Tomohito Arao

Root-to-shoot Cd translocation via the xylem is the major process determining shoot and grain cadmium accumulation in rice

Physiological properties involved in divergent cadmium (Cd) accumulation among rice genotypes were characterized using the indica cultivar ‘Habataki’ (high Cd in grains) and the japonica cultivar ‘Sasanishiki’ (low Cd in grains). Time-dependence and concentration-dependence of symplastic Cd absorption in roots were revealed not to be responsible for the different Cd accumulation between the two cultivars because root Cd uptake was not greater in the Cd-accumulating cultivar ‘Habataki’ compared with ‘Sasanishiki’. On the other hand, rapid and greater root-to-shoot Cd translocation was observed in ‘Habataki’, which could be mediated by higher abilities in xylem loading of Cd and transpiration rate as a driving force. To verify whether different abilities in xylem-mediated shoot-to-root translocation generally account for the genotypic variation in shoot Cd accumulation in rice, the world rice core collection, consisting of 69 accessions which covers the genetic diversity of almost 32 000 accessions of cultivated rice, was used. The results showed strong correlation between Cd levels in xylem sap and shoots and grains among the 69 rice accessions. Overall, the results presented in this study revealed that the root-to-shoot Cd translocation via the xylem is the major and common physiological process determining the Cd accumulation level in shoots and grains of rice plants.

Effects of water management on cadmium and arsenic accumulation and dimethylarsinic acid concentrations in Japanese rice.

Rice consumption is a major source of cadmium and arsenic for the population of Asia. We investigated the effects of water management in rice paddy on levels of cadmium and arsenic in Japanese rice grains. Flooding increased arsenic concentrations in rice grains, whereas aerobic treatment increased the concentration of cadmium. Flooding for 3 weeks before and after heading was most effective in reducing grain cadmium concentrations, but this treatment increased the arsenic concentration considerably, whereas aerobic treatment during the same period was effective in reducing arsenic concentrations but increased the cadmium concentration markedly. Flooding treatment after heading was found to be more effective than flooding treatment before heading in reducing rice grain cadmium without a concomitant increase in total arsenic levels, although it increased inorganic arsenic levels. Concentrations of dimethylarsinic acid (DMA) in grain were very low under aerobic conditions but increased under flooded conditions. DMA accounted for 3-52% of the total arsenic concentration in grain grown in soil with a lower arsenic concentration and 10-80% in soil with a higher arsenic concentration. A possible explanation for the accumulation of DMA in rice grains is that DMA translocates from shoots/roots to the grains more readily than does inorganic arsenic.

Characterizing the role of rice NRAMP5 in Manganese, Iron and Cadmium Transport

Metals like manganese (Mn) and iron (Fe) are essential for metabolism, while cadmium (Cd) is toxic for virtually all living organisms. Understanding the transport of these metals is important for breeding better crops. We have identified that OsNRAMP5 contributes to Mn, Fe and Cd transport in rice. OsNRAMP5 expression was restricted to roots epidermis, exodermis, and outer layers of the cortex as well as in tissues around the xylem. OsNRAMP5 localized to the plasma membrane, and complemented the growth of yeast strains defective in Mn, Fe, and Cd transport. OsNRAMP5 RNAi (OsNRAMP5i) plants accumulated less Mn in the roots, and less Mn and Fe in shoots, and xylem sap. The suppression of OsNRAMP5 promoted Cd translocation to shoots, highlighting the importance of this gene for Cd phytoremediation. These data reveal that OsNRAMP5 contributes to Mn, Cd, and Fe transport in rice and is important for plant growth and development.

