliar Si plays an active role in alleviating the effects of Cd exposure in rice. In particular, foliar Si may alter the expression pattern of genes associated with transport, biosynthesis and metabolism, and oxidation reduction.

Comparative physiological and transcriptomic analyses illuminate common mechanisms by which silicon alleviates cadmium and arsenic toxicity in rice seedlings.

The inessential heavy metal/loids cadmium (Cd) and arsenic (As), which often co-occur in polluted paddy soils, are toxic to rice. Silicon (Si) treatment is known to reduce Cd and As toxicity in rice plants. To better understand the shared mechanisms by which Si alleviates Cd and As stress, rice seedlings were hydroponically exposed to Cd or As, then treated with Si. The addition of Si significantly ameliorated the inhibitory effects of Cd and As on rice seedling growth. Si supplementation decreased Cd and As translocation from roots to shoots, and significantly reduced Cd- and As-induced reactive oxygen species generation in rice seedlings. Transcriptomics analyses were conducted to elucidate molecular mechanisms underlying the Si-mediated response to Cd or As stress in rice. The expression patterns of the differentially expressed genes in Cd- or As-stressed rice roots with and without Si application were compared. The transcriptomes of the Cd- and As-stressed rice roots were similarly and profoundly reshaped by Si application, suggesting that Si may play a fundamental, active role in plant defense against heavy metal/loid stresses by modulating whole genome expression. We also identified two novel genes, Os01g0524500 and Os06g0514800, encoding a myeloblastosis (MYB) transcription factor and a thionin, respectively, which may be candidate targets for Si to alleviate Cd and As stress in rice, as well as for the generation of Cd- and/or As-resistant plants. This study provides valuable resources for further clarification of the shared molecular mechanisms underlying the Si-mediated alleviation of Cd and As toxicity in rice.

Different effects of foliar application of silica sol on arsenic translocation in rice under low and high arsenite stress.

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.

Dynamics of gene expression associated with arsenic uptake and transport in rice during the whole growth period.

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.

A transcriptomic (RNA-seq) analysis of genes responsive to both cadmium and arsenic stress in rice root.

Cadmium (Cd) and arsenic (As) are nonessential and toxic elements in rice that often occur together in contaminated paddy field soils. To understand whether rice has a common molecular response mechanism against Cd and As toxicity, 30-day seedlings (Oryza sativa L. indica) were exposed separately to Cd and As3+ in hydroponic cultures for up to 7 days. Root transcriptomic analysis of plants exposed to Cd and As for 3 days revealed that a total of 2224 genes in rice roots responded to Cd stress, while 1503 genes responded to As stress. Of these, 841 genes responded to both stressors. The genes in common to Cd and As stress were associated with redox control, stress response, transcriptional regulation, transmembrane transport, signal transduction, as well as biosynthesis and metabolism of macromolecules and sulfur compounds. In plants exposed to Cd and As separately or in combination for 3 and 7 days, qRT-PCR verification revealed that the glutathione metabolism associated gene Os09g0367700 was significantly up-regulated with respect to unexposed controls and had a positive synergistic effect under combined Cd and As stress. In addition, the redox control related genes Os06g0216000, Os07g0638300 and Os01g0294500, the glutathione metabolism related gene Os01g0530900, the cell wall biogenesis related genes Os05g0247800, Os11g0592000 and Os03g0416200, the expression regulation related genes Os07g0597200 and Os02g0168200, and the transmembrane transport related genes Os04g0524500, also varied significantly with respect to an unexposed control and displayed synergistic effects after 7 days of simultaneous exposure to Cd and As. Our identification of a novel set of genes in rice which responded to both Cd and As3+ stress may be of value in mitigating the toxicity of co-contaminated soils. These results also provide a deeper understanding of the molecular mechanisms involved in response to multi-metal/loids stress, and may be used in the genetic improvement of rice varieties.

Identification of a NFκB Inhibition Site on the Proximal Promoter Region of Human Organic Anion Transporting Polypeptide 1A2 Coding Gene SLCO1A2.

