OsNRAMP1 transporter contributes to cadmium and manganese uptake in rice

Rice is a major dietary source of the toxic metal, cadmium (Cd). Previous studies reported that the rice transporter, OsNRAMP1, (Natural resistance-associated macrophage protein 1) could transport iron (Fe), Cd and arsenic (As) in heterologous yeast assays. However, the in planta function of OsNRAMP1 remains unknown. Here, we showed that OsNRAMP1 was able to transport Cd and manganese (Mn) when expressed in yeast, but did not transport Fe or As. OsNRAMP1 was mainly expressed in roots and leaves and encoded a plasma membrane-localized protein. OsNRAMP1 expression was induced by Cd treatment and Fe deficiency. Immunostaining showed that OsNRAMP1 was localized in all root cells, except the central vasculature, and in leaf mesophyll cells. The knockout of OsNRAMP1 resulted in significant decreases in root uptake of Cd and Mn and their accumulation in rice shoots and grains, and increased sensitivity to Mn deficiency. The knockout of OsNRAMP1 had smaller effects on Cd and Mn uptake than knockout of OsNRAMP5, while knockout of both genes resulted in large decreases in the uptake of the two metals. Taken together, OsNRAMP1 contributes significantly to the uptake of Mn and Cd in rice, and the functions of OsNRAMP1 and OsNRAMP5 are similar but not redundant.     

OsNRAMP3 Is a Vascular Bundles-Specific Manganese Transporter That Is Responsible for Manganese Distribution in Rice

Manganese (Mn) is an essential trace element for plants. Recently, the genes responsible for uptake of Mn in plants were identified in Arabidopsis and rice. However, the mechanism of Mn distribution in plants has not been clarified. In the present study we identified a natural resistance-associated macrophage protein (NRAMP) family gene in rice, OsNRAMP3, involved in Mn distribution. OsNRAMP3 encodes a plasma membrane-localized protein and was specifically expressed in vascular bundles, especially in phloem cells. Yeast complementation assay showed that OsNRAMP3 is a functional Mn-influx transporter. When OsNRAMP3 was absent, rice plants showed high sensitivity to Mn deficiency. Serious necrosis appeared on young leaves and root tips of the OsNRAMP3 knockout line cultivated under low Mn conditions, and high Mn supplies could rescue this phenotype. However, the necrotic young leaves of the knockout line possessed similar levels of Mn to the wild type, suggesting that the necrotic appearance was caused by disturbed distribution of Mn but not a general Mn shortage. Additionally, compared with wild type, leaf Mn content in osnramp3 plants was mostly in older leaves. We conclude that OsNRAMP3 is a vascular bundle-localized Mn-influx transporter involved in Mn distribution and contributes to remobilization of Mn from old to young leaves.     

Role of the iron transporter OsNRAMP1 in cadmium uptake and accumulation in rice

The heavy metal cadmium (Cd) is toxic to humans, and its accumulation in rice grains is a major agricultural problem. Rice has seven putative metal transporter NRAMP genes, but microarray analysis showed that only OsNRAMP1 is highly up-regulated by iron (Fe) deficiency. OsNRAMP1 localized to the plasma membrane and transported Cd as well as Fe. OsNRAMP1 expression was observed mainly in roots and was higher in the roots of a high-Cd-accumulating cultivar (Habataki) than in those of a low-Cd-accumulating cultivar (Sasanishiki). The amino acid sequence of OsNRAMP1 in the Sasanishiki and Habataki cultivars was found to be 100% identical. These results suggest that OsNRAMP1 participates in cellular Cd uptake and that the differences observed in Cd accumulation among cultivars are because of differences in OsNRAMP1 expression levels in roots.     

An overview of rhizosphere processes related with plant nutrition in major cropping systems in China

