Overexpression of vacuolar transporters OsVIT1 and OsVIT2 reduces cadmium accumulation in rice.

Excessive cadmium in rice grain in agricultural production is an important issue to be addressed in some southern regions of China. In this study, we constructed transgenic rice overexpressing OsVIT1 and OsVIT2 driven by 35S promoter in the cultivar ZH11. Compared with ZH11, OsVIT1 expression in leaves was significantly increased by 3-6.6 times and OsVIT2 expression in leaves was significantly increased by 2-2.5 times. Hydroponic experiments showed that overexpression of OsVIT1 and OsVIT2 increased the tolerance to Fe deficiency, significantly reduced Cd content in shoot and xylem sap, and had no effect on Cd tolerance in rice. Two years of field trials showed that the Fe content in the grain of OsVIT1 and OsVIT2 overexpressed materials was significantly reduced by 20-40% and the straw Fe content was significantly increased by 10-45%, and the grain Fe content distribution ratio was significantly decreased and the straw Fe distribution ratio was significantly increased compared with the wild type. The OsVIT1 and OsVIT2 overexpressed materials significantly reduced the Cd content of grain by 40-80% and the Cd content of straws by 37-77%, and the bioconcentration factor of Cd was significantly reduced in both grains and straw of OsVIT1 and OsVIT2 overexpressed materials. Overexpression of OsVIT1 and OsVIT2 did not affect the concentration of other metal ions in rice straw and grain. qRT-PCR analysis showed that the expression of the low affinity cation transporter OsLCT1 was significantly downregulated in the OsVIT1 and OsVIT2 overexpressed materials. In conclusion, overexpression of OsVIT1 and OsVIT2 reduced Cd accumulation in straw and grains, providing a strategy for Cd reduction in rice.

CHLORIDE CHANNEL-b mediates vacuolar nitrate efflux to improve low nitrogen adaptation in Arabidopsis.

The vacuole is an important organelle for nitrate storage, and the reuse of vacuolar nitrate under nitrate starvation helps plants adapt to low-nitrate environments. CHLORIDE CHANNEL-b (CLC-b) in the vacuolar membrane is a nitrate transporter; however, its regulation and effects on nitrate efflux have not been established. Here, we evaluated CLC-b expression and its effects on physiological parameters under low nitrate conditions. CLC-b expression increased significantly in the roots of wild-type Arabidopsis (Arabidopsis thaliana) Col-0 under nitrate starvation. Under low nitrate, clcb mutants showed reductions in chlorophyll content and xylem sap nitrate concentration, shoot/root nitrate ratios, shoot/root total N ratios, and biomass. CLC-b-overexpression yielded opposite phenotypes and increased nitrogen use efficiency. CLC-b mutants showed elevated chlorate tolerance and an increased proportion of vacuolar nitrate relative to the total protoplast nitrate content as compared to the wild type. Yeast 1-hybrid, EMSA, and chromatin immunoprecipitation (ChIP) experiments showed that HRS1 HOMOLOG2 (HHO2), the expression of which is downregulated under low nitrate, binds directly to the promoter of CLC-b. clcb/hho2 double mutants and HHO2-overexpressing clcb plants had similar phenotypes under low nitrate to those of clcb single mutants. Thus, CLC-b mediates vacuolar nitrate efflux and is negatively regulated by HHO2, providing a theoretical basis for improving plant adaptability to low nitrate.

Genome and Transcriptome Identification of a Rice Germplasm with High Cadmium Uptake and Translocation.

The safe production of food on Cd-polluted land is an urgent problem to be solved in South China. Phytoremediation or cultivation of rice varieties with low Cd are the main strategies to solve this problem. Therefore, it is very important to clarify the regulatory mechanism of Cd accumulation in rice. Here, we identified a rice variety with an unknown genetic background, YSD, with high Cd accumulation in its roots and shoots. The Cd content in the grains and stalks were 4.1 and 2.8 times that of a commonly used japonica rice variety, ZH11, respectively. The Cd accumulation in the shoots and roots of YSD at the seedling stage was higher than that of ZH11, depending on sampling time, and the long-distance transport of Cd in the xylem sap was high. Subcellular component analysis showed that the shoots, the cell wall, organelles, and soluble fractions of YSD, showed higher Cd accumulation than ZH11, while in the roots, only the cell wall pectin showed higher Cd accumulation. Genome-wide resequencing revealed mutations in 22 genes involved in cell wall modification, synthesis, and metabolic pathways. Transcriptome analysis in Cd-treated plants showed that the expression of pectin methylesterase genes was up-regulated and the expression of pectin methylesterase inhibitor genes was down-regulated in YSD roots, but there were no significant changes in the genes related to Cd uptake, translocation, or vacuole sequestration. The yield and tiller number per plant did not differ significantly between YSD and ZH11, but the dry weight and plant height of YSD were significantly higher than that of ZH11. YSD provides an excellent germplasm for the exploration of Cd accumulation genes, and the cell wall modification genes with sequence- and expression-level variations provide potential targets for phytoremediation.