Organic anion transporting polypeptides (OATPs; gene symbol SLCO) are membrane transporters that mediate the transport of wide ranges of compounds. The expression of different OATP members has been reported in the kidney, liver, placenta, brain, and intestine. Because of their broad substrate spectra and wide distribution within the human body, these transporters have been proposed to play key roles in the influx transport of many oral drugs. Inflammation is known to regulate the expression and functions of many drug-metabolizing enzymes and drug transporters. As a proinflammatory cytokine, tumor necrosis factor-α (TNFα) has been shown to affect the expression of different drug transporters, including OATP family members. In the present study, a putative nuclear factor-κB (NFκB) binding site ranging from -1845 to -1836 was identified at the proximal promoter region of OATP1A2 coding gene SLCO1A2 Electrophoretic mobility shift assays and chromatin immunoprecipitation showed that nuclear extracts from both breast cancer cell MCF7 and liver cancer cell HepG2 interacted with an oligonucleotide probe containing the putative NFκB binding site and that the DNA-protein complexes contained both p65 and p50 subunits of NFκB. Further study revealed that the binding site may be responsible in part for the suppression effect of TNFα toward SLCO1A2 expression because the treatment of TNFα significantly increased. Treatment of TNFα significantly increased formation of the DNA-protein complexes and mutations at essential bases of the putative NFκB binding site abolished responsiveness to the TNFα neutralizing antibody, suggesting that the binding site may be responsible in part for the suppression effect of TNFα towars SLCO1A2 expression.

Comparative genomic hybridization and transcriptome sequencing reveal that two genes, OsI_14279 (LOC_Os03g62620) and OsI_10794 (LOC_Os03g14950) regulate the mutation in the γ-rl rice mutant.

We previously established the genetic locus of the rolled-leaf mutant, γ-rl, to chromosome 3. In this study, we performed a comparative genomic hybridization (CGH) analysis to identify the genes responsible for the γ-rl mutant phenotype. This was combined with RNA transcriptome sequencing (RNA-seq) to analyze differences in the mRNA expression in seeds 12 h after germination. Using the reference genome of the "indica type" rice from GenBank, we created a chip with 386,000 high density DNA probes designed to target chromosome 3. The genomic DNA from γ-rl and Qinghuazhan (the wild-type) was used for hybridization against the chip to compare signal differences. We uncovered 49 regions with significant differences in hybridization signals including deletions and insertions. RNA-seq analysis between γ-rl and QHZ identified 1060 differentially expressed genes, which potentially regulate numerous biological activities. Moreover, we identified 72 annotated genes in the 49 regions discovered in CGH. Among these, 44 genes showed differential expression in RNA-seq. qRT-PCR validation of the candidate genes confirmed that seven of the 44 genes showed a significant change in their expression levels. Among these, four genes [OsI_10125 (LOC_Os03g06654), OsI_14045 (LOC_Os03g62490), OsI_14279 (LOC_Os03g62620) and OsI_14326 (LOC_Os03g63250)] were down regulated and three genes [(OsI_10794 (LOC_Os03g14950), OsI_11412 (LOC_Os03g21250) and OsI_14152 (LOC_Os03g61360)] were up regulated with a fold change ≥2.0 and a P value ≤ 0.01. Finally, we constructed transgenic plants to study the in vivo functions of these genes. RNAi knock down of LOC_Os03g62620 resulted in rolled-leaf, lower seed-setting and decreased seed growth phenotypes. Transgenic plants with LOC_Os03g14950 over-expression showed dwarf plants with a shortened leaf phenotype. Our results, LOC_Os03g62620 and LOC_Os03g14950 as the essential genes responsible for creating the γ-rl mutant phenotypes suggested that these genes may play crucial roles in regulating rice leaf development and seed growth.

Silica nanoparticles alleviate cadmium toxicity in rice cells: Mechanisms and size effects.

