Polar localization of a rice silicon transporter requires isoleucine at both C- and N-termini as well as positively charged residues.

Silicon (Si) is important for stable and high yields in rice (Oryza sativa), a typical Si hyperaccumulator. The high Si accumulation is achieved by the cooperation of 2 Si transporters, LOW SILICON 1 (OsLsi1) and OsLsi2, which are polarly localized in cells of the root exodermis and endodermis. However, the mechanism underlying their polar localization is unknown. Here, we identified amino acid residues critical for the polar localization of OsLsi1. Deletion of both N- and C-terminal regions resulted in the loss of its polar localization. Furthermore, the deletion of the C-terminus inhibited its trafficking from the endoplasmic reticulum to the plasma membrane. Detailed site-directed mutagenesis analysis showed that Ile18 at the N-terminal region and Ile285 at the C-terminal region were essential for the polar localization of OsLsi1. Moreover, a cluster of positively charged residues at the C-terminal region is also required for polar localization. Phosphorylation and Lys modifications of OsLsi1 are unlikely to be involved in its polar localization. Finally, we showed that the polar localization of OsLsi1 is required for the efficient uptake of Si. Our study not only identified critical residues required for the polar localization of OsLsi1, but also provided experimental evidence for the importance of transporter polarity for efficient nutrient uptake.

Knockout of a rice K5.2 gene increases Ca accumulation in the grain.

Rice is a staple food for half of the world's population, but it is a poor dietary source of calcium (Ca) due to the low concentration. It is an important issue to boost Ca concentration in this grain to improve Ca deficiency risk, but the mechanisms underlying Ca accumulation are poorly understood. Here, we obtained a rice (Oryza sativa) mutant with high shoot Ca accumulation. The mutant exhibited 26%-53% higher Ca in shoots than did wild-type rice (WT) at different Ca supplies. Ca concentration in the xylem sap was 36% higher in the mutant than in the WT. There was no difference in agronomic traits between the WT and mutant, but the mutant showed 25% higher Ca in the polished grain compared with the WT. Map-based cloning combined with a complementation test revealed that the mutant phenotype was caused by an 18-bp deletion of a gene, OsK5.2, belonging to the Shaker-like K+ channel family. OsK5.2 was highly expressed in the mature region of the roots and its expression in the roots was not affected by Ca levels, but upregulated by low K. Immunostaining showed that OsK5.2 was mainly expressed in the pericycle of the roots. Taken together, our results revealed a novel role for OsK5.2 in Ca translocation in rice, and will be a good target for Ca biofortification in rice.

Boron uptake in rice is regulated post-translationally via a clathrin-independent pathway.

Uptake of boron (B) in rice (Oryza sativa) is mediated by the Low silicon rice 1 (OsLsi1) channel, belonging to the NOD26-like intrinsic protein III subgroup, and the efflux transporter B transporter 1 (OsBOR1). However, it is unknown how these transporters cooperate for B uptake and how they are regulated in response to B fluctuations. Here, we examined the response of these two transporters to environmental B changes at the transcriptional and posttranslational level. OsBOR1 showed polar localization at the proximal side of both the exodermis and endodermis of mature root region, forming an efficient uptake system with OsLsi1 polarly localized at the distal side of the same cell layers. Expression of OsBOR1 and OsLsi1 was unaffected by B deficiency and excess. However, although OsLsi1 protein did not respond to high B at the protein level, OsBOR1 was degraded in response to high B within hours, which was accompanied with a significant decrease of total B uptake. The high B-induced degradation of OsBOR1 was inhibited in the presence of MG-132, a proteasome inhibitor, without disturbance of the polar localization. In contrast, neither the high B-induced degradation of OsBOR1 nor its polarity was affected by induced expression of dominant-negative mutated dynamin-related protein 1A (OsDRP1AK47A) or knockout of the mu subunit (AP2M) of adaptor protein-2 complex, suggesting that clathrin-mediated endocytosis is not involved in OsBOR1 degradation and polar localization. These results indicate that, in contrast to Arabidopsis thaliana, rice has a distinct regulatory mechanism for B uptake through clathrin-independent degradation of OsBOR1 in response to high B.