The OsNRAMP1 iron transporter is involved in Cd accumulation in rice

Cadmium (Cd) is a heavy metal toxic to humans and the accumulation of Cd in the rice grain is a major agricultural problem, particularly in Asia. The role of the iron transporter OsNRAMP1 in Cd uptake and transport in rice was investigated here. An OsNRAMP1:GFP fusion protein was localized to the plasma membrane in onion epidermal cells. The growth of yeast expressing OsNRAMP1 was impaired in the presence of Cd compared with yeast transformed with an empty vector. Moreover, the Cd content of OsNRAMP1-expressing yeast exceeded that of the vector control. The expression of OsNRAMP1 in the roots was higher in a high Cd-accumulating cultivar (Habataki) than a low Cd-accumulating cultivar (Sasanishiki) regardless of the presence of Cd, and the amino acid sequence of OsNRAMP1 showed 100% identity between Sasanishiki and Habataki. Over-expression of OsNRAMP1 in rice increased Cd accumulation in the leaves. These results suggest that OsNRAMP1 participates in cellular Cd uptake and Cd transport within plants, and the higher expression of OsNRAMP1 in the roots could lead to an increase in Cd accumulation in the shoots. Our results indicated that OsNRAMP1 is an important protein in high-level Cd accumulation in rice.

Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice

Rice (Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon ion-beam irradiation produced three rice mutants with <0.05 mg Cd⋅kg−1 in the grain compared with a mean of 1.73 mg Cd⋅kg−1 in the parent, Koshihikari. We identified the gene responsible for reduced Cd uptake and developed a strategy for marker-assisted selection of low-Cd cultivars. Sequence analysis revealed that these mutants have different mutations of the same gene (OsNRAMP5), which encodes a natural resistance-associated macrophage protein. Functional analysis revealed that the defective transporter protein encoded by the mutant osnramp5 greatly decreases Cd uptake by roots, resulting in decreased Cd in the straw and grain. In addition, we developed DNA markers to facilitate marker-assisted selection of cultivars carrying osnramp5. When grown in Cd-contaminated paddy fields, the mutants have nearly undetectable Cd in their grains and exhibit no agriculturally or economically adverse traits. Because mutants produced by ion-beam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide.

Genotypic differences in cadmium uptake and distribution in soybeans

In order to investigate the genetic differences in uptake and distribution of cadmium in soybeans, 17 varieties of soybean were grown first in soil and then four or five varieties of soybean were grown in nutrient solution with different levels of cadmium.Significant genotypic differences in seed cadmium levels were found. The seed cadmium concentration was lowest for the En-b0-1-2 soybean variety, and highest for Harosoy, in both field and pot experiments. The seed cadmium levels of Tohoku 128, a cross between Enrei and Suzuyutaka, were intermediate between those of the parents. For four soil types, containing from 0.2 to 6.5 mg kg−1 extractable cadmium, the ranking of soybean genotypes based on seed cadmium level was similar, indicating that there is a genetic factor involved in the varietal differences in cadmium concentration. Among the four soybean varieties tested in one experiment in the present study, the cadmium concentrations in leaves, stems and pods as well as the total cadmium uptake were lowest for En-b0-1-2. These results suggest that cadmium uptake and/or translocation from root to shoot are low in En-b0-1-2. In solution culture containing 100 μg L−1 cadmium, the cadmium concentrations in seeds, stems and pods at the seed maturation stage were also the lowest for En-b0-1-2. In a second experiment, the cadmium concentrations in the leaves, stem and petiole were lower at both 7 and 15 days after the addition of cadmium to the nutrient solution for En-b0-1-2 and Enrei than for Tohoku 128, Suzuyutaka and Harosoy; however, the cadmium concentrations of roots for En-b0-1-2 and Enrei were higher than for the other varieties. We propose that the lower levels of cadmium found in the seeds of certain varieties of soybean result from the combination of lower initial uptake and retention of higher levels of cadmium in the roots, thus limiting its translocation to the shoot.

Heavy metal contamination of agricultural soil and countermeasures in Japan

Many heavy metals exist in minute amounts in natural agricultural soil. However, when their amounts exceed a certain level due to pollutants brought from outside, soil contamination occurs and agricultural products become contaminated. There have been many cases in Japan of heavy metal contamination originating from old mines and smelters, and soil contamination of agricultural land has become a social issue. In particular, cadmium (Cd) is one of the most harmful heavy metals. If agricultural products absorb an excessive amount of Cd, they may adversely affect people’s health, and therefore allowable concentrations are regulated by law. If agricultural land has become contaminated with Cd, measures for minimizing the absorption of Cd by agricultural crops are necessary; these include: (1) soil dressing, (2) water management (paddy field), (3) chemical cleaning of soil, (4) phytoextraction, and (5) use of different varieties and rootstock. Other heavy metals such as arsenic, lead, copper, zinc, and mercury also sometimes cause contamination of agricultural soil.

Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum

Solanum torvum Sw. cv. Torubamubiga (TB) is a low cadmium (Cd)-accumulating plant. To elucidate the molecular mechanisms of the Cd acclimation process in TB roots, transcriptional regulation was analysed in response to mild Cd treatment: 0.1 μM CdCl2 in hydroponic solution. A unigene set consisting of 6296 unigene sequences was constructed from 18 816 TB cDNAs. The distribution of functional categories was similar to tomato, while 330 unigenes were suggested to be TB specific. For expression profiling, the SuperSAGE method was adapted for use with Illumina sequencing technology. Expression tag libraries were constructed from Cd-treated (for 3 h, 1 d, and 3 d) and untreated roots, and 34 269 species of independent tags were collected. Moreover, 6237 tags were ascribed to the TB or eggplant (aubergine) unigene sequences. Time-course changes were examined, and 2049 up- and 2022 down-regulated tags were identified. Although no tags annotated to metal transporter genes were significantly regulated, a tag annotated to AtFRD3, a xylem-loading citrate transporter, was down-regulated. In addition to induction of heavy metal chaperone proteins, antioxidative and sulphur-assimilating enzymes were induced, confirming that oxidative stress developed even using a mild Cd concentration. Rapid repression of dehydration-related transcription factors and aquaporin isoforms suggests that dehydration stress is a potential constituent of Cd-induced biochemical impediments. These transcriptional changes were also confirmed by real-time reverse transcription-PCR. Further additions of TB unigene sequences and functional analysis of the regulated tags will reveal the molecular basis of the Cd acclimation process, including the low Cd-accumulating characteristics of TB.

Genotypic Variation in Shoot Cadmium Concentration in Rice and Soybean in Soils with Different Levels of Cadmium Contamination

A pot experiment was conducted to investigate whether the shoot cadmium (Cd) concentration in 11 rice and 10 soybean cultivars varied among 4 soils with different levels of Cd contamination. Significant differences in shoot Cd concentration were found among rice or soybean cultivars grown in the 4 soils. The ranking of the rice cultivars for the shoot Cd concentration varied considerably among the soils. On the other hand, the soybean cultivars were ranked similarly in terms of shoot Cd concentration in the 4 soils. Significant and positive correlations were found between the Cd and Zn concentrations and between the Cd and Mn concentrations in the shoot of rice cultivars, when they were grown in 2 soils with relatively moderate levels of Cd contamination. The shoot Cd concentration in the soybean cultivars, however, was not correlated with the concentrations determined for any of the metals (Zn, Mn, Cu, and Fe) across the 4 soils. Significant and positive correlations between the concentrations of Cd in younger shoots and mature seeds were detected among the soybean cultivars in 2 soils used, unlike among the rice cultivars, indicating that it may be difficult to evaluate the genotypic variation in seed Cd concentration using relatively younger shoots in the case of rice. These results revealed that genotypic differences in shoot Cd concentration in rice or soybean are variable or invariable among soils, respectively.

Arsenic Distribution and Speciation near Rice Roots Influenced by Iron Plaques and Redox Conditions of the Soil Matrix

Elevated arsenic (As) concentrations in rice and the soil solution result from changes in soil redox conditions, influenced by the water management practices during rice cultivation. Microscale changes in redox conditions from rhizosphere to soil matrix affect the As speciation and Fe plaque deposition. In order to focus on the rhizosphere environment, we observed microscale distribution and speciation of As around the rhizosphere of paddy rice with X-ray fluorescence mapping and X-ray absorption spectroscopy. When the soil matrix was anaerobic during rice growth, Fe-plaque did not cover the entire root, and As(III) was the dominant arsenic species in the soil matrix and rhizosphere. Draining before harvest led the conditions to shift to aerobic. Oxidation of As(III) to As(V) occurred faster in the Fe-plaque than the soil matrix. Arsenic was scavenged by iron mottles originating from Fe-plaque around the roots. The ratio of As(V) to As(III) decreased toward the outer-rim of the subsurface Fe mottles where the soil matrix was not completely aerated. These results provide direct evidence that speciation of As near rice roots depends on spatial and temporal redox variations in the soil matrix.