Cell Wall Polysaccharide-Mediated Cadmium Tolerance Between Two Arabidopsis thaliana Ecotypes.

Cadmium (Cd) is a toxic metal element and the mechanism(s) underlying Cd tolerance in plants are still unclear. Increasingly more studies have been conducted on Cd binding to plant cell walls (CW) but most of them have focused on Cd fixation by CW pectin, and few studies have examined Cd binding to cellulose and hemicellulose. Here we found that Cd binding to CW pectin, cellulose, and hemicellulose was significantly higher in Tor-1, a Cd tolerant A. thaliana ecotype, than in Ph2-23, a sensitive ecotype, as were the concentrations of pectin, cellulose, and hemicellulose. Transcriptome analysis revealed that the genes regulating CW pectin, cellulose, and hemicellulose polysaccharide concentrations in Tor-1 differed significantly from those in Ph2-23. The expressions of most genes such as pectin methyl esterase inhibitors (PMEIs), pectin lyases, xyloglucan endotransglucosylase/hydrolase, expansins (EXPAs), and cellulose hydrolase were higher in Ph2-23, while the expressions of cellulose synthase-like glycosyltransferase 3 (CSLG3) and pectin ethyl esterase 4 (PAE4) were higher in Tor-1. The candidate genes identified here seem to regulate CW Cd fixation by polysaccharides. In conclusion, an increase in pectin demethylation activity, the higher concentration of cellulose and hemicellulose, regulated by related genes, in Tor-1 than in Ph2-23 are likely involved in enhanced Cd CW retention and reduce Cd toxicity.

Overexpression of a Defensin-Like Gene CAL2 Enhances Cadmium Accumulation in Plants.

Accumulation and detoxification of cadmium in rice shoots are of great importance for adaptation to grow in cadmium contaminated soils and for limiting the transport of Cd to grains. However, the molecular mechanisms behind the processes involved in this regulation remain largely unknown. Defensin proteins play important roles in heavy metal tolerance and accumulation in plants. In rice, the cell wall-localized defensin protein (CAL1) is involved in Cd efflux and partitioning to the shoots. In the present study, we functionally characterized the CAL2 defensin protein and determined its contribution to Cd accumulation. CAL2 shared 66% similarity with CAL1, and its mRNA accumulation is mainly observed in roots and is unaffected by Cd stress, but its transcription level was lower than that of CAL1 based on the relative expression of CAL2/Actin1 observed in this study and that reported previously. A promoter-GUS assay revealed that CAL2 is expressed in root tips. Stable expression of the CAL2-mRFP fusion protein indicated that CAL2 is also localized in the cell walls. An in vitro Cd binding experiment revealed that CAL2 has Cd chelation activity. Overexpression of CAL2 increased Cd accumulation in Arabidopsis and rice shoots, but it had no effect on the accumulation of other essential elements. Heterologous expression of CAL2 enhanced Cd sensitivity in Arabidopsis, whereas overexpression of CAL2 had no effect on Cd tolerance in rice. These findings indicate that CAL2 positively regulates Cd accumulation in ectopic overexpression lines of Arabidopsis and rice. We have identified a new gene regulating Cd accumulation in rice grain, which would provide a new genetic resource for molecular breeding.

The plant defensin gene AtPDF2.1 mediates ammonium metabolism by regulating glutamine synthetase activity in Arabidopsis thaliana.