Reducing phosphorus accumulation in rice grains with an impaired transporter in the node.

Phosphorus is an important nutrient for crop productivity. More than 60% of the total phosphorus in cereal crops is finally allocated into the grains and is therefore removed at harvest. This removal accounts for 85% of the phosphorus fertilizers applied to the field each year. However, because humans and non-ruminants such as poultry, swine and fish cannot digest phytate, the major form of phosphorus in the grains, the excreted phosphorus causes eutrophication of waterways. A reduction in phosphorus accumulation in the grain would contribute to sustainable and environmentally friendly agriculture. Here we describe a rice transporter, SULTR-like phosphorus distribution transporter (SPDT), that controls the allocation of phosphorus to the grain. SPDT is expressed in the xylem region of both enlarged- and diffuse-vascular bundles of the nodes, and encodes a plasma-membrane-localized transporter for phosphorus. Knockout of this gene in rice (Oryza sativa) altered the distribution of phosphorus, with decreased phosphorus in the grains but increased levels in the leaves. Total phosphorus and phytate in the brown de-husked rice were 20-30% lower in the knockout lines, whereas yield, seed germination and seedling vigour were not affected. These results indicate that SPDT functions in the rice node as a switch to allocate phosphorus preferentially to the grains. This finding provides a potential strategy to reduce the removal of phosphorus from the field and lower the risk of eutrophication of waterways.

A pericycle-localized silicon transporter for efficient xylem loading in rice.

Rice is able to accumulate high concentrations of silicon (Si) in the shoots, and this ability is required for the mitigation of abiotic and biotic stresses. Although transporters for Si uptake have been identified, a transporter for the xylem loading of Si has not been found. We functionally characterized a Si transporter, OsLsi3, in terms of tissue-specific localization, knockout line phenotype and mathematic simulation. OsLsi3 was shown to be an efflux Si transporter. OsLsi3 was mainly expressed in the mature root region, and its expression was downregulated by Si. Immunostaining with a specific antibody showed that OsLsi3 was localized to the pericycle in the roots, without polarity. However, when it was expressed under the control of the OsLsi2 promoter, OsLsi3 became polarly localized to the proximal side of both the exodermis and endodermis. Knockout of this gene resulted in decreased Si uptake and concentration in the xylem sap under low Si supply, but not under high Si supply. Mathematical modeling showed that localization of OsLsi3 to the pericycle accounts for c. 30% of the total Si loading to the xylem under low Si concentrations. In summary, OsLsi3 was involved in the xylem loading of Si in rice roots, which is required for the efficient root-to-shoot translocation of Si.

A crucial role for a node-localized transporter, HvSPDT, in loading phosphorus into barley grains.

Grains are the major sink of phosphorus (P) in cereal crops, accounting for 60-85% of total plant P, but the mechanisms underlying P loading into the grains are poorly understood. We functionally characterized a transporter gene required for the distribution of P to the grains in barley (Hordeum vulgare), HvSPDT (SULTR-like phosphorus distribution transporter). HvSPDT encoded a plasma membrane-localized Pi/H+ cotransporter. It was mainly expressed in the nodes at both the vegetative and reproductive stages. Furthermore, its expression was induced by inorganic phosphate (Pi) deficiency. In the nodes, HvSPDT was expressed in both the xylem and phloem region of enlarged and diffuse vascular bundles. Knockout of HvSPDT decreased the distribution of P to new leaves, but increased the distribution to old leaves at the vegetative growth stage under low P supply. However, knockout of HvSPDT did not alter the redistribution of P from old to young organs. At the reproductive stage, knockout of HvSPDT significantly decreased P allocation to the grains, resulting in a considerable reduction in grain yield, especially under P-limited conditions. Our results indicate that node-based HvSPDT plays a crucial role in loading P into barley grains through preferentially distributing P from the xylem and further to the phloem.