Rhizosphere processes of individual plants have been widely investigated since 1904 when the term “rhizosphere” was first put forward. However, little attention has been paid to rhizosphere effects at an agro-ecosystem level. This paper presents recent research on the rhizosphere processes in relation to plant nutrition in main cropping systems in China. In the peanut (Arachis hypogaea L.)/maize (Zea mays L.) intercropping system, maize was found to improve the Fe nutrition of peanut through influencing its rhizosphere processes, suggesting an important role of phytosiderophores released from Fe-deficient maize. Intercropping between maize and faba bean (Vicia faba L.) was found to improve nitrogen and phosphorus uptake in the two crops compared with corresponding sole crop. There was a higher land equivalent ratio (LER) in the intercropping system of maize and faba bean than the treatment of no root interactions between the two crops. The increased yield of maize intercropped with faba bean resulted from an interspecific facilitation in nutrient uptake, depending on interspecific root interactions of the two crops. In the rotation system of rice (Oryza sativa L.)-wheat (Triticum aestivum L.) crops, Mn deficiency in wheat was caused by excessive Mn uptake by rice and Mn leaching from topsoil to subsoil due to periodic cycles of flooding and drying. However, wheat genotypes tolerant to Mn deficiency tended to distribute more roots to deeper soil layer and thus expand their rhizosphere zones in the Mn-deficient soils and utilize Mn from the subsoil. Deep ploughing also helped root penetration into subsoil and was propitious to correcting Mn deficiency in wheat rotated with rice. In comparison, oilseed rape (Brassica napus L.) took up more Mn than wheat through mobilizing sparingly soluble soil Mn due to acidification and reduction processes in the rhizosphere. Thus, oilseed rape was tolerant to the Mn-deficient conditions in the rice-oilseed rape rotation. Oxidation reactions on root surface of rice also resulted in the formation of Fe plaque in the rice rhizosphere. Large amounts of Zn were accumulated on the Fe plaque. Zinc uptake by rice plants increased as Fe plaque formed, but decreased at high amounts of Fe plaque. It is suggested that to fine-tune cropping patterns and optimize nutrient management based on a better understanding of rhizosphere processes at an agro-ecosystem level is crucial for increasing nutrient use efficiency and developing sustainable agriculture in China.     

缺Cu~（2＋）和Zn~（2＋）对水稻OsNRAMP家族基因表达与主要金属离子吸收的影响

对野生型水稻进行缺Cu2＋和Zn2＋处理,利用定量RT-PCR技术分析OsNARAMP家族不同基因的表达情况,并采用ICP-MS技术检测在水稻根、茎叶中Fe、Zn、Cu和Mn等元素的积累情况。结果表明水稻中OsNRAMP家族有9个基因,可分为3个亚类;OsNRAMP5可能参与了Cu2＋的吸收和转运,而OsNRAMP2和OsNRAMP3可能参与Zn2＋吸收和转运。同时缺Cu2＋和Zn2＋能刺激Fe2＋离子在水稻根和茎叶中的积累,减缓Mn2＋离子在根中的吸收;而缺Cu2＋减缓水稻中Zn2＋离子的吸收。这些金属离子吸收的情况和OsNRAMP家族可能存在密切关系。     

Expression in Arabidopsis and cellular localization reveal involvement of rice NRAMP,OsNRAMP1, in arsenic transport and tolerance

Irrigation of paddy fields to arsenic (As) containing groundwater leads to As accumulation in rice grains and causes serious health risk to the people worldwide. To reduce As intake via consumption of contaminated rice grain, identification of the mechanisms for As accumulation and detoxification in rice is a prerequisite. Herein, we report involvement of a member of rice NRAMP (Natural Resistance‐Associated Macrophage Protein) transporter, OsNRAMP1, in As, in addition to cadmium (Cd), accumulation through expression in yeast and Arabidopsis. Expression of OsNRAMP1 in yeast mutant (fet3fet4) rescued iron (Fe) uptake and exhibited enhanced accumulation of As and Cd. Expression of OsNRAMP1 in Arabidopsis provided tolerance with enhanced As and Cd accumulation in root and shoot. Cellular localization revealed that OsNRAMP1 resides on plasma membrane of endodermis and pericycle cells and may assist in xylem loading for root to shoot mobilization. This is the first report demonstrating role of NRAMP in xylem mediated loading and enhanced accumulation of As and Cd in plants. We propose that genetic modification of OsNRAMP1 in rice might be helpful in developing rice with low As and Cd content in grain and minimize the risk of food chain contamination to these toxic metals.     