BACKGROUND: In plants, ammonium metabolism is particularly important for converting absorbed nitrogen into amino acids. However, the molecular mechanism underlying this conversion remains largely unknown. RESULTS: Using wild type Arabidopsis thaliana (Col-0) and AtPDF2.1 mutants (pdf2.1-1 and pdf2.1-2), we found that the small cysteine-rich peptide AtPDF2.1, a plant defensin, is involved in regulating ammonium metabolism in the shoot. Ammonium significantly induced the expression of AtPDF2.1 in the shoot and root, particularly in root xylem vascular bundles, as demonstrated by histochemical analysis. Subcellular localization analysis revealed that AtPDF2.1 was localized to the cell wall. Ammonium concentration was higher in the shoot of mutants than in the shoot of Col-0, but no differences were found for total nitrogen content, root ammonium concentration, and the expression of the ammonium transporter gene AtAMT2.1. The activity of glutamine synthetase was significantly decreased in mutants, and the glutamine synthetase family genes GLN1.3 and GLN1.5 were significantly downregulated in mutants compared to Col-0. The activity of nitrate reductase showed no difference between mutants and Col-0. CONCLUSIONS: Overall, these data suggest that AtPDF2.1 affects ammonium metabolism by regulating the expression of GLN1.3 and GLN1.5 through a yet unidentified mechanism.

Proteomic changes in the xylem sap of Brassica napus under cadmium stress and functional validation.

BACKGROUND: The xylem sap of vascular plants primarily transports water and mineral nutrients from the roots to the shoots and also transports heavy metals such as cadmium (Cd). Proteomic changes in xylem sap is an important mechanism for detoxifying Cd by plants. However, it is unclear how proteins in xylem sap respond to Cd. Here, we investigated the effects of Cd stress on the xylem sap proteome of Brassica napus using a label-free shotgun proteomic approach to elucidate plant response mechanisms to Cd toxicity. RESULTS: We identified and quantified 672 proteins; 67% were predicted to be secretory, and 11% (73 proteins) were unique to Cd-treated samples. Cd stress caused statistically significant and biologically relevant abundance changes in 28 xylem sap proteins. Among these proteins, the metabolic pathways that were most affected were related to cell wall modifications, stress/oxidoreductases, and lipid and protein metabolism. We functionally validated a plant defensin-like protein, BnPDFL, which belongs to the stress/oxidoreductase category, that was unique to the Cd-treated samples and played a positive role in Cd tolerance. Subcellular localization analysis revealed that BnPDFL is cell wall-localized. In vitro Cd-binding assays revealed that BnPDFL has Cd-chelating activity. BnPDFL heterologous overexpression significantly enhanced Cd tolerance in E. coli and Arabidopsis. Functional disruption of Arabidopsis plant defensin genes AtPDF2.3 and AtPDF2.2, which are mainly expressed in root vascular bundles, significantly decreased Cd tolerance. CONCLUSIONS: Several xylem sap proteins in Brassica napus are differentially induced in response to Cd treatment, and plant defensin plays a positive role in Cd tolerance.

A non-secreted plant defensin AtPDF2.6 conferred cadmium tolerance via its chelation in Arabidopsis.

KEY MESSAGE: Plant defensin AtPDF2.6 is not secreted to the apoplast and localized in cytoplasm. AtPDF2.6 is mainly expressed in root vascular bundles of xylem parenchyma cell, and significantly induced by Cd stress. AtPDF2.6 detoxicate cytoplasmic Cd via chelation, thus enhanced Cd tolerance in Arabidopsis. In order to detoxify the heavy metal cadmium (Cd), plants have evolved several mechanisms, among which chelation represents the major Cd-detoxification mechanism. In this study, we aimed to identify a new defensin protein involved in cytoplasmic Cd detoxification by using plant molecular genetics and physiological methods. The results of bioinformatic analysis showed that the Arabidopsis thaliana defensin gene AtPDF2.6 has a signal peptide that may mediate its secretion to the cell wall. Subcellular localization analysis revealed that AtPDF2.6 is localized to the cytoplasm and is not secreted to the apoplast, whereas histochemical analysis indicated that AtPDF2.6 is mainly expressed in the root xylem parenchyma cells and that its expression is significantly induced by Cd. An in vitro Cd-binding assay revealed that AtPDF2.6 has Cd-chelating activity. Heterologous overexpression of AtPDF2.6 increased Cd tolerance in Escherichia coli and yeast, and AtPDF2.6 overexpression significantly enhanced Cd tolerance in Arabidopsis, whereas functional disruption of AtPDF2.6 decreased Cd tolerance. These data suggest that AtPDF2.6 detoxifies cytoplasmic Cd via chelation and thereby enhances Cd tolerance in Arabidopsis. Our findings accordingly challenge the commonly accepted view of defensins as secreted proteins.

The Arabidopsis defensin gene AtPDF2.5 mediates cadmium tolerance and accumulation.