The ZIP Transporter Family Member OsZIP9 Contributes To Root Zinc Uptake in Rice under Zinc-Limited Conditions.

Zinc (Zn) is an important essential micronutrient for plants and humans; however, the exact transporter responsible for root zinc uptake from soil has not been identified. Here, we found that OsZIP9, a member of the ZRT-IRT-related protein, is involved in Zn uptake in rice (Oryza sativa) under Zn-limited conditions. OsZIP9 was mainly localized to the plasma membrane and showed transport activity for Zn in yeast (Saccharomyces cerevisiae). Expression pattern analysis showed that OsZIP9 was mainly expressed in the roots throughout all growth stages and its expression was upregulated by Zn-deficiency. Furthermore, OsZIP9 was expressed in the exodermis and endodermis of root mature regions. For plants grown in a hydroponic solution with low Zn concentration, knockout of OsZIP9 significantly reduced plant growth, which was accompanied by decreased Zn concentrations in both the root and shoot. However, plant growth and Zn accumulation did not differ between knockout lines and wild-type rice under Zn-sufficient conditions. When grown in soil, Zn concentrations in the shoots and grains of knockout lines were decreased to half of wild-type rice, whereas the concentrations of other mineral nutrients were not altered. A short-term kinetic experiment with stable isotope 67Zn showed that 67Zn uptake in knockout lines was much lower than that in wild-type rice. Combined, these results indicate that OsZIP9 localized at the root exodermis and endodermis functions as an influx transporter of Zn and contributes to Zn uptake under Zn-limited conditions in rice.

Node-Localized Transporters of Phosphorus Essential for Seed Development in Rice.

About 60-85% of total phosphorus (P) in cereal crops is finally allocated to seeds, where it is required for seed development, germination and early growth. However, little is known about the molecular mechanisms underlying P allocation to seeds. Here, we found that two members (OsPHO1;1 and OsPHO1;2) of the PHO1 gene family are involved in the distribution of P to seeds in rice. Both OsPHO1;1 and OsPHO1;2 were localized to the plasma membrane and showed influx transport activities for inorganic phosphate. At the reproductive stage, both OsPHO1;1 and OsPHO1;2 showed higher expression in node I, the uppermost node connecting to the panicle. OsPHO1;1 was mainly localized at the phloem region of diffuse vascular bundles (DVBs) of node I, while OsPHO1;2 was expressed in the xylem parenchyma cells of the enlarged vascular bundles (EVBs). In addition, they were also expressed in the ovular vascular trace, the outer layer of the inner integument (OsPHO1;1) and in the nucellar epidermis (OsPHO1;2) of caryopses. Knockout of OsPHO1;2, as well as OsPHO1;1 to a lesser extent, decreased the distribution of P to the seed, resulting in decreased seed size and delayed germination. Taken together, OsPHO1;2 expressed in node I is responsible for the unloading of P from the xylem of EVBs, while OsPHO1;1 is involved in reloading P into the phloem of DVBs for subsequent allocation of P to seeds. Furthermore, OsPHO1;1 and OsPHO1;2 expression in the caryopsis is important for delivering P from the maternal tissues to the filial tissues for seed development.

Functional evolution of nodulin 26-like intrinsic proteins: from bacterial arsenic detoxification to plant nutrient transport.

Nodulin 26-like intrinsic proteins (NIPs) play essential roles in transporting the nutrients silicon and boron in seed plants, but the evolutionary origin of this transport function and the co-permeability to toxic arsenic remains enigmatic. Horizontal gene transfer of a yet uncharacterised bacterial AqpN-aquaporin group was the starting-point for plant NIP evolution. We combined intense sequence, phylogenetic and genetic context analyses and a mutational approach with various transport assays in oocytes and plants to resolve the transorganismal and functional evolution of bacterial and algal and terrestrial plant NIPs and to reveal their molecular transport specificity features. We discovered that aqpN genes are prevalently located in arsenic resistance operons of various prokaryotic phyla. We provided genetic and functional evidence that these proteins contribute to the arsenic detoxification machinery. We identified NIPs with the ancestral bacterial AqpN selectivity filter composition in algae, liverworts, moss, hornworts and ferns and demonstrated that these archetype plant NIPs and their prokaryotic progenitors are almost impermeable to water and silicon but transport arsenic and boron. With a mutational approach, we demonstrated that during evolution, ancestral NIP selectivity shifted to allow subfunctionalisations. Together, our data provided evidence that evolution converted bacterial arsenic efflux channels into essential seed plant nutrient transporters.

