A weak allele of OsNRAMP5 confers moderate cadmium uptake while avoiding manganese deficiency in rice.

Decreasing cadmium (Cd) concentrations in rice grains can effectively reduce potential risks to human health because rice is the major contributor to Cd intake in many diets. Among several genes involved in rice Cd accumulation, the loss of function of OsNRAMP5 is known to be effective in reducing grain concentration by inhibiting root uptake. However, disruption of this gene simultaneously decreases manganese (Mn) uptake because OsNRAMP5 is a major Mn transporter. With the aim of improving Mn uptake in OsNRAMP5 mutants while still restricting the grain Cd concentration below the upper limit of international standards, we identified a novel OsNRAMP5 allele encoding a protein in which glutamine (Q) at position 337 was replaced by lysine (K). The mutant carrying the OsNRAMP5-Q337K allele showed intermediate Cd and Mn accumulation between that of the wild-type and OsNRAMP5-knockout lines, and exhibited more resistance to Mn deficiency than the knockout lines. Different amino acid substitutions at position Q337 significantly affected the Cd and Mn transport activity in yeast cells, indicating that it is one of the crucial sites for OsNRAMP5 function. Our results suggest that the OsNRAMP5-Q337K allele might be useful for reducing grain Cd concentrations without causing severe Mn deficiency in rice cultivars through DNA marker-assisted breeding.

OsNRAMP5 Is a Major Transporter for Lead Uptake in Rice.

Lead (Pb) is one of the most toxic metals affecting human health globally. Food is an important source of chronic Pb exposure in humans. How Pb is taken up by rice, a staple food for over half of the global population, remains unknown. In the present study, we investigated the role of OsNRAMP5, a member of the NRAMP (Natural Resistance-Associated Macrophage Protein) transporter family, in Pb uptake by rice roots. Heterologous expression of OsNRAMP5 in yeast increased Pb uptake and sensitivity toward Pb. Knockout of OsNRAMP5 in rice by CRISPR/Cas9 gene editing resulted in significant decreases in root uptake of Pb and accumulation in rice shoots. The maximum influx velocity (Vmax) for Pb uptake of the knockout mutants was 70% lower than that of wild-type plants. When grown in Pb-contaminated paddy soil, OsNRAMP5 knockout mutants accumulated approximately 50 and 70% lower Pb concentrations in the grain and straw, respectively, than the wild type. OsNRAMP5 expression in rice roots was not affected by Pb exposure. These results indicate that OsNRAMP5 is a major transporter for Pb uptake in rice, in addition to its role in the uptake of manganese and cadmium. This study provides a mechanistic understanding of Pb uptake in rice plants and a potential strategy to limit Pb accumulation in rice grains.

Lateral roots but not root hairs contribute to high uptake of manganese and cadmium in rice.

Rice (Oryza sativa L.) can accumulate high manganese (Mn) in the shoots through uptake by the roots, which consist of crown roots, lateral roots and root hairs. We investigated the role of lateral roots and root hairs in Mn and cadmium (Cd) uptake by using two indica rice mutants defective in formation of lateral roots (osiaa11) and root hairs (osrhl1). The uptake of Mn and Cd in osiaa11 was significantly lower than that in wild type 'Kasalath', but there was no difference between wild type and osrhl1. Furthermore, a kinetic study showed that Mn uptake in osiaa11 was much lower than that in wild type and osrhl1 across a wide range of Mn concentrations. The role of lateral roots in Mn and Cd uptake was further confirmed in a japonica rice mutant defective in lateral root formation. We found that expression of Mn transporter gene Natural Resistance-Associated Macrophage Protein 5 (OsNRAMP5), but not of Metal Tolerance Protein 9 (OsMTP9), was lower in osiaa11 than in wild type; however, there were no differences between osrhl1 and the wild type. Immunostaining showed that OsNRAMP5 and OsMTP9 were localized in the exodermis and endodermis of crown roots and lateral roots, but not in the root hairs. Taken together, our results indicate that lateral roots, but not root hairs, play an important role in high Mn and Cd uptake in rice.

Overexpression of the manganese/cadmium transporter OsNRAMP5 reduces cadmium accumulation in rice grain.