Although it was recently determined that silicon can alleviate cadmium (Cd) toxicity in rice, the effects of silicon properties and the molecular mechanisms are still unclear. Here, the effect of silica nanoparticles (SiNPs) on Cd toxicity in rice was examined using cells cultured in suspension in the presence or absence of SiNPs (19 nm, 48 nm and 202 nm). The results showed that the presence of SiNPs substantially enhanced the proportion of live cells to 95.4%, 78.6% and 66.2%, respectively, suggesting that the extent of alleviation of Cd toxicity decreased gradually with size of SiNPs. The morphological results showed that dramatic damage and severe structural changes in the organelle integrity of cells occurred in the absence of SiNPs, whereas the cells exposed to the SiNPs remained nearly intact even in the presence of high concentrations of Cd. Furthermore, the SiNPs accumulated on the surface of the rice cells. Using inductively coupled plasma mass spectroscopy, Cd accumulated preferentially in plant cells with cell walls. In addition, noninvasive microtest technology showed that the average Cd2+ influx in those treated with SiNPs (19 nm, 48 nm and 202 nm) decreased by 15.7-, 11.1- and 4.6-fold, respectively. The gene expression of Cd uptake and transport (OsLCT1 and OsNramp5) was inhibited by SiNPs, but the gene expression of Cd transport into vacuole (OsHMA3) and Si uptake (OsLsi1) was enhanced by the SiNPs. These results indicate that the presence of SiNPs increased at least 1.87-fold the Si uptake capacity and inhibited the Cd uptake capacity, which together resulted in the alleviation of the toxicity of Cd in rice. This study provided a molecular-scale insight into the understanding of the SiNPs-induced alleviation of the toxicity of Cd in rice.

The availabilities of arsenic and cadmium in rice paddy fields from a mining area: The role of soil extractable and plant silicon.

Adequate silicon (Si) can greatly boost rice yield and improve grain quality through alleviating stresses associated with heavy metals and metalloids such as arsenic (As) and cadmium (Cd). The soil plant-available Si is relatively low in South China due to severe desilicification and allitization of the soils in this region. Conversely, pollution of heavy metals and metalloids in the soils of this region occurs widely, especially As and Cd pollution in paddy soil. Therefore, evaluating the plant availability of Si in paddy soil of South China and examining its correlation with the availability of heavy metals and metalloids are of great significance. Accordingly, in our study, 107 pairs of soil and rice plant samples were collected from paddy fields contaminated by As and Cd in South China. Significantly positive correlations between Si in rice plants and Si fractions in soils extracted with citric acid, NaOAc-HOAc buffer, and oxalate-ammonium oxalate buffer suggest that these extractants are more suitable for use in extracting plant-available Si in the soils of our present study. Significantly negative correlations between different Si fractions and As or Cd in rice plant tissues and negative exponential correlations between the molar ratios of Si to As/Cd in rice roots, straws, husks or grains and As/Cd in rice grains indicate that Si can significantly alleviate the accumulation of As/Cd from soils to the rice plants. Finally, a contribution assessment of soil properties to As/Cd accumulation in rice grains based on random forest showed that in addition to Si concentrations in soil or rice plants, other factors such as Fe fractions and total phosphorus also contributed largely to As/Cd accumulation in rice grains. Overall, Si exhibited its unique role in mitigating As or Cd stress in rice, and our study results provide strong field evidence for this role.

Cloning, characterization and expression of OsFMO(t) in rice encoding a flavin monooxygenase.

Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of OsFMO(t), a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. OsFMO(t) was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a 'GxGxxG' characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO(t) and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of OsFMO(t) in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO(t) protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of OsFMO(t) was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by OsFMO(t) is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.

Preliminary Studies on the Amino-acid Residues at Active Center of the Lysozyme from Raphanus sativus Leaves.

A 1ysozyme from Raphanus sativus leaves was purified by the method of affinity chromatography on a deaminated regenerated crab chitin column. The purified enzyme was crystallized and showed a single band on polyacrylamide gel disc electrophoresis. The functional groups at the active center of the enzyme were studied by the method of pH dynamics and chemical modification. It was found that carboxyl groups(Glu/Asp), tryptophanyl and histidyl residues were probably essential groups for the catalytic activity. The enzymatic activity was not affected when the enzyme was modified by reagents which could specifically react with tyrosyl, cysteinyl, arginyl and seryl/threonyl residues. It was inhibited by histamine and GlcNAc. The difference among Raphanus sativus lysozyme, HEWI and papaya lysozyme was discussed.