Tomato roots have a functional silicon influx transporter but not a functional silicon efflux transporter.

Silicon (Si) accumulation in shoots differs greatly with plant species, but the molecular mechanisms for this interspecific difference are unknown. Here, we isolated homologous genes of rice Si influx (SlLsi1) and efflux (SlLsi2) transporter genes in tomato (Solanum lycopersicum L.) and functionally characterized these genes. SlLsi1 showed transport activity for Si when expressed in both rice lsi1 mutant and Xenopus laevis oocytes. SlLsi1 was constitutively expressed in the roots. Immunostaining showed that SlLsi1 was localized at the plasma membrane of both root tip and basal region without polarity. Furthermore, overexpression of SlLsi1 in tomato increased Si concentration in the roots and root cell sap but did not alter the Si concentration in the shoots. By contrast, two Lsi2-like proteins did not show efflux transport activity for Si in Xenopus oocytes. However, when functional CsLsi2 from cucumber was expressed in tomato, the Si uptake was significantly increased, resulting in higher Si accumulation in the leaves and enhanced tolerance of the leaves to water deficit and high temperature. Our results suggest that the low Si accumulation in tomato is attributed to the lack of functional Si efflux transporter Lsi2 required for active Si uptake although SlLsi1 is functional.

The grapevine NIP2;1 aquaporin is a silicon channel.

Silicon (Si) supplementation has been shown to improve plant tolerance to different stresses, and its accumulation in the aerial organs is mediated by NIP2;1 aquaporins (Lsi channels) and Lsi2-type exporters in roots. In the present study, we tested the hypothesis that grapevine expresses a functional NIP2;1 that accounts for root Si uptake and, eventually, Si accumulation in leaves. Own-rooted grapevine cuttings of the cultivar Vinhão accumulated >0.2% Si (DW) in leaves when irrigated with 1.5 mM Si for 1 month, while Si was undetected in control leaves. Real-time PCR showed that VvNIP2;1 was highly expressed in roots and in green berries. The transient transformation of tobacco leaf epidermal cells mediated by Agrobacterium tumefaciens confirmed VvNIP2;1 localization at the plasma membrane. Transport experiments in oocytes showed that VvNIP2;1 mediates Si and arsenite uptake, whereas permeability studies revealed that VvNIP2;1 expressed in yeast is unable to transport water and glycerol. Si supplementation to pigmented grape cultured cells (cv. Gamay Freáux) had no impact on the total phenolic and anthocyanin content, or on the growth rate and VvNIP2;1 expression. Long-term experiments should help determine the extent of Si uptake over time and whether grapevine can benefit from Si fertilization.

Polar Localization of the NIP5;1 Boric Acid Channel Is Maintained by Endocytosis and Facilitates Boron Transport in Arabidopsis Roots.

Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in µ subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots.

Orchestration of three transporters and distinct vascular structures in node for intervascular transfer of silicon in rice.

