Gene identification and transcriptome analysis of low cadmium accumulation rice mutant (lcd1) in response to cadmium stress using MutMap and RNA-seq.

BACKGROUND: Cadmium (Cd) is a widespread toxic heavy metal pollutant in agricultural soil, and Cd accumulation in rice grains is a major intake source of Cd for Asian populations that adversely affect human health. However, the molecular mechanism underlying Cd uptake, translocation and accumulation has not been fully understood in rice plants. RESULTS: In this study, a mutant displaying extremely low Cd accumulation (lcd1) in rice plant and grain was generated by EMS mutagenesis from indica rice cultivar 9311 seeds. The candidate SNPs associated with low Cd accumulation phenotype in the lcd1 mutant were identified by MutMap and the transcriptome changes between lcd1 and WT under Cd exposure were analyzed by RNA-seq. The lcd1 mutant had lower Cd uptake and accumulation in rice root and shoot, as well as less growth inhibition compared with WT in the presence of 5 µM Cd. Genetic analysis showed that lcd1 was a single locus recessive mutation. The SNP responsible for low Cd accumulation in the lcd1 mutant located at position 8,887,787 on chromosome 7, corresponding to the seventh exon of OsNRAMP5. This SNP led to a Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 in the lcd1 mutant. A total of 1208 genes were differentially expressed between lcd1 and WT roots under Cd exposure, and DEGs were enriched in transmembrane transport process GO term. Increased OsHMA3 expression probably adds to the effect of OsNRAMP5 mutation to account for the significant decreases in Cd accumulation in rice plant and grain of the lcd1 mutant. CONCLUSIONS: An extremely low Cd mutant lcd1 was isolated and identified using MutMap and RNA-seq. A Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 is likely responsible for low Cd accumulation in the lcd1 mutant. This work provides more insight into the mechanism of Cd uptake and accumulation in rice, and will be helpful for developing low Cd accumulation rice by marker-assisted breeding.

Inhibition of nitrate transporter 1.1-controlled nitrate uptake reduces cadmium uptake in Arabidopsis.

Identification of mechanisms that decrease cadmium accumulation in plants is a prerequisite for minimizing dietary uptake of cadmium from contaminated crops. Here, we show that cadmium inhibits nitrate transporter 1.1 (NRT1.1)-mediated nitrate (NO3 (-)) uptake in Arabidopsis (Arabidopsis thaliana) and impairs NO3 (-) homeostasis in roots. In NO3 (-)-containing medium, loss of NRT1.1 function in nrt1.1 mutants leads to decreased levels of cadmium and several other metals in both roots and shoots and results in better biomass production in the presence of cadmium, whereas in NO3 (-)-free medium, no difference is seen between nrt1.1 mutants and wild-type plants. These results suggest that inhibition of NRT1.1 activity reduces cadmium uptake, thus enhancing cadmium tolerance in an NO3 (-) uptake-dependent manner. Furthermore, using a treatment rotation system allowing synchronous uptake of NO3 (-) and nutrient cations and asynchronous uptake of cadmium, the nrt1.1 mutants had similar cadmium levels to wild-type plants but lower levels of nutrient metals, whereas the opposite effect was seen using treatment rotation allowing synchronous uptake of NO3 (-) and cadmium and asynchronous uptake of nutrient cations. We conclude that, although inhibition of NRT1.1-mediated NO3 (-) uptake by cadmium might have negative effects on nitrogen nutrition in plants, it has a positive effect on cadmium detoxification by reducing cadmium entry into roots. NRT1.1 may regulate the uptake of cadmium and other cations by a common mechanism.

OsCOPT7 is involved in copper accumulation and transport through xylem.

Copper (Cu) is an essential micronutrient for humans, but excessive Cu in rice grains causes health risks. Currently, the mechanisms underlying Cu accumulation in rice are unclear. Here, we identified a novel member of the high-affinity copper transporter (Ctr)-like (COPT) protein family in rice, OsCOPT7, which controls Cu accumulation in rice grains. Mutation in the coding sequence of OsCOPT7 (mutant lc1) leads to inhibition of Cu transport through the xylem, contributing to lower Cu concentrations in the grain of lc1. Knockout or modulation of the expression of OsCOPT7 significantly impacts Cu transportation in the xylem and its accumulation in rice grains. OsCOPT7 localizes at the multi-pass membrane in the cell and the gene is expressed in the exodermis and stele cells, facilitating Cu loading into the xylem. OsCOPT7 expression is upregulated under Cu deficiency and in various organs, implying its contribution to Cu distribution within the rice plant. The variable expression pattern of OsCOPT7 suggests that OsCOPT7 expression responds to Cu stress in rice. Moreover, assays reveal that OsCOPT7 expression level is suppressed by the SQUAMOSA promoter-binding protein-like 9 (OsSPL9) and that OsCOPT7 interacts with Antioxidant Protein1 (OsATX1). This study elucidates the involvement of OsCOPT7 in Cu loading into the xylem, its subsequent distribution within the rice plant, and the potential of this protein in reducing the risk of high Cu concentrations in rice grain grown on Cu-contaminated soil.