Rice is a major dietary source of the toxic metal cadmium (Cd), and reducing its accumulation in the grain is therefore important for food safety. We selected two cultivars with contrasting Cd accumulation and generated transgenic lines overexpressing OsNRAMP5, which encodes a major influx transporter for manganese (Mn) and Cd. We used two different promoters to control the expression, namely OsActin1 and maize Ubiquitin. Overexpression of OsNRAMP5 increased Cd and Mn uptake into the roots, but markedly decreased Cd accumulation in the shoots, whilst having a relatively small effect on Mn accumulation in the shoots. The overexpressed OsNRAMP5 protein was localized to the plasma membrane of all cell types in the root tips and lateral root primordia without polarity. Synchrotron X-ray fluorescence mapping showed that the overexpression lines accumulated more Cd in the root tips and lateral root primordia compared with the wild-type. When grown in three Cd-contaminated paddy soils, overexpression of OsNRAMP5 decreased concentration of Cd in the grain by 49-94% compared with the wild type. OsNRAMP5-overexpression plants had decreased Cd translocation from roots to shoots as a result of disruption of its radial transport into the stele for xylem loading, demonstrating the effect of transporter localization and polarity on ion homeostasis.

Silicon reduces cadmium accumulation by suppressing expression of transporter genes involved in cadmium uptake and translocation in rice.

Silicon (Si) alleviates cadmium (Cd) toxicity and accumulation in a number of plant species, but the exact molecular mechanisms responsible for this effect are still poorly understood. Here, we investigated the effect of Si on Cd toxicity and accumulation in rice (Oryza sativa) by using two mutants (lsi1 and lsi2) defective in Si uptake and their wild types (WTs). Root elongation was decreased with increasing external Cd concentrations in both WTs and mutants, but Si did not show an alleviative effect on Cd toxicity in all lines. By contrast, the Cd concentration in both the shoots and roots was decreased by Si in the WTs, but not in the mutants. Furthermore, Si supply resulted in a decreased Cd concentration in the root cell sap and xylem sap in the WTs, but not in the mutants. Pre-treatment with Si also decreased Cd accumulation in the WTs, but not in the mutants. Silicon slightly decreased Cd accumulation in the cell wall of the roots. The expression level of OsNramp5 and OsHMA2 was down-regulated by Si in the WTs, but not in the mutants. These results indicate that the Si-decreased Cd accumulation was caused by down-regulating transporter genes involved in Cd uptake and translocation in rice.

OsNRAMP5 contributes to manganese translocation and distribution in rice shoots.

Manganese (Mn) is an essential micronutrient for plants playing an important role in many physiological functions. OsNRAMP5 is a major transporter responsible for Mn and cadmium uptake in rice, but whether it is involved in the root-to-shoot translocation and distribution of these metals is unknown. In this work, OsNRAMP5 was found to be highly expressed in hulls. It was also expressed in leaves but the expression level decreased with leaf age. High-magnification observations revealed that OsNRAMP5 was enriched in the vascular bundles of roots and shoots especially in the parenchyma cells surrounding the xylem. The osnramp5 mutant accumulated significantly less Mn in shoots than the wild-type plants even at high levels of Mn supply. Furthermore, a high supply of Mn could compensate for the loss in the root uptake ability in the mutant, but not in the root-to-shoot translocation of Mn, suggesting that the absence of OsNRAMP5 reduces the transport of Mn from roots to shoots. The results suggest that OsNRAMP5 plays an important role in the translocation and distribution of Mn in rice plants in addition to its role in Mn uptake.

Mutation of OsNRAMP5 reduces cadmium xylem and phloem transport in rice plants and its physiological mechanism.

Natural resistance associated macrophage protein 5 (NRAMP5) is a key transporter for cadmium (Cd) uptake by rice roots; however, the effect of OsNRAMP5 on Cd translocation and redistribution in rice plants remains unknown. In this study, an extremely low Cd-accumulation mutant (lcd1) and wild type (WT) plants were utilized to investigate the effect of OsNRAMP5 mutation on Cd translocation and redistribution via the xylem and phloem and its possible physiological mechanism using field, hydroponic and isotope-labelling experiments. The results showed that OsNRAMP5 mutation reduced xylem and phloem transport of Cd, due to remarkably lower Cd translocation from roots to shoots and from the leaves Ⅰ-Ⅲ to their corresponding nodes, as well as lower Cd concentrations in xylem and phloem sap of lcd1 compared to WT plants. Mutation of OsNRAMP5 reduced Cd translocation from roots to shoots in lcd1 plants by increasing Cd deposition in cellulose of root cell walls and reducing OsHMA2-and OsCCX2-mediated xylem loading of Cd, and the citric acid- and tartaric acid-mediated long-distance xylem transport of Cd. Moreover, OsNRAMP5 mutation inhibited Cd redistribution from flag leaves to nodes and panicles in lcd1 plants by increasing Cd sequestration in cellulose and vacuoles, and decreasing OsLCT1-mediated Cd phloem transport in flag leaves.