Requirement of mineral elements in different plant tissues is not often consistent with their transpiration rate; therefore, plants have developed systems for preferential distribution of mineral elements to the developing tissues with low transpiration. Here we took silicon (Si) as an example and revealed an efficient system for preferential distribution of Si in the node of rice (Oryza sativa). Rice is able to accumulate more than 10% Si of the dry weight in the husk, which is required for protecting the grains from water loss and pathogen infection. However, it has been unknown for a long time how this hyperaccumulation is achieved. We found that three transporters (Lsi2, Lsi3, and Lsi6) located at the node are involved in the intervascular transfer, which is required for the preferential distribution of Si. Lsi2 was polarly localized to the bundle sheath cell layer around the enlarged vascular bundles, which is next to the xylem transfer cell layer where Lsi6 is localized. Lsi3 was located in the parenchyma tissues between enlarged vascular bundles and diffuse vascular bundles. Similar to Lsi6, knockout of Lsi2 and Lsi3 also resulted in decreased distribution of Si to the panicles but increased Si to the flag leaf. Furthermore, we constructed a mathematical model for Si distribution and revealed that in addition to cooperation of three transporters, an apoplastic barrier localized at the bundle sheath cells and development of the enlarged vascular bundles in node are also required for the hyperaccumulation of Si in rice husk.

Identification of a mammalian silicon transporter.

Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.

The Tonoplast-Localized Transporter MTP8.2 Contributes to Manganese Detoxification in the Shoots and Roots of Oryza sativa L.

Manganese (Mn) cation diffusion facilitators (Mn-CDFs) play important roles in the Mn homeostasis of plants. In rice, the tonoplast-localized Mn-CDF metal tolerance protein 8.1 (MTP8.1) is involved in Mn detoxification in the shoots. This study functionally characterized the Mn-CDF MTP8.2 and determined its contribution to Mn tolerance. MTP8.2 was found to share 68% identity with MTP8.1 and was expressed in both the shoots and roots, but its transcription level was lower than that of MTP8.1. Transient expression of the MTP8.2:green fluorescent protein (GFP) fusion protein and immunoblotting studies indicated that MTP8.2 was also localized to the tonoplast. MTP8.2 expression in yeast conferred tolerance to Mn but not to Fe, Zn, Co, Ni or Cd. MTP8.2 knockdown caused further growth reduction of shoots and roots in the mtp8.1 mutant, which already exhibits stunted growth under conditions of excess Mn. In the presence of high Mn, the MTP8.2 knockdown lines of the mtp8.1 mutant showed lower root Mn concentrations, as well as lower root:total Mn ratios, than those of wild-type rice and the mtp8.1 mutant. These findings indicate that MTP8.2 mediates Mn tolerance along with MTP8.1 through the sequestration of Mn into the shoot and root vacuoles.

A node-localized transporter OsZIP3 is responsible for the preferential distribution of Zn to developing tissues in rice.

Developing tissues such as meristem with low transpiration require high Zn levels for their active growth, but the molecular mechanisms underlying the preferential distribution to these tissues are poorly understood. We found that a member of the ZIP (ZRT, IRT-like protein), OsZIP3, showed high expression in the nodes of rice (Oryza sativa). Immunostaining revealed that OsZIP3 was localized at the xylem intervening parenchyma cells and xylem transfer cells of the enlarged vascular bundle in both basal and upper nodes. Neither OsZIP3 gene expression nor encoded protein was affected by either deficiency or toxic levels of Zn. Knockdown of OsZIP3 resulted in significantly reduced Zn levels in the shoot basal region containing the shoot meristem and elongating zone, but increased Zn levels in the transpiration flow. A short-term experiment with the (67) Zn stable isotope showed that more Zn was distributed to the lower leaves, but less to the shoot elongating zone and nodes in the knockdown lines compared with the wild-type rice at both the vegetative and reproductive growth stages. Taken together, OsZIP3 located in the node is responsible for unloading Zn from the xylem of enlarged vascular bundles, which is the first step for preferential distribution of Zn to the developing tissues in rice.

High Silicon Accumulation in the Shoot is Required for Down-Regulating the Expression of Si Transporter Genes in Rice.