Sulfide alleviates cadmium toxicity in Arabidopsis plants by altering the chemical form and the subcellular distribution of cadmium.

Several sulfur compounds are thought to play important roles in the plant tolerance to cadmium (Cd), but the role of inorganic sulfide in Cd tolerance remains largely unknown. In this study, we found that Cd exposure increased the accumulation of soluble sulfide in Arabidopsis plants. When exogenous sulfide, in the form of NaHS, was foliarly applied, Cd-induced growth inhibition and oxidative stress were alleviated. In addition, although the foliar application of sulfide did not affect the total Cd levels, it significantly decreased the soluble Cd fractions in plants. Furthermore, foliar applications of sulfide decreased Cd distribution in the cytoplasm and organelles, but increased Cd retention in the cell wall, which is a less sensitive compartment. These results suggest that the Cd-induced accumulation of soluble sulfide alleviates Cd toxicity in plants by inactivating Cd and sequestering it into the cell wall.

Effects of split applications of nitrogen fertilizers on the Cd level and nutritional quality of Chinese cabbage.

Cadmium (Cd) contamination in soil is an increasingly serious problem. Management of plant nutrients has been proposed as a potentially promising strategy for minimizing Cd accumulation in crops grown in contaminated soil. This study investigated the effects of split applications of nitrogen (N) fertilizers on the Cd concentration in Chinese cabbage (Brassica chinensis L.) plants grown in Cd-contaminated soil. Compared with single applications, split applications of ammonium or urea resulted in significantly lower Cd concentrations, and higher biomass production and antioxidant-associated nutritional quality in the edible plant parts. However, when nitrate was used as the N fertilizer, there were no significant differences between the split and single applications for the same parameters. We conclude that a split application could be more beneficial than a single application method when ammonium or urea is used as the N fertilizer for vegetable cultivation in Cd-contaminated soil.

Modification of nitrate uptake pathway in plants affects the cadmium uptake by roots.

NRT1.1 is a dual-affinity nitrate (NO3(-)) transporter involved in both high- and low-affinity NO3(-) uptake in Arabidopsis plants. In a recent study, we showed that, under cadmium (Cd) exposure, blocking the NRT1.1-mediated NO3(-) uptake reduces Cd entry into roots, thus lowing Cd levels in plants and improving plant growth. In addition, we also found that the Cd levels in edible parts of 11 Chinese cabbage (Brassica rapa L. ssp. pekinensis) cultivars correlated well with the NO3(-) uptake rates of their roots. These results suggested that the NO3(-) uptake of roots negatively regulate Cd uptake. Modification of NO3(-) uptake in crops by modulating NO3(-) uptake pathway might provide a biological engineering approach to reducing Cd accumulation in edible organs, thus improving food safety.

Exogenous abscisic acid application decreases cadmium accumulation in Arabidopsis plants, which is associated with the inhibition of IRT1-mediated cadmium uptake.

Cadmium (Cd) contamination of agricultural soils is an increasingly serious problem. Measures need to be developed to minimize Cd entering the human food chain from contaminated soils. We report here that, under Cd exposure condition, application with low doses of (0.1-0.5 µM) abscisic acid (ABA) clearly inhibited Cd uptake by roots and decreased Cd level in Arabidopsis wild-type plants (Col-0). Expression of IRT1 in roots was also strongly inhibited by ABA treatment. Decrease in Cd uptake and the inhibition of IRT1 expression were clearly lesser pronounced in an ABA-insensitive double mutant snrk2.2/2.3 than in the Col-0 in response to ABA application. The ABA-decreased Cd uptake was found to correlate with the ABA-inhibited IRT1 expression in the roots of Col-0 plants fed two different levels of iron. Furthermore, the Cd uptake of irt1 mutants was barely affected by ABA application. These results indicated that inhibition of IRT1 expression is involved in the decrease of Cd uptake in response to exogenous ABA application. Interestingly, ABA application increased the iron level in both Col-0 plants and irt1 mutants, suggesting that ABA-increased Fe acquisition does not depend on the IRT1 function, but on the contrary, the ABA-mediated inhibition of IRT1 expression may be due to the elevation of iron level in plants. From our results, we concluded that ABA application might increase iron acquisition, followed by the decrease in Cd uptake by inhibition of IRT1 activity. Thus, for crop production in Cd contaminated soils, developing techniques based on ABA application potentially is a promising approach for reducing Cd accumulation in edible organs in plants.