Amino Acid Residues of the Metal Transporter OsNRAMP5 Responsible for Cadmium Absorption in Rice.

The transport of metals such as iron (Fe), manganese (Mn), and cadmium (Cd) in rice is highly related. Although Fe and Mn are essential elements for plant growth, Cd is a toxic element for both plants and humans. OsNRAMP5-a member of the same family as the Fe, Mn, and Cd transporter OsNRAMP1-is responsible for the transport of Mn and Cd from soil in rice. Knockout of OsNRAMP5 markedly reduces both Cd and Mn absorption, and this OsNRAMP5 knockout is indispensable for the development of low-Cd rice. However, in low-Mn environments, such plants would exhibit Mn deficiency and suppressed growth. We generated random mutations in OsNRAMP5 via error-prone PCR, and used yeast to screen for the retention of Mn absorption and the inhibition of Cd absorption. The results showed that alanine 512th is the most important amino acid residue for Cd absorption and that its substitution resulted in the absorption of Mn but not Cd.

Mitigating cadmium accumulation in rice without compromising growth via modifying the regulatory region of OsNRAMP5.

Cadmium (Cd) intake poses a significant health risk to humans, and the contamination of rice grains with Cd is a major concern in regions where rice is a staple food. Although the knockout of OsNRAMP5, which encodes a key transporter responsible for Cd and manganese (Mn) uptake, can significantly reduce Cd accumulation in rice grains, recent studies have revealed that this knockout adversely affects plant growth, grain yield, and increases vulnerability to abiotic and biotic stresses due to reduced Mn accumulation. In this study, we employed CRISPR/Cas9 technology to modify the regulatory region of OsNRAMP5 with the aim of reducing Cd accumulation in rice grains. Our findings demonstrate that mutations in the regulatory region of OsNRAMP5 do not impact its expression pattern but result in a reduction in translation. The decreased translation of OsNRAMP5 effectively decreases grain Cd accumulation while leaving Mn accumulation and important agronomic traits, including yield, unaffected. Thus, our study presents a practical and viable strategy for reducing Cd accumulation in rice grains without compromising Mn accumulation or overall rice production.

Knockout of OsNRAMP5 enhances rice tolerance to cadmium toxicity in response to varying external cadmium concentrations via distinct mechanisms.

OsNRAMP5 is a transporter responsible for cadmium (Cd) and manganese (Mn) uptake and root-to-shoot translocation of Mn in rice plants. Knockout of OsNRAMP5 is regarded as an effective approach to minimize Cd uptake and accumulation in rice. It is vital to evaluate the effects of knocking out OsNRAMP5 on Cd and Mn accumulation, as well as Cd tolerance of rice plants in response to varying environmental Cd concentrations, and to uncover the underlying mechanism, which until now, has remained largely unexplored. This study showed that knockout of OsNRAMP5 decreased Cd uptake, but simultaneously facilitated Cd translocation from roots to shoots. The effect of OsNRAMP5 knockout on reducing root Cd uptake weakened, however its effect on improving root-to-shoot Cd translocation was constant with increasing environmental Cd concentrations. As a result, its mutation dramatically reduced Cd accumulation in shoots under low and moderate Cd stress, but inversely increased that under high Cd conditions. Interestingly, Cd tolerance of its knockout mutants was persistently enhanced, irrespective of lower or higher Cd concentrations in shoots, compared with that of wild-type plants. Knockout of OsNRAMP5 mitigated Cd toxicity by dramatically diminishing Cd uptake at low or moderate external Cd concentrations. Remarkably, its knockout effectively complemented deficient mineral nutrients in shoots, thereby indirectly enhancing rice tolerance to severe Cd stress. Additionally, its mutation conferred preferential delivery of Mn to young leaves and grains. These results have important implications for the application of the OsNRAMP5 mutation in mitigating Cd toxicity and lowering the risk of excessive Cd accumulation in rice grains.

Metarhizium robertsii as a promising microbial agent for rice in situ cadmium reduction and plant growth promotion.