Rice requires high silicon (Si) for its high and sustainable yield. The efficient uptake of Si in rice is mediated by two transporters OsLsi1 and OsLsi2, which function as influx and efflux transporters, respectively. Our previous studies showed that the mRNA expression levels of these transporter genes were down-regulated by Si. Herein we investigated the mechanism underlying regulation of OsLsi1 and OsLsi2 expression. There was a negative correlation between the expression level of OsLsi1 and OsLsi2 and shoot Si accumulation when the rice seedlings were exposed to different Si supply conditions. A split root experiment showed that the expression of both OsLsi1 and OsLsi2 was also down-regulated in half the roots without direct Si exposure when the other half of the roots were exposed to Si. Analysis with transgenic rice carrying different lengths of OsLsi1 promoter regions fused with green fluorescent protein (GFP) as a reporter gene revealed that the region responsible for the Si response of OsLsi1 expression is present between -327 to -292 in the promoter. However, this region was not associated with the tissue and cellular localization of OsLsi1. In conclusion, the Si-induced down-regulation of Si transporter genes is controlled by shoot Si, not root Si, and the region between -327 and -292 in the OsLsi1 promoter is involved in this regulation of OsLsi1 expression in rice.

An Aluminum-Inducible IREG Gene is Required for Internal Detoxification of Aluminum in Buckwheat.

Buckwheat (Fagopyrum esculentum Moench) is able to detoxify aluminum (Al) both externally and internally, but the molecular mechanisms underlying its high Al tolerance are not understood. We functionally characterized a gene (FeIREG1) belonging to IRON REGULATED/ferroportin in buckwheat, which showed high expression in our previous genome-wide transcriptome analysis. FeIREG1 was mainly expressed in the roots, and its expression was up-regulated by Al, but not by other metals and low pH. Furthermore, in contrast to AtIREG1 and AtIREG2 in Arabidopsis, the expression of FeIREG1 was not induced by Fe deficiency. Spatial expression analysis showed that the Al-induced expression of FeIREG1 was found in the root tips and higher expression was detected in the outer layers of this part. Immunostaining also showed that FeIREG1 was localized at the outer cell layers in the root tip. A FeIREG1-green fluorescent protein (GFP) fusion protein was localized to the tonoplast when transiently expressed in onion epidermal cells. Overexpression of FeIREG1 in Arabidopsis resulted in increased Al tolerance, but did not alter the tolerance to Cd, Co and Fe. The tolerance to Ni was slightly enhanced in the overexpression lines. Mineral analysis showed that the accumulation of total root Al and other essential mineral elements was hardly altered in the overexpression lines. Taken together, our results suggest that FeIREG1 localized at the tonoplast plays an important role in internal Al detoxification by sequestering Al into the root vacuoles in buckwheat.

Root and shoot transcriptome analysis of two ecotypes of Noccaea caerulescens uncovers the role of NcNramp1 in Cd hyperaccumulation.

The Zn/Cd hyperaccumulator, Noccaea caerulescens, has been studied extensively for its ability to accumulate high levels of Zn and Cd in its leaves. Previous studies have indicated that the Zn and Cd hyperaccumulation trait exhibited by this species involves different transport and tolerance mechanisms. It has also been well documented that certain ecotypes of N. caerulescens are much better Cd hyperaccumulators than others. However, there does not seem to be much ecotypic variation for Zn hyperaccumulation in N. caerulescens. In this study we employed a comparative transcriptomics approach to look at root and shoot gene expression in Ganges and Prayon plants in response to Cd stress to identify transporter genes that were more highly expressed in either the roots or shoots of the superior Cd accumulator, Ganges. Comparison of the transcriptomes from the two ecotypes of Noccaea caerulescens identified a number of genes that encoded metal transporters that were more highly expressed in the Ganges ecotype in response to Cd stress. Characterization of one of these transporters, NcNramp1, showed that it is involved in the influx of Cd across the endodermal plasma membrane and thus may play a key role in Cd flux into the stele and root-to-shoot Cd transport. NcNramp1 may be one of the main transporters involved in Cd hyperaccumulation in N. caerulescens and copy number variation appears to be the main reason for high NcNramp1 gene expression underlying the increased Cd accumulation in the Ganges ecotype.