The HvNramp5 Transporter Mediates Uptake of Cadmium and Manganese,But Not Iron

The Natural Resistance Associated Macrophage Protein (Nramp) represents a transporter family for metal ions in all organisms. Here, we functionally characterized a member of Nramp family in barley (Hordeum vulgare), HvNramp5. This member showed different expression patterns, transport substrate specificity, and cellular localization from its close homolog in rice (Oryza sativa), OsNramp5, although HvNramp5 was also localized to the plasma membrane. HvNramp5 was mainly expressed in the roots and its expression was not affected by Cd and deficiency of Zn, Cu, and Mn, but slightly up-regulated by Fe deficiency. Spatial expression analysis showed that the expression of HvNramp5 was higher in the root tips than that in the basal root regions. Furthermore, analysis with laser microdissection revealed higher expression of HvNramp5 in the outer root cell layers. HvNramp5 showed transport activity for both Mn²⁺ and Cd²⁺, but not for Fe²⁺ when expressed in yeast. Immunostaining with a HvNramp5 antibody showed that this protein was localized in the root epidermal cells without polarity. Knockdown of HvNramp5 in barley resulted in a significant reduction in the seedling growth at low Mn supply, but this reduction was rescued at high Mn supply. The concentration of Mn and Cd, but not other metals including Cu, Zn, and Fe, was decreased in both the roots and shoots of knockdown lines compared with the wild-type barley. These results indicate that HvNramp5 is a transporter required for uptake of Mn and Cd, but not for Fe, and that barley has a distinct uptake system from rice.Transport of mineral elements from soil to different organs and tissues of plants requires different types of transporters (Hall and Williams, 2003; Nevo and Nelson, 2006; Yokosho et al., 2009; Olsen and Palmgren, 2014; Sasaki et al., 2016), which include the zinc-regulated transporters, iron-regulated transporter-like protein family; the natural resistance-associated macrophage protein (Nramp) family of transporters; the multidrug and toxic compound extrusion protein transporters; the heavy metal ATPase transporters; the oligopeptide transporters family; the ATP-binding cassette family of transporters; and the cation-diffusion facilitator family of transporters. Among them, Nramp represents a transporter family for metal ion in all organisms including bacteria, animals, and plants (Curie et al., 2000; Nevo and Nelson, 2006). Some members of this family in plants have been functionally characterized, especially in model plants such as Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). In Arabidopsis, there are six members of Nramp transporter proteins. AtNramp1 is localized to the plasma membrane of root cells and functions as a high-affinity transporter for Mn uptake (Cailliatte et al., 2010). Both AtNramp3 and AtNramp4 are localized to the tonoplast and play redundant roles in Fe, exporting from the vacuole during seed germination and in Mn homeostasis at the adult stage (Thomine et al., 2000; Lanquar et al., 2005, 2010). AtNramp6 is targeted to a vesicular-shaped endomembrane compartment and is implicated in the distribution or availability of Cd within cells (Cailliatte et al., 2009). However, the function of AtNramp2 and AtNramp5 has not been characterized.On the other hand, there are seven members of Nramp transporter family in the rice genome, of which four have been functionally characterized. They all are localized to the plasma membrane but show different roles. OsNramp1 shows transport activity for Fe and Cd in yeast and is proposed to be involved in Cd accumulation (Takahashi et al., 2011). OsNramp3 is localized at the vascular tissues of nodes and plays an important role in distribution of Mn, but not Fe and Cd (Yamaji et al., 2013). On the other hand, OsNrat1 (OsNramp4) transports trivalent Al ion (Xia et al., 2010) and is required for high Al tolerance in rice roots. Finally, OsNramp5 functions as a major transporter responsible for root Mn and Cd uptake (Ishimaru et al., 2012; Ishikawa et al., 2012; Sasaki et al., 2012). However, the function of OsNramp2, OsNramp6, and OsNramp7 is unknown.In addition to Nramp members characterized in rice and Arabidopsis, some members in other plant species have also been characterized. For example, a soybean (Glycine max) Nramp transporter, GmDMT1 is implicated in the ferrous iron transport (Kaiser et al., 2003). Nramp1 isolated from Noccaea caerulescens, a Zn/Cd hyperaccumulator, is involved in the influx of Cd across the endodermal plasma membrane and plays a key role in Cd flux into the stele and root-to-shoot Cd transport (Milner et al., 2014). In Malus baccata, Nramp1 is capable of mediating the distribution of ions as well as transport of Fe, Mn, and Cd (Xiao et al., 2008). Besides, Nramp1 and Nramp3 in tomato (Solanum lycopersicum) have also been suggested to be involved in Mn transport (Bereczky et al., 2003). When NcNramp3 and NcNramp4 from Noccaea caerulescens were expressed in yeast, NcNramp3 transported Fe, Mn, and Cd, while NcNramp4 also transported Zn in addition to Fe, Mn, and Cd (Oomen et al., 2009). However, Nramp4 isolated from Thlaspi japonicum, a Ni hyperaccumulator, showed transport activity for Ni but not for Zn, Cd, or Mn in yeast (Mizuno et al., 2005). These findings indicate that Nramp members have a diverse role in metal transport in plants.Barley (Hordeum vulgare) is the fourth most important cereal crop in the world; however, less progress has been made in understating of molecular mechanisms on mineral element transport in barley due to its large genome size. For example, no Nramp members in barley have been functionally characterized so far. In this study, we first isolated barley Nramp member, HvNramp5, which is a close homolog of rice OsNramp5. Detailed functional analysis revealed that HvNramp5 is involved in the uptake of both Mn and Cd, but not of Fe in barley roots. Furthermore, we found that different from OsNramp5, HvNramp5 showed a distinct pattern in the gene expression, cellular localization, and transport substrate.     

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.     