Although excess cadmium (Cd) accumulation is harmful to plants, the molecular mechanisms underlying Cd detoxification and accumulation in Arabidopsis thaliana remain largely undetermined. In this study, we demonstrated that the A. thaliana PLANT DEFENSIN 2 gene AtPDF2.5 is involved in Cd tolerance and accumulation. In vitro Cd-binding assays revealed that AtPDF2.5 has Cd-chelating activity. Site-directed mutagenesis of AtPDF2.5 identified eight cysteine residues that were essential for mediating Cd tolerance and chelation. Histochemical analysis demonstrated that AtPDF2.5 was mainly expressed in root xylem vascular bundles, and that AtPDF2.5 was significantly induced by Cd. Subcellular localization analysis revealed that AtPDF2.5 was localized to the cell wall. The overexpression of AtPDF2.5 significantly enhanced Cd tolerance and accumulation in A. thaliana and its heterologous overexpression in rice increased Cd accumulation; however, the functional disruption of AtPDF2.5 decreased Cd tolerance and accumulation. Physiological analysis suggested that AtPDF2.5 promoted Cd efflux from the protoplast and its subsequent accumulation in the cell wall. These data suggest that AtPDF2.5 promotes cytoplasmic Cd efflux via chelation, thereby enhancing Cd detoxification and apoplastic accumulation.

A defensin-like protein drives cadmium efflux and allocation in rice.

Pollution by heavy metals limits the area of land available for cultivation of food crops. A potential solution to this problem might lie in the molecular breeding of food crops for phytoremediation that accumulate toxic metals in straw while producing safe and nutritious grains. Here, we identify a rice quantitative trait locus we name cadmium (Cd) accumulation in leaf 1 (CAL1), which encodes a defensin-like protein. CAL1 is expressed preferentially in root exodermis and xylem parenchyma cells. We provide evidence that CAL1 acts by chelating Cd in the cytosol and facilitating Cd secretion to extracellular spaces, hence lowering cytosolic Cd concentration while driving long-distance Cd transport via xylem vessels. CAL1 does not appear to affect Cd accumulation in rice grains or the accumulation of other essential metals, thus providing an efficient molecular tool to breed dual-function rice varieties that produce safe grains while remediating paddy soils.

[Hematopoietic reconstitution of recipient mice after mFasL-cDNA transfected hematopoietic cell transplantation].

OBJECTIVE: The study was designed to explore the influence of transfected bone marrow mononuclear cells (BMMNC) transplantation on the hematopoietic cells activity and the recipient mice hematopoietic reconstitution. METHODS: The exogenous mFasL-cDNA gene was transferred to Balb/C mouse BMMNC by liposomes. Then the transferred BMMNC was co-cultured with BMMNC from BAC mouse (H-2d x b, male) at a ratio of 0.625 to 1 for 6 days. In the experimental group (the 3rd group), 1 x 10(7) (0.5 ml) mixed viable cells were injected into whole bodily irradiated ((60)Co-r) mice (6 to 8 week old female Balb/C) via the tail vein. The following grafted mice were simultaneously used in the study, the mice transplanted with 0.5 ml of culture medium, the mice transplanted with the mixture of untransferred Balb/C mouse BMMNC and BAC mouse BMMNC, the mice transplanted with the mixture of transferred Balb/C mouse BMMNC and BAC mouse BMMNC and the mice transplanted with Balb/C mouse BMMNC. The hematopoietic reconstitution, the origin of bone marrow cells responsible for the reconstitution, the graft versus host disease (GVHD), the survival rate for the recipient mice were observed after bone marrow transplantation (BMT). RESULTS: The counts of leukocytes and platelets in recipient blood of group four on +10 d and +20 d after BMT were higher than those in group three and group two (P < 0.01), but on +30 d after BMT the counts of leukocytes and platelets in recipient blood of group two, group three and group four were at their normal levels. The Y chromosome from donor mice was discovered in BMMNC of recipient mice having survived for over two months after BMT in group two and group three. The survival rate of the recipient mice two mouths after BMT in all groups were 0% for group one, 30% for group two, 80% for group three, and 100% for group four, respectively. The total survival rate of recipient mice in the experimental group was obviously higher than that of group two (P < 0.01). Grade II to III GVHD signs were found on the histology from dead mice after BMT in group three and group two, and the mice having survived for over two months in the group two. Grade I GVHD signs were found on histology from 7 out of 8 mice which survived for over two months after BMT in group three. CONCLUSIONS: The transplantation of mixed cells into recipient mice made the recipient mice achieve hematopoietic reconstitution from donor BMMNC.