The toxic chemical element cadmium (Cd) in paddy fields triggered increasing problems of growth inhibition and food security in rice consistently. In this study, we found Metarhizium robertsii, which is widely used as a bioinsecticide and biofertilizer in agriculture and recently found to be resistant to Cd, developed intraradical and extraradical symbiotic hyphae in rice seedlings, and successfully colonized in the rice rhizosphere soil to more than 103 CFUs g-1 soil at harvesting. M. robertsii colonization significantly reduced Cd accumulations in both hydroponically cultured seedlings and the matured rice cultured in Cd contaminated potting soil (2 ppm). Notably, Cd accumulation reduction of the roots, stems, leaves, husks and grains of the matured rice induced by the fungus were 44.3%, 32.1%, 35.3%, 31.9% and 24.7%, respectively. It was caused by the M. robertsii-induced suppression of Cd intake transporter gene osNramp5 in the rice roots, and the chemical stabilizing of Cd to the residual fraction in the rhizosphere soil. In addition, the colonization of M. robertsii significantly promoted the growth characters and the photosynthesis of the rice plants. This is achieved by the increase of endogenous hormone levels of indole-3-acetic, gibberellin A3 and brassinolide induced by M. robertsii. Furthermore, the fungus enhanced the antioxidative capacities via increasing enzyme activities of catalase, peroxidase and the production of glutathione, ascorbic acid, proline in the rice plants. Our work provides theoretical basis for expanding the use of M. robertsii as in situ Cd accumulation reduction and detoxification agents for rice in contaminated paddy fields.

Effects of external Mn2+ activities on OsNRAMP5 expression level and Cd accumulation in indica rice.

Manganese (Mn) transporter OsNRAMP5 was widely reported to regulate cadmium (Cd) uptake in rice. However, the relationship between OsNRAMP5 expression level and Cd accumulation, impacts of external ion activities on OsNRAMP5 expression level and Cd accumulation are still unclear. Investigations of the relationship between OsNRAMP5 expression level and Cd accumulation in three indica rice genotypes were conducted under various external Mn2+ activities ranging from Mn deficiency to toxicity in EGTA-buffered nutrient solution. Results in this work indicated that OsNRAMP5 expression level in roots significantly up-regulated at Mn phytotoxicity compared to that at Mn deficiency, which may stimulate by the increasing uptake of Mn. Our work also demonstrated that root Cd concentration of all the tested rice decreased notably when external Mn2+ activity reached the level of toxicity. This may explain by the increasing competition between the excess Mn2+ and Cd2+ as well as the disorder of element absorption caused by root damage at Mn toxicity. Our work also revealed that the relationship between OsNRAMP5 expression level in roots and Cd accumulation in roots was insignificant for all the tested genotypes. Besides, OsNRAMP5 expression level in roots seemed more related to root Mn accumulation. The fact that function of OsNRAMP5 mainly focuses on Mn uptake, together with the fact that many transporter genes involved in Cd uptake might result in the insignificant correlation between OsNRAMP5 expression level and Cd accumulation in roots. At last, multi-level regulating and processing of the process from gene expression to protein translation might account for the inconsistent relationship between root OsNRAMP5 expression level and Cd accumulation in roots.

Mutation at Different Sites of Metal Transporter Gene OsNramp5 Affects Cd Accumulation and Related Agronomic Traits in Rice (Oryza sativa L.).

OsNramp5 is a key gene involved in the control of the uptake of Cd, Mn, and other metal ions by rice root cells. The functional deficiency of this gene can significantly reduce the accumulation of Cd in rice grains, but the effects of its mutation on agronomic traits such as yield and quality have not been investigated comprehensively yet. In the present study, three Huanghuazhan-based OsNramp5 mutants [LCH1 (Low Cadmium Huanghuazhan 1), LCH2 (Low Cadmium Huanghuazhan 2), and LCH3 (Low Cadmium Huanghuazhan 3)] were obtained using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. The mutation-type analysis showed that LCH1, LCH2, and LCH3 encoded defective OsNramp5 protein sequences containing only 76aa, 176aa, and 266aa, respectively. The determination of metal content and the statistics of related agronomic traits revealed that the functionally deficient OsNramp5 not only significantly reduced the accumulation of Cd in the grains of the mutants but also affected rice yield and quality. However, with the decrease of OsNramp5 mutation degree, its effects on chlorenchyma Mn accumulation, yield, and quality were also diminished. Additionally, we also found that the increase in the concentration of Mn in the soil restored the phenotype of the declined yield and quality due to the functional deficiency of OsNramp5. Our findings provide novel insights into and new materials for breeding rice varieties with low Cd accumulation and excellent agronomic traits under severe Cd pollution environment.