Biochemical and molecular changes in rice seedlings (Oryza sativa L.) to cope with chromium stress

Chromium (Cr) is very toxic to both humans and plants. This investigation aimed to understand the physiological and molecular responses of rice seedlings to Cr stress. Cr toxicity did not significantly affect morphological features and Cr accumulation in roots and shoots in Pokkali but not in BRRI 51, although there was a reduction in chlorophyll concentration in leaves of both genotypes. These results imply that Pokkali has mechanisms to cope with Cr supplementation. We therefore performed quantitative real‐time PCR on the expression pattern of two chelator genes, OsPCS1 and OsMT1, but there were no significant changes in expression in roots and shoots of Pokkali and BRRI 51 following Cr stress. This suggests that there was no metal sequestration following heavy metal stress in roots of these genotypes. Moreover, no expression of two heavy metal transporter genes, OsHMA3 and OsNRAMP1, was induced after Cr stress in roots and shoots, suggesting that these transporter genes are not induced by Cr stress or might not be involved in Cr uptake in rice. We also performed a targeted study on the effect of Cr on Fe uptake mechanisms. Our studies showed a consistent reduction in Fe uptake, Fe reductase activity and expression of Fe‐related genes (OsFRO1 and OsIRT1) under Cr stress in both roots and leaves of Pokkali. In contrast, these parameters and genes were significantly increased in Cr‐sensitive BRRI 51 under Cr stress. The results confirm that limiting Fe uptake through the down‐regulation of Fe reductase and Fe transporter genes is the main strategy of Cr‐tolerant Pokkali to cope with Cr stress. Finally, increased CAT, POD and GR activity and elevated glutathione and proline synthesis might provide strong antioxidant defence against Cr stress in Pokkali. Taken together, our findings reveal that Cr stress tolerance in rice (Pokkali) is not related to metal sequestration but is associated with reduced Fe transport and increased antioxidant defence.     

In Silico Analysis of Natural Resistance-Associated Macrophage Protein (NRAMP) Family of Transporters in Rice

In Oryza sativa (rice) there are seven members in the NRAMP (natural resistance- associated macrophage protein) family of transporter proteins. They have been identified as OsNRAMP1, OsNRAMP2, OsNRAMP3, OsNRAMP4, OsNRAMP5, OsNRAMP6 and OsNRAMP7. Several metal ions like Zn²⁺, Mn²⁺, Fe²⁺, Cd²⁺ etc. have been studied to be transported via NRAMP transporter proteins in rice plant. In spite of this, very little information is available regarding these transporters. Hence it is important to computationally predict and characterize the OsNRAMP family of transporters for studying and understanding their molecular insights in future studies. For this purpose, various in silico methods and tools were used for the characterization of OsNRAMP family of transporter proteins. Physico-chemical properties of the protein sequences were calculated, putative transmembrane domains (TMDs) and conserved motif signatures were determined and their interaction partners were predicted. 3D models of all the members of OsNRAMP transporters were generated using online structure prediction tool followed by their analysis. In silico microarray analysis was done to understand the expression pattern of these transporters in rice plant. Currently, only limited knowledge is available about the structural and functional aspects of these transporters, hence this study would provide more theoretical information about them.     

Exploiting new tools for iron bio-fortification of rice

Rice is one of the most important staple crops and efficient iron (Fe) adsorption during growth not only improves rice yield, but also enriches this essential micronutrient in rice grains to address Fe deficiency in humans. In this article, we review updates on research into the molecular mechanisms regulating Fe uptake from soil and its transport from roots to shoots to seeds in rice plants. Understanding the regulation and expression of genes involved in Fe homeostasis will benefit the development of variants with enhanced Fe utilization to improve rice output and quality.     

Physiological and Molecular Responses Under Fe Deficiency in Two Rice (Oryza sativa) Genotypes Differing in Iron Accumulation Ability in Seeds

Fe is an essential mineral element that plants need for their growth. When there is low soil availability of Fe, plants show severe deficiency symptoms. Under Fe-deficiency conditions, plants alter a number of processes to acquire Fe from soil. Genes involved in these mechanisms have been identified from different model plants, including Arabidopsis and rice. Fe transport within plants is also tightly regulated. In this study, we used H9405, a cultivar of rice with high Fe accumulation in seeds, and Yangdao 6, a cultivar with low seed Fe accumulation, to study their responses under different Fe conditions. Our results showed that genes involved in acquisition of Fe from soil in these two cultivars were both up-regulated in roots under Fe-deficiency conditions, and the elevation of the expression was much higher in Yangdao 6 than in H9405. However, remobilization-related genes in shoot vasculature were expressed in an opposite way between the two cultivars. In H9405, the expression of these genes was up-regulated; but in Yangdao 6, their expression was reduced. Our results showed that the differential expression of root-uptake and shoot-remobilization genes in the two cultivars is correlated to the Fe content in roots, shoots, and seeds. Strategies to biofortify rice cultivars with different characteristics were also discussed based